The effect of Prede~ 2X and Flucort® on blood metabolites, immune function and milk composition in

Holstein dairy cows

Dy

Madhu Rani Sindhwani

Department of Animal Science McGiII University

February,2007

A thesis submitted to the Faculty of Graduate Studies and Research in partial fultlUment of the requirements for the degree of

MASTER OF SCIENCE

~adhu Rani Sindhwani, 2007

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Abstract

Glucocorticoids are commonly used to treat cows with clinical ketosis and fa~ liver disease. This study investigated the effects of 10 mg/mL of Flucort and Predefi' 2X on the day of calving on blood metabolites, immune function and milk composition on 30 transitional Holstein cows. Sample of blood and milk were analyzed for energy metabolites (glucose, NEF A, BHB and insulin), mineraI metabolites (Ca, P, Na, K, Cl and Mg), energy function parameters (antibody, lymphocyte), milk compositional parameters (protein, fat, lactose, SCC). There were no differences in glucose, Na, Cl, Mg, antibody, lymphocyte and milk fat, were observed among treatments. Flucort® treated cows had significantly lower NEFA on Dl, higher BHB on D21 and D28, lower insulin on D 14, higher Ca on Dl and lower P on Dl. Predefi' 2X treated cows had significantly higher BHB on D21, higher insulin on D7, lower Ca on Dl, higher sec on Dl and higher milk protein on DI. With respect to the significant data in this study, the use of glucocorticoids Flucort® and Prede~ 2X in a single intramuscular injection on dl for the treatment of ketosis is not warranted.

II

Résumé

Les glucocorticoIdes sont couramment utilisés pour traiter les vaches atteintes avec la cétose clinique ainsi qu'avec le syndrome du foie gras. Cette étude avait pour but de déterminer les effets de l'administration de 10 mg/ml de Flucort® et Prede~ 2X suite au vêlage sur les métabolites sanguins, le système immunitaire et la composition du lait de 30 vaches Holstein en période de transition. Les échantillons de sérum et de lait ont été analysés pour les métabolites énergétiques (glucose, AGL, acides B-hydroxybutyriques et l'insuline), les métabolites minéraux (Ca, P, Na, K, Cl et Mg), les paramètres fonctionnels énergétiques (anticorps, lymphocytes) et les paramètres de la composition du lait (protéines, gras, lactose, taux de cellules somatiques). Aucune différence dans le taux de glucose, Na, Cl, Mg, anticorps, lymphocytes et gras dans le lait n'a pu être détectée entre les traitements administrés. Les vaches traitées au Flucort® avaient une concentration d'AGL significativement plus basse au jour 1, un taux d'acides B-hydroxybutyriques plus élevé au jour 21 et 28, une concentration d'insuline plus basse aujour 14, un taux de Ca plus élevé au jour 1 et un taux de P plus bas au jour 1. Les vaches traitées avec Prede:f!' 2X avaient une concentration d'acide B­hydroxybutyrique significativement plus élevée au jour 21, une concentration d'insuline plus élevée au jour 7, un taux de Ca plus bas au jour 1, un taux de cellules somatiques plus élevé au jour 1 et un taux de protéines plus élevé dans le lait au jour 1. Basé sur les données statistiquement significatives obtenues lors de cette étude, l'utilisation des glucocorticoïdes Flucort® et Prede~ 2X en une seule injection intramusculaire ne peut garantir le traitement d'une cétose chez la vache.

III

Acknowledgements

1 would like to express my great gratitude to those who generously provided their supervision and assistance. They were:

Dr. Xin Zhao, my thesis supervisor, for his valuable guidance, patience, and advice during this study.

Dr. Ken Leslie, Dr. Arif Mustafa, Dr. Ng-Kwai-Hang, my committee members, for their advice, opinions and critical evaluations.

Dr. Roger Cue and Dr. Todd Duffield, for their help with the statistical analysis.

Nicole Perkins, Erin Vemooy, Cindy Todd, technicians at the AHL of Guelph University, Hélène Lalande, Jai-Wei Lee, Ming-Kuei Lee, and PATLQ for their help with my experiments.

Paul Meldrum, Phil Lavoie, Nancy Lavigne, Natasha, Abou, Judy, Ali, Lauren, Stephane, Eric, Brad, Alain, José, Martin, Milène, Isabella and Jason at the Macdonald Campus Farm, for their tremendous help with the animaIs.

Barbara Stewart, Sandra Nagy, Cinthya Horvath and Leslie Ann Laduke for their encouraging smiles.

AlI other prof essors, staff, and students at Macdonald Campus who have contributed to the completion of my M.Sc. thesis.

Annie, Josée, Pascale, Christian, Fadi, Malek, Charbel, Ming-Kai, Dana, Marsha and aU the "Girls", for their friendships and encouragement.

My girls at the barn, Viper (6898), Chives (881), Nala (1168), Ruby (6899), Feria (879), Saab (3734), High Society (1618), Happy Time (1613), Babel (893), Mistletoe (6537), Honey Suckle (1188), Muskoka (882), Saraby (1581), Meli Melo (894), Devil (8481), Lotessie (7484), Rocalle (2552), Heather (113), Boo (902), Hokus (1179), Macarena (3448), Mini Me (1626), Muffin (891), Fate (886), Pam (885), Justine (1155), JobeU (1160), Punch (858), Salsa (8401) and Dolly (1612) for being part of my Masters research and being so wonderful to work with.

My amazing parents, my brothers Rav and Maughan and their families for their love and encouragement.

Finally, my wonderful Fiancé, Jag, whom without his support, encouragement and love, this would not be possible.

IV

Table of Contents

Page

Abstract ................................................................................. .11

Résumé ................................................................................. .111

Acknowledgments .................................................................... .IV

Table of Contents ....................................................................... V

List of Tables ............................................................................ X

