Bovine somatotropin supplementation during the transition period and early lactation as a management strategy to improve...

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Bovine somatotropin supplementation during the transition period and early lactation as a management strategy to improve physiological adaptations, liver function, health and productivity of dairy cows
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xvii, 267 leaves : ill. ; 29 cm.
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Liboni, Marcio
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Thesis (Ph. D.)--University of Florida, 2004.
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Includes bibliographical references.
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by Marcio Liboni.
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Printout.
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BOVINE SOMATOTROPIN SUPPLEMENTATION DURING THE TRANSITION
PERIOD AND EARLY LACTATION AS A MANAGEMENT STRATEGY TO
IMPROVE PHYSIOLOGICAL ADAPTATIONS, LIVER FUNCTION, HEALTH AND
PRODUCTIVITY OF DAIRY COWS













By

MARCIO LIBONI


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004





























Copyright 2004

by

Marcio Liboni






























To Kellym, with love.















ACKNOWLEDGEMENTS


First, I would like to express my deep gratitude and appreciation to Dr. H. Herbert

Head, my committee chairman and advisor, who gave me all imaginable support to

achieve this goal. In addition, I would like to thank all other members of my doctoral

committee, Dr. Lokenga Badinga, Dr. Kermit C. Bachman, Dr. Michael J. Fields and Dr.

Carlos A. Risco, for the help that I received from them. Special appreciation is extended

to Dr. Lokenga Badinga for his guidance with regard to laboratory work associated with

mRNA for enzymes, and for his willingness to allow me use his laboratory facilities to

accomplish my studies.

Special thanks go to my parents Sidnei and Aurea, to my brother and sister

Marcos and Fernanda and to Neusa for the continuous support given during this doctoral

program. I also would like to give special thanks and recognition to Ms. M. Joyce Hayen,

our laboratory chemist, for the advice and help in conducting the research at the Dairy

Research Unit (DRU), and for helping me like a mother would. I also thank Dr. Mehmet

S. Gulay and Dr. Tomas I. Belloso for the help given during farm work at the DRU, and

to Mary Russell, David Armstrong, James Lindsey, the milkers and feeders and other

personnel at the DRU for the attention and care of the dairy cows used in my experiment.

My thanks also go to Elizabeth S. Johnson for her help and attention during my

laboratory work in Dr. Badinga's laboratory.









I also extend special appreciation to the Department of Animal Sciences of the

University of Florida for giving me the financial support to achieve this goal. I also

would like to acknowledge all other persons who in one way or another have helped me.

Finally, I would like to give thanks to the Lord for all things.















TABLE OF CONTENTS

page


ACKNOWLEDGMENTS............................................................................................ iv

LIST O F TA BLES ....................................................................................................... ix

LIST O F FIG U RE S .......................................................................................................... xiii

A B STRA C T ............................................ ................................................................ xvi

CHAPTER

1 INTRODUCTION................. ................................................................................. 1

2 LITERATURE REVIEW.......................................................................................... 7

Dry Matter (Feed) Intake during the Transition Period ............................................. 7
Changes in Body Weight and Body Condition Score during the Transition Period.. 12
Hormonal and Metabolic Adaptations during the Transition Period......................... 18
Liver Function during the Transition Period ......................................................... 28
Mammary Gland Function during the Transition Period..................................... 36
Diseases and the Transition Period .......................................................................42
Recombinant Bovine Somatotropin (bST) and Lactation in Dairy Cows.................49
Strategies to Improve Metabolic Adaptations during the Transition Period.............. 55

3 MATERIALS AND METHODS ............................................................................... 63

A nim als ............................................................................................................... 63
Treatm ents........................................... ............................................................. .. 64
Experimental Design ........................................................................................ 64
Feeding ................. ................. ......................... ........................................ ........... 65
Body Weight and Body Condition Score....... ............ ........................................ 65
Colostrum and Milk Samples...................................................................... 65
Blood Collection, Handling and Storage.................................... ............ ............... 67
Liver Biopsies, Handling and Storage................................................................... 68
Radioim unoassays................................... ...... ................................................. 69
Second Antibody Preparation............................................................................. 69
Insulin, Somatotropin and IGF-I lodination and Protein Separation ..................70
Insulin A ssay ........................................................ ................. ....................... 72









Somatotropin Assay .............................................................................................74
IG F-I A ssay ................................................................................................................ 76
Extraction of IGF-I from binding proteins........ ......... ........................................... 76
A ssay ............................................................................................................... 77
C calcium A ssay...................................................................................................... 78
G lucose A ssay ...................................................................................................... 80
Non-esterfied Fatty Acids (NEFA) Assay ........................................... .............. 81
P-Hydroxybutyrate (B-HBA) Assay ............................................................... 83
Total Lipid Determination of Liver Samples ..................................... ............ .. 84
Triacylglycerol (TAG) Determination of Liver Samples........................................ 85
Messenger Ribonucleic Acid (mRNA) Determination by DOT BLOT Procedure... 87
Extraction of mRNA ..................................................................................... 87
Dot Blot Membrane Transfer ........................................................................ 88
H ybridization.......................... ......................................................................... 89
Incubation, Film Development and Membrane Stripping................................... 92
Calving Variables, Health Status and Reproductive Performance........................... 92
Statistical A nalyses ............................................................................................... 95
Productive Responses, Physiological Changes, Calving Information and
Reproductive Performance......................................................................... 95
Health information and reproductive performance........................................... 100

4 RESU LTS........................................................................................................... 102

Physiological Adaptations during the Prepartum Period ....................................... 102
Body Weight and Body Condition Score....................................................... 102
Hormones and Growth Factor.......................................................................... 103
Calcium and Metabolites................................................................................. 109
Physiological Adaptations during the Postpartum Period...................................... 116
Body Weight and Body Condition Score....................................................... 116
Hormones and Growth Factor.......................................................................... 126
Calcium and Metabolites.................................................................................. 143
Liver Metabolism of Lipids and Carbohydrates ................................................ 145
C alving V ariables.................................................................................................. 155
Health Information and Reproductive Performance .............................................. 167
Milk Yield and Milk Composition........................................................................ 171

5 D ISC U SSIO N ..................................................................................................... 187

APPENDIX

A RESIDUAL CORRELATION COEFFICIENTS AMONG HORMONES AND
METABOLITES MEASURED FROM 21 D PREPARTUM THROUGH
CALVING IN PLASMA OF HOLSTEIN COWS......................................... 244

B RESIDUAL CORRELATION COEFFICIENTS AMONG HORMONES AND
METABOLITES MEASURED DURING THE EARLY POSTPARTUM
PERIOD (0-63 DIM) IN PLASMA OF HOLSTEIN COWS................................... 245









C SUMMARY OF HETEROGENEITY OF REGRESSION FOR VARIABLES
MEASURED DURING EXPERIMENT ................................................................ 246

D RESIDUAL CORRELATION COEFFICIENTS AMONG LIVER MEASURES
DURING THE TRANSITION PERIOD OF HOLSTEIN COWS......................... 247

E SUMMARY OF TREATMENT EFFECTS OF MULTIPAROUS HOLSTEIN
COWS SUPPLEMENTED WITH bST DURING PREPARTUM OR DURING
POSTPARTUM PERIODS COMPARED TO NON-SUPPLEMENTED
C O N TR O LS ............... .......................................................................................... 248

F SUMMARY OF TREATMENT EFFECTS OF MULTIPAROUS HOLSTEIN
COWS SUPPLEMENTED WITH bST DURING PREPARTUM AND
POSTPARTUM PERIODS COMPARED TO NON-SUPPLEMENTED
C O N TR O LS ......................................................................................................... 249

LIST OF REFERENCES ................................................................................................250

BIOGRAPHICAL SKETCH .................................................................................... 269















LIST OF TABLES


Table Page

1. Dry matter concentrations and chemical composition of anionic close-up diet
(CUD) and fresh-cow TMR fed to Holstein Cows ......................................... ...66

2. Arrangement of tubes for radioimmunoassay of insulin..................................... ...73

3. Arrangement of tubes for radioimmunoassay of ST................................... ....76

4. Arrangement of tubes for radioimmunoassay of IGF-I.......................................78

5. Standards for determination of calcium by flame atomic absorption spectometry.....79

6. Standard dilutions used in the glucose assay ............................................................80

7. Arrangement of tubes and solutions used to make plasma protein-free filtrates........81

8. Standard dilutions for NEFA determination ............................................................82

9. Standard dilutions used for determination of TAG...................................................86

10. Least squares analyses of variance for body weight (BW) and body condition
score (BCS) of Holstein cows during the prepartum period................................... 104

11. Least squares means and SEM for body weight (BW) and body condition score
(BCS) of Holstein cows for treatments and season of calving during the
prepartum period ....................................................................................................... 105

12. Least squares analyses of variance for concentrations of insulin, somatotropin
and insulin-like growth factor-I (IGF-I) in plasma of Holstein cows during the
prepartum period ....................................................................................................... 110

13. Least squares means and SEM for concentrations of insulin(INS), somatotropin
(ST) and insulin-like growth factor-I (IGF-I) for treatments and season of calving
in plasma of Holstein cows during the prepartum period ....................................... 111

14. Least squares analyses of variance for concentrations of calcium and glucose in
plasma of Holstein cows during the prepartum period ............................................. 117









15. Least squares analyses of variance for concentrations of non-esterified fatty acids
and 0-hydroxybutyrate in plasma of Holstein cows during the prepartum period.... 118

16. Least squares means and SEM for concentrations of calcium (Ca), glucose (GLC),
non-esterified fatty acids (NEFA) and 0-hydroxybutyrate (B-HBA) for treatments
and season of calving during the prepartum period ................................................ 119

17. Least squares analyses of variance for body weight (BW) and body condition
score (BCS) of Holstein cows from 0-63 DIM ....................................................... 127

18. Least squares analyses of variance for body weight (BW) and body condition
score (BCS) of Holstein cows from 64-150 DIM ................................................... 128

19. Least squares analyses of variance for body weight (BW) and body condition
score (BCS) of Holstein cows from 0-150 DIM ..................................................... 129

20. Least squares means and SEM for body weight (BW) and body condition score
(BCS) of Holstein cows for treatments and season of calving during various time
periods postpartum .................................................................................................... 130

21. Cubic regression coefficients that describe treatment effects on body weight
(BW) of Holstein cows from 0-150 DIM................................................................ 131

22. Cubic regression coefficients that describe treatment effects on body condition
score (BCS) of Holstein cows from 0-150 DIM ..................................................... 133

23. Least squares analyses of variance for concentrations of insulin, somatotropin
and insulin-like growth factor-I (IGF-I) in plasma of Holstein cows during
0-63 D IM ................................................................................................................... 137

24. Least squares means and SEM for concentrations of insulin (INS), somatotropin
(ST) and insulin-like growth factor-I (IGF-I) in plasma of Holstein cows for
treatments and season of calving during 0-63 DIM................................................ 138

25. Cubic regression coefficients that describe treatment effects on concentration of
somatotropin in plasma of Holstein cows from 0-63 DIM ..................................... 139

26. Cubic regression coefficients that describe treatment effects on concentrations of
IGF-I in plasma of Holstein cows from 0-63 DIM ................................................. 141

27. Least squares analyses of variance for concentrations of glucose and calcium in
plasma of Holstein cows during postpartum period................................................ 146

28. Least squares analyses of variance for concentrations of non-esterified fatty acids
and p-hydroxybutyrate in plasma of Holstein cows during postpartum period........ 147








29. Least squares means and SEM for concentrations of glucose (GLC), calcium (Ca),
non-esterified fatty acids (NEFA) and p-hydroxybutyrate (B-HBA) for treatments
and season of calving during the postpartum period............................................... 148

30. Cubic regression coefficients that describe treatment effects on concentrations
of P-hydroxybutyrate (B-HBA) in plasma of Holstein cows from 0-28 DIM......... 149

31. Least squares analyses of variance for percentages of fat (LFAT) and
triacylglycerol (TAG) in liver of Holstein cows (wet weight basis) during
prepartum and postpartum periods............................................................................ 156

32. Least squares analyses of variance for mRNA expression in liver of Holstein cows
for pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and
microsomal triacylglycerol transfer protein-1 (MTP)............................................ 157

33. Least squares means and SEM for percentages of fat (LFAT) and triacylglycerol
(TAG) in liver of Holstein cows on a wet weight basis, and for liver mRNA
expression of pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase
(PEPCK) and microsomal triacylglycerol transfer protein-i (MTP) for treatments
and day from 21 d prepartum to +28 DIM.............................................................. 158

34. Least squares means and SEM for percentages of fat (LFAT) and triacylglycerol
(TAG) in liver of Holstein cows on a wet weight basis for each day of biopsy ....... 159

35. Least squares means and SEM for mRNA expression in liver of Holstein cows for
pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK)
for each day of biopsy ............................................................................................... 160

36. Least squares means and SEM for mRNA expression in liver of Holstein cows for
microsomal triacylglycerol transfer protein 1 (MTP) for each day of biopsy.......... 161

37. Least squares analyses of variance for colostrum immunoglobulin content, calf
birth weight and calving difficulty of Holstein cows.............................................. 168

38. Least squares means and SEM for colostrum immunoglobulin content (Colost),
calf birth weight (CLFWT) and calving difficulty (DCV) of Holstein cows for
treatment groups and season of calving .................................................................. 169

39. Disease incidence and reproductive performance of multiparous Holstein cows
supplemented or not with bST .................................................................................. 172

40. Summary of logistic regression analyses of the associations between TRT, SEA
and diseases, and variables of reproductive performance of multiparous Holstein
cow s during 0-60 D IM ............................................................................................. 173

41. Least squares analyses of variance for milk yield of Holstein cows during 3-63
and 64-150 DIM............................................................................................................ 178








42. Least squares analyses of variance for milk yield of Holstein cows during 3-150
DIM and for Actual 305-d M Y ............................................................................ 179

43. Least squares means and SEM for milk yield (MY) of Holstein cows for treatments
and season of calving ........................................................................................... 180

44. Cubic regression coefficients that describe treatment effects on milk yield of
Holstein cows during 3-150 DIM ......................................................................... 182

45. Least squares analyses of variance for percentages of milk fat and milk protein
and somatic cell count (SCC) in milk of Holstein cows from 3-63 DIM ................ 185

46. Least squares means and SEM for percentages of milk fat (FAT) and milk protein
(PROT), and somatic cell count in milk (SCC) of Holstein for cows treatments and
season of calving ........................................................ ...................................... 186















LIST OF FIGURES


Figure Page

1. Some metabolic pathways of adipocytes and their adaptations during transition
period ......................................................................................................................... 2 1

2. Schematic drawing of rat liver tissue...................................................... .............. 29

3. Relationships among metabolic disorders............................................................. 48

4. Backbone structure of the hGHx(hGHbp)2 complex .................................... ...50

5. Least squares means depicting body weights (BW) of multiparous Holstein cows
supplemented or not with bST during the prepartum and/or postpartum periods.
Vertical dashed line indicates calving. Shaded area represents time during which
full dose of bST was supplemented to all cows........................................................ 106

6. Least squares means depicting body condition scores (BCS) of multiparous Holstein
cows supplemented or not with bST during the prepartum and/or postpartum periods.
Vertical line indicates calving. Shaded area represents time during which full dose
of bST was supplemented to all cows......................................................................... 107

7. Least squares means depicting insulin concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or postpartum
periods.Vertical dashed line indicates calving. Shaded area represents period after
calving......................................................................................................................... 112

8. Least squares means depicting somatotropin concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or the
postpartum periods. Vertical dashed line indicates calving. Shaded area represents
period after calving ................................................................................................ 113

9. Least squares means depicting IGF-I concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or postpartum
periods. Vertical dashed line indicates calving. Shaded area represents period after
calving ......................................................................................................... ......... 114









10. Least squares means depicting calcium concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or during the
postpartum periods. Vertical dashed line indicates calving. Shaded area represents
period after calving.......................................... .................................................. 120

11. Least squares means depicting glucose concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or during
the postpartum periods. Vertical dashed line indicates calving. Shaded area
represents period after calving. ................................................................................. 121

12. Least squares means depicting NEFA concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or during
the postpartum periods. Vertical dashed line indicates calving. Shaded area
represents period after calving. ................................................................................ 122

13. Least square means depicting p-hydroxybutyrate (B-HBA) concentrations in
plasma of multiparous Holstein cows supplemented or not with bST during the
prepartum and/or during the postpartum periods. Vertical dash line indicates
calving. Shaded area represents period after calving.............................................. 123

14. Cubic regression curves depicting body weight changes of multiparous Holstein
cows supplemented or not with bST during the prepartum and/or postpartum periods.
Model: bST, season, bST*season effect, using cow(bST*season) as error, with wk
to the third order as covariate. Shaded area represents time during which full dose
of bST was supplemented to all cows ..................................................................... 132

15. Cubic regression curves depicting body condition score of multiparous Holstein
cows supplementedor not with bST during the prepartum and/or postpartum periods.
Model: bST, season, bST*season effect, using cow(bST*season) as error, with wk
to the third order as covariate. Shaded area represents time during which full dose
of bST was supplemented to all cows..................................................................... 134

16. Cubic regression curves depicting somatotropin concentrations in plasma of
multiparous Holstein cows supplemented or not with bST during the prepartum
and/or postpartum periods. bST, season, bST*season effect, using
cow(bST*season) as error, with day to the third order as covariate....................... 140

17. Cubic regression curves depicting IGF-I concentrations in plasma of multiparous
Holstein cows supplemented or not with bST during the prepartum and/or postpartum
periods. Model: bST, season, bST*season effect, using cow(bST*season) as error,
with day to the third order as covariate................................................................... 142

18. Cubic regression curves depicting p-hydroxybutyrate (B-HBA) concentrations in
plasma of multiparous Holstein cows supplemented or not with bST during the
prepartum and/or postpartum periods. Model: bST, season, bST*season using
cow(bST*season) as error, with day to the third order as covariate ....................... 150









19. Least squares means depicting percentages of total fat in liver (LFAT, wet weight
basis) of multiparous Holstein cows supplemented or not with bST during the
prepartum and/or postpartum periods. a'b Superscripts within day from calving
indicate means that differed at P<0.05.................................................................... 162

20. Least squares means depicting percentage of triacylglycerol in liver (TAG, wet
weight basis) of multiparous Holstein cows supplemented or not with bST during
the prepartum and/or postpartum periods. a,b Superscripts within day from
calving indicate means that differed at P<0.05....................................................... 163

21. Least squares means depicting mRNA expression ofpyruvate carboxylase (PC)
of multiparous Holstein cows supplemented or not with bST during the prepartum
and/or postpartum periods. ab Supercripts within day from calving indicate means
that differed at P<0.05............................................................................................... 164

22. Least squares means depicting mRNA expression of phosphoenolpyruvate
carboxykinase (PEPCK) of multiparous Holstein cows supplemented or not with
bST during the prepartum and/or periods. ab Superscripts within day from calving
indicate means that differed at P<0.05.................................................................... 165

23. Least squares means depicting mRNA expression of microsomal triacylglycerol
transfer protein 1 (MTP) of multiparous Holstein cows supplemented or not with
bST during the prepartum and/or postpartum periods. a,b Superscripts within day
from calving indicate means that differed at P<0.05 .............................................. 166

24. Least squares means depicting daily milk yield of multiparous Holstein cows
supplemented or not with bST during the prepartum and/or postpartum periods.
Shaded area represents time during which full dose of bST was supplemented to
all cow s............................................................................................................. ......... 181

25. Cubic regression curves depicting mean daily milk yield of multiparous Holstein
cows supplemented or not with bST during the prepartum and/or postpartum
period. Model: bST, season, and the two-way interaction of bST*season effect,
using cow nested with bST*season as error, with day to the third order as covariate.
Shaded area represents time during which full dose of bST was supplemented to
all cow s............................................................................................................. ......... 183

26. Cubic regression curves depicting mean daily milk yield (MY) of multiparous
Holstein cows supplemented or not with bST (500 mg/14 d) from 64-150 DIM.
Model: bST, season, bST*season effect, using cow(bST*season) as error, with
day to the third order as covariate. ....................................................................... 184















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

BOVINE SOMATOTROPIN SUPPLEMENTATION DURING THE TRANSITION
PERIOD AND EARLY LACTATION AS A MANAGEMENT STRATEGY TO
IMPROVE PHYSIOLOGICAL ADAPTATIONS, LIVER FUNCTION, HEALTH AND
PRODUCTIVITY OF DAIRY COWS

By

Marcio Liboni

August, 2004

Chair: H. Herbert Head
Major Department: Animal Sciences

Objectives were to evaluate effects of supplemental bST (0.4 mL/14d, 10.2 mg/d,

POSILAC) during prepartum and/or postpartum periods and early lactation on

physiological adaptations [changes in body weight (BW), body condition score (BCS)

and concentrations of insulin, somatotropin, IGF-I, calcium, glucose, non-esterified fatty

acids and P-hydroxybutyrate in plasma], on metabolism of lipids and carbohydrates in

liver (fat deposition and gene expression of key enzymes), on health, and on milk yield

(MY) and composition. Multiparous Holstein cows were assigned randomly to a 2x2

factorial arrangement of treatments (TRT) to give four groups (I=no bST, n=26; II=bST

postpartum, n=25; I=bST prepartum, n=27; IV= bST prepartum and postpartum, n=25).