1. Introduction ........................................................................... 1

II. Literature Review ................................................................... 3

1. Transition Period ............................................................. 3

1.1. Hormone Changes during the Transition Period ............. 3

1.1.1. Estrogen ................................................. 4

1.1.2. Progesterone ........................................... 4

1.1.3. Insulin ................................................... 4

1.1.4. Growth Hormone ...................................... 5

1.1.5. Thyroxine and 3,5,3'-triiodothyronine ............ .5

1.1.6. Glucocorticoid .......................................... 6

1.2. Feed intake and energy balance during the transition

period ............................................................... 6

1.2.1. Nutrient Requirements ................................ 6

1.2.2. Dry matter intake (DMI) ............................. 6

1.2.3. Energy balance ........................................ 7

1.3. Glucose demand .................................................. 7

1.4. Gluconeogenesis in the cow ..................................... 8

1.4.1. Propionate ............................................... 9

1.4.2. Amino acids ............................................. 9

1.4.3. Lactate and glycerol.. ............................... 10

1.5. Metabolism ofnon-esterified fatty acids (NEFAs) ......... 1O

1.6. Fatty Liver ....................................................... 12

1.7. Effect ofBCS on fat mobilization ............................ 14

1.8. Further metabolism ofNEFAs ............................... .15

1.9. Ketosis ............................................................ 17

V

2. The immune system ofthe cow ............. , ............................. 18

2.1. Nonspecific Immune Response ............................... 19

2.2. Specifie Immune Response .................................... 19

2.2.1. B Lymphocytes: Antibody-Mediated

Immunity .................................................... 20

2.2.2. T Lymphocytes: Cell-Mediated Immunity ....... 20

2.3. Changes in the immune system during the peripartum

period .................................................................. 21

2.4. Major diseases during the transitional period ............... 24

2.4.1. Mastitis ................................................. 25

2.4.2. Udder Defense ........................................ 26

2.4.3. Compromised Reproduction ....................... .26

3. Adrenal Cortex ............................................................. 28

3.1. Corticosteroids ................................................... 28

3.1.1. Mineralocorticoids ................................... 29

3.1.2. Glucocorticoids ....................................... 29

3.1.2.1. CortisoL .................................... 29

4. Synthetic Glucocorticoids ................................................. 30

4.1. Flucort® ........................................................... 34

4.2. Predef!> 2X ........................................................ 35

III. Hypothesis and Objectives .................................................... .39

IV. Materials and Methods .......................................................... 40

1. Experimental design ....................................................... 40

2. Feeding ...................................................................... 41

2.1. Close-Up Dry Ration ........................................... 41

2.2. Fresh Cow Ration ............................................... 41

3. Blood Serum Collection .................................................. .42

4. Milk Sampling .............................................................. 42

S. Milk Sodium and Milk Potassium Analysis ........................... .42

6. Body Condition Scoring ................................................... 43

7. Serum Biochemical Analysis ............................................ .43

8. Antibody Production (ELISA) .......................................... .43

9. ConA-Induced Lymphocyte Proliferation .............................. .44

10. Serum Insulin Analysis .................................................. .45

VI

11. Statistical Analysis ...................................................... .45

V. Results and discussion ............................................................ 48

1. Flucort® ..................................................................... 49

1.1. Energy Status .................................................... 49

1.1.1. Glucose ................................................ 49

1.1.2. Insulin ................................................. 54

1.1.3. Non-esterified fatty acids ........................... 54

1.1.4. p-hydroxybutyrate .................................... 56

1.2. Mineral status .................................................... 59

1.2.1. Calcium ................................................ 59

1.2.2. Phosphorus ........................................... 61

1.2.3. Potassium .............................................. 61

1.2.4. Sodium ................................................. 62

1.2.5. Chloride ............................................... 63

1.2.6. Magnesium ........................................... 64

1.3. Immune Function ................................................ 64

1.3.1. Antibody Production ................................ 64

1.3.2. Lymphocyte proliferation ........................... 65

1.4. Milk Status ........................................................ 67

1.4.1. Milk Yield (kg) ....................................... 67

1.4.2. Protein .................................................. 68

1.4.2.1. Protein (%) ................................. 68

1.4.2.2. Protein Yield (kg) .............. " ......... 70

1.4.3. Fat ...................................................... 70

1.4.3.1. Fat (%) ..................................... 70

1.4.3.2. Fat Yield (kg) ............................. 71

1.4.4. Lactose ................................................ 71

1.4.4.1. Lactose (%) ................................ 71

1.4.4.2. Lactose Yield (kg) ........................ 71

1.4.5. Milk potassium (mgIL) .............................. 72

1.4.6. Milk sodium (mgIL) ................................. 72

1.4.7. Somatic cell count ................................... 73

2. Prede~ 2X .................................................................. 74

2.1. Energy Status .................................................... 74

VII

2.1.1. Glucose ................................................ 74

2.1.2. Insulin ................................................. 75

2.1.3. Non-esterified fatty acids ........................... 76

2.1.4. p-hydroxybutyrate .................................... 77

2.2. Serum Mineral status ............................................ 79

2.2.1. Calcium ................................................ 79

2.2.2. Phosphorus ........................................... 79

2.2.3. Potassium .............................................. 80

2.2.4. Sodium ................................................. 81

2.2.5. Chloride ............................................... 81

2.2.6. Magnesium ........................................... 82

2.3. Immune Function ................................................ 82

2.3.1. Antibody Production ................................ 82

2.3.2. Lymphocyte proliferation ........................... 83

2.4. Milk Status ........................................................ 84

2.4.1. Milk Yield (kg) ....................................... 84

2.4.2. Protein .................................................. 84

2.4.2.1. Protein (%) ................................. 84

2.4.2.2. Protein Yield (kg) ......................... 85

2.4.3. Fat ...................................................... 86

2.4.3.1. Fat (%) ..................................... 86

2.4.3.2. Fat Yield (kg) .............................. 86

2.4.4. Lactose ................................................ 87

2.4.4.1. Lactose (%) ................................ 87

2.4.4.2. Lactose Yield (kg) ........................ 87

2.4.5. Milk potassium (mgIL) .............................. 88

2.4.6. Milk sodium (mgIL) ................................. 88

2.4.7. Somatic cell count ................................... 88

3. Flucort® versus Prede~ 2X ............................................... 89

3.1. Energy Status .................................................... 89

3.2. Serum Mineral Status .......................................... 89

3.3. Immune Function ................................................ 90

3.4. Milk Status ....................................................... 90

VI. CODclusioD .......................................................................... 92

VIII

VII. References ........................................................................ 94

Appendices ............................................................................ 111

Animal Use Protocol. ....................................................... 112

Amendment to Animal Use Protocol. .................................... 113

Certificate of Radiation Safety ............................................. 114

McGill University InternaI Radioisotope Permit.. ...................... 115

IX

List of Tables

Table 1. Comparison of Drug Duration and Action Potency* (Dowling, 2004) .......................................................................................................... 33

Table 2. Frequency ofparities (1 to >5) within treatment ....................... .40

Table 3. Frequency of calving seasons within treatment.. ....................... .40

Table 4. Diet Composition (% of DM) ofClose-Up Ration ..................... .41

Table 5. Diet Composition (% of DM) ofFresh Cow Ration ................... .41

Table 6. F value from mixed models for aU parameters in cows that were either treated day of calving with a Flucort®, Prede~ 2X or served as negative controls1

................................................................................. 48

Table 7. Overall least squares means from mixed models for serum biochemical parameters, immune function parameters and milk parameters in cows that were either treated day of calving with a Flucort®, Prede~ 2X or placebo control l

........................................................................ 50

Table 8. Least squares means from mixed models for serum energy parameters in cows on D-IO, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort®, Predef!> 2X or placebo l

.......................................................................................... 52

Table 9. Frequency of cows with high NEFA levels at D-IO, D-5, DO, Dl, D7, D14, D21 and D28 by treatment group ............................................ .55

Table 10. Frequency of multiparous cows with high NEF A levels at D-IO, D-5, DO, Dl, D7, D14, D21 and D28 by treatment group ............................... .56

Table Il. Frequency ofprimiparous cows with high NEFA levels at D-I0, D-5, DO, Dl, D7, D14, D21 and D28 by treatment group ........................... 56

Table 12. Frequency of body condition score within treatment and within day ........................................................................................ 56

Table 13. Distribution of subclinical ketosis at D-IO, D-5, DO, Dl, D7, D14, D21 and D28 by treatment group .................................................... 57

Table 14. Distribution of subclinical ketosis in multiparous cows at D-IO, D-5, DO, Dl, D7, D14, 021 and D28 by treatment group ........................... .57

Table 15. Distribution ofsubclinical ketosis in primiparous cows at D-IO,D-5, DO, Dl, D7, D14, D21 and D28 by treatment group ..................................... 57

Table 16. Least squares means from mixed models for average dry matter intake in cows on D-I0, D-5, DO, Dl, week 1, week 2, week 3, and week 4

x

post-calving that were either treated day of calving with a Flucort®, Prede~ 2X or served as negative controls l

................................................... 58

Table 17. Least squares means from mixed models for serum minerai parameters in cows on D-IO, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort®, Predef!1 2X or placebo! ............................................................................... 60

Table 18. Least squares means from mixed models for serum minerai parameters in cows on D-IO, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort®, Predef!1 2X or placebo! ............................................................................... 63

Table 19. Least squares means from mixed models for antibody production values in cows DO, D7, D14, D21 and D28 relative to calving that were either treated day of calving with a Flucort®, Predef!1 2X or served as negative controls! .................................................................................. 65

Table 20. Least squares means from mixed models for lymphocyte proliferation values in cows DO and D7 relative to calving that were either treated day of calving with a Flucort®, Predef!1 2X or served as negative controIs· ........................ " ............... " ........ " .... " ......................................................................................... 66

Table 21. Least squares means from mixed models for average milk yield in cows on week 1, week 2, week 3, and week 4 post-calving that were either treated day of calving with a Flucort®, PredefM> 2X or served as negative controls1

.................................................................................. 68

Table 22. Least squares means from mixed models for milk component parameters in cows DI, D7, D14 and D21 relative to calving that were either treated day of calving with a Flucort®, Predef!1 2X or served as negative controls1

................................................................................... 69

Table 23. Frequency of mastitis, as denoted by sec > 283,000 cells/ml (Guidry, 1985; Reneau, 1986), percentages of cows by treatment group ....... 74

XI

1. Introduction

The transition period is a critical time for the dairy cow as the

periparturient period is characterized by tremendous metabolic adaptation of

the cow from the demands of late pregnancy to those of early lactation

(Grummer, 1993). Dairy cows increase their total nutrient requirements and

their demand for energy supply to satisfy the requirements of the uterus, fetal

development and the onset of lactation (Bell, 1995), therefore changes are

necessary in body tissue metabolism to meet the requirements of energy (Bell,

1995; Overton et al., 2001). During the final 3 weeks of gestation, a 20 to 40%

decline in dry matter intake (DMI) occurs which may initiate a negative

energy balance (Hayirli et al., 2002). If energy intake is insufficient to meet

energy requirements, one of the major metabolic adaptations involves the

mobilization of fatty acids from adipose tissue. Mobilization of adipose tissue

results in the release of non-esterified fatty acids (NEFAs) into the blood

stream. The plasma NEF A are used as a fuel source by muscle and taken up by

the liver. The liver takes up NEF A in proportion to their concentrations in

plasma, but typically does not have sufficient capacity to completely dispose

ofNEFA through export into the blood or catabolism for energy (Grum et al.,

1996). When nutrient intake is insufficient and large amounts of NEF A are

released into the blood, the liver begins to accumulate and store NEF A which

may be esterified to form triglycerides (TG) and the acetyl-CoA that is not

into the tricarboxylic acid (TCA) cycle is converted to ketone bodies, such as

p-hydroxybutyrate (BHB) (Goff and Horst, 1997) resulting in increased ketone

production which can cause ketosis and is detrimental to overall cow health

and performance (Grummer, 1993; Drackley et al., 2001). Elevated plasma

NEF A is associated with hepatic triglyceride accumulation, consequently it

can cause fatty liver which can ultimately lead to prolonged recovery from

other disorders, increased incidence of health problems, and compromised

Iiver function (Drackley, 1999; Grummer, 1993; Veenhuizen et al., 1991).

Failure of the transition cow to appropriately adjust her metabolism to support

increased nutrient requirements of early lactation may result in the occurrence

of metabolic disorders, poor reproductive performance, and decreased milk

production during the upcoming lactation (Bell, 1995; Grummer, 1995;

1

Drackley, 1999). In addition, the risk of displaced abomasum, retained

placenta and clinical mastitis is significantly increased (Duffield, et al., 2002).

It is hypothesized that preventive therapy could be administered to

promote reversai of the ketogenic processes in the liver and assist with the

needed increase in glucose supply. Administration of oral propylene glycol,

intravenous dextrose, exogenous insulin and various glucocorticoids have been

experimented for this purpose. However, to date, no consistant successful

therapeutic options have been reported. G1ucocorticoid therapy has been used

widely in dairy cattle for the treatment of ketosis as they enhance the

mobilization of glucose precursors (amino acids) and stimulate the rate of

gluconeogenesis. However, use of synthetic glucocorticoids in cows as a

preventive measure is limited since a great number of veterinarians share the

common perception that these drugs are contraindicated due to their potential

to induce immunosuppression.

Flumethasone sterile suspension (Flucort®, Wyeth Animal Health) is

approved for the treatment of ketosis in dairy cattle in Canada. Flucort®

contains a potent corticosteroid with reportedly 60 to 80 times greater

glucocorticoid activity than prednisolone (Flucort® package insert). On the

other hand, Prede:t«' 2X (9-alpha-fluoroprednisolone) is an isoflupredone

acetate sterile suspension made for injection by Pharmacia & Upjohn. It

contains potent corticosteroid and has greater glucocorticoid activity (10 times

more potent) than an equal quantity of prednisolone in its ability to elevate

blood glucose level. Prede:t«' 2X is also long-lasting (48 h gluconeogenic

activîty). It îs reported that blood glucose levels retum to normal or above

within 8 to 24 h after injection, following a reduction in blood and urine

ketone levels by increasing its gluconeogenic and glycogen disposition

activity.