Biweekly supplementation of bST began 21 d before expected calving through 63 d in

milk (DIM). From 64 DIM through the end of lactation, all cows were supplemented








biweekly with a full bST dose (1.2 m/14d, 35.7 mg/d). Blood samples were collected

from all cows thrice weekly, and liver biopsies were taken from -9 cows per TRT group

at --21, +2, +14 and +28 d from calving. Cows were fed an anionic diet prepartum and a

cationic diet during the postpartum period for ad libitum consumption. Supplementing

bST only prepartum (III) did not impact physiological adaptations, but in TRT II cows

(bST Post only), supplementation increased concentrations of P-hydroxybutyrate in

plasma and accumulation of triacylglycerol in liver during the first 28 DIM. Milk

production, incidence of diseases and reproductive performance of TRT II cows were

similar to cows on TRT I and III (control and bST Prepartum). For cows on TRT IV (bST

Prepartum and Postpartum), milk production was increased by 12.8% (4.64 kg) during

the first 150 DIM, and milk production was higher until the end of lactation, and the cows

were healthier than controls or cohorts. Compared to controls, cows on TRT IV had

similar BW and BCS changes, showed increased concentrations of IGF-I in plasma

during experiment, similar concentrations of hormones and metabolites in plasma, and

improved liver function. These observations indicate that supplementation of low doses

of bST improves metabolic adaptations and health of dairy cows, which is reflected in

increased milk production.


xvii















CHAPTER I
INTRODUCTION

For dairy cows, the period around parturition has been considered the most

critical phase during the whole lactation cycle (Chilliard, 1999). During this phase, there

are physiological priorities that require metabolic adaptations of many tissues and organs

to allow cows to meet general maintenance of body and their productive needs (Bauman

and Elliot, 1983). These adaptations provide adequate nourishment to the fetus during its

final phase of gestational development (Bell, 1995), allow the initiation of lactogenesis

and parturition (Head, 2000), and thereafter copious milk production (Capuco et al.,

1997).

Late pregnancy imposes a substantial cost to the dairy cow. The total

requirements for nutrients at the end of pregnancy are about 75% greater than in

nonpregnant animals of the same body weight (Bauman and Currie, 1980). At 250 d of

pregnancy, a 35 kg bovine fetus requires daily a total of 2,335 Kcal of energy, and about

220 g of metabolizable amino acids (AA) for its growth. In addition, the growth of the

uteroplacental tissues (placentomes, endometrium, myometrium) also imposes an extra

requirement for nutrients. It has been estimated that a gravid uterus (64 kg at 250 d of

pregnancy) utilizes 46, 72 and 12% of the total maternal supply of glucose, amino acids

and acetate, a substantial demand for nutrients during late pregnancy (Bell, 1995).

In addition to development of uterine tissues and the fetus, there is a marked

increase in mammary gland activity as parturition approaches, a period termed








lactogenesis. Lactogenesis has been divided into two phases (I and II), and in dairy cows

it begins about 30-14 d before calving. Phase I is characterized by a renewal of damaged

and senescent mammary epithelial cells in the gland (Capuco et al., 1997), by

morphological and biochemical changes in these cells, and by the synthesis of a

precolostral fluid (secretion composed of proteins, lipids and carbohydrates). Phase II

begins immediately before parturition and is characterized by copious secretion of

colostrum (colostrogenesis; Oliver and Sordillo, 1989). For the latter process to occur,

there must be an increase in uptake of nutrients such as glucose, amino acids and

nonesterified fatty acids (NEFA) by the mammary gland to synthesize lactose (Holt,

1983), milk proteins (Boisgard et al., 2001) and milk lipids (Shennan and Peaker, 2001).

This is accomplished by the increased flow of blood to the gland, that has been observed

in both small and large ruminants. In dairy goats, beginning at d 2 prepartum and

continuing through d 1 and 0.5 prepartum, there was a 400% increase in blood flow to the

gland and 150% increase in uptake of glucose when compared to levels observed during

d 3 prepartum (Davis et al., 1979). In dairy cows, it was observed that the increase in

blood flow began at d 2 prepartum (Malven et al., 1987). The galactopoietic activity of

the mammary gland imposes a significant demand to the dairy cow during this very

critical phase. For example, the production of just 10 kg of colostrum on the day of

calving requires an extra 11 Mcal of energy, 140 g of protein, 23 g of calcium, 9 g of

phosphorus and 1 g of magnesium that must be obtained from the diet or by mobilization

of body stores (Goff and Horst, 1997).

After calving (following the onset of lactation) the function of the mammary

gland is the physiological priority for the dairy cow. The gland has a massive requirement








for glucose to produce lactose and provide energy, and also for other nutrients that are

used to synthesize other milk components. Estimates are that at d 4 of lactation, a dairy

cow producing 30 kg milk/d has a mammary uptake of 85 and 75% of the daily predicted

postabsorptive glucose and amino acid supply (Bell et al., 1995). These data agreed with

earlier estimates reported by Bickerstaffe and Annison (1974); they also estimated that

the mammary uptake of glucose during lactation could reach about 85% of the total

available sources.

Because of the increased and very substantial demand for nutrients during the

peripartum period, the dairy cow faces great nutritional challenges as parturition

approaches to meet these demands. During the prepartum period there is a decrease in

feed intake, and during the early postpartum period, feed intake does not meet the

requirements for milk production (Grummer, 1995). To obtain adequate quantities of

nutrients for maintenance and milk production, the dairy cow must mobilize its body

reserves to meet the demands (Drackley, 1999), a physiological process of adaptation

termed homeorhesis. It occurs in conjunction with the maintenance of the physiological

equilibrium, termed homeostasis (Bauman and Currie, 1980).

During periods of homeorhesis there is overall adaptation of the body to meet

nutritional needs. Significant changes occur in the metabolism of body tissues responsible

for storage of nutrients, and in organ(s) where the biochemical reactions responsible for

synthesis of nutrients occurs (e.g., liver). Some of these changes include increased

lipolysis and decreased lipogenesis in the adipose tissue, which leads to the release of

NEFA (McNamara, 1991); increased hepatic gluconeogenesis and glycogenolysis

(Greenfield et al., 2000) that leads to increased synthesis of glucose; decreased use of








glucose and, in general, increased use of lipids as energy sources in all body tissues

(Bauman and Currie, 1980); the mobilization of protein reserves from muscle and other

body tissues, which gives rise to use of lactate and AA in the process of gluconeogenesis

(Bauman and Elliot, 1983); and increased calcium absorption from the intestines and

mobilization from bone tissue (Kronfeld, 1971).

These described adaptations are mediated by hormonal changes that occur to

initiate parturition and lactogenesis (Grummer 1995). Insulin and glucagon have primary

roles in the process of homeostatic regulation. Their principal function is to maintain the

constancy of glucose supply. In addition to the homeostatic hormones, there are changes

in concentrations of other hormones such as estrogen (E2), progesterone (P4),

parathormone (PTH), vitamin D3 (VIT D3), prolactin (PRL), placental lactogen (PL) and

somatotropin (ST; Tucker, 1985). Subsequent studies indicated that some of these

hormones specifically target tissues involved in metabolic adaptations, and therefore they

were considered to be homeorhetic hormones (Chilliard, 1999; Ingvartsen and Andersen,

2000).

The peripartum period is characterized by the occurrence of many commonly

known metabolic disturbances/diseases of dairy cows (Drackley, 1999). Fatty liver,

clinical and subclinical ketosis, and milk fever (MF) are just three of the disorders that

are caused by imbalances in the metabolic adaptations described previously (Morrow,

1976; Goff and Horst, 1997; Heuer et al., 1999). In addition, these primary disorders also

predispose the animals to other disorders such as metritis (MET), mastitis (MAST),

displaced abomasum (DA) and poor reproductive performance (Gearhart et al., 1990;









Staples et al., 1990). As a consequence, they lead to poor productive performance during

the lactation that follows parturition.

Due to the metabolic complexity, physiological priorities and changes in DMI

observed during the periparturient period, some authors referred to this phase as the

"transitional phase" (Gerloff, 1988) or "transition period" (Grummer, 1995). By the

definition of both authors, this period begins at 3 wk prepartum and extends through

calving. In addition to this prepartum period, Grummer (1995) also suggested a

postpartum period that would begin right after parturition and last until 3 wk of lactation;

thus the total period is 6 wk. Drackley (1999) also stressed and reinforced the importance

of defining the periparturient phase as the transition period, and supported the length as

defined by Grummer (1995), because of the greater occurrence of health problems that

had their genesis during this relatively short period of time (Curtis et al., 1983; Thompson

et al., 1983; Goff and Horst, 1997; Kelton et al., 1998).

One management strategy that has been considered to improve the metabolic

adaptations during the transition period is supplementing exogenous bovine somatotropin

(bST). The well-known effects of bST on feed intake, on metabolic and lactational

hormones, and on the ability to increase the availability of glucose precursors in the

lactating cow may be an important way to bring about desired effects during the

transition period (Gluckman and Breier, 1987; Pell and Bauman, 1987; Bauman and

Vernon, 1993; Eppard et al., 1996). It is known from previous studies (Stanisiewski et al.,

1992; Garcia-Gavidia et al., 2000, Gulay et al., 2003b) that supplementing dairy cows

with low doses of bST around parturition (prepartum and postpartum injections) had

small but positive effects on metabolism, hormones and milk production.









Garcia-Gavidia et al. (1998) showed that supplementation of cows with low doses

of bST during the prepartum and postpartum periods tended to allow them to better

maintain their feed intake during the prepartum period and they also had more rapid

increase in feed intake postpartum. Although changes observed were not significant,

bST-supplemented cows produced more milk during the first 65 d postpartum, and these

supplemented cows also showed changes in important hormones and metabolites that

favored this greater milk yield.

Therefore, the objectives of this research were to evaluate the influence of

supplementing multiparous Holstein cows fed ad libitum, with low doses of recombinant

bST (10.2 mg/day, 0.4 ml/14 d of POSILAC, Monsanto Company, St. Louis, MO)

during the peripartum and/or postpartum periods. Evaluation criteria for supplementation

were milk production and composition, changes in BW and BCS, calving variables,

plasma concentrations of specific hormones and metabolites, changes in liver metabolism

of lipids and carbohydrates, and the incidences of calving-related diseases.















CHAPTER 2
LITERATURE REVIEW

Dry Matter (Feed) Intake during the Transition Period

Adequate nutrition during the transition period is of paramount importance for the

dairy cow, as previously mentioned. Among the many aspects related to nutrition, dry

matter intake (DMI) is fundamentally important because it establishes the amount of

nutrients available to an animal to maintain health and to support milk production

(National Research Council [NRC], 2001), and the magnitude and duration of the need of

the animal to draw upon labile body reserves to meet her requirements for specific

nutrients and energy. Despite the increased requirements for energy and nutrients,

prepartum and postpartum DMI of dairy cows is not optimal. During the prepartum

period, DMI decreases gradually as cows approach time to calve (Bell, 1995), and after

calving DMI does not meet the requirements for the copious production of milk during

the early weeks of lactation (Bauman and Currie, 1980; Drackley, 1999).

A number of studies have been carried out during the past several decades that

evaluated the pattern of DMI during the transition period (Coppock et al., 1972;

Marquardt et al., 1976; Bertics et al., 1992; Vazquez-Anon et al., 1994; Grum et al.,

1996; Greenfield et al., 2000). General observations were that a gradual decline in DMI

began about 3 wk prepartum, with the most dramatic decrease occurring during the final

week preceding calving (Grummer, 1995). Marquardt et al. (1976) observed a decrease in

DMI beginning at 7 d before calving in both young (first calf heifers, average age 28.4









mo) and aged multiparouss, average age 69.8 mo) cows. The DMI for heifers and cows

on d 1 prepartum was 13% less than the DMI (% BW) observed at 7 d prepartum. These

results indicated that DMI decreases in dairy cows occurred irrespective of their age

(heifer and cows) around calving. Observations by Ingvartsen and Andersen (2000) also

showed that significant decreases in DMI occurred in both heifers and dairy cows around

parturition. The percentage decrease observed was 20% in both animal classes when

compared to -21 d DMI.

Greenfield et al. (2000) reported a greater depression in prepartum DMI of

Holstein dairy cows. In their experiment, the decrease was about 30% and began at 7 to

10 d before calving. However, in a study conducted by Vazquez-Anon et al. (1994), the

decrease in prepartum DMI was not seen until 2 d before calving; extent of depression

was 40% when compared to the previous day DIM (d 3 prepartum). Recent results

(Gulay et al., 2003a) reported that the decrease in DMI began 8 d prepartum, but the

decrease only was significant during the last 4 d before calving. The mean DMI

depression among the groups of cows was from 32 to 45% compared to levels observed

at d 8 prepartum.

With regard to the postpartum DMI, many observations indicated a substantial

increase following parturition (Grummer et al., 1995; Grum et al., 1996; Garcia-Gavidia,

1998; Santos et al., 1999; Greenfield et al., 2000; Doepel et al., 2002; Gulay et al., 2003a;

McNamara et al., 2003, Selberg et al., 2004; Gulay et al., 2004), but the requirements for

milk production in these studies also were far greater than the amount of nutrients

ingested. Overall, during the early postpartum period, dairy cows show a sudden and

marked increase in nutrient requirements at a time when DMI lags far behind (Bell, 1995;








Drackley, 1999). This leads to a period of negative energy balance (NEB), during which

the dairy cow must rely on the mobilization of body tissue reserves (adipose tissue and

protein from muscles) to meet her nutrient demands (Staples et al., 1990; de Vries and

Veerkamp, 2000; Reist et al., 2002; Gulay et al., 2003a).

In a comprehensive study designed to investigate the general pattern of energy

balance (EB) during early lactation in dairy cattle, de Vries et al. (1999) observed that

maximum energy requirements for milk production occurred at wk 7, 5 and 6 for first,

second and third lactation cows. On the other hand, averaged among all cows, maximum

energy intake that would be sufficient to meet the demands occurred only by 72 DIM. In

other studies, positive EB was achieved by d 22 (Santos et al., 2000), d 85 (Walters et al.,

2002) and d 42 (Gulay et al., 2003a) of lactation. Despite divergent results with regard to

time to achieve positive EB, all studies indicated that dairy cows experienced a period of

NEB after calving.

The causes of the prepartum decrease in DMI are not well described or well

understood. Due to the dramatic physiological changes occurring during the transition

period, Forbes (1971) speculated that significant endocrine changes, such as the increase

in estrogen around calving, were the likely causes for the depression in DMI. To evaluate

this, he studied the effect of estrogen injections on voluntary intake of hay and

concentrate diets by sheep. When animals were injected with quantities of estrogens that

produced similar blood concentrations of the hormone to those found during late

pregnancy, no significant effects were observed for hay intake; however, there were

significant decreases in concentrate intake. This was the first evidence that estrogens

could be responsible, at least in part, for the decrease in DMI around parturition.








To evaluate if elevated estrogen and other traits of late pregnancy were associated

with depressed DIM during the transition period of Holstein cows, Erb et al. (1982)

conducted an extensive statistical analysis using data from 86 multiparous cows. Their

objective was to establish some associations between DMI (as percentage of BW) during

the last week before calving with many variables, including the hormones estrone and

estradiol a and p. Cow age and quantitative indices of seasonality (temperature,

photoperiod, and plasma PRL) were negatively associated with percentage DMI, but

none of the other hormones in plasma were associated with DMI (as percentage of BW).

More recently, Grummer et al. (1990) reported interesting results of a field study

designed to evaluate the role of estrogen on ruminant DMI. Their experiment consisted of

injecting nonpregnant lactating (260 d of lactation) and nonlactating Holstein cows with

estradiol-17P benzoate during 14 d of feeding, followed by 3 d of fasting, to mimic the

prepartum transition period. In the lactating group, estrogen injected cows showed

decreased DMI, but not the controls. The decrease was 39% compared to baseline pre-

injection period values of the lactating cows. Because milk production was negatively

impacted by estrogen (decrease of 65% in production compared to baseline values), it

was not possible to determine if the DMI depression was due to direct effects of estrogen;

however, the non-lactating group of cows showed no effects of estrogen injections.

Goff and Horst (1997) proposed a different reason for the decrease in DMI

observed shortly before calving. Their proposed reason for decrease was based on the

concentrations of endogenous opioids (P-endorphin) seen around parturition. Plasma

concentrations of these peptides typically increase prepartum to reduce the perception of

pain by the cow during this phase. Because these compounds are used in treatment of









diarrheal disease in humans to decrease the motility of the gastrointestinal tract, they may

have a similar effect on the dairy cow, with one effect reflected as decreased DMI.

Other possibilities for causes of depression in DMI were evaluated by Hayirli et

al. (2002). In their study, data from 699 Holsteins fed 49 diets during the final 3 wk of

gestation were compiled from 16 experiments conducted during the 1990s. Objectives

were to assess associations among animal factors (parity and BCS) and dietary factors

(concentrations of organic macronutrients NEL, CP, RUP, RDP, NDF, NFC, ADF, EE

and ash concentrations) with the DMI depression observed during the prepartum phase of

the transition period. The DMI (% of BW) decrease prepartum was positively correlated

with parity and NFC, and negatively correlated with BCS, NDF and EE. Among all

causes, day of gestation, other animal factors and dietary factors accounted with 56.1%,

19.7% and 24.2%, respectively, in the variation of DMI. The DMI decreased 32.2%

during final 3 wk of gestation, and 88.9% of that decline occurred during the final week

of gestation, which essentially agreed with previous findings. Average daily DMI during

the final 3 wk of gestation for cows was greater than for heifers (1.88 vs. 1.69% of BW,

respectively). These results indicated that pregnancy and animal factors (physiological

factors) were the main reasons for decreased DMI observed approaching parturition; and

that those from nutritional origin accounted for only part of the decrease and certainly

played a less important role.

Clearly, results described and evaluated previously indicated that the transition

period is marked by nutritional challenges. The hallmarks were the decrease of DMI

when parturition was imminent (last week before calving), and the sluggish increase in

DMI during the first weeks of lactation, which is a period of tremendous demands for








nutrients. Despite extensive efforts to understand the physiological basis for these events

during the last several decades, the causes for the depression in DMI during the

prepartum transition period still are unknown. Some significant associations between

variables of seasonality, animal factors (BCS) and a few dietary factors were identified,

but the real underlining reasons) for such behavior remains to be discovered.