In summary, an effective preventive therapy for bovine ketosis could

reduce and avoid metabolic disorders and production losses. Prede~ 2X and

Flucort® are two synthetic glucocorticoids that were investigated in the present

study. The effects of these veterinary drugs injected on the day of calving in

Holstein primiparous and multiparous cows were evaluated by monitoring

parameters of blood metabolites, immune function and milk composition

during the transition period.

2

II. Literature Review

1. Transition Period

The transition period is defined as the period 2 to 4 weeks prior to

calving (close-up) through 2 to 4 weeks after calving (fresh cow) and is a

critical period that detennines both productivity and profitability in a dairy

herd. During the transition period, the cow experiences a number of changes

such as the initiation of lactation, ration shifts, location and social group

changes and rapid changes in both hormonal and metabolic systems. AH of

these may increase the level of stress in the cow during this period and special

attention to nutrition and management practices may help minimize the level

of stress.

The transition period can also be detined as the transition of feeding

regimens. Changes in nutrient demand require cows to make metabolic

adaptations to meet the demands of late pregnancy to those of early lactation

(Bell, 1995; Overton et al., 2001), essentiaHy a cow must make a transition

from consuming a high fiber ration for dry cows to a lactation diet that is high

in energy and lower in fiber (Radostits et al., 1994).

1.1. Hormone Changes during Transition Period

Major changes in honnonal status in particular estrogen, progesterone

and insulin occur during the transition period and these changes contribute to

changes in blood metabolites. Changes in honnones around calving such as

estradio~ progesterone and growth honnone are illustrated in Figure 1.

3

Glucocorllcoids 12

(fl9ImI serum) e 4

Growln 9 HoI"mone

6 (1IQ/mlsel'l.mt

Proloclln 200 (rI\1lm1 serum) 100

vt:========:::::::::::- -----1--------Progesterone

(rlQlml serum)

Eltrodlol-17 p (po/ml .erum)

·26 -22 -19 -15 -12 -9 Days 'rom Pa,IuriNon

Figure 1. Hormonal changes around calving (Bell, 1995)

1.1.1. Estrogen

Estrogen, primarily estrone of placental origin, increases in plasma

during late gestation but decreases immediately at calving (Chew et al., 1979).

The surge of estrogen that occurs on the day prior to calving may act as a

regulator of hepatic fatty acid metabolism in ruminants (Green et al.,1999). It

has been demonstrated that changes in blood estrogen or estrogen:

progesterone ratio may influence feed intake (Grummer et al.,1990).

1.1.2. Progesterone

Progesterone concentrations during the dry period are elevated for

maintenance of pregnancy but decline rapidly approximately 2 d before

calving (Chew et al., 1979). In lactating dairy cows, energy balance during the

first few weeks post-calving is positively related to concentrations of plasma

progesterone (P4) during the first post-calving estrous cycle (Villa-Godoy et

al., 1988; Spicer et al., 1990).

1.1.3. Insulin

Insulin is important for proper function of the reproductive processes

as it facilitates the partitioning of nutrients between the rapidly growing fetus

and the mammary tissue (Ebling et al., 1990). Insulin stimulates the synthe sis

of glycogen, increases uptake of glucose by muscle and adipose tissues,

4

promotes the uptake of branched-chain ammo acids by muscle, which

promotes muscle tissue synthesis and reduces protein catabolism (Jim

Quiqley, 2001). Insulin reduces ketogenesis and gluconeogenesis in the liver

but stimulates muscle tissue uptake of glucose, amino acids and ketone bodies.

In ruminants, acetate is the main precursor of lipogenesis and its uptake is

stimulated by insulin (Sterbauer, 2005). As a cow progresses from late

gestation to early lactation, plasma insulin decreases (Grummer 1995), with an

acute surge at calving (Kunz et al., 1985). During the transition period, the

adipose tissue becomes highly resistant to insulin (Bell, 1995) leading to

lowered responsiveness and sensitivity of extra hepatic tissues to insulin (Sano

et al., 1991). The decrease in sensitivity of adipose tissue to insulin may also

explain increased lipolysis and mobilization of NEF A from the adipose tissue

(Bell, 1995).

1.1.4. Growtb Hormone

Unlike insulin. endogenous plasma concentration of growth hormone

rises during late pregnancy, with a peak at calving (Bell, 1995). Therefore, as

growth hormone levels rise, insulin levels decrease. Under conditions of

negative energy balance, it has been shown that growth hormone affects fat

mobilization (Etherton and Bauman, 1998). Growth hormone has been shown

to decrease lipogenic enzyme activity and lipogenesis, thus opposing the

actions of insulin (Bell, 1995). Furthermore it can reduce glucose pool and

distribution space by enhancing glucose transport to the uterus and the udder

(Arieli et al., 2001).

1.1.5. Tbyroxine and 3, 5, 3'-trüodothyronine

Concentrations of some other hormones also change during the

transitional period, including thyroid hormones and glucocorticoids. The

thyroid gland principaUy synthesizes thyroxine (T4), considered a prohormone

which is transformed into the metabolically active 3,5,3'-triiodothyronine (T3)

by enzymatic 5'-deiodination in the liver (Chopra et al., 1978). Thyroid

hormones play a role in maintaining energy expenditure for high priority

functions (Bauman and Currie, 1980). Thyroxine (T4) concentrations gradually

increase during late gestation and then decrease approximately 50% at calving

(Kunz et al., 1985). Similar, but less pronounced, changes occur in 3,5,3'­

triiodothyronine (Tj). There is an inverse relationship between milk yield and

5

the levels of these thyroid hormones during early lactation, hence decreased

levels ofT3 and T4 result in high milk levels.

1.1.6. Glucocorticoid

Cortisol and lesser amounts of corticosterone are the most important

glucocorticoid hormones secreted by the bovine adrenal gland. Cortisol is

generally considered a powerful immune suppressive agent and likely

exacerbates the immune suppression normally observed in the periparturient

period (Goff and Kimura, 2002). Cortisol exacerbates the immune suppression

rather than causes it, because most studies suggest that immune suppression

begins 1-2 weeks before calving (Kehrli et al., 1989 a, b), and the cortisol

surge occurs on the day of calving and the day after calving (Goff and Kimura,

2002). Edgerton and Hafs (1973) reported glucocorticoid concentrations

increase on the day of calving and return to near pre-calving concentrations

the following day. The actions of glucocorticoids on carbohydrate, protein,

and lipid metabolism result in sparing of glucose and a tendency to

hyperglycemia and increased glucose production. In addition, they decrease

lipogenesis and increase lipolysis in adipose tissue, which results in the release

of glyceroi and free fatty acids.