Changes in Body Weight and Body Condition Score during the Transition Period

The decrease in DMI seen during the transition period triggers a cascade of

metabolic responses in dairy cows. Among these is the mobilization of nutrient

precursors from body reserves (adipose tissue [NEFA] and muscles [AA]) to meet the

demands (Bell, 1995). If dairy cows are either mobilizing (decreasing) or increasing their

body reserves, changes in body weight will occur. However, as Klosterman (1972)

indicated, the assessment of body weight changes alone does not account for factors other

than weight and frame size, and it is not the most suitable way to observe changes in cow

metabolism. Therefore, in addition to BW measurements, it is important to evaluate body

condition (BC), or status of cow fitness pertaining to degree of body fat (Wildman et al.,

1982).

Useful systems developed to define means to score body condition, or define

body condition score (BCS) of dairy cows in the US have been proposed by Wildman et

al. (1982), Edmonson et al. (1989) and Ferguson et al. (1994). The first system proposed

use of a scale from 1 to 5 to score thin (underconditioned) and fat (overconditioned)

cows, respectively. Subsequent systems used the same scale with the inclusion of 0.25

point increments between scores, thus functioning as a 17-point scale. Despite these

differences in the scoring systems, all essentially achieved the same objective of








permitting BCS of dairy cows to be described during all phases of lactation, including the

dry period.

The assessment of BW and BCS changes in dairy cows has been the focus of

attention in a great number of experiments. Investigators were interested to observe

whether these changes were positively or negatively associated with milk yield (Wildman

et al., 1982; Stanisiewki et al., 1992; Domeq et al., 1997), reproductive performance

(Studer, 1998; Formigoni and Trevisi, 2003) or health (Gearhart et al., 1990; Smith et al.,

1997). With regard to transition cow studies, there also was a great interest in evaluating

BW and BCS changes, because the BCS assesses the amount of metabolizable energy

stored in fat and muscles (Edmonson et al., 1989), an important qualitative method to

indirectly monitor nutritional status of transition cows.

Due to the nutritional challenges that dairy cows face during the transition period,

changes in BW and BCS are expected during this period. General observations are that

both BW and BCS would be lost beginning at calving and this loss continues through 4-8

wk of lactation, a period when dairy cows reach positive energy balance (Fronk et al.,

1980; Doepel et al., 2002; Walters et al., 2002; Pushpakumara et al., 2003). Expulsion of

the calf and fetal membranes at time of calving and the dramatic decrease in DMI

observed around calving are two of the causes responsible for these losses (Smith et al.,

1997). The extent of BCS loss reported by some authors varied from 0.2 to 1.05 pt, and it

was correlated with BW loss. It has been estimated that a change in one unit of BCS

corresponds to 56 kg of live weight change in the dairy cow (Otto et al., 1991). Because

each 1-kg BW loss corresponds to -4.92 Mcal of NEL (National Research Council [NRC],

1989), the energy loss from body reserves can be calculated by monitoring BCS scores.








Typically, cows lose from 55 to 289 Mcal of NEL (BCS loss of 0.2 and 1.05 pt) during

the first 4-8 wk of lactation, as mentioned previously.

It is after these first weeks postpartum that dairy cows begin to recover the BCS

(energy) and BW lost during early lactation. Most authors (Staples et al., 1990;

Rukkwamsuk et al., 1999; Pushpakumara et al., 2003) have shown that moderate changes

occur after cows reach a state of positive energy balance (de Vries et al., 1999). Wildman

et al. (1982) showed that relative to days in milk (DIM), dairy cows at less than 80

(n=570), between 80 and 159 (n=595), between 160-239 (649), and greater than 239 DIM

(n=797) had average BCS of 2.51, 2.70, 2.95 and 3.39 pt, respectively. During the dry

period, average BCS was 3.37 pt. for a total of 462 cows evaluated.

However, it is important to mention that it is during the dry period, of which

about one-half is the prepartum phase of the transition period, that dairy cows recover

from the extensive metabolic load imposed during the previous lactation. It also is during

this time period that they must adjust their physiological systems to have a smooth

transition from parturition to the next lactation (Bauman and Elliot, 1983). Therefore,

recommendations are that dairy cows should maintain a BCS between 3.25 and 3.75 pt

(average of 3.5 pt) during the -60 d dry period and to recover about 0.25-050 pt that was

lost during the early lactation period (Wildman et al., 1982; National Research Council

[NRC], 2001). This recommended calving BCS would indicate that they have replenished

their body reserves, and also would avoid excess deposition of adipose tissue, which

often has been linked with the increased incidence of health disturbances postpartum

(Gerloff, 1986; Maisey et al., 1993; Laven and Andrews, 1998).








If dairy cows are underconditioned during the dry period, losses in BCS actually

can begin to occur before calving, as demonstrated by Domeq et al. (1997). In their study,

early dry period dairy cows, with an average BCS of 2.77 pt (0.58 pt less than the

recommended [Wildman et al., 1982]) began to lose BCS during the last 2 wk before

calving. The BCS at parturition averaged 2.66 pt, and mean loss of BCS during the first 4

wk of lactation was 0.62 pt, or = 170.82 Mcal of NEL. This showed that dry-period

undercondition was not desirable for transition dairy cows and led to loss of BW during

the prepartum transition period. This also led to dairy cows being more vulnerable to

metabolic disorders, such as fatty liver (Morrow, 1976; Grummer, 1993).

The extent of BCS loss after calving varied among cows with different BCS at

parturition. Gerloff et al. (1986) observed that dairy cows with greater BCS at calving

(3.29, 3.03 and 2.97) had greater BCS loss after parturition (1.17, 0.8 and 0.72, pt,

respectively). Pedron et al. (1993) observed a similar pattern of BCS loss after

parturition. In their study, dairy cows with BCS of 4.0, 3.5 and 3.0 at calving had greater

loss of BCS during the first 8 wk of lactation (1.05, 0.8 and 0.6 pt, respectively).

However, Smith et al. (1997) did not find any correlation between BCS at parturition and

BCS loss during the early postpartum of obese cows (BCS 4.3) and cows with the

recommended BCS (3.5). Even under their treatment regimens, which included restricted

feeding of obese and normal BCS groups from 14-45 DIM, overall BCS losses were

about 0.90 pt during the first 45 d of lactation.

Losses of BCS during the early postpartum period did not seem to impact milk

yield. Pedron et al. (1993) obtained results that suggested that overconditioned dairy

cows at calving (4.5 BCS) did produce more milk from 15-60 DIM than cows with BCS









of 3.0 and 3.5. The milk yields were 37.54, 39.86 and 45.07 kg/d for cows with BCS of

3.0, 3.5 and 4.5, respectively. Despite the numerical differences, the milk yield

differences across groups were not significant. Others reported that overconditioned cows

or those with recommended BCS at calving produced the same amount of milk during

early lactation, and also that loss of BCS did not impact milk production. In a study

conducted by Fronk et al. (1980), control and overconditioned cows at calving (BCS 3.0

and 3.8 pt., respectively) had BCS losses of 0.36 and 0.26 pt. during 14-63 DIM. The MY

for both groups was = 34.5 kg/d during this period. In addition, Smith et al. (1997)

reported that average MY did not differ among obese and normal-conditioned cows

during the first 42 DIM. The BCS loss and average MY across groups during the period

was -0.9 pt and 38 kg/d. Similarly, Busato et al. (2002) also observed that significant

BCS loss during the first 56 DIM (> 0.75 pt.) did not impact MY of fat and lean cows

(BCS > or < than 3.25 prepartum). Average MY of treatment groups was 28.1 kg/d

throughout experimental period.

At the physiological level, BCS losses have been correlated with concentrations

of NEFA in plasma (Bauman and Currie, 1980). During the transition period, there was

increased mobilization of adipose tissue from body stores beginning one week before

parturition, regardless of the BCS status (underconditioned, normal or overconditioned;

Santos et al., 2000; Busato et al., 2002; Gulay et al, 2003a). Another important

observation was that postpartum concentrations of NEFA in plasma of overconditioned

cows were significantly greater during the first weeks following calving than in cows

with adequate BCS. Possible reasons for differences were because these cows had greater

stores of body fat available to be mobilized, and/or due to differences in the metabolic









adaptations that had occurred during the transition period, particularly beginning at the

days (2-3) before parturition.

With regard to BCS and health, risks of disease incidences are strongly associated

with BCS during the prepartum period and with losses of BCS during the early

postpartum period. Morrow (1976) was one of the first investigators to observe that

overfeeding dairy cows during the dry period elevated the risk factor for the incidence of

"fat cow syndrome". This syndrome is related to the excessive mobilization of adipose

tissue during the transition period, and always has disastrous consequences for the dairy

cow (Grummer et al., 1993). Cows with this syndrome usually had one or more of the

following disorders: milk fever, ketosis, displaced abomasum, indigestion, retained fetal

membranes, metritis, mastitis or salmonelosis (Morrow, 1976).

Subsequent studies (Gearhart et al., 1990; Heuer et al., 1999) reported that when

cows were overconditioned at dry-off they had more reproductive and foot problems.

Overconditioning during early lactation was related to higher incidence of metritis and

milk fever, and that BCS loss during the dry period also was associated with dystocia.

For subsequent reproductive efficiency, it has been proposed that serious energy deficit

during the postpartum transition period and early lactation negatively impacted the

conception rate at first insemination, number of days open, and number of inseminations

to conception for dairy cows. This energy deficit led to decreased blood concentrations of

insulin, insulin-like growth factor-I, and progesterone, which had carryover effects on

postpartum ovarian activity (Staples et al., 1990; Formigoni and Trevisi, 2003;

Pushpakumara et al., 2003). Because the BCS/weight loss seen during the early








postpartum period occurs due to an energy deficit, it has been directly correlated with

reproductive performance of dairy cattle during the early postpartum period.

The assessment of BCS during the transition period is an important method to

critically evaluate the nutritional status of dairy cows. The metabolic changes of the

transition period imposes changes in BCS, and by the monitoring these, it has been

possible to manage cows accordingly to improve productivity, reproductive performance

and minimize the incidences of health disturbances.

Hormonal and Metabolic Adaptations during the Transition Period

The loss in BCS during the transition period is a reflection of the metabolic

adaptations of many systems, organs and tissues of the dairy cow to cope with the

increased requirements for nutrients. At the onset of calving, the mammary gland

becomes the physiological priority, and because the nutrient input via feed intake is not

optimal to meet the demands for milk production (pattern of DMI and nutrient

requirements), a series of metabolic adaptations occurs to allow the cow to meet the

needs. Loss in BCS indicates mobilization of energy sources from adipose tissue, the

main energy storage tissue in the body, and lactate/ amino acids from other body tissues,

all to be used primarily as gluconeogenic precursors by the liver. However, the

mobilization of these precursors does not occur as a random event, rather, it is highly

coordinated and is orchestrated by hormonal changes that occur during this phase

(Bauman and Currie, 1980; Chilliard, 1999).

The key event in the metabolic adaptation seen during the transition period is the

change in function in the adipose from anabolism (synthesis of triacylglycerol from

glucose, and storage of triacylglycerol), to catabolism [breakdown of triacylglycerol, and








release of glycerol and fatty acids (NEFA) into the blood stream; Bauman and Elliot,

1983]. These metabolic changes, which begin before calving (Grummer, 1995), also were

observed in adipose tissue collected from dairy cows 2 wk after calving (early lactation).

The general observations observed in vitro were that adipocytes (functional unit of the

adipose tissue) obtained from lactating cows were smaller in size, had increased glycerol

release when stimulated by adrenaline, and also had decreased uptake of glucose from the

media when compared to the cells obtained 2 wk before parturition. In addition, blood

samples obtained from cows during the early lactation period had increased

concentrations of NEFA, confirming the suspicion of increased mobilization of lipids

from adipose cells (Pike and Roberts, 1980).

In a series of experiments designed to evaluate adipose tissue metabolism of dairy

cows overfed or restricted fed during the dry period, Rukkwamsuk et al. (1998, 1999)

observed that basal in vitro lipolytic rate, as measured by glycerol production per gram of

adipose tissue of restricted fed cows, was greater at 1 wk before parturition compared to

0.5, 1, 2 and 3 wk after parturition. This increase was accompanied by greater mean

plasma concentrations of NEFA during wk 0.5 and 1 than during wk 2, 3 and 6. In

addition, the in vitro rate of esterification (measured by the formation of labeled TAG

using labeled oleate) was significantly less at wk 1, 2 and 3 after parturition than at I wk

before parturition. The decrease in basal rate of esterification at 0.5 wk was 69% and

81% for cows that were restricted fed and overfed, respectively. These results

corroborated previous observations, which showed increased lipolytic activity and a

decreased lipogenic activity in adipocytes of dairy cows during the transition period.








In addition to the direct changes observed in the adipose tissue activity, others

also observed changes in concentrations of NEFA in plasma around parturition (Simmons

et al., 1994; Smith et al., 1997; Santos et al., 2000). In these studies, concentrations of

NEFA in plasma began to increase at 1 wk before parturition, peaked I wk after

parturition, and decreased sharply as lactation progressed. Mean concentrations of NEFA

in plasma were about 200, 1,000-1,200, and 400 Ateq/L during prepartum, at 1 wk

postpartum and during 14-50 d after parturition, respectively.

To better understand what might be controlling the described changes, it is

important to highlight the basic metabolic pathways that occur in the adipocyte with

regard to lipid metabolism. The adipocyte synthesizes lipid (TAG) using fatty acids that

are synthesized either de novo within the cells (mostly from glucose or acetate) or from

fatty acids released from blood TAG of chylomicrons and very low density lipoproteins

(VLDL) by the action of lipoprotein lipase (LPL). Fatty acids are released from stored

TAG within the adipocyte (lipolysis) by the action of the hormone sensitive lipase

enzyme. Some of the fatty acids released are re-esterified within the adipocytes, but the

remainder are released and transported into the blood for use elsewhere in the body

(Figure 1).

The controllers of these pathways are hormones and other humoral factors. The adipose

tissue of dairy cows is mainly lipogenic when stimulated by insulin and in the presence of

glucose (Bauman and Elliot, 1983; Kersten, 2001), and lipolytic when stimulated by

glucagon, catecholamines such as epinephrine (P-adrenergic receptor stimulator), and

adenosine (McNamara, 1991; McNamara et al., 1992; Vernon and Pond, 1997; Chilliard,

2000).








VLDL-TG
VLDLG Fatty acids (NEFA) Glycerol



Acetate -. Fatty acds

f ^ ,Fatty acid& \

/ ..---.-- Triacylglycerls
Glucose i r

''G--+ Glyceral 3-mphate


Adipocyte

Figure 1. Some metabolic pathways within adipocytes and their adaptations during the
transition period. Enzymes: ACC, acetyl CoA carboxylase; LPL lipoprotein lipase;
G3PDH, glycerol 3-phosphate dehydrogenase; GPAT, glycerol 3-phosphate
acyltransferase; HSL, hormone-sensitive lipase. Enzyme activity: t or 4, (increased or
decreased, respectively). Metabolic fluxes: bold line, flux increased; dashed line, flux
decreased (Adapted from Vernon and Pond, 1997).


Attempts to identify the role of these hormones in controlling lipid metabolism in

the adipocyte and in overall body began during early 1970's. Smith et al. (1975)

conducted an experiment to monitor changes in blood concentrations of insulin and

somatotropin beginning at parturition through 56 DIM, to relate these changes to the

nutritional status of dairy cows during the early postpartum period. Observations were

that mean concentrations of insulin and glucose were at their lowest from 1-20 DIM (8.8

pU/mL and 37.3 mg/dL, respectively) and had a constant increase thereafter. From 21 to

40 DIM mean concentrations were 10.3 pU/mL and 40.6 mg/dL, and from 41 to 56 DIM,

mean plasma concentrations of insulin and glucose were 11.9 pU/mL and 43.3 mg/dL,

respectively. However, mean concentrations of somatotropin were greatest at calving









(19.3 ng/mL), and decreased as lactation progressed through 56 DIM (13.6 ng/mL). In

another study, Lomax et al. (1979) showed that arterial and portal-venous concentrations

of insulin, and the pancreatic output and hepatic uptake of insulin were approximately 2,

3, 3 and 5-fold greater in normally fed non-lactating cows than in lactating cows. This

confirmed that differences in the secretion of insulin existed during the two physiological

states.

Similar results also were observed in subsequent studies conducted to evaluate

metabolic profiles of dairy cows during the transition period (Athanasiou and Phillips,

1978; Putnam and Varga, 1999; Busato et al., 2002, Block et al., 2003). In those studies,

mean concentrations of insulin during the prepartum and postpartum phases of the

transition period were about 0.7-0.8 and 0.4 ng/mL; and for somatotropin, mean

prepartum and postpartum concentrations were 8.0 and 14.0 ng/mL, respectively. With

regard to concentrations of somatotropin after parturition, they continued to increase, or

were greater as lactation progressed compared to those concentrations seen prepartum.

The decrease in concentration of insulin during the prepartum period seems to be

responsible for increased lipolysis in the adipose tissue. However, Rukkwamsuk et al.

(1999) also observed that adipocytes collected from dairy cows at 0.5, 1 and 3 wk

postpartum had significantly lower in vitro rates of esterification than prepartum cells

even when both types of cells (prepartum and postpartum) were treated with insulin and

glucose in the media. In addition, rates of lipolysis increased significantly when

adrenaline was added to the media (Pike and Roberts, 1980). This indicated that there

also was altered responsiveness of adipose tissue to insulin and other effectors around

parturition.








In an elegant experiment, Jaster and Wegner (1981) observed that the lipolytic

responsiveness seen during early lactation was greater than during the dry period due to

the increased number of P-adrenergic receptors (lipolytic) located in the cell surface.

Subsequently, Bauman and Elliot (1983) also reported that the in vivo lipolytic response

of adipose tissue to adrenaline during early pregnancy was greater than during late

pregnancy in dairy cows. The changes in lipolytic responsiveness were attributed to the

hormonal changes observed around parturition, which included decreased P4 and

increased estradiol, prolactin, and PGF2a concentrations during the prepartum period

(Thatcher et al., 1980; Bauman and Elliot, 1983). In addition to these hormones, the

increase in somatotropin also can be considered as a regulator of lipolysis because the

hormone increases adipose tissue response to catecholamines, and it is a potent inhibitor

of lipogenesis (Bauman and Vernon, 1993). Therefore, changes in catecholamine

response indicates that the system is sensitive to the hormonal changes described, and

when it is driven towards lipolysis, it becomes resistant (less responsive) to insulin

stimulation of lipogenesis (Vernon and Pond, 1997).

Another key event in the metabolic changes seen during the transition period is

the adaptation seen in carbohydrate and protein metabolism. Bell (1995) estimated that

for dairy cow producing 30 kg of milk/d at 4 d postpartum there is a deficit of 500 g/d of

gluconeogenic precursors propionatee, dietary amino acids, lactate, and glycerol from

adipose tissue) that are needed to meet daily demands for glucose. When the supply of

propionate for hepatic gluconeogenesis is limiting, as seen during the time there is a

decrease in DMI, lactate and AA released from muscles are used as gluconeogenic

substrates (Baird et al., 1980).









To observe the mobilization of AA from body tissues around parturition, Blum et

al. (1985) assessed blood concentrations of 3-methylhistidine (3-MH) during the

transition period. The 3-MH is a by-product formed by methylation of histidine in actin

and in myosin of muscles during the accretion of protein that occurs physiologically.

When there is a breakdown of these macromolecules, 3-MH is liberated into the AA pool

in the blood. Results reported were that plasma 3-MH increased rapidly before calving

(20 gmol/L) to maximal concentrations at 1 wk postpartum (32 Lmol/L), thereafter

decreasing to a low of 14 lmol/L at 5 wk after calving. All indicated that there was

increased mobilization of proteins from body reserves during this period. Bell and

Bauman (1997) observed a similar pattern in changes of 3-MH concentrations of mature

Holstein cows around parturition. Despite differences in magnitude of absolute values

observed during the previous study, 3-MH concentrations were about 4, 13 and 4 pmol/L

at d 17 prepartum, and at d 4 and 17 postpartum, respectively. Although these results

cannot determine if AA mobilized are from skeletal muscles or from other tissues, such

as the involuting uterus, information reviewed by Bell (1995) suggested that the skeletal

muscle tissue strongly contributed to the blood plasma AA pool. He also indicated that

the research reported by Reid et al. (1980) showed a significant reduction (25%) in

muscle fiber diameter of dairy cows immediately after calving. Therefore, there was

strong evidence to indicate that there would be an increase in lactate and AA availability

in the blood from muscles for potential use by the liver for glucose production.

To evaluate if there was increased use of lactate and AA as gluconeogenic

precursors by the liver, Greenfield et al. (2000) assessed the abundance of pyruvate

carboxylase (PC) mRNA and its activity in livers of dairy cows during the transition








period. They evaluated the PC enzyme because it is a rate limiting enzyme for hepatic

gluconeogenesis from the precursors lactate, pyruvate, alanine and other AA. Liver

biopsies were conducted at d -28, -14, +1, +28 and +56 relative to parturition. Abundance

of mRNA for PC increased significantly (7.5-fold) the day after calving compared to d

-28 levels. Also, the activity of the enzyme increased linearly as mRNA abundance

increased. It is not possible to quantify how much of the AA mobilized contributed to

hepatic gluconeogenesis (Bell and Bauman, 1997), but there is strong evidence indicating

that the liver adapts around calving to utilize AA and other sources to synthesize glucose

via gluconeogenesis.

Among the hormones that play a role in controlling these metabolic adaptations

around parturition, insulin, corticoids and somatotropin can be considered to have

important functions. The decrease in insulin concentration during early lactation

diminishes the capacity for glucose uptake by tissues that are insulin-dependent, such as

skeletal muscles, and this can initiate a cascade of events leading to protein breakdown

and AA release into the blood stream (Bauman and Vernon, 1993). Corticoids are potent

stimulators of hepatic gluconeogenesis in lactating and non-lactating cows. Because the

concentration of corticoids increases around calving, it may be associated with the

increase in gluconeogenic activity. In fact, these assumptions were made based upon

results of studies conducted during the late 1940's and early 1950's that addressed the

treatment of ketotic cows with cortisone (steroid hormone; Dye et al., 1953).

Observations were that after the administration of the steroid to ketotic cows, there was

mobilization of amino acids from muscles, increased liver deamination of these amino









acids, increased urea production, and increased gluconeogenesis from some of the

resultant ketoacids.

With regard to somatotropin, results from numerous studies have evaluated the

effects of exogenous recombinant bovine somatotropin (rbST) in lactating dairy cows and

other ruminants. Results indicated that somatotropin exerted direct and/or indirect actions

(through insulin-like growth factors) to regulate the changes described previously (Bell

and Bauman, 1997). Because there is an increase in the concentration of somatotropin as

lactation progresses, it is a strong candidate as mediator of these changes.

The onset of lactation also requires metabolic adaptations of tissues other than

adipose, muscle and liver to help meet the demands for another important nutrient,

calcium (Ca; Horst, 1986; Horst et al., 1994; Goff and Horst, 2003). The initiation of

copious milk production includes secretion of great quantities of calcium in the milk

(National Research Council [NRC], 2001). In addition to the sharp increase in demand

for calcium as lactation is initiated and progresses, the slow increase in DMI has a

negative impact on mineral metabolism during this time period (Gerloff, 1988). When

this occurs the dairy cow must rely on the mobilization of calcium from bones to meet its

needs (Bauman and Elliot, 1983). However, following the onset of lactation, there is a

delay of more than a week before bone calcium is mobilized extensively (Ramberg et al.,

1970). The great output of calcium in the milk leads to decreased blood concentration of

the mineral, a condition that is clinically manifested as parturient paresis, or milk fever

(Kronfeld, 1971).

Recent research involving calcium metabolism of transition dairy cows has

explored the potential of using prepartum transition diets varying in the cation-anion








(DCAD) difference to improve the adaptation described as lactation occurs (Moore et al.,

2000; Melendez et al., 2002; Gulay et al., 2003a; Melendez et al., 2003). The dietary

DCAD relates to the difference between total fixed (nonmetabolizable and bioavailable)

cations and total fixed anions in the diet, which most often is calculated as meq (Na + K)

- (Cl + S)/100 g of dietary DM. Negative and positive DCAD diets are denominated

anionic and cationic, respectively. Feeding prepartum diets with negative DCAD causes

mild metabolic acidosis, which increases calcium excretion via urine (Gaynor et al.,

1989). This, in turn, stimulates the hormonal system that controls calcium metabolism

(parathormone (PTH), and vitamin D) to increase absorption of calcium from the

intestines and bone resorption of calcium (Horst, 1986; Wang et al., 1994).

Anionic diets are efficient in maintaining blood calcium levels around parturition

in mature cows and in heifers, as demonstrated by Moore et al. (2000). In their study,

heifers and cows fed anionic (-15 meq/100 g of dietary DM) or neutral (0) DCAD diets

had higher concentrations of ionized calcium (iCa, the biologically active form of the

mineral; Dauth et al., 1984) in blood than those fed a cationic diet (+15 meq/100 g DM)

during prepartum period and at calving. The iCa concentrations ranged from 3.67 to 4.95

mg/dL, or about 50% of the total concentration of calcium in blood, which ranges from 9

to 11 mg/dL (Kronfeld, 1971). Despite differences in calcium concentration, no

differences in plasma hydroxyproline (blood metabolite released from bones when there

is tissue resorption) were observed among experimental groups, which indicated that their

anionic diet did not cause extensive bone resorption. However, Gaynor et al. (1989)

demonstrated that anionic diets caused calcium mobilization from bone, as well as an

increase in excretion of calcium in the urine.








These are just a few examples of the many studies carried out to assess the

metabolic adaptations that take place during the transition period. In general, all agree

that major changes in lipid, carbohydrate, protein and mineral metabolism occur in the

dairy cow to accommodate the demands brought about by the abrupt onset and rapid

increase in milk production. The changes in hormonal concentrations seen around

parturition likely orchestrated the adaptations, which, it seemed occurred to the benefit of

the new born at the expense of the dairy cow.

Liver Function during the Transition Period

The primary fate of NEFA mobilized from adipose tissue and lactate/AA from

muscles is uptake and metabolism in the liver. The liver plays a unique role in processing

these precursors to produce glucose to support mammary metabolism and milk synthesis

and also other molecules that can be used as energy sources by the peripheral tissues,

such as ketone bodies (Grummer, 1993). In the dairy cow, it was estimated that 85% of

all glucose produced in the body came from the liver, with the remainder being

synthesized in the kidney (Bauman and Elliot, 1983). This underlined the importance of

this organ in the metabolism of carbohydrates, especially during the transition period.

Besides being the center of metabolism of carbohydrates, lipids and proteins, the liver is

the center of defense, it is the control station of the hormonal system (it executes the

clearance of hormones secreted by other endocrine glands), and it has an important role

in maintaining blood pH and immunoregulatory functions (KmieC et al., 2001).

At the microscopic level, the liver is composed of hepatocytes, non-hepatocyte

cells sinusoidall endothelial, Kupffer, stellate and pit cells), and an extracellular space

compartment sinusoidall lumen, disse space and biliary canaliculi). In the rat, these








structures occupy 77.8, 6.3 and 15.9%, respectively, of the total volume of the organ. The

hepatocytes execute the major metabolic functions described previously, Kupffer cells

are hepatic macrophages, stellate cells are fat-storage cells or vitamin A rich cells, and pit

cells represent liver-specific natural killer cells (NK, cancer killer; Figure 2).


Figure. 2. Schematic drawing of the rat liver tissue. The parenchyma is built up of a
trabecular network of cell plates made up of hepatocytes (H). The wall of sinusoids,
formed by fenestrated endothelial cells (E) and Kupffer cells (K), does not show basal
membrane; however, stellate cells (F) are present in the perisinusoidal space of Disse
(DS). Pit cells (P) also stay attached to the sinusoid wall (Sasse et al., 1992; with
permission).


In the dairy cow, the stage of production and the feeding behavior exert extensive

changes in blood flow and types of metabolites that reach the liver, as observed by

Lomax and Baird (1983). In their experiment, blood flow to the liver of normally fed

lactating cows at 44 DIM was 52% greater than in non-lactating cows (average 11 mo

after last parturition). Another observation was that after short-term fasting (4 d), a








significant reduction (50%) in liver blood flow occurred, regardless of the milk

production stage. During a normal fed state 46, 16, 8.6 and 0.8% of assimilated

precursors propionate, lactate, AA and glycerol were converted to glucose, respectively,

in the liver. After a 4-d period of fasting, contributions of these precursors to glucose

production were 2.2, 81.6, 36.8 and 21.9%, respectively. In addition, after the 4-d fast,

NEFA was 98.9% of the assimilated precursor converted to ketones, compared with 0.0%

of the butyrate. It is likely, that to some extent, these changes also occur in transition

dairy cows around parturition, a period characterized by a significant decrease in DMI

and energy and precursor availability.

Liver gluconeogenesis is not a simple process. The pathway has irreversible

glycolytic reactions, which requires specific, rate-limiting enzymes to catalyze them. The

reactions are (1) the mitochondrial conversion of pyruvate to oxalacetate; (2) cytosolic

conversion of oxalacetate to phosphoenolpyruvate; (3) cytosolic hydrolysis of fructose-

1,6-biphosphate to form fructose-6-phosphate; and (4) cytosolic hydrolysis of glucose-6-

phosphate to produce glucose. The rate-limiting enzymes are (1) pyruvate carboxylase

(PC, EC 6.4.1.1; Greenfield et al., 2000); (2) phosphoenolpyruvate carboxykinase

(PEPCK, EC 4.1.1.32, Lomax et al., 1986); (3)fructose-l,6-diphosphatase (FBPase, EC

3.1.3.11; Rukkwamsuk et al., 1999); and (4) glucose-6-phosphatase (EC 3.1.3.9;

Rukkwamsuk et al., 1999). The precursors utilized in these pathways are glycerol, lactate,

AA and the product of complete oxidation of NEFAs, acetyl CoA (Beitz, 1993; Zubay,

1998).

Once transported into the hepatocyte, NEFAs have three possible paths of

metabolism. The first is complete oxidation in the tricarboxylic acid cycle (TCA cycle)








reactions by the process of p-oxidation, generating C02, H20 and ATP. The second is

partial oxididation to acetyl-CoA, and then diversion via TCA cycle to form ketone

bodies (acetoacetate, P-hydroxybutyrate and acetone). The third is re-esterification to

form TAG molecules and storage in the cell or excreted as very low-density lipoprotein

cholesterol (VLDL; Drackley, 1999). The steps of NEFA metabolism (in the hepatocytes)

are regulated by a group of enzymes including carnitine palmitoyltransferase I (CPT-1,

EC 2.3.1.21), which transfers NEFA into mitochondria for complete P-oxidation and

ketogenesis. The other enzymes in this group are 3-hydroxy-3-methylglutaryl CoA

synthase (HMG-CoA, EC 4.1.3.5), the regulatory enzyme in mitochondrial ketogenesis,

and enzymes of the esterification pathway, glycerol-3-phosphate acyltransferase (GPAT,

EC 2.3.1.15), phosphatidate phosphohydrolase (PAPase, EC 3.1.3.4) and diacylglycerol

acyltransferase (DGAT, EC 2.3.1.20). The third destination is highly regulated by

microsomal triacylglycerol transfer protein (MTP, EC 5.3.4.1; White et al., 1998), which

has a critical role in coordinating the assembly of VLDL in the liver, so that it can be

secreted from the hepatocyte.

Lipid metabolism is a key area in the biology of the transition period of the dairy

cow. Normal lipid mobilization from adipose tissue seems to cause no problems for liver

function; however, excessive lipid mobilization is linked to greater incidences of

periparturient health problems (Drackley, 1999). Histologically, detectable fat is present

in the liver of virtually all dairy cows during the first weeks after calving, but the

condition is considered severe if the percent of fat in liver cell is greater than 20% on a

DM basis (Reid and Collins, 1980). In a survey study, Reid (1980) observed that 37, 48

and 15 % of Friesian cows, and 62, 33, and 5 % of Guernsey cows had mild (0-20),









moderate (20-40) or severe (40-70) percentage of fat in liver on a DM basis, respectively.

In addition, Pearson and Maas (2002) indicated that moderate to high amounts of fat (15-

30%, wet weight basis) were present in the liver of all postparturient, high-producting

dairy cows, even those that were healthy.

To determine whether the accumulation of fat in the liver is accompanied by

significant changes in cell and organelle structure, Reid and Collins (1980) conducted an

experiment to correlate percentage of fat in liver, indicators of cell function, and

observations of histological and stereological analysis of the liver of dairy cows with

mild (<20% fat) or severe fatty liver (>30% fat on a DM basis). Their observations

showed that cows with severe fatty liver had a significant increase in plasma

concentrations of the enzyme aspartate aminotransferase (AST, EC 2.6.1.1) compared to

cows with mild fatty liver (92.7 vs. 49.9 IU/L), indicating liver damage. In addition, cows

with severe fatty liver had deposition of fat cysts (lipogranulomas, that originated from

ruptured stellate cells) in the hepatocyes, increased cell volume, decreased volume of

RER per liver cell, and evidence of mitochondrial damage. All of these changes indicated

significant morphological and structural changes in hepatocyte organelles due to

excessive deposition of fat.

Because there is no limiting step in the uptake of circulating NEFA by the liver,

excessive mobilization of fat from adipose tissue can lead to subsequent hepatic

deposition of the metabolite in the form of TAG. Gaal et al. (1983) found a significant

positive correlation between serum concentration of NEFAs and the total lipid

concentration in liver (+0.67; P<0.01) of Friesian dairy cows during the transition period.

However, no significant correlation was found between P-HBA and total liver lipid








content, but there was a significant correlation between P-HBA and NEFA (+.032,

P<0.05). Concentrations of total lipids in liver (wet basis), and NEFA and 3-HBA

concentrations in blood ranged from 3.5-20.1%, 249-1,888 pmol/L and 0.19-1.03

mmol/L, respectively. Ono et al. (1988) observed that peak concentrations of NEFAs of

healthy Holstein cows at day of calving were significantly less than in diseased cows.

The concentration of NEFA in plasma and the percentage of fat in liver (DM basis) of

normal and diseased cows were 800 tEq/L and 1,400 giEq/L, 18.9 and 25.3%,

respectively. Van Den Top et al. (1995) and Grummer (1993) agreed that rate of fatty

acid esterification into TAG in liver was dependent upon the supply of fatty acids, as

mentioned previously; however, Grummer (1993) also suggested that fatty liver occurred

when the rate of hepatic fatty acid esterification exceeded the rate of TAG disappearance

via hydrolysis plus export as a constituent of VLDL.

Information cited previously indicates that the slow rate of TAG export as a

constituent of VLDL may be one cause of fatty liver. In fact, Bauchart (1993) observed

that hepatic VLDL secretion in calves was five-times less than in young adults ( 24 yr.

old). Pullen et al. (1990) earlier conducted a study to compare the ability of liver slices

from eight species to synthesize TAG from the NEFA in the incubation media. The rate

of liver TAG synthesis was similar among species studied; however, liver slices from

species in which the liver contribution to lipogenesis is minor (sheep, cattle, pig and

guinea pig) secreted less TAG than liver slices from species in which lipogenesis occurs

predominantly in the liver (chicken and fish) or in liver and adipose tissue (rat and

rabbit). These results suggested that the ability of liver to secrete VLDL was proportional








to the capacity of the liver to synthesize lipids, and that ruminants have an inherently low

capacity to export TAG as a constituent of VLDL.

To test the hypothesis that rates of hepatic TAG esterification were positively

correlated to concentration of NEFAs in plasma, Van Den Top et al. (1996) conducted an

experiment to obtain information about the activities of the enzymes of the esterification

pathway (GPAT, PAPase and DGAT) in Holstein x Dutch Friesian that developed fatty

liver during postpartum. A surprising observation was that microsomal GPAT, the

enzyme that catalyzes an early step in the first part of the pathway leading to TAG and

phospholipid formation, was depressed in overfed cows, despite the higher concentrations

of NEFA in plasma of these cows compared to restricted fed cows (1.50 vs. 0.75 mmol/L

at 1.5 wk postpartum). Although GPAT activity was lower, TAG liver accumulation of

overfed cows was greater than in restricted fed cows during the first 8 wk postpartum.

The authors suggested that this depression in GPAT was a mechanism used to divert fatty

acids from esterification to the P-oxidation pathway, so that further fatty liver

development, and perhaps any associated liver damage, were diminished.

Neutral lipids and phospholipids comprise the total lipid deposited in the liver.

Neutral lipids are mainly cholesterol esters, TAG, free fatty acids (FFA) and cholesterol.

Phospholipids are composed by phosphatidyl ethanolamine, phosphatidyl serine,

phosphatidyl choline and sphingomyelin. Ono et al. (1988) observed that the TAG

portion of liver of Holstein cows that had 18.9 and 23.5% fat in liver (DM basis) was 4.5

and 9.1%, respectively. An interesting finding was that, in addition to these increase in

the neutral lipids in the TAG component of diseased livers, there also was an increase in

phosphatidyl serine in phospholipids. These results agreed with observations of Hippen et








al. (1999). In their study clinically diseased cows with fatty liver had higher liver TAG

than non-diseased cows, as expected. The percentage of TAG in diseased cows was

greater than 8% of the total liver volume on a wet weight basis.

Some recent research involving fatty liver syndrome explored the use of glucagon

to improve the condition in diseased Holstein cows (Bobe et al., 2003). The authors

believed that glucagon would improve health status of diseased cows by decreasing

lipolysis due to indirect actions via increased concentrations of plasma glucose and

insulin in the adipocyte. Glucagon injections (5 mg every 8 hr [15 mg/d]) began at d 8

postpartum and continued through 22 DIM. Their results showed that injections of

glucagon decreased plasma concentrations of NEFA, increased concentrations of glucose

and insulin, and decreased liver TAG of older cows (>3.5 yr), but not of younger cows.

No effects of glucagon injections on MY were observed. These results indicated that

glucagon significantly decreased TAG accumulation in liver, by decreasing the

mobilization of NEFA from adipose tissue, which agreed with the hypotheses they

previously described.

Clearly, the liver has a major role throughout the transition period of dairy cows.

The decrease in DMI and changes in metabolism around parturition have both direct and

indirect effects on liver function. Decrease in blood flow and changes in the assimilation

of precursors require that an adaptation in the hepatic gluconeogenic and lipolytic-

ketogenic pathways occurs in order to use the available precursors. However, if fat

mobilization exceeds that used by the liver, bad consequences likely will occur.

Excessive fat deposition can cause structural damage in the liver, can lead to excessive

production of ketone bodies, and can impair overall liver function. Studies undertaken to








evaluate the basic mechanism of fat deposition in the cow liver indicated that the inability

to limit fat uptake and the inherent decrease in capacity to excrete TAG via VLDL in

ruminants were the likely causes for the deposition of fat in the liver. These changes

ultimately will exert negative effects on overall health of the cow during the transition

period.