1.2. Feed intake and energy balance during the transition period

Like hormone changes, changes in dry matter intake during the

transition period also influences metabolism.

1.2.1. Nutrient Requirements

Dairy cows increase their total nutrient requirements and their demand

for energy supply by 23% for maintenance and pregnancy during the last

month of gestation to satisfy the increased requirements of the uterus and fetal

development (Bell, 1995). Cows carrying twins have higher fetai

requirements. Additionally, there is an increase in energy demand by the onset

of lactation at calving for milk synthesis.

1.2.2. Dry matter intake (DMI)

Due to metabolic and fill constraints, intake of feed and energy by

transition cows are limited (Bertics et al., 1992). The causes for decreased pre­

calving DMI are not known but may be endocrine-related For example,

changes in blood estrogen or estrogen:progesterone ratio may influence feed

intake (Grummer et al., 1990). Dry matter intake decreases by 20-30%, 1 or 2

6

d before calving, and does not recover until 1 to 2 d after calving. Cows

carrying twins start to reduce their dry matter intake earlier. It has also been

observed that heifers have a lower dry matter intake than cows.

Previous studies have implicated leptin as one of the modulators of

feed intake (lngvartsen and Andersen, 2000; Meister, 2000). Leptin is

produced by adipocytes, and leptin concentrations rise in parallel with body

condition score (BCS) (Delavaud et al., 2000; Ehrhardt et al., 2000).

Kadokawa et al. (2000) and Block et al. (2001) reported that plasma leptin

concentrations decrease dramatically during the periparturient period, increase

slightly during the tirst 4 weeks post-calving, and remain relatively unchanged

after week 4 post-calving in dairy cows.

1.2.3. Energy balance

The inability to consume adequate amounts of feed causes cows to

enter a state of negative energy balance Le. energy requirements exceed

energy intake. Therefore transitional cows need to make necessary changes in

body tissue metabolism by mobilizing body reserves to meet nutrient and

energy requirements for maintenance, gestation and milk synthesis during

early lactation (Bell, 1995; Overton et al., 2001). Primiparous cows exhibit

negative energy balance in early lactation similar to that of multiparous cows

(Lin et al., 1984).

Bertics et al (1992) demonstrated the effects of reduced dry matter

intake with cows that were force-fed via ruminai fistulas to maintain feed

intake versus control cows before calving. Control cows having lower feed

intake pre-calving had higher liver triglyceride (35 vs. 3%, DM basis of liver

sample) and NEFA (1,392 vs. 667 pEqlL) and lower blood glucose (74 vs. 55

mg/dL) at d 1 post-calving than the force-fed cows. At d 14 post-calving, the

control cows had higher plasma BHB than the force-fed cows (15 vs. 9

mg/dL). A decrease in pre-calving intake seems unavoidable, but the

magnitude and duration of deerease ean vary (Berties et al., 1992; Vazquez­

Anon et al., 1994; Grummer et al., 1995).

1.3. Glucose demand

Glucose is required in large amounts during the transition period as a

fuel (energy) for the uterus, mammary gland, peripheral tissues, central

nervous system, red blood cells, gastrointestinal tract, and is also required for

7

the synthe sis of lactose, which largely controls milk volume. During late

pregnancy, the gravid uterus consumes 46% of the maternaI glucose

production, whereas the glucose demands associated with lactation account for

85% of the glucose entry (Bell, 1995).

Two weeks before calving, the drive for glucose transport into the

uterus and the mammary gland is strong (Arieli et al., 2001). Plasma glucose

concentrations remain stable or increase slightly during the pre-calving

transition period, increase dramatically at calving, and then decrease

immediately post-calving (Kunz et al., 1985; Vazquez-Anon et al., 1994).

The estimates of whole-body glucose demand of gestating dairy cows

were approximately 1000 to 1100 gld during the last 21 days pre-calving and

the demand increased sharply after calving and was approximately 2.5 times

greater at d 21 post-calving compared with that during the three weeks

preceding calving (Overton, 2001). However, glucose demand during early

lactation is greater than that which can he supported from diet during that

time. Estimated dietary supply of glucose and precursors to support

gluconeogenesis is sufficient for much of the dry period in well-fed cows.

Before calving, dry matter and glucose uptake from the gut decreases, glucose

supply is almost equal to demanda After calving, glucose supply is insufficient

(about -500 gld) to support the demand hecause dry matter intake increases

more slowly than nutrient demand (Bell, 1995).

At the onset of lactation, the cow compensates for this situation in part

by decreasing glucose oxidation by tissues that do not absolutely require it

(Le., muscle). Furthermore, synthesis offat in adipose tissue is essentially shut

down and glucose is not required to make glycerol and provide energy in

support of fat synthesis in adipose tissue. The cow also increases

gluconeogenesis to meet this increased glucose demand (Overton, 2001).

1.4. Gluconeogenesis in the cow

Glucose, amino acids, and fatty acids can be used as energy sources for

maintenance of the cow, fetal growth and milk production. Furthermore, the

sources of each may vary widely through the course of the transition period.

Thus, these three nutrients will he considered.

The liver of the cow must more than double its glucose production in

the immediate postooealving period in order to meet the demand for glucose

8

(Overton and Waldron, 2004). Gluconeogenesis, the formation of glucose

ftom nonhexose precursors, occurs largely in the liver and to a smaller extent

in the renal cortex. The glucose produced passes into the blood to supply other

tissues.

The increase in plasma glucose at calving may result from increased

glucagon and glucocorticoid concentrations that promote depletion of hepatic

glycogen stores (Grummer, 1995). Although the demand for glucose by the

mammary tissue for lactose synthesis continues after calving, hepatic glycogen

stores begin to replete and are increased by d 14 post-calving (Vazquez-Anon,

1994). This probably reflects an increased gluconeogenic capacity to support

lactation.

The substrates for gluconeogenesis are propionate, amino acids, lactate

and glycerol.

1.4.1. Propionate

The contribution ofpropionate to gluconeogenesis is 32-73% (Seal and

Reynolds, 1993). Lomax and Baird (1983) reported that propionate produced

by ruminaI fermentation as the primary substrate for hepatic gluconeogenesis

in the dairy cow accounts for 50 to 60% of total glucose entry in fed animaIs.