Mammary Gland Function during the Transition Period

The metabolic adaptations during the transition period occur almost entirely to

support the function of the mammary gland following parturition. However, it has been

recognized that physiological changes that take place only during the dry period also are

required for the appropriate lactogenic function of the mammary gland (Capuco and

Akers, 1999). These changes occur during distinct phases or functional transitions in

response to the cessation of milking, and to the dramatic variations in the concentrations

of reproductive and metabolic hormones observed during the prepartum transition period

(Tucker, 2000). For the dairy cow, the functional transitions are from lactation to forced

involution (dry-off), from involution to colostrogenesis, and from colostrogenesis to

initiation of the new lactation (Oliver and Sordillo, 1989). Early studies indicated that a

dry period length of 40-60 d was necessary for these transitions to occur. However,

results of more recent research suggests that a 30-35 d dry-period likely is sufficient for

the optimal function of the mammary gland during the lactation that follows (Bachman,

2002; Gulay et al., 2003a).

The beginning of the dry period is characterized by a dramatic shift in the

function of the mammary epithelial cells. This is reflected by changes in concentrations

of constituents in mammary secretions, as were observed by Athie et al. (1996). In their









experiment, Holstein cows assigned to a 60 d dry period showed a loss of differentiated

function and overall functionality of the mammary epithelial cells (decrease in the

volume and in the concentration of a-lactalbumin, lactose and citrate in mammary

secretions), as well as an increase in the capacity of immune defense in the organ

(increase in immunoglobulins, lactoferrin and somatic cell count in mammary secretions),

as the dry period progressed. Changes were more evident after 7-10 d dry and were

maintained through the end of the experimental period (30 d dry). This shift in function

likely was initiated by the increase in alveolar pressure and by accumulation of the

feedback inhibitor of lactation caused by milk stasis (Fleet et al., 1978; Wilde et al.,

1995).

In addition to the changes in the function of mammary epithelial cells, there also

are significant effects of dry-off on the structure of the mammary parenchymal tissue, as

observed by histological studies. In non-pregnant crossbred beef cows, non-suckling of

an udder-half for 21 or 42 d (mimicking a dry-off) caused a 50 and 64 % reductions of

total parenchymal DNA compared to the continuously suckled udder-half. Despite the

histological evidence of alveolar structure in non-suckled glands, numbers of cells per

alveolar cross-section were reduced compared to suckled counterparts (22 vs. 32 cells).

Also, it was observed that regression of the mammary gland was not uniform throughout

all regions. Greater involution occurred in the parenchymal tissue localized just above the

gland cistern compared to the tissue adjacent to the ventral body wall (Akers et al., 1990).

Observations from a study completed by Capuco et al. (1997) using prepartum

Holstein cows differed to some extent from that of Akers et al. (1990). Their objectives

were to observe involution, growth and differentiation of epithelial cells in mammary









glands of experimental cows that were milked during the last 60-d of gestation (no dry

period), and to compare the findings with mammary glands of cows that had a regular

60-d dry period. Important and significant findings were that no net loss of mammary

cells involutionn) and no regression of alveolar structure occurred in cows during the dry

period. In addition, at 7 d prepartum the proportion of epithelial cells was greater in dry

cows than in lactating cows, indicating a greater renewal of damaged or senescent cells

during the dry phase. One similar observation to that observed in the study of Akers et al.

(1990) was that only the lower region of the mammary gland (adjacent to gland cistern)

appeared to be most sensitive to stimuli promoting renewal, not involution of epithelial

cells.

The renewal of damaged cells is facilitated because of remodeling of the

parenchymal tissue during the dry period. During the first 30 d of a 60-d dry period Athie

et al. (1997) observed that the plasminogen and plasmin system seemed to be responsible

for the remodeling. Plasminogen is the inactive zymogen of plasmin, a proteolytic

enzyme that carries out the breakdown of components of the extracellular matrix that

surrounds the alveoli (collagen type IV). The researchers observed that there was a

decrease in plasminogen:plasmin ratio, an increase in plasmin concentration, and in the

proteolytic activity of plasmin in mammary secretions as the dry period progressed. The

secretion of plasminogen activators by somatic cells (macrophages) and myoepithelial

cells was considered to be the cause of these physiological events.

Capuco et al. (1997) also observed that none of the epithelial cells were in a

secretary stage at 35 d prepartum in mammary glands of Holstein cows that had a 60-d

dry period. At 20 d prepartum, the number of cells in a secretary stage increased









substantially, and by 7 d prepartum alveolar luminal area was at its maximum, reflecting

the initial accumulation of colostral secretions. These changes in secretion pattern

indicated that by 20 d prepartum (beginning of the prepartum transition period) there was

a significant increase in mammary gland activity toward milk secretion. Oliver and

Sordillo (1989) characterized this phase as phase I lactogenesis, whereas phase II began

shortly before calving and was characterized by copious secretion of colostrum.

It is important to mention that the study conducted by Akers et al. (1990) utilized

non-pregnant beef cows at 59 d postpartum. The Holstein cow study (Capuco et al.,

1997) utilized late pregnant cows during late lactation. Contrasting results obtained may

be due, in part, to breed differences, although they more likely were due to reproductive

and lactational stage of the experimental cows. These observations indicated the possible

involvement of hormones in regulating mammary tissue renewal during the dry period. In

fact, the prepartum transition period is marked by tremendous changes in concentrations

of reproductive and non-reproductive hormones that directly and/or indirectly impact

mammary gland function of ruminants (Anderson et al., 1981; Neville et al., 2002).

Knowledge about lactogenesis was gained from studies that dealt with hormonal

induction of lactation in dairy ruminant species (Head, 1999). One of the early studies to

address this issue was conducted by Smith and Schanbacher (1973). In their experiment,

various breeds of nonlactating dairy cows (n=9) and one nulliparous heifer were injected

with a mixture of 17 j$-estradiol (E2) and progesterone (P4) twice a day for 7 to 10

consecutive days. The treatments were successful in inducing lactation in 7 of the 10

animals (including the heifer) by about 19.3 d after termination of the steroid injection









period. This strongly indicated that these steroid hormones were necessary to initiate

mammary growth, cell differentiation and milk production.

In addition to E2 and P4, prolactin, corticoids and somatotropin also are required

for initiation of lactation, as demonstrated in numerous studies summarized in reviews

(Delouis et al., 1980; Head, 1999; Tucker, 2000). In cows, total estrogens begin to

increase at 10 d prepartum to a maximum of 5 ng/mL, but concentrations decrease

sharply soon after calving. At the same time, there is a progressive decrease in

concentration of P4 from 7 ng/mL to a minimum concentration (< 1.0 ng/mL) at

parturition. Concentrations of prolactin, somatotropin and corticoids generally increase

significantly beginning at 5-7 d before parturition, with peak concentrations around

calving. Concentrations at peak were about 150, 25 and 10 ng/mL for prolactin,

corticoids and somatotropin, respectively (Head, 1999).

Ovarian steroids (E2 and P4) are considered necessary to induce lobulo-alveolar

formation in mammary tissue, mainly in heifers during the last trimester of their first

pregnancy. In multiparous cows, E2 has a mammogenic role, which is potentiated by P4.

Progesterone is a well-known inhibitor of lactogenesis, and its withdrawal during the

peripartum time period is related to the initiation of lactogenesis. Prolactin has an

important role in the formation of the lobulo-alveolar structure, but high concentrations at

parturition are more important to induce copious milk secretion. With regard to

corticoids, studies conducted with ewes have shown that injections of hydrocortisone

acetate were required to stimulate appropriate lactogenic response in nulliparous animals

(secretion of p-casein). In addition, corticoids potentiate action of prolactin on

lactogenesis (Delouis et al., 1980). Early studies recognized that somatotropin ensured








maintenance of lactation, but it was not critical for mammogenesis and lactogenesis (Peel

and Bauman, 1987) because the hormone did not exert a direct effect on mammary

growth or lactogenesis. This latter action likely was due the absence of the somatotropin

receptor in epithelial cells (Keys and Djiane, 1988).

Recently, Sinowatz et al. (2000) found evidence of immunoreactive somatotropin

receptor (irSTR) in the epithelial and stromal compartments during different stages of

mammary gland development (mammogenesis, lactation and involution). The ductular

epithelium showed irSTR during most stages, whereas the alveolar epithelium contained

a modest amount of irSTR during pregnancy, but this increased during lactation. In dry

cows, irSTR in the alveolar epithelium was very weak or negative, confirming the

observations that somatotropin could not have a direct effect on mammary epithelial cells

during the dry period. Effects of direct action of somatotropin in mammary cells during

lactation remain to be determined.

Despite the absence of somatotropin receptor in mammary epithelial cells,

somatotropin has a strong indirect effect on dry period mammogenesis through actions of

insulin-like growth factor-I (IGF-I) secreted by the liver, and by other tissues that have

somatotropin receptor, such as adipocytes (Thissen et al., 1994) that also are present in

the mammary gland (Chilliard, 1999). There is an abundance of IGF-I receptors in

mammary epithelial cells during the dry period, and it is well known that the growth

factor and its binding proteins exerts significant regulation (inhibition) of mammary

epithelial cell apoptosis during the dry-period (Accorsi et al., 2002; Blum and

Baumrucker, 2002).









Review of previous research leads to the conclusion that a dry period is necessary

for the renewal of epithelial mammary tissue, and that the hormonal events of the

prepartum transition period are necessary to stimulate the tissue regeneration

(remodeling). Milk stasis initiates a cascade of events in the parenchymal tissue that

culminates with the replacement of damaged and senescent cells. Estrogen, P4 and IGF-I

are the most likely hormones or growth factor responsible for the stimulation of cell

renewal. Prolactin, withdrawal of P4 and corticoids are importantly responsible for

lactogenesis, which is divided into phases I and I. Phase I begins about 30-14 d before

calving, and phase H (colostrogenesis) begins just a few days before calving. Finally,

somatotropin is not directly required for mammogenesis and lactogenesis, but IGF-I

secreted under somatotropin stimulation, is an important indirect effector of somatotropin

for mammogenesis and lactogenesis.

Diseases and the Transition Period

A successful transition from the dry period into lactation is achieved only when

adequate metabolic changes occur. However, despite the physiological efforts by the cow

to carry out these changes, some functions are often impaired, that lead to diseases. It is

during the first 2 wk of lactation (postpartum transition period) that most diseases of

dairy cows occur (metabolic [milk fever MF, ketosis KETO, displacement of

abomasum LDA], infectious [clinical mastitis MAST] and reproductive [retained

placenta RP and metritis MET]). In addition, some disorders that are not diagnosed

during this early lactation phase, such as laminitis (LAME), have causes (etiology) that

can be traced back to insults that occurred during early lactation (Erb and Grohn, 1988;

Goff and Horst, 1997).








To better understand the epidemiology of the diseases mentioned, a number of

studies were conducted to identify causes, risk factors, and also to look for associations

among the disorders (epidemiological parameters; Belyea et al., 1974; Roine and

Saloniemi, 1978; Erb et al. 1985; Grohn et al., 1989; Rajala-Schultz et al., 1999;

Fleischer et al., 2001). All information gathered proved to be useful for the development

of strategies for disease prevention and necessary, not only to promote animal well-being,

but also to minimize the enormous negative economic impact that these diseases have on

the dairy industry. In the US, economic losses from MF, RP, KETO, LDA, and LAME

have been estimated to be $ 335.00, 285.00, 145.00, 340.00, and 302.00 per case,

respectively. Overall, these losses include prophylactic or clinical treatment, lost milk

production, increased culling, and cost of surgery in case of LDA (Kelton et al., 1998).

Reports of epidemiological parameters for the diseases mentioned previously

were made based upon experiments that studied lactation incidence rates (LIR, number of

cases per lactation divided by 100 lactations) of diseases in a specific population, or by

carrying out analyses of results from many published studies to draw more broad

conclusions. Using the first approach, Erb et al. (1985) conducted a study to develop and

evaluate models and relationships among productive variables and disorders of the

transition period using data from 784 primiparous heifers and 2,066 multiparous Holstein

cows. The LIR for RP, MET, MF and MAST in heifers and cows were 3.6 and 12.1, 10.2

and 12.3, 0 and 6.5, and 6.3% and 10.5%, respectively. In another study, Gr6hn et al.

(1989) conducted an extensive analysis of 61,124 Finish Ayshire lactations. Among

heifers and cows, the LIR and time of diagnosis during the lactation of MF, LDA, KETO,








RP, MET (early) and MAST (acute) were 4.0, 0.5, 6.0, 4.4, 2.3 and 6.2 % and 1, 21, 28,

2, 16, 44 DIM, respectively.

Erb and Grohn (1988) and Kelton et al. (1998) used analyses of results from

published research to draw conclusions about disease incidence. In the first study,

information was gathered from 70 epidemiological (observational) studies conducted

with Holstein-Friesians and Finnish Ayrshire dairy cows. For these studies, the LIR for

MF, LDA and KETO observed varied from 1.2 to 14.1, 1.22 to 2.5 and 1.1 to 9.2%,

respectively. Diagnosis of MF, LDA and KETO occurred between 0-15, 0-13, and 21-35

DIM, respectively. In the second study (Kelton et al., 1998), data from 195 research

publications that appeared between 1970 and 1996 for various dairy breeds were used to

draw conclusions about LIR. The overall LIR ranges for MF, RP, MET, KETO, LDA,

LAME and MAST were 0.03 to 22.3, 1.3 to 39.2, 2.2 to 37.3, 1.3 to 18.3, 0.3 to 6.3, 1.8

to 30, and 1.7 to 54.6%, respectively. The median LIR for the diseases were 6.5, 8.6,

10.1,4.8, 1.7, 7.0, and 14.2%, respectively. The information presented indicated that LIR

for all diseases were similar, and that most diseases were diagnosed during the early

postpartum period (0-50 DIM).

In addition to incidence rates, some studies evaluated impact of these diseases in

the reduction in milk production. In an experiment conducted using 500 Holstein cows

(Deluyker et al., 1994), those with Metritis, MAST, and KETO had MY reductions of

266, 281, and 253 kg during the first 120 DIM, respectively. Displaced abomasum caused

a reduction of 402 kg during the first 49 DIM. An interesting observation was that

decrease in MY production occurred even before diagnosis of some diseases, such as

MET. The LIR and diagnosis of MET, MAST, KETO and DA were 5.6, 26.1, 18.3 and








2.0%, and 24, 26, 21 and 21 DIM, respectively. In a more comprehensive study, Rajala-

Schultz (1999) analyzed the effects of disease and parity (adjustment) on milk yield loss

of 22,242 Finnish-Ayrshire cows. For primiparous and 2, 3 and 4+ parity cows, MY

losses due to MF were 47.6, 37.8, 79.8 and 33.6 kg during the first 56 d after the

diagnosis. For KET, 126.0, 126.0, 67.2 and 535.4 kg of milk were lost between 28 d

before and 42 d after diagnosis (70 d time frame). The LIR for MF and KETO for 1,2, 3

and 4+ parity cows were 0.2, 1.1, 5.7, 16.7%, and 2.5, 3.2, 4.2 and 4.1%, respectively.

The average MY loss for KETO reported by Rajala-Schultz (1999) was 213.7 kg, similar

to the 253.4 kg reported by Deluyker et al. (1994). In older cows, greater MY loss

probably was due to their greater capacity for milk production as they grow older (Head,

2000). The LIR of both studies reported were in the range previously mentioned.

Others used different approaches to evaluate associations between MY and LIR of

diseases. In a study conducted using 2,197 lactations of Holstein-Friesian cows, Fleischer

et al. (2001) hypothesized that MY could be the cause of disease. Indeed, trends of

correlations between previous lactation MY and RP, MAST and MF were observed

(P<0.10). In addition, there were significant (P<0.05) negative correlations between

current lactation MY and claw diseases (LAME) or cystic ovaries (CYST). The estimated

probability of appearance (EPA) for all diseases increased as lactation MY increased

from 6,000 to 12,000 kg. The 6,000 and 12,000 kg MY EPA for RP, MAST, MF, LAME

and CYST were 6.4 and 17%, 17.9 and 44.2%, 4.0 and 13.2%, 16.2 and 32.2 %, and 9.0

and 27.5 %, respectively.

In addition to the previously described associations, others have evaluated

possible relationships among diseases. One of the first researchers to indicate possible








correlations was Morrow (1976) when characterizing fat cow syndrome. This was

described previously in the BCS section of this review (page 17). Cows with this

syndrome had ketonuria (clinical sign of ketosis) that usually was associated with MF,

DA, digestive problems, RP, MET, MAST and salmonelosis (diarrheas). Following these

observations, others (Curtis et al., 1983; Thompson et al., 1983; Markusfeld, 1984, 1985;

Gerloff et al., 1986; Correa et al., 1990; Kaneene et al., 1997; Melendez et al., 2003)

evaluated possible correlations among diseases, as well as the correlation between

etiological factors of one disease with other disorders. In most studies, the odds ratio

(OR, the antilogarithm of the coefficient for an independent variable in a logistic

regression) was used as an approximate measure of relative risk. An OR greater or

smaller than 1 implied increased or decreased risk of contracting a disease with

increasing value of independent variable (cause), respectively (Grohn et al., 1989).

Using the approach described above to establish associations between diseases,

Curtis et al. (1983) evaluated associations between MF and 7 periparturient disorders

(dystocia, RP, MET, LDA, LAME, KETO and MAST) in 33 Holstein dairy herds (2,190

cows). Significant (P<0.01) associations between MF and dystocia, RFM, KETO and

MAST were detected. The OR were 6.5, 3.2, 8.9 and 8.1, meaning that cows that had MF

were 6.5, 3.2, 8.9 and 8.1 times more likely to have each of the other disorders. In a

subsequent study, Markusfeld (1984) observed that Israeli-Friesian cows with long dry

period, and that had RP or MET, were 3.0 and 3.1 times more likely to develop ketosis

than healthy cows. In addition, among all ketotic cows in the population (LIR 18%),

82.4% had at least one concurrent disease (RP, MET, DA). The long dry period was

observed to be a predisposing factor because cows (usually low-yielders) became









overconditioned during the long period dry. This indicated there was a relationship

between management and disease incidence.

Gerloff et al. (1986) observed that higher culling rate of postpartum dairy cows

was associated with severe hepatic lipidosis (HL) caused by a greater loss of BCS during

the early postpartum period. For severe and mild HL cows, BCS at calving was similar,

but BCS loss during early postpartum period was 1.17 and 0.72 pt., respectively. As a

consequence of HL, serum NEFA and liver TAG (wet basis) concentrations at 3 wk

postpartum and culling rate were ~400 and 750 [lEq/L, 2 and 11%, and 15 and 42%, for

mild and severe HL cows, respectively. Although there only were trends of significance

between culling rates, they did indicate the possibility of existence of this association. In

another study, Kaneene et al. (1997) also observed trends of the possible relation of

metabolic events associated with energy insufficiency (increased fat mobilization and

serum lipoprotein metabolism) with increased risk of MET and RP.

The results of all experiments described are just examples of some relationships

among milk production and periparturient disorders. The wealth of information in some

studies (large data sets), allows researchers to draw broad and significant conclusions

about the relationships found. To better understand these, path analysis (regression

models) models were proposed. An example of a path analysis is in Figure 3. It

represents significant relationships (OR) among metabolic disorders obtained from a

study using 61,124 Finnish-Ayrshire cows (Grohn et al., 1989). A disease indicated by

the arrow means that it is a likely outcome when the preceding disorder (risk factor, at the

beginning of the arrow) occurs. In the model presented, it is important to mention that








most of the 10 disorders lead to ketosis and abomasal disorder, and also that ketosis is a

predisposing factor for some diseases.


RETAINED
PLACENTA
1
UDDE
I EDEMA


FOOT/LEG


OUTIOORI R
HYPOMG

1


I
EARLY
METRIM
1
INDOOR


PARESIS


-- MASTITIS N I /
___ --_ ABOMASAL TRAUATAIC
DISORDER R---- ETICULO-
ParTONITrs


Figure 3. Relationships among metabolic disorders. Hypomg stands for hypomagnesemia
(Adapted from Grohn et al., 1989).


It is during or following the transition period of dairy cows that most metabolic,

reproductive and infectious diseases occur. Diseases included are MF, KETO, DA,

MAST, RP and MET. Results of many experiments indicated that these diseases occur to

a similar extent in various dairy populations. Another important observation is that the

likely outcome of any disease is ketosis. This underlines the importance of improving the

metabolic adaptations involving liver and fat metabolism during the transition period.

This would require appropriate dietary management during the prepartum and postpartum

periods, through strict control of BCS status, and through the maintenance of adequate

DMI of a well-balanced ration.








Recombinant Bovine Somatotropin (bST) and Lactation in Dairy cows

The scientific and management information arising from dairy research involving

somatotropin (ST) has progressed far beyond understanding the role of somatotropin in

the metabolic adaptations seen during the transition period. Since the discovery that

"growth hormone" or somatotropin (the greek derivative) was the substance in crude

extract of bovine pituitary glands responsible for stimulating the growth of rats, and for

increasing milk yield of goats and hundreds of dairy cows in the late 1920s, the

galactopoietic potential of somatotropin gained more attention (Etherton and Bauman,

1998). Through the late 1970s research studies using bST were limited because adequate

amounts of hormone required for large scale study could not be provided from the only

source at that time (slaughtered animals). However, the advent of recombinant DNA

technology in the early 1980s enabled an impressive number of researchers to investigate

the galactopietic effects of somatotropin in dairy cows, once significant quantities of the

hormone could be produced in vitro (recombinant bST, rbST; Peel and Bauman, 1987;

Bauman and Vernon, 1993).

Somatotropin is a large protein hormone (190 or 191 amino acids; mol. wt.

22,000 Daltons) produced by the acidophilic cells (somatotrophs) in the anterior pituitary.

Secretion of somatotropin is regulated by two hypothalamic peptides that act to stimulate

(growth hormone-releasing factor, GRF) or inhibit somatostatinn) release of somatotropin

(Etherton and Bauman, 1998). The hormone has four major variants in cattle. These arise

from the combination of two possible N-terminal amino acids (alanine or phenylalanine)

and two possible amino acids at position 127 of somatotropin leucinee or valine). In the

dairy breeds Brown Swiss, Holstein, Guernsey, Ayrshire and Jersey, the frequencies of








leucine-127 and valine-127 alleles detected were 1.0 and 0, .93 and .07, .92 and .08, .79

and .21, .56 and .44, respectively (Lucy et al., 1993). Valine-127 variant is known to

elicit greater increase in milk yield than the leucine variant (Etherton and Bauman, 1998).

At the cellular level, somatotropin has a unique effect compared to other protein

hormones. The somatotropin membrane receptor (cytokine-type oftransmembrane

receptor) can recognize two different sites of a somatotropin molecule, allowing one

molecule to bind two receptors at the same time, forming a dimer (Figure 4). After

binding and dimerization, a cascade of intracellular signal transduction pathways is

initiated (PKC pathway), ending with the binding of signaling molecules in the target

genes (Maharajan and Maharajan et al., 1993).






















Figure 4. Backbone structure of the hGH-(hGHbp)2 complex. The hormone, receptor I
and receptor H are shown as red, green and blue 13 strands and loops, respectively (De
Vos, A. M., M. Ultsch and A. A. Kossiakoff. 1992. Science 255(5042): 306-312.
AAAS).








Investigations of rbST effects in dairy cows began with short-term studies (5-21 d)

conducted during the early 1980s. These evaluated daily injections of the hormone, and

results agreed that gradual increase in milk yield occurred after a few days of rbST

treatment. Maximum production was reached during the first week after the first

injection. When treatment was terminated, milk yield gradually returned to pretreatment

levels over a similar period of time. No increase in feed intake was observed in these

studies. Increases in MY varied from 5 to 40% (Peel and Bauman, 1987; Bachman et al.,

1999). The variations in response occurred due to duration and dose, lactation stage,

management conditions, and other unidentified factors.

One of the first studies to address the effects of long-term use of rbST on

production responses of dairy cows was conducted by Bauman et al. (1985). In their

experiment, 30 multiparous Holstein cows supplemented with 0, 13.5, 27 and 40.5 mg/d

of methionyl bovine somatotropin (MBS) or with 27 mg/d of pituitary bovine

somatotropin (PBS) produced 27.5, 34.4, 38.0, 39.4 and 32.5 kg/d of FCM during the

period from 84 10 through 188 DIM. Increases in milk yield were 23.3 to 41.2% over

the dose ranges tested. Increases in net energy intake occurred 5-9 wk after the initiation

of treatments. The average energy balance across the treatment period for all

somatotropin supplemented groups was positive and sufficient to replenish body stores

that had been mobilized and used during early lactation. In addition, treatment with

somatotropin showed a trend to increase feed efficiency to produce milk (ratio of FCM

yield/NE Mcal). Increases in milk yield with different bST doses also were reported by

many researchers including Hansen et al. (1994). In their study, supplementation with

bST began at 28 to 35 d postpartum, and they observed increases of 10.5 and 9.0% of








3.5% FCM in multiparous cows and primiparous heifers injected with 5.15 and 16.5 mg/d

of bST during a whole lactation, respectively, compared to non-supplemented controls.

Bachman et al. (1999) summarized results of a large number of long-term field

studies (15) conducted to evaluate the effects of daily or bi-weekly (14 d, sustained

release formula) injections of rbST on production responses of dairy cows. The number

of cows, dose used, duration of experiments, and time of treatment initiation ranged from

6-63, 20.6-50.0 mg/d, 70-266 d and 28-56 d postpartum, respectively. Average control

MY (kg/d) and DMI (kg/d) ranged from 19.8-29.8 and 15.5-23.7, respectively. Average

increase in MY (kg/d) and DMI (kg/d) in treated cows ranged from 2.2-10.1 and 0-3.7.

The average increase in MY was from 9.2 to 36.2 % in rbST treated cows. Although

significant increases in DMI were observed, in 13 of the 15 studies the cows were in

negative EB balance during the period of supplementation.

Subsequently, Bauman et al. (1999) examined production responses in northeast

commercial dairy herds after POSILAC [prolonged-release formulation of the rbST

(500 mg/14d; 35.7 mg/d) made by Monsanto Co.] was approved for commercialization in

the US in 1994. They conducted an extensive study using 200,000 individual DHI

lactation records obtained from herds that utilized or did not utilize bST supplementation.

Data were collected over an 8-yr period (4-yr pre-approval of bST and 4-yr post-approval

commercial availability of POSILAC ). During the 4-yr post-approval period, dairy cows

in bST herds on average produced more milk (2.93 kg/d), fat (88 g/d), protein (100 g/d)

and had slightly higher SCC in milk than control non-supplemented cows in the same

herd. No effect on milk composition was observed due to bST. Milk response to bST was

minimal during the first 60 d post-injection, and then gradually increased until reaching a








plateau that was maintained over the last one-half of the lactation. In addition, lactation

persistency was greater for bST injected cows. No adverse effects of bST on health were

observed, because herd-life of animals was not altered by bST treatment.

The increases in milk production observed in bST-supplemented cows resulted

from a favorable adaptation of the diverse physiological processes that support milk

production, and involve the metabolism of all nutrient classes. These adaptations are

directly and indirectly mediated by somatotropin. Because adipocytes and hepatocytes

have somatotropin receptors, these cells are direct targets of the hormone. Indirect actions

in cells are mediated by somatotropin-dependent insulin-like growth factors (IGF-I, IGF-

II and the IGF binding proteins [IGFBPs; I-VI]), which exert most somatotropin actions

in the mammary gland due to the extensive presence of specific IGF-I receptors in the

organ and in other body tissues that give support to milk production, such as muscles

(Bauman, 1999).

In a 1993 review, Bauman and Vernon summarized the effects of bST on specific

tissues and physiological adaptations in lactating cows. In the mammary gland,

somatotropin (mainly through IGF-I) increased blood flow and maintenance/activity of

secretary cells. These events led to increased uptake of nutrients for milk synthesis,

which was substantially enhanced and had normal composition. At the whole body level,

somatotropin increased glucose availability for lactose (milk) synthesis by decreasing

muscle use and overall body oxidation of glucose, and by increasing the rates of liver

gluconeogenesis. This latter metabolic event occurs because of the inability of insulin to

inhibit hepatic gluconeogenesis when somatotropin is supplemented and concentrations

are greater, and also by the increase in voluntary feed intake to match the need of the









mammary gland and other organs and tissues for glucose that has been observed

following long-term use of bST. In addition, somatotropin increases immune response,

circulating IGF-I, IGF binding protein 3 (IGFBP-3, which acts as one of the regulators of

the biological action of IGF-I; Jones and Clemmons, 1995), NEFA oxidation, if cows are

in negative energy balance, energy expenditure/cardiac output consistent with increase in

milk yield, productive efficiency, and also decreases AA oxidation. With regard to the

adipose tissue, the adipocyte is a major target for somatotropin action. It regulates the

biochemical pathways depicted in Figure 1. Under somatotropin supplementation, dairy

cows in positive energy balance have decreased lipogenesis, because the hormone

reduces the lipogenic action of insulin and adenosine, an autocrine/paracrine factor. If

dairy cows are in negative energy balance under somatotropin supplementation, there is

an increase in basal lipolytic rate due to enhanced sensitivity of adipocytes to

catecholamines (Jaster and Wegner, 1981; Vernon and Pond, 1997; Rukkwamsuk et al.,

1999).

In a comparison to the genetically superior cow (GS cows), bST supplemented

cows (bST cows) did not differ for most of the variables evaluated (Peel and Bauman,

1987). Digestibility of feed, maintenance, partial efficiency of milk synthesis, and overall

efficiency (kg milk/kg feed) did not differ between the two groups. However, GS cows

better utilized their body reserves during early lactation, and they also had higher feed

intake than bST cows. In bST cows, increased mobilization of body reserves to provide

needed nutrients and increased feed intake occurred 5 wk after beginning of bST

supplementation. The mammary gland of GS cows had greater quantities of secretary

tissue, but activity per secretary cell was not known. In bST-supplemented cows there is









an increase in synthetic rate/activity per cell in the mammary gland, but not in number of

secretary cells (Capuco et al., 2001). However, in both GS and bST-supplemented groups

of cows there is a need for improved management to optimize reproductive performance,

and to reduce the calving interval, which otherwise is increased. These comparisons

indicated that any dairy cow with good productive potential can become more like a

genetically superior animal by supplementing it with bST.

The advent of recombinant DNA technology during the early 1980s also has led

to the greatest rate of advance in the field of lactation biology. Recombinant bST allowed

a great number of researchers to adequately explore overall potential to increase milk

production of dairy animals, and also to understand the physiological mechanisms that

elicited such responses. The increase in mammary gland activity due to the direct actions

of IGF-I and IGFBPs, changes in adipose tissue and liver function due to direct actions of

somatotropin, and indirect actions on overall body metabolism are key events that led to

increased productive performance. These latter findings also improved knowledge about

the possible roles of somatotropin in metabolic adaptations observed during the transition

period.

Strategies to Improve Metabolic Adaptations during the Transition Period

Among all metabolic adaptations seen during the peripartum phase, those related

to lipid metabolism appear to be the most important. The modem dairy cow relies heavily

upon the mobilization of lipids that then are used as an energy source for peripheral

tissues (ketone bodies), and also as a precursor for synthesis of milk fat by the mammary

gland (NEFAs) during early lactation. Increases in lipid metabolism push hepatic

functions to the limit, and any imbalance in the synchrony of metabolic events can lead to









a greater increase in fat mobilization, a change that can lead to disease ketosiss). Attempts

to improve overall functionality during the transition period should focus on increasing

the availability of energy sources for liver gluconeogenesis, to minimize mobilization of

lipids, and to improve export of fat from the liver (Grummer, 1993).

In a review article, Grummer (1995) summarized and evaluated some findings

that could be used to improve metabolic adaptations during the transition period. One of

the first observations was that DMI, as a percentage of BW, at d 21 postpartum was

positively correlated with DMI at d 1 prepartum. One likely explanation for this is that

overconditioned cows during the prepartum had a poor appetite postpartum (high body

availability of energy precursors [fat]). Therefore, management strategies to avoid

overconditioning of cows during the dry period should be used. Another approach would

be to offer diets differing in energy (NEL) and CP contents during the prepartum period.

In general, observations were that cows fed diets with increased nutrient concentrations

(increased proportion of concentrate in the diet) had increased DMI during the time

period from d 21 to 7 prepartum. As expected, these differences decreased and were lost

as parturition approached. Furthermore, the higher intake of nutrients during that 2 wk

period prepartum caused a more severe decrease in DMI during the final week before

calving.

Despite the potential negative effects of DMI, the benefits of feeding high energy

diets may offer greater benefits than simply providing more calories. For example, the

increase in prepartum dietary energy content initiates the appropriate adaptations in the

rumen to the postpartum diet, which is high in overall and specific nutrient content to

support milk production (National Research Council [NRC], 2001). Changes include








those in the microflora population and an increase in length of papillae that, in times

needed, increase the production and absorption of gluconeogenic VFA (proprionate)

compared to non-adapted cows (Goff and Horst, 1997). Increased gluconeogenesis

minimizes the depletion of hepatic glycogen stores, increases concentration of glucose

and insulin in serum, decreases serum concentrations of NEFA and TAG deposition in

liver, and potentially reduces ketogenesis (Grummer, 1993). In fact, these improvements

in metabolic adaptations were observed by Doepel et al. (2002). In their experiment,

Holstein cows fed a high energy (HE) diet (1.65 vs. 1.30 Mcal/kg of NEL) during the

prepartum transition period had increased postpartum DMI and improved energy balance

during both periods. In addition, HE diet reduced plasma concentrations of NEFA at

calving and reduced hepatic TAG content.

Other methods that could be used to increase quantities of gluconeogenic

precursors include oral supplementation of propylene glycol (PG) as a drench (Grummer,

1995) or including ionophores in the TMR (Vallimont et al., 2001). The use of these

compounds during the transition period increased concentrations of glucose in blood and

decreased concentrations of NEFA.

Another strategy that could be used to improve extent and timing of metabolic

adaptations in cows during the transition period is the supplementation of recombinant

bovine somatotropin (bST). The overall effects of bST on the metabolism of lactating

dairy cows (Bauman, 1999) could be beneficial to the transition dairy cow. During the

early 1990s, researchers supplemented dairy cows with bST before calving or during the

early postpartum period (transition period) to evaluate effects on MY (Bachman et al.,

1992; Santos et al., 1999; Putnam et al, 1999), on improvement of recovery of diseased








animals with fat cow syndrome (Maisey et al., 1993; Laven and Andrews, 1998), and on

mineral metabolism (Law et al., 1994; Eppard et al., 1996).

Results of previous studies were variable. For example, Bachman et al. (1992) did

not observe any positive or negative effects of bST-supplementation (25 mg/d) from d 21

to 7 prepartum on MY of cows during the subsequent lactation. However, Santos et al.

(1999) did observe significant effects of bST (500 mg/14 d) on daily MY of Holstein

cows injected from 5 through 45 DIM. Despite an increase in MY (about 10%), DMI was

slightly less in bST-supplemented cows. Results indicated there was increased efficiency

of nutrient utilization to produce milk in the bST-supplemented cows. Putnam et al.

(1999) observed similar results with regard to production responses. In their study,

Holstein cows supplemented with bST (500 mg/14 d) from d 28 prepartum through d 42

postpartum had substantial increases in MY. Cows supplemented with bST produced 3.3

kg/d more milk than control cows through 42 DIM, but DMI was slightly higher in

supplemented cows, in contrast to results of Putnam et al. (1999).

Studies of Maisey et al. (1993), Laven and Andrews (1998), and Eppard et al.

(1996) obtained contrasting results. In the first experiment (Maisey et al., 1993), cows

with fatty liver syndrome that were supplemented with 640 mg of bST showed no

increase of plasma concentrations of glucose or B-HBA, indicating that bST did not lead

to clinical ketosis. In the study by Laven and Andrews (1998), Jersey cows with signs of

fatty liver syndrome and supplemented with 640 mg of bST had greater plasma

concentrations of NEFA and B-HBA than diseased non-treated cows. In the third study

(Eppard et al., 1996), transition Jersey cows injected with 500 mg bST/14 d from 28 d

prepartum through 14 d postpartum had an increase in duration but not in the incidence of








clinical ketosis. The bST led to greater concentrations of B-HBA in blood of

supplemented cows than non-supplemented cows.

The research of Maisey et al. (1993), Laven and Andrews (1998), and Eppard et

al. (1996) indicated that bST-supplementation caused both positive or negative effects on

dairy cows during the transition period and early lactation. However, it is important to

mention that in each study they supplemented high doses of bST (22.8-35.7 mg/d).

Others have explored the potential of using low doses of bST (5-15 mg/d) during the

postpartum period (Stanisiewski et al., 1992) or during the dry period (Simmons et al.,

1994) to evaluate the effects on production, overall metabolism, health and reproduction.

In the first study (Stanisiewski et al., 1992), multiparous Holstein cows injected with 5 or

14 mg of bST from 0-60 DIM and from 61-130 DIM had significant increases in FCM/d

(1.2 and 1.3 kg for 5 and 14 mg/d of bST, respectively) compared to non-supplemented

control cows. From 61-130 DIM, increases were 2.7 to 4.1 kg/d. Groups of cows

supplemented with low doses of bST had increased pregnancy rates and first conception

rates, and no adverse effects on health were observed. In the second study (Simmons et

al., 1994), Holstein cows were supplemented with 0, 5 or 14 mg/d of bST during the last

46 6 d before expected parturition. Supplementation with low doses of bST

significantly increased concentrations of somatotropin in blood (6.5 vs. 22.7 ng/mL),

better maintained IGF-I concentrations as parturition approached, and did not impact

serum concentrations of NEFA during the treatment period. These studies indicated that

supplementing low doses of bST may positively impact production, reproduction and

health of transition cows, without showing the adverse effects on metabolic adaptations

that were observed when full doses were used during similar time period.









In subsequent studies (Garcia-Gavidia et al., 2000; Gulay et al., 2000),

multiparous Holstein cows were supplemented with bST at 0, 5.1, 10.2 and 15.3 mg/d

during the prepartum and postpartum periods. Objectives were to evaluate the effects on

productive and metabolic responses. In both experiments, injections began at 3 wk

prepartum and extended through -65 DIM. In the study of Garcia-Gavidia (2000), groups

of control (non-supplemented) and bST-supplemented cows (5.1 mg/d of bST) were fed

postpartum diets containing 0 or 15% whole cottonseeds. During the prepartum, bST-

supplementation increased concentrations of glucose, but no effects were observed on

mean concentrations of insulin or IGF-I in plasma. The prepartum DMI of bST-

supplemented cows was slightly greater than non-supplemented cows. In addition, no

significant effects of bST, diet or their interactions were observed on any variables during

the first -65 DIM. The milk production was numerically but not significantly greater in

bST-supplemented cows (39.27 vs. 37.69 kg/d, an increase of 4.2%).