Propionate is produced ftom the breakdown of grains by rumen fermentation

and remains the principal gluconeogenic substrate during the transition period

(Overton et al., 1998). Propionate is the principal substrate used to make

glucose by Iiver followed under normal conditions by amino acids, lactate, and

glycerol (Overton, 2001) and can contribute up to 60% of the substrate

necessary for gluconeogenesis in ruminants (DiCostanzo et al., 1999).

Furthermore, the capacity of the liver to make glucose from propionate

appears to he supply related, especially during the ftrst 21 days of lactation

(Overton et al., 1998).

1.4.2. Amino acids

The contribution of atnino acids to gluconeogenesis is 10-30% (Seal

and Reynolds, 1993). The amino acids used for gluconeogenesis in the Iiver

after calving come from skeletal muscle as weIl as dietary amino acids (Bell

1995). Data from Overton et al. (1998) and Simmons et al. (1994) support

increased degradation of skeletal muscle protein during the ftrst 21 days of

lactation. This may explain why feeding diets containing more CP than the

9

NRC requirements (2001) (18% CP) in early lactation cows has proven to be

effective. The rate of gluconeogenesis from amino acids peaks near calving

(Bell et al., 2000). AIl amino acids except leucine and lysine can make a net

contribution to gluconeogenesis (Bergman and Heitmann, 1978). Alanine and

glutamine have been reported to be the most gluconeogenic of aIl amino acids

(Bergman and Heitmann, 1978). Similar to propionate, utilization of amino

acids for gluconeogenesis may he supply-dependent.

1.4.3. Lactate and glycerol

Lactate and glycerol contribute a small amount to gluconeogenesis

(Seal and Reynolds, 1993). The maximal contribution of lactate to

gluconeogenesis is 15%. Glycerol, released from adipose tissue as a

consequence of lipolysis, may contribute as much as 15 to 20% of the glucose

demand around calving (Bell, 1995) or during feed deprivation (Baird et al.,

1980).

Amino acids, lactate and glycerol contribute a greater percentage of

total glucose synthesis when DMI or propionate availability declines (Danfaer

et al., 1995; Donkin and Armentano, 1993; Reynolds et al., 1988; Lomax and

Baird, 1983). Propionate and amino acids are considered supply related during

the first 21 days of lactation. Therefore, when glucose synthesis is inadequate

due to insufficient amounts of propionic acid, altemate sources of energy must

be found and body fat stores hegin to he broken down.

1.5. Metabolism ofnon..esterified fatty acids (NEFAs)

There is a normal breakdown of body fat around calving time because

ofthe hormonal changes associated with calving. Concentrations of circulating

lipolytic hormones increase near the time of calving (Bremmer et al., 1998) and

contribute to fatty acid mobilization from adipose tissue. Stress situations also

increase the mobilization ofbody fat stores. In addition, triacylglycerols stored

in adipose tissue are mobilized to be used as an altemate energy source due to

the reduction in energy intake. Low levels of glucose in the blood cause

hormones (epinephrine and glucagon) to activate the enzyme adenylyl cyclase

in the adipocyte plasma membrane which produces an intracellular second

messenger, cyclic AMP (cAMP). A cAMP-dependent protein kinase

phosphorylates and thereby activates hormone-sensitive triacylglycerol lipase,

which catalyzes hydrolysis of the ester linkages of triacylglycerols. The fatty

10

acids are mobilized and released into the bloodstream in the form of NEF As

where they bind to blood prote in serum albumin. These bound NEF As are

carried to body tissues such as skeletal muscle, heart, and renal cortex where

they will dissociate from albumin and transported into ceUs to he oxidized for

energy production (Grummer, 1993).

Fatty acids are utilized as fuels for skeletal muscle, liver, and other

organs of the cow. Approximately 50% of fatty acids found in milk fat come

from either the diet or from lipoprotein TG in blood. Fatty acids used by the

mammary gland during synthesis of milk fat can also be provided by NEF A in

the blood, which are released during mobilization of adipose tissue (Overton,

2001). Bell (1995) suggested that 40% offatty acids in milk fat during the first

week of lactation may come from blood NEF A.

During the last week before calving, the concentration of NEF A

increase slowly as the cow approaches calving, and usually range from 200 to

300 f.1M. Values increase sharply from 2 to 3 days hefore calving and

generally peak at 800 to 1200 f.1M on the day of calving (Grummer, 1995). The

rapid rise in NEF A at calving is presumably due to the stress of calving

(Grummer, 1995). It is not known how much of the initial increase in plasma

NEF A can he accounted for by changing endocrine status versus energy

restriction (Grummer, 1995). Bertics et al (1992) demonstrated that force

feeding cows during the transition period reduced the magnitude of NEF A

increase but did not completely eliminate it. An increase in plasma NEF A was

observed d 1 pre-calving in cows that did not experience dry matter intake

depression (Vazquez-Anon et aL, 1994). These observations indicate at least

part of the pre-calving increase in plasma NEFA is hormonally induced.

After calving, NEF A concentrations decrease rapidly, but

concentrations remain higher than they were before calving (Vasquez-Anon et

al.,1994). By 3 weeks after calving, values should again be below 300 f.1M.

Heifers experience a higher tevet ofNEFA which may be associated with their

greater nutrient demands for growth. Values greater than 700 f.1M heyond d 7

after calving, indicate severe negative energy balance or health problems

(Drackely, 1999).

11

1.6. Fatty Liver

When nutrient intake is insufficient and large amounts of NEF A are

released into the blood, the liver begins to accumulate and store NEF A in

proportion to their concentrations in plasma. In liver, NEF A will be esteritied

to form TG, which can either he exported as part of very low-density

lipoprotein (VLDL), or stored. In ruminants, export to blood or disposai of

NEF A occurs at a very slow rate (low capacity) relative to many other species

and mechanisms regulating this export are unknown (Grummer, 1993). Under

conditions of increased hepatic NEF A uptake and esteritication, triglyceride

accumulation occurs, causing fatty liver disease. Fatty liver is a major

metabolic disease that affects up to 50% of dairy cows in early lactation. High

concentrations of liver TG promote the synthesis of additional triglyceride and

decrease the oxidation of NEF A (Grum et al., 1996), thereby further increasing

accumulation of triglyceride in the liver. Fatty liver occurs when the rate of

hepatic triglyceride synthesis exceeds the rate of triglyceride disappearance

through either hydrolysis or secretion via very low density lipoproteins

(VLDL), (i.e. export oftriglyceride as VLDL from the liver cannot keep pace

with increased NEF A uptake and triglyceride synthesis by the liver)

(Grummer, 1993).