In the study conducted by Gulay et al. (2000), treatments included prepartum and

postpartum supplementation with various amounts of bST (0, 5.1, 10.2 and 15.3 mg/d;

groups I to IV, respectively) that began at the same time prepartum as in study of Garcia-

Gavidia (2000). Effects of bST-supplementation on DMI, BCS, BW, MY, metabolic

hormones and metabolites were evaluated. During prepartum, the two greater amounts of

bST (10.2 and 15.3 mg/d) caused increases in plasma concentrations of somatotropin, but

only the greatest amount (15.3 mg/d) increased plasma concentration of IGF-I. During

the postpartum, the 10.2 and 15.3 mg/d amounts of bST caused increases in plasma

concentrations of somatotropin, IGF-I, T3 and numerically greater MY (30.93, 31.23,

32.03 and 34.93 kg/d for groups I to IV, respectively). However, for BCS, cows in groups









Im and IV better maintained BCS than cows in groups I and I. No adverse effects of

bST-supplementation were observed during the experimental period.

Subsequently, Gulay et al. (2000, 2003b) evaluated the effects of bST

supplemented at 10.2 mg/d during the prepartum and postpartum transition period on

DMI, BW, BCS, MY and plasma concentrations of IGF-I, insulin and NEFA of Holstein

cows. Supplementation began about 21 d prepartum and was extended through 42 DIM.

No effects of bST supplementation were detected on DMI, BW and BCS during the

prepartum and postpartum periods. Supplemented cows had numerically but not

statistically greater MY (FCM 3.5%) than non-supplemented cows. During prepartum, no

differences were detected for mean concentrations of glucose or NEFA, and not for

somatotropin, as was expected. During the postpartum, no differences were detected for

mean concentration of insulin, glucose or NEFA. Plasma concentrations of somatotropin

and IGF-I were greater in bST-supplemented cows than non-supplemented cows, as

expected.

The numerically greater increases in MY observed for bST-supplemented cows in

these studies, without an adverse effect on postpartum DMI, BCS, or concentration of

NEFA in plasma, indicated that daily amounts of bST had a significant positive impact

on transition dairy cows. Because milk production is highly associated with health rates,

as mentioned in a previous section, bST may also have played a role in improving the

metabolic adaptations to avoid disorders, mainly related to liver metabolism.

Somatotropin is a metabolic hormone that has an important role in regulating some of the

adaptations that involve adipose tissue metabolism and liver function. Therefore, use of

bST-supplementation has the potential of stimulating desirable positive changes.






62


However, to date, no study has been carried out to critically evaluate the effects of

supplementing low doses of bST (10.2 mg/d) during the transition period, or only during

the prepartum or during the early lactation on changes in metabolic adaptations involving

overall metabolism, on liver metabolism of carbohydrates and lipids, on health, and any

possible effects of these changes on productive responses of multiparous Holstein cows.















CHAPTER 3
MATERIALS AND METHODS


All cows used in this experiment were housed at the Dairy Research Unit (DRU)

of the Department of Animal Sciences of the University of Florida, Hague, FL where the

experiment was conducted. Treatments, measurements and sample collections began in

August 2001 and ended August 2002. This experiment was approved by the Institutional

Animal Care and Use Committee (IACUC) of the University of Florida.

Animals

One hundred and three multiparous Holstein cows were assigned randomly to the

experiment when they were about 21 d before expected parturition. Cows were assigned

to four treatment groups (I to IV) as they become available to enter trial during the

Summer of 2001. The number of previous lactations of experimental cows at the time

they were assigned ranged from 1 to 5. Average number of previous lactations for each of

the four groups was 2.32, 1.84, 2.16 and 2.16 for groups I, II, III and IV, respectively.

The actual days that cows assigned to the four groups spent during the prepartum period

were 20, 19, 19 and 19 1.46 d, respectively. The body weight (BW) and body condition

score (BCS) of the cows at the time that they were assigned to treatment ranged from 598

to 889 kg, and 2.5 to 5.0 pt., respectively. The average BW and BCS were 735 kg and

3.41 pt. among all cows, and neither measures differed among TRT groups (P=0.8881 for









BW and P=0.5201 for BCS). After calving, all cows remained on the experiment through

150 DIM.

Treatments

Experimental design. Cows were assigned randomly to a 2x2 arrangement of

treatments based upon period of bST supplementation during the transition period and/or

early lactation. The bST used was prepared as an oil emulsion (500 mg/1.4 mL) that is

marketed under the name POSILAC for enhancement of lactation (Monsanto Co., St.

Louis, MO). The amount of bST supplemented was 0.4 mL/14 d; 1/3 of the bST dose

(-1.2 mLU14 d) recommended for the enhancement of lactation. This quantity provided

approximately 10.2 mg/d of bST for the 2-wk injection time periods. Prepartum

injections began at about 21 d (3 wk) before parturition and were extended through

calving day, a total of 2 injections. Postpartum injections began approximately 24 h after

calving (regardless of time of last injection) and extended through 70 DIM, a total of 5

injections. The last injection was at 56-58 DIM. The arrangements of treatments and cow

numbers per TRT group follows:


Treatment I (Control) 26 cows No bST Prepartum, No bST Postpartum
Treatment II (Post) 25 cows No bST Prepartum, bST Postpartum
Treatment III (Pre) 27 cows bST Prepartum, No bST Postpartum
Treatment IV (Pre/Post) 25 cows bST Prepartum, bST Postpartum


Injections were in the left or right ischiorectal fossa and were administered after

blood collection, but prior to a.m. feeding and milking. After 63 DIM, all cows, including

the controls, were supplemented with a full dose of bST (500 mg/14d) during the

remainder of their lactation, according to management practice at the DRU.









Feeding. During the experimental period, prepartum and postpartum cows of all

treatment groups were managed and fed in the same calving pen and fresh cow free-stall

barn, respectively. Diets were fed ad libitum to allow 5-10% daily feed refusals.

Prepartum cows were fed once daily between 10:00-12:00h, and postpartum cows were

fed twice a day between 10:00-1200h and again between 16:00-18:00h. Feed adjustments

were made daily to allow desired feed refusals. Prepartum and postpartum diets were

prepared following National Research Council (NRC, 2001) recommendations for both

animal classes, and were anionic close-up diet (CUD) and TMR fresh cow diet cationicc),

respectively. Diet composition and formulations are in Table 1. Diets were prepared as

total mixed rations (TMR) and were group fed from a mixer wagon. No individual

records of feed consumption were made through the experimental period, except group

intakes were estimated daily by the feeding crew so amount offered exceeded amount

consumed.

Body Weight and Body Condition Score

Body weight (BW) and body condition scores [BCS; 1 (thin, underconditioned)

and 5 (fat, overconditioned); Edmonson et al.,1989] were recorded during the

experiment. Each cow was weighed and BCS was estimated at time of group assignment

(~ 21 d prepartum) and weekly on the same day each week (Friday) before

a.m. feeding or milking (8:00-12:00h) through 70 d postpartum. Thereafter, BW and BCS

were estimated biweekly until cows completed the experiment (- 150 DIM).

Colostrum and Milk Samples

A sample of colostrum was collected from all cows at first milking (n=103), and

samples were analyzed for quality using a commercial hygrometer (Colostrometer,









Table 1. Dry matter concentrations and Chemical Composition of Anionic close-up diet
(CUD) and Fresh-Cow TMR fed to Holstein Cows.
Item Anionic CUD2 Fresh-Cow TMR3
Ingredient ----------DM basis----------

Corn Silage 44.2 21.7
Sorghum Silage --- 8.3
Alfalfa Hay --- 7.3
Cottonseed Hulls 8.8 5.5
Whole Cottonseeds 6.7 7.7
Citrus Pulp 8.5 7.7
Corn Meal 13.0 20.1
Molasses --- 2.6
Soy Plus4 2.2 6.5
Soybean Meal 7.0 9.4
Springer Minerals 7.4 ---
Lactating Cow Minerals --- 3.3
White salt 0.3 ---
Zinc Chloride 1.2 ---
Magnesium Sulfate 0.7 ---

Nutrient Percentage5

DM 49.41 56.66
CP 13.80 17.45
Sol CP6 29.42 30.51
NDF 37.42 34.74
ADF 24.49 22.36
NSC 36.12 39.88
EE7 4.18 4.56
TDN(%) 66.60 72.75
NEL (Mcal/kg) 1.47 1.60
Ca+2 1.66 0.65
P+ 0.34 0.38
Na+ 0.21 0.30
K+ 1.02 1.33
Cl1 1.09 0.23
S-2 0.40 0.18
Calculated DCAD8 -5.78 +38.75
'Values expected for diets formulated (National Research Council [NRC], 2001) on dry matter
basis. 253:47 forage:concentrate. '43:57 forage:concentrate. 4Trademark of West Central, Ralton,
IA. 5 DM Basis. 6 Soluble protein as percentage of the CP. 7 Ether extract. 8 Meq (Na + K) (Cl +
S)/100 g of diet DM.








Nasco, Fort Atkinson, WI). Values obtained were recorded as mg of

immunoglobulins/mL of colostrum. Milk samples also were collected bi-weekly from all

cows (n=103) during three consecutive milkings (08:30, 15:00 and 01:30h) on the same

day of the week for analyses of milk constituents during the first 10 wk of lactation.

Samples (50 mL) were analyzed for fat, protein and SCC contents at Southeast

Milk Laboratory, Inc. (Belleview, FL). Milk yield was recorded at each daily milking

from 3 d after parturition through 150 DIM.

Blood Collection, Handling and Storage

Blood samples were collected from the tail vein of all cows (n=103) three times a

week before a.m. feeding or milking (06:30-10:00 h) through 70 DIM. Prepartum and

postpartum cows were bled in a holding area walk through or in the free-stall barn,

respectively, after elevating the tail without any other restraint. For blood collection,

Vacutainer brand needles (2.54 cm; 20 gauge) and tubes containing sodium heparin

were used (10 x 100 mm blood collection tubes, Becton-Dickinson, Fairlawn, NJ). Blood

samples were placed in an ice-bath immediately after collection and processed within 2 h.

The order in which cows were sampled on a given day was random and differed from

bleeding to bleeding. After sampling, prepartum cows were released to the dry lot, and

postpartum cows were milked and then returned to the free-stall barn.

All samples of blood were centrifuged at 3,000 RPM at 50 C for 30 min (RC-3B

refrigerated centrifuge, H 600A rotor, Sorvall Instruments, Wilmington, DE) to separate

plasma. Plasma from each sample was aliquoted into labeled 5 mL (12x75 mm)

polypropylene tubes (Sarstedt Inc., Newton, NJ), capped, and frozen at -200 C until

analyzed. The plasma samples were used for analysis of insulin, somatotropin (ST),









insulin-like growth factor 1 (IGF-I), calcium (Ca), glucose, non-esterified fatty acids

(NEFAs) and P-hydroxybutyrate (B-HBA).

Liver Biopsies, Handling and Storage

Liver biopsies were conducted in a subset of 8, 10, 10 and 11 cows randomly

selected from TRT groups I, II, III and IV, respectively. For each cow in the four subsets,

puncture biopsies were conducted at about 21 d prepartum, +2, +14 and +28 d

postpartum, a total of 4 biopsies per cow, as described by Hughes (1962). The procedure

was conducted with cows in a restraint chute as follows. An 8x8 cm area in the medium-

dorsal region of the 9, 10 or 11th intercostal space of the right side of the cow was clipped

and cleaned with Betadine Surgical Scrub (10% povidone iodine, 1% glycerin, 0.25%

igepal, and 88.75 % purified H20; Purdue Manufacturing Company, Norwalk, CT) and

70% Alcohol (Dal-Vet Phamaceudicals, Savannah, MO). The skin region to be

punctured was anesthetized subcutaneously and intra-muscularly with = 5 mL of

Lidocaine Hydrochloride with Epinephrine (1.0-2.0 % lidocaine hydrochloride, 0.001%

L-epinephrine and purified H20, Elkins-Sinn, INC., Cherry Hill, NJ). Following

anesthesia, a 2.5 cm incision was made in the skin with an Axicut Sterile Disposible

Scapel (Dynarex Corporation, Brewster, NY), and the liver was punctured with a custom-

made biopsy needle (30 cm long by 0.6 cm wide). The liver obtained from the puncture

(3-5 g) was rinsed with 0.9% saline solution, blotted dry on filter paper, placed in a

cryogenic vial (Fisher Scientific, Pittsburgh, PA, R#05-669-64) and submerged in liquid

nitrogen (-192 oC) until transferred to a -80 C freezer (Bio Freezer, Mallinckrodt, INC.,

Marietta, OH). Following puncture, the incision was sutured using a skin stapler (3MTM

PreciseTM PGX Disposable Skin Stapler, St. Paul, MN), and treated once with









Nitrofurasone (NAPP Chemicals, Lodi, NJ). Liver samples were subsequently analyzed

for total fat (FAT) and triacylglycerol (TAG) content, and also to quantify the abundance

of messenger ribonucleic acid (mRNA) of genes in liver encoding enzymes pyruvate

carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and microsomal

triacylglycerol transfer protein (MTP).

Radioimmunoassays

Second antibody preparation. Florida native sheep managed at the DRU were

used to develop specific second antibodies for use in specific radioimmunoassays (RIA)

of hormones and IGF-I. Rabbit gamma globulin (Sigma Chemical Co., St. Louis, MO. #

R-9133) and guinea pig gamma globulin (Sigma Chemical Co., St. Louis, MO. # R-9135)

were injected into sheep to induce production of second antibodies. Guinea pig gamma

globulin (20-25 mg) or rabbit gamma globulin (30-40 mg) were added to 5 to 15 mL

distilled water in an Erlenmyer flask and stirred on a magnetic stir plate until solubilized.

Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO) was added in equal

amounts to each solution and mixed using an OMNI 2000 micro-blender (OMNI

International, Waterbury, CT) for approximately 1 min. About 5 ml of this homogenized

mixture were injected subcutaneously (SC) over the left and right shoulders of each sheep

in three to four separate locations. Ten to 14 d after initial injection the procedure was

repeated, except Freund's incomplete adjuvant was mixed with the gamma globulins

prepared as described above. After 14 d the sheep were bled by jugular venipuncture;

approximately 400 ml of blood were taken using an 18 gauge butterfly needle and 60 cc

syringe to withdraw the blood. Blood was dispensed into 40 ml screw top centrifuge

tubes, placed on ice and left overnight to clot (16 h at 4 OC). Tubes then were centrifuged









at 3000 RPM for 30 min (RC-3B refrigerated centrifuge, H 600A rotor, Sorvall

Instruments, Wilmington, DE). Serum was pooled and frozen. Sheep were bled again

after an additional 28 d and thereafter at irregular intervals. Sheep were re-injected with

gamma globulins in Freund's incomplete adjuvant (Sigma Chemical Co., St. Louis MO)

at about 3 to 6 mo intervals. This procedure provided large batches of sheep anti-rabbit

gamma globulin serum (SAR) or sheep anti-guinea pig gamma globulin serum (SAGP)

for use as the second antibody in the specific RIA procedures.

Insulin, somatotropin and IGF-I iodination and protein separation. The

iodination procedure used was similar for all hormones. For insulin, bovine insulin (100-

300 lig # 118F-4826; Sigma Immunochemicals, St Louis, MO) was weighed and mixed

with an equal quantity of 5 mM HCI (pH=2.5) to yield solutions that contained 1 lg/pL.

For somatotropin (ST), bovine ST (100-300 gug; USDA Reproduction Lab, # AFP 5200)

was weighed and mixed with an equal quantity of 0.01 M NaHCO3 (pH=9.5) to yield

solutions that contained 1 lg/piL. Then, an equal amount of 0.01 M phosphate buffer

(2 mL of 0.5 M phosphate buffer [23.04 g sodium phosphate monobasic, 70.98 g sodium

phosphate dibasic and 1.0 g sodium azide in 1 L distilled H20, pH=7.5] in 100 mL

distilled H20, pH=7.5) was added to give a final concentration of 0.5 ug insulin or 0.5 jug

ST/pL solution, respectively. For insulin-like growth factor-I (IGF-I), bovine IGF-I

(25 gg Cat# 01-189; Upstate Biotechnology, Lake Placid, NY.) was dissolved in 250 uL

of 0.1 M acetic acid to give stock 0 (0.1 gig/juL). Then, following procedures outlined, 10

gL of each solution containing insulin, ST or IGF-I were frozen in microcentrifuge vials

(Fisher Scientific, 1.0 mL, flat top) for use in the iodination procedure.








To separate iodinated insulin (INS1125) and IGF-I (IGF-I 125) from free iodine, a 16

mL (20 cm long) chromatography column (Kontes Glass Company, Vineland, NJ, #

420400-1020) was used. To separate iodinated ST (ST 25) from free iodine, a 40 mL (50

cm) chromatography column (Kontes Glass Company, Vineland, NJ, # 420401-1050)

was used. For insulin and IGF-I, and for ST, Sephadex G-50 and G-75 (Sigma Chemical

Co., St. Louis MO) dispersed in distilled H20 was added to the column until the

Sephadex packing was about 2.5 cm of the top of the column.

Subsequently, the insulin column was washed with 5 mL of borate/BSA buffer

(16.50 g boric acid, 5.40 g NaOH, 6.0 mL 12 N HC1, 200 mg Merthiolate and 0.5% BSA

in 2 L distilled H20, pH=8.0); ST column was washed with 5 mL of 0.1 M

phosphate/BSA buffer (9.216 g sodium phosphate monobasic, 28.392 g sodium

phosphate dibasic, 2 g sodium azide, 16.8 g NaCI and 5 g BSA in 2 L distilled H20,

pH=7.5); and IGF-I column was washed with 5 mL of phosphate/BSA buffer (200 mg

protamine, 4.14 g sodium phosphate, 10 mL of a 2% solution of sodium azide, 3.72 g

EDTA and 2.5g BSA in 1 L distilled H20, pH=7.5). Following this first wash, columns

for each hormone were re-washed (5 mL) with 0.01 M phosphate buffer. Phosphate

buffer was retained at the top of the column until it was used for protein separation.

Immediately prior to the iodination, 3 mg of chloramine-T and 5 mg sodium

metabisulfite were weighed into 12x75 mm borosilicate glass tubes (Fisher Scientific,

Pittsburgh, PA) then each chemical was dissolved individually in 1 mL 0.5 M phosphate

buffer. Sequentially, radioactive iodine (1125 10 gL, I mci/10 tL), 0.5 M phosphate buffer

(20 tL), and 10 pL chloramine-T were added to the vial that contained the insulin, ST or

IGF-I. The iodination reaction was allowed to proceed for 15-20 sec, during which time









contents were mixed by finger tapping; then 10 gL sodium metabisulfite were added to

allow complete oxidation of chloramine-T. The final reaction mixture then was

transferred to the top of the Sephadex column. Following the transfer, the reaction vial

was rinsed with 50 pL 0.01 M phosphate buffer (for all hormones or growth factor) to

ensure complete transfer of the iodinated proteins.

To collect iodinated insulin, borosilicate tubes (13x100 mm) numbered 1 to 35

were filled with 500 .gL of borate/BSA buffer; for ST, tubes were numbered 1 to 50 and

500 pL of 0.1 M phosphate/BSA buffer were added; and for IGF-I, tubes were numbered

1 to 35 and 500 gL of phosphate/BSA buffer were added. Tubes were placed in a fraction

collector to receive 20 drops from the Sephadex column. Aliquots (10 pL) of each eluted

fraction were transferred to a second set of tubes to identify elution peaks by counting

radioactivity using a Tracor Analytic Gamma Counter (Nuclear-Chicago, Gamma Trac

1191, G. D. Searle and Co., Des Plaines, IL). Tubes that contained the first large peak,

which corresponded to 1125 bound to protein, were pooled. The second large peak was

free 1125. The I'25-INS, -ST and -IGF-I were stored at 4 oC and used within 15 d of

iodination.

Insulin Assay

A double antibody radioimmunoassay procedure, as described by Soeldner and

Sloane (1965), modified and validated by Malven et al. (1987), was used for assay of

insulin in plasma samples. Purified insulin (Sigma Immunochemicals, St. Louis, MO.

R#118F-4826) was weighed (-100 gg) as stock insulin and dissolved in 5 mM HCL

(pH=2.5). The stock INS (10 gg/10 pL) was diluted in borate/BSA buffer to give a final

concentration of 100 ng/ml for preparation of standards that contained 0.1, 0.2, 0.3, 0.5,









0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 15.0 ng INS/mL. The first antibody (primary), guinea

pig anti-bovine insulin, was obtained from Sigma Chemical Co. (St. Louis, MO). It was

dissolved in borate/BSA buffer (1:20,000). The second antibody, sheep anti-guinea pig

(SAGP) gammaglobulin, was prepared as described, and diluted 1:2 to 1:5, as

appropriate, for complete precipitation of the RIA reaction complex, in borate/EDTA

buffer (16.50 g Boric acid, 5.40 g NaOH, 6.0 mL 12 N HCI, 200 mg Merthiolate and 1.86

g EDTA disodium salt diluted in 100 mL of borate buffer, pH=8.0). Normal guinea pig

serum (NGPS) was diluted 1:200 in borate/BSA buffer for use in the assay.

Aliquots of insulin standards (100 gLL) and plasma samples (125 gL) were diluted

with 200 and 175 pL borate/BSA buffer, and assayed in triplicate and duplicate,

respectively, in 12x75 mm borosilicate tubes. Immediately after samples were pipetted,

100 jiL diluted primary antiserum were added to all tubes, except for total count, NSB-B

and NSB-P tubes. Eight to 12 h later 100 gL iodinated insulin (INS""25 30,000 CPM)

were added to all tubes. Tube arrangements and the volumes used in all individual assay

tubes are in Table 2.

Table 2. Arrangement of tubes for radioimmunoassay of insulin.
Tubes' Buffer Sample 1st Ab 1125 Plasma
TCT --- --- --- 100 ---
NSB-B 400 --- --- 100 ---
NSB-P 275 --- --- 100 125
Zero 300 --- 100 100 ---
Standard 200 100 100 100 ---
Sample 175 125 100 100 ---
'All volumes in gL. TCT is the total count tube. NSB-B is the buffer nonspecific binding
tube. NSB-P is the plasma nonspecific binding tube. Zero is reference tube, no insulin
added.









After pipetting, all assay tubes were incubated for a total of 24 h at 4 oC.

Following incubation period, 100 g.L each of SAGP and NGPS were added to all tubes,

except the total count tubes, and then incubated for additional 10 min. Then, 750 gL of

15% polyethylene glycol (PEG, Carbowax PEG 8000, Fisher Scientific, Pittsburgh, PA)

in borate buffer (16.50 g boric acid, 5.40 g NaOH, 6.0 mL 12 N HCI and 200 mg

Merthiolate in 2 L distilled H20, pH=8.0) were added to all tubes except the total count

tubes, and vortexed. Following 5 to 10 min incubation the tubes were centrifuged at 3000

RPM for 30 min at 4 C (RC-3B refrigerated centrifuge, H 600A rotor, Sorvall

Instruments, Wilmington, DE), decanted, and inverted on absorbent paper to drain. After

tubes had dried at room temperature, bound radioactivity in tubes was measured using a

Packard auto gamma counter (model B-5005). Results were calculated using the spline

curve fit for radioimmunoassay data processing procedure with 6.67 as a correction

multiplier to correct concentrations to ng/ml. The intra- and inter-assay variations for

insulin radioimmunoassay were 5.78 and 3.6%, respectively.

Somatotropin Assay

A double antibody radioimmunoassay procedure, as described by Garcia-Gavidia

(1998), was used for assay of ST in plasma samples. Purified bovine ST, supplied by

USDA (AFP-5200), was weighed (1 mg) as stock ST and diluted in 10 mL of 0.01 M

NaHCO3 (pH=9.5) to give a concentration of 100,000 ng/mL. Two (2) mL of the stock

ST were was diluted with 18 mL of 0.1M phosphate/BSA buffer to give a concentration

of 10,000 ng/mL. Then, 1 mL of this solution was diluted with 99 mL of 0.1 M

phosphate/BSA buffer to give a solution with final concentration of 100 ng/mL for

preparation of standards. A set of 12 standards was prepared to contain 0.1, 0.2, 0.4, 0.6,









0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, and 5.0 ng ST/100 gL. For first antibody preparation,

Rabbit anti-ovine ST (National Hormone and Pituitary Program) was diluted in 0.1 M

phosphate/BSA buffer (1:40,000). Second antibody (sheep anti-rabbit serum [SAR]) was

diluted 1:4 in phosphate/EDTA buffer without BSA. Normal rabbit serum (NRS) was

diluted 1:100 in 0.1 M phosphate/BSA buffer for use.

Aliquots of ST standards and plasma samples (100 glL) were diluted with 200 gtL

0.1 M phosphate/BSA buffer, and assayed in triplicate and duplicate in 12x75 mm

borosilicate tubes, respectively. Immediately after samples were pipetted, 100 jtL of first

antibody and 100 pL of iodinated (ST"25 = 25,000 cpm) were pipetted into all tubes,

except for total count, NSB-B and NSB-P, tubes that received no first antibody. After

incubation for 24 h at 40C, 100 RL each of SAR and NRS were added to all tubes, except

the total count tubes, and mixed (Table 3). Then, 1.0 mL of 6.0% polyethylene glycol

(PEG, Carbowax PEG 8000, Fisher Scientific, Pittsburgh, PA) in 0.1 M phosphate buffer

(without BSA) was added to all tubes, except total count, and vortexed for 1 min.

Following a 5 to 10 min incubation the tubes were centrifuged at 3000 RPM for 30 min at

4 C (RC-3B refrigerated centrifuge, H 600A rotor, Sorvall Instruments, Wilmington,

DE), decanted, and inverted on absorbent paper to drain.

After tubes had dried at room temperature, bound radioactivity in tubes was

measured using a Packard auto gamma counter (model B-5005). Results were calculated

using the spline curve fit for radioimmunoassay data processing procedure with 10 as a

correction multiplier to correct concentrations to ng/ml. The intra- and inter-assay

variations for somatotropin insulin radioimmunoassay were 5.69 and 3.55%, respectively.









Table 3. Arrangement of tubes for radioimmunoassay of ST.

Tubes' Buffer Sample 1st Ab 1125 Plasma
TCT --- --- --- 100 ---
NSB-B 400 --- --- 100 ---
NSB-P 300 --- --- 100 100
Zero 300 --- 100 100 ---
Standard 200 100 100 100 ---
Sample 200 100 100 100 ---
SAll volumes in gL. TCT is the total count tube. NSB-B is the buffer nonspecific binding
tube. NSB-P is the plasma nonspecific binding tube. Zero is reference tube, no ST added.

IGF-I Assay

A double antibody radioimmunoassay, as described by Abribat et al. (1990) and

modified for sample extraction by method of Daughaday et al. (1980) and Enright et al.

(1990), was used for determination of IGF-I in plasma samples.

Extraction of IGF-I from binding proteins. The method of Enright et al. (1990)

was used for the extraction of IGF-I from its binding proteins in plasma samples. An

extraction mixture of ethanol, acetone and acetic acid (EAA 60:30:10 by volume) was

used for extraction. Exactly 100 giL plasma and 100 gL pool plasma for NSB-P were

pipetted into 12x75 mm borosilicate tubes, then 400 ptL of extraction mixture were

pipetted into these tubes. The mixture was vortexed for 15 sec and then allowed to stand

for 30 min at room temperature. After this wait, tubes were centrifuged at 3000 RPM for

30 min at 4 oC (RC-3B refrigerated centrifuge, H 600A rotor, Sorvall Instruments,

Wilmington, DE). Then, 125 giL of the supernatant were transferred into 12x75 mm

polystyrene tubes (Sarstedt Inc., Newtown, NJ), and 50 itL of 0.855 M TRIZMA base









and 175 IpL of the phosphate/BSA buffer were added to each tube to make the final

plasma dilution of 1:14.

Assay. Highly purified recombinant bovine IGF-I (25ig), obtained from Upstate

Biotechnology (Lake Placid, NY, Cat# 01-189) was dissolved in 250 IL of 0.1 M acetic

acid to give stock 0 (100 ng/pL), then aliquoted into microcentrifuge tubes (lig/10 (XL)

and frozen. To make stock 1, 10 plL of stock 0 were added to 490 p.L of phosphate/BSA

buffer to give 2 ng/4L. Stock 2 was prepared by adding 10 jgL of stock 1 to 990 iL of

phosphate/BSA buffer to give a final concentration of 20 pg IGF-1/pL. Standards were

prepared from stock 2 to contain 50, 100, 200, 400, 600, 800, 1000, 1500, 2000, 3000,

4000 and 5000 pg IGF-I/mL. The first antibody, rabbit anti-bovine IGF-I (Lot#

AFP4892898) was diluted 1:160,000 in phosphate/BSA buffer; second antibody (sheep

anti-rabbit serum [SAR]) was diluted to 1:3 in phosphate/BSA buffer; and normal rabbit

serum (NRS) was diluted 1:50 in phosphate/BSA buffer.

Aliquots of plasma extract (10 gXL) and standards (100 pL) were mixed with 190

and 100 pL, respectively, of phosphate/BSA buffer in 12x75 polystyrene tubes, and

assayed in duplicate and triplicate. Immediately after pipetting, 100 gL of first antibody

were added to all tubes, except total count, and NSB-B and NSB-P tubes. Following the

addition of first antibody, 100 pL iodinated IGF-I (IGF-I"25 = 20,000 cpm) were added to

all tubes. After a 24 h incubation at 4 C incubation, 50 pL each of SAR and NRS were

added to all tubes, except the total count tubes (Table 4), mixed and allowed to stand 30

min, and then 1 mL of 6% polyethylene glycol in phosphate/BSA buffer (PEG,

Carbowax PEG 8000, Fisher Scientific, Pittsburgh, PA) was added. Tubes were vortexed

and allowed to stand for an additional 15 min. Following incubation, tubes were









centrifuged at 3000 RPM for 30 min at 4 C (RC-3B refrigerated centrifuge, H 600A

rotor, Sorvall Instruments, Wilmington, DE), decanted, and inverted on absorbent paper

to drain.


Table 4. Arrangement of tubes for radioimmunoassay of IGF-I.

Tubes1 Buffer Sample Is Ab 1125 Plasma
Extract
TCT --- --- --- 100 ---
NSB-B 300 --- --- 100 ---
NSB-P 290 --- --- 100 10
Zero 200 --- 100 100 ---
Standard 100 100 100 100 ---
Sample 190 10 100 100 ---

SAll volumes in giL. TCT is the total count tube. NSB-B is the buffer nonspecific binding
tube. NSB-P is the plasma extract nonspecific binding tube. Zero is reference tube, no
IGF-I added.

After tubes had dried at room temperature, bound radioactivity in tubes was

measured using a Packard auto gamma counter (model B-5005). Results were calculated

using the spline curve fit for radioimmunoassay data processing procedure with 1.4 as a

correction multiplier to correct concentrations to ng/ml of plasma. The intra- and inter-

assay variations for somatotropin insulin radioimmunoassay were 7.08 and 3.6%,

respectively.

Calcium Assay

The method of Miles et al. (2001) for mineral analysis by flame atomic absorption

spectrophotometry was used to determine concentration of calcium in plasma samples.

Calcium reference standard solution (1000 ppm, Fisher Scientific, Pittsburgh, PA, #

SC191-500) was diluted 10-fold in a volumetric flask to prepare 100 ppm Ca stock









solution. From the stock Ca, standards containing 5, 10 and 15 ppm were prepared (Table

5).

To prepare 5% Lanthanum (La), 117.3 g Lanthanum Oxide (La203) were added to

a 2 liter pyrex beaker and dissolved in 500 mL of concentrated HCI in a fume hood. After

the solution released 02 and Cl2 and all La was in solution, it was transferred to a 2 L

volumetric flask and brought to 2 L with distilled water to prepare the 5% La solution.

Table 5. Standards for determination of calcium by flame atomic absorption spectometry.
Standard, ppm
Ingredient 0 5 10 15
100 ppm Ca stock, mL 0 5 10 15
50% TCA, mL 18 18 18 18
5% La, mL 18 18 18 18
TCA=Trichloracetic acid, La=Lanthanum

To prepare a 50% TCA solution, 500 g of trichloracetic acid (TCA) was dissolved in

distilled water in a 1 L volumetric flask. Then, 200 mL 5% La and 200 mL 50% TCA

were transferred to a I L volumetric flask and distilled water added to give 1 liter of a 1%

La-10% TCA solution.

The assay consisted of pipetting 500 gL of plasma samples into polypropylene

tubes (Sarstedt Inc., Newton, NJ). To precipitate proteins, 4.5 mL of the 1% La-10%

TCA solution were added to each tube containing sample, then they were vortexed for 20

sec and finally all tubes centrifuged at 3000 RPM for 20 min at 4 C (RC-3B refrigerated

centrifuge, H 600A rotor, Sorvall Instruments, Wilmington, DE). About 4 mL of filtrate

were transferred into polypropylene tubes and samples were analyzed for concentrations

of Ca using a Flame Atomic Absorption Spectrophotometer (Perkin-Elmer Model 5000).









Glucose Assay

The method of Raabo and Terkildsen (1960) was used for assay of glucose in the

plasma samples. Concentrations of glucose were determined using a Sigma Diagnostics

Glucose Enzymatic kit (# 510) purchased from Sigma Chemical Co. (St. Louis, MO).

Specific concentrations of standards used were: 0, 25, 50, 75 and 100 mg of glucose/dL.

These were prepared by diluting the glucose standard solution provided (100 mg/dL) with

distilled water to achieve the desired standard concentrations at the same time plasma

samples or standards were deproteinized. Standard dilutions used in the assay are in

Table 6.

Table 6. Standard dilutions used in the glucose assay.
Concentration of Standards GLC Standard Solution Amount of distilled
(mg/dL) (p1L) water (gL)
0 0 1000
25 25 975
50 50 950
75 75 925
100 100 900

After preparing the solutions, 100 gL of standards and plasma were added to

12x75 mm borosilicate tubes containing 900 gL of distilled H20. The precipitation of

proteins was effected by adding 500 gL of barium hydroxide solution (0.3N) and 500 gL

of zinc sulfate solution (5%) to all tubes, including standards. The tubes were vortexed

then centrifuged at 3000 RPM for 15 min at 4 OC (RC-3B refrigerated centrifuge, H 600A

rotor, Sorvall Instruments, Wilmington, DE).

Aliquots of the 1:20 protein-free plasma filtrate (20 pL of standards and samples)

were added to wells in a 96-well (0.45 ml well capacity) polypropylene plate (Fisher









Scientific, Pittsburgh, PA) in triplicate and duplicate, respectively. Then 200 gL of

combined enzyme-color reagent solution [(1 capsule of PGO enzymes + 1.6 ml o-

dianisidine dihydrochloride color reagent solution) + 100 ml of distilled water] were

added to each well. The plasma protein-free filtrate and combined reactants were

incubated in a constant temperature oven (Model DN-41, American Scientific Products,

Japan) for 30 min at 370C. After incubation, the absorbance was read in an Automated

Microplate Reader (model EL 309, Bio-Tek Instruments, INC., Laboratory division,

Winooski, VT) using blank as reference at 450 nm wavelength. Arrangement of tubes for

this assay is described in Table 7. Linear regression of absorbance and glucose

concentration was used to determine the concentration (glucose/dL) in plasma samples;

final concentrations were expressed as mg/dL.

Table 7. Arrangement of tubes and solutions used to make plasma protein-free filtrates.
Barium
Barium Zinc Sulfate
Distilled Hydroxide c s ulfate TOTAL
Tubes Sample (uL) solution
Water (pL) solution (L)
(AL)I (RL)2
Standard 900 100 500 500 2
Sample 900 100 500 500 2
'Barium hydroxide, 0.3 N; 2Zinc Sulfate, 5% solution.

Nonesterified Fatty Acid (NEFA) Assay

An in vitro enzymatic method described by Johnson and Peters (1993) was used

for assay of NEFA in all plasma samples. Concentrations of NEFA were determined

using a WAKO NEFA C Test kit (# 994-75409E) purchased from Wako Chemicals

INC., Richmond, VA. Color reagent solution A, provided in the kit, was prepared by

combining 10 mL of diluted color reagent A with 13.3 mL 50 mM phosphate buffer (4.6g

sodium phosphate monobasic, 14.2 g sodium phosphate dibasic in 500 mL distilled








water, pH -6.9): Color reagent solution B was prepared by combining 20 mL of diluted

color reagent B provided in the kit with 33.3 mL of 50 mM phosphate buffer. Vials

containing reagents A and B were mixed gently and stored at 5 oC for up to 2 wk.

Specific concentrations of standards (0, 200, 400, 600, 800 and 1000 pEq

NEFA/L) were prepared by diluting the NEFA standard solution provided (1000 pEq/L)

with 0.9% saline solution (9 g of NaCI in 1000 mL distilled water) to achieve the desired

standard concentration (Table 8).

Table 8. Standard dilutions for NEFA determination.
Concentration of Standards Oleic Stock Solution
(pEq/L) (1.0 mM, LL)0.9% Saline L)
0 0 500
200 100 400
400 200 300
600 300 200
800 400 100
1000 500 0


For the assay, 96 well (0.45 ml well capacity) polypropylene plates (Fisher

Scientific, Pittsburgh, PA) were used. Aliquots (3.0 pL of standards and samples) were

pipetted in triplicate and duplicate, respectively. Following pipetting, 60 gL of Wako

Reagent solution A were added to each well and plates were incubated for 30 min at 25

C in a constant temperature oven (Model DN-41, American Scientific Products, Japan).

After incubation, 120 gL of Wako Reagent solution B were added to each well, followed

by an additional incubation period of 30 min at 25 OC. After this step, the microplate was

allowed to stand for 5 min at room temperature then absorbance for each well was read in

an Automated Microplate Reader (Model EL 309, Bio-Tek Instruments, INC., Laboratory








Division, Winooski, VT), using blank as reference at 550 nm wavelength. Linear

regression of absorbance and NEFA concentration was used to determine the

concentration (NEFA/L) in plasma samples with final concentrations expressed as gEq/L.

P-Hydroxybutyrate (B-HBA) Assay

An in vitro enzymatic method, as described, by Hansen and Freier (1978) was

used for assay of B-HBA in plasma samples. Concentrations of B-HBA were determined

using a Pointe Scientific P-Hydroxybutyrate Kit (# H7587-01; H7587-02) purchased

from Pointe Scientific, Inc., Lincoln Park, MI. Specific standard concentrations used

(2.08, 10.41 and 41.66 mg/dL) were provided in the kit. Preparation of the working

reagent consisted of adding 10 parts of RI with 1.5 parts of R2, both were provided in the

kit.

Aliquots of 3 gL of standard and samples were added to wells in a 96-well (0.45

ml well capacity) polypropylene plate (Fisher Scientific, Pittsburgh, PA) in triplicate and

duplicate, respectively. Following pipetting, 120 pL of working reagent solution were

added to all wells and plates were incubated for 5 min at 37 C in a constant temperature

oven (Model DN-41, American Scientific Products, Japan). After incubation, the

absorbance was read in an Automated Microplate Reader (Model EL 309, Bio-Tek

Instruments, INC., Laboratory division, Winooski, VT) using blank as reference at 490

nm wavelength. Linear regression of absorbance and B-HBA concentration was used to

determine the concentration (B-HBA/dL) in plasma samples with final concentrations

expressed as mg/dL.