Apolipoprotein BI00 (Apo BI00) is the major prote in of VLDL, and

its concentration in the liver is inversely related to hepatic triglyceride

concentration. Gruffat et al. (1997) examined stage of lactation-dependent

regulation of Apo B 100 in high producing dairy cows during the tirst 12

weeks of lactation. Cows were fattened during gestation and were underfed

just after calving to increase fat mobilization and induce hepatic lipidosis.

Concentration of Apo BI 00 in liver was approximately 25% lower during the

tirst 4 weeks of lactation than during late pregnancy. Hepatic Apo BI 00

concentrations retumed to pre-calving levels by 12 weeks of lactation.

Accumulation of lipid in the liver commonly occurs prior to or at

calving (Bertics et al.,1992) although the greatest increase in liver triglyceride

typically occurs at calving. In a study by Vasquez-Anon et al. (1994), lipid did

not accumulate in the liver until after the concentration of NEF A in plasma

increased at calving. By d 1 after calving, the largest increase in hepatic

triglyceride has occurred and concentration in the liver remained constant or

12

increased slightly during the post-calving transition period (Bertics et al.,

1992; Vasquez-Anon et al., 1 994). Heifers seem to be less susceptible to fatty

liver at d 1 post-calving, however reasons are unknown (Grummer et al.,

1995).

The extent to which feed intake is depressed before and after calving or

during disease moderates the degree of infiltration of TG. Fatty liver can

develop within 24 h of an animal going off feed. Because of the slow rate of

triglyceride export as lipoprotein, once fatty liver has developed, it will persist

for an extended period of time. Depletion of TG from the liver usually begins

when the cow reaches a state of positive energy balance about 5 to 10 weeks

after calving and may take several weeks to be completed.

Fatty liver is a consequence of negative energy balance, not positive

energy balance. Energy consumption above requirements for maintenance and

production purposes will not directly result in deposition of triglyceride in

hepatic tissue. Triglyceride deposition will occur only if the cow becomes

overconditioned and, consequently, reduces feed intake. Fatty liver is likely to

develop concurrently with other diseases, typically disorders that are seen at or

shortly after calving, including metritis, mastitis, displaced abomasum,

acidosis, and hypocalcemia. Cows that are slow to increase in milk production

and feed intake after calving are likely to have fatty liver. However, fatty liver

is the result of poor feed intake, which is a condition that leads to low blood

glucose, which also contributes to fatty liver because insulin suppresses fat

mobilization from adipose tissue. Fatty liver is often associated with obese

cows and downer cows.

Fatty liver syndrome (> 20% fat) impairs the function of the liver,

increases disease incidence, pro longs recovery from other disorders, reduces

fertility, and sometimes leads to death (Drackley, 1999; Grummer, 1993;

Veenhuizen et al., 1991; Overton and Waldron, 2004). There are no known

clinical signs that are unique to cows with fatty liver. Fatty liver has been

associated with low milk production, increased clinical mastitis, and poor

reproductive performance. However, cause and effect have not been

established, and the metabolic consequence oftriglyceride accumulation in the

liver has not been determined. Field observations suggest that response to

13

treatment of concurrent disorders is poor if triglyceride infiltration of the liver

is extensive.

Liver biopsy is the only reliable method to detennine severity of fatty

liver in dairy cattle. Measurement of total lipid or triglyceride content by

analytical methods after extraction from tissue by organic solvents is

necessary for quantitative assessment of fatty liver; however, these assays are

not routinely conducted in commerciallaboratories.

Blood metabolites, urine metabolites or blood enzyme activity have

been proposed as diagnostic tools for the detennination of fatty liver. When

conditions are conducive to the development of fatty liver, blood glucose

concentrations are low and blood NEF A and BHB concentrations are high.

Blood cholesterol concentration is usually low when fatty liver occurs, and

this may reflect impairment in the ability of the liver to secrete lipoproteins.

However, blood metabolites or enzymes are poor indices of fatty liver because

baseline (nonnal) concentrations vary tremendously among animais.

Microscopie evaluation can he used to estimate the volume of the

tissue occupied by fat. Mild, moderate, and severe fatty Iiver are often defined

as <20%, 20-40%, and >40% fat (percentage of cell volume), respectively.

However, these values have litt le meaning relative to impact of physiological

functions or clinical signs of fatty liver.

1.7. EtTect ofBeS on fat mobilization

The current National Research Council (NRC, 2001) recommendation

is that cows should not gain B W during the dry period, except for B W

associated with growth of the fetus and fetai membranes. Furthermore, cows

should end lactation with the same body condition score (BCS) as desired at

the start of the next lactation (3.5 to 3.75 on a five-point scale, 1 =- thin to 5 = fat). This will allow the cow to calve with an adequate but not excessive body

fat reserve.

Early studies (Garnsworthy, 1988) using lactating dairy cows showed

that the delay between maximum milk yield and maximum DMl might be

related to body condition at calving. Cows calving with high body condition

slowly increased DMl and then reached maximum DMllater than did those

cows calving with poor body condition. Over-conditioned cows are more

likely to have poor appetites post-calving (Holter et al., 1990). A fat cow is

14

more prone to mobilize fat and to have a depressed appetite causing a greater

loss of body condition in early lactation (de Ondarza, 1998). This high lipid

mobilization after calving could lengthen the delay to reach maximum DMI

(Bareille and Faverdin, 1996). Concentrations of NEF A and BHB in plasma

and TG in liver were elevated in primiparous cows calving at heavier BW and

higher BCS indicating a positive correlation between high BCS and BW with

increased levels of BHB and NEFA (Grummer et al., 1995). Excessive

deposition of adipose tissue pre-calving is highly correlated to post-calving

metabolic disorders, such as ketosis and fatty liver syndrome (Baird, 1982).

On the other hand, cows that begin lactation with a BCS <3.25 may not

be capable of mobilizing enough energy to support maximal milk production

(Otto et a1.,1991). Thin cows have greater mobilization of body fat due to

decreased insulin levels and a greater conversion of the resulting NEF As to

TG and ketones. Under-conditioned cows have insufficient energy reserves.

Rapid and/or excessive body weight losses can increase the incidence of

metabolic disorders.

Failure of the transition cow to appropriately adjust her metabolism to

support increased nutrient requirements of early lactation may result in the

occurrence of metabolic disorders, poor reproductive performance, and

decreased milk production during the upcoming lactation (Bell, 1995;

Grummer, 1995; Drackley, 1999).

1.8. Furtber metabolism orNEFAs

The liver is a major site of long-chain fatty acid metabolism, especially

during feed deprivation or early lactation when mobilization of adipose tissue

TG Ieads to increased NEF A concentrations in blood. The mechanism for the

disposaI of NEF A during excessive lipid mobilization is the increased hepatic

uptake ofNEFA (Grum et al., 1996). In the liver, NEFAs are transported into

mitochondria where the enzymes for acid oxidation are located. The free fatty

acids that enter the cytosol from the blood cannot pass directly through the

mitochondrial membranes and therefore must undergo a series of enzymatic

reactions to he activated, and the activated fatty acid can then enter the

mitochondria via an acyl-camitine/camitine transporter. Mitochondrial J3-oxidation takes place in three stages. The first stage is composed of four steps;

first, dehydrogenation; second, the addition of water to the resulting double

15

bond; third, oxidation of the f3-hydroxyacyl-CoA to a ketone; fourth, thiolytic

cleavage by coenzyme A. During stage one, fatty acids undergo oxidative

removal of successive two-carbon units in the form of acteyl-CoA starting

from the carboxyl end of the fatty acyl chain. In the second stage, the acetyl­

CoA produced from the oxidation of fatty acids can be oxidized to C02 and

H20 by the citric acid cycle, which also takes place in the mitochondrial

matrix. The frrst two stages of fatty acid oxidation produce the reduced

electron carriers NADH and F ADH2' which in the third stage donate electrons

to the mitochondrial respiratory chain, through which electrons pass to oxygen

with the concomitant phosphorylation of ADP to ATP. Therefore, the energy

released by fatty acid oxidation is used for A TP synthesis. The number of

reaction required for f3-oxidation will vary depending on the nature of the fatty

acid (ie. saturated, degree ofunsaturation, odd-number fatty acids).

Although the major site of fatty acid oxidation is the mitochondrial

matrix, peroxisomes contain enzymes capable of oxidizing fatty acids to

acetyI-CoA by a similar pathway. Peroxisomes are membrane-enclosed

cellular compartments in which fatty acid oxidation produces H202, which is

then enzymatically destroyed. The difference between peroxisomal and

mitochondrial oxidation is in the frrst step. In peroxisomes, the flavoprotein

dehydrogenase that introduces the double bond passes electrons directly to O2

producing H20 2, which is immediately cleaved to H20 and O2. The resulting

energy produced from peroxisomal oxidation is dissipated as heat and not

conserved as ATP such as in mitochondrial oxidation. Liver peroxisomes do

not contain the enzymes of the citric acid cycle and cannot catalyze the

oxidation of acetyl-CoA to CO2• The fatty acid produced from peroxisomal

oxidation can enter the mitochondria to be oxidized.

Acetyl-CoA formed in the liver during oxidation of fatty acids can

enter the citric acid cycle or can be converted to ketone bodies (acetone,

acetoacetate and J3-hydroxybutyrate) for export to other tissues. Acetone is

produced in smaller quantities than other ketone bodies. Acetoacetate and 13-hydroxybutyrate are transported to the blood and extrahepatic tissues, where

they are oxidized in the citric acid cycle to provide most of the energy required

by skeletal muscle, heart muscle, and renai cortex. The brain, which usually

16

uses glucose for energy, can adapt to the use of acetoacetate or ~­

hydroxybutyrate when glucose is not available or under starvation conditions.

The production and export of ketone bodies from the liver to extrahepatic

tissues allows continued oxidation of fatty acids from the liver when acetyl­

CoA is not being oxidized in the citric acid cycle. During starvation,

gluconeogenesis depletes citric acid cycle intermediates, diverging acetyl-CoA

to ketone body production. Fatty acids therefore enter the mitochondria to be

degraded to acteyl-CoA, which cannot pass through the citric acid cycle

because cycle intermediates (such as oxaloacetate) have been drawn off for

use as substrates in gluconeogenesis. The rate of fatty acid mobilization from

adipose tissue exceeds that of their oxidation. The accumulation of acetyl­

CoA, which is not incorporated into the citric acid cycle builds up in the liver

and accelerates the formation of ketone bodies beyond the capacity of

extrahepatic tissues to oxidize them. The increased blood levels of

acetoacetate and ~-hydroxybutyrate, lowers the blood pH causing acidosis.

Extreme acidosis can lead to coma and in sorne cases death. Severe starvation

leads to high concentrations of ketone bodies in the blood and urine causing

ketosis.

1.9. Ketosis

Ketone bodies production is favored when blood glucose

concentrations are low and is detrimental to overall cow health and

performance (Grummer, 1993; Drackely et al., 2001). The characteristics of

ketosis include reduced milk yield, loss of body weight, loss of appetite, and

occasionally, signs of nervousness. Sometimes these signs are clearly

recognized (clinical) but, often, they are not easily seen (subclinical).

Although symptoms of subclinical ketosis are not detectable, it is potentially

serious because it usually remains undetected, untreated, and could progress to

a clinical condition (Baird, 1982). Subclinical ketosis has also been associated

with a 10ss of milk production of 1.0 to 1.4 kg/d (Doohoo & Martin, 1984).

This loss in production is economically significant to the dairy producer, since

the reported prevalence for subclinical ketosis ranges from 8.9 to 34%

(Kauppinen, 1984).

Subclinical ketosis is a disorder that is associated with increased levels

of circulating ketone bodies and can he identified by the concentrations of

17

ketone bodies in the serum, milk and urine (Grum et al., 1996). Reported

threshold concentrations of serum BHB that are used to define subclinical

ketosis range from 1000 to 1400 mmol/L (Nielen et al., 1994). The primary

risk period for subclinical ketosis is the first 2 months of lactation, but the

peak prevalence occurs during the Ist month (Andersson & Emanuelson,

1985; Dohoo & Martin, 1984).

In genera~ fatter cows (Bes > 3.75) will experience more ketosis (de

Ondarza, 1998). Duffield et al. (1998) reported that higher BHB levels were

observed in fat cows and that the risk of clinical ketosis was three times

greater for fat cows than for thin or fair-conditioned cows. Several other

factors influence the prevalence of hyperketonemia or ketosis, including age

(Andersson, 1988; Dohoo & Martin, 1984), season (Tveit et al., 1992), and

breed (Andersson, 1988). However, the influence of heritability is thought to

be relatively low (Tveit et al., 1992).

Ketosis has been found to occur after a cow develops a fatty liver.

Liver triglyceride:glycogen ratio at calving may be an indicator of a cow's

susceptibility to ketosis (Veenhuizen et al.,1991). The etiology of fatty liver

and ketosis are similar, and in both cases liver function is impaired (DeBoer et

al., 1985; Drackley et al., 1992). In addition, fatty liver and ketosis are

common metabolic disorders in transition cows and increase predisposition to

other post-calving health problems.

2. The immune defense system of the cow

Immunity refers to the body's ability to resist or eliminate potentially

harmful materials or abnormal cells. The immune defense system of the

ruminant plays a key role in recognizing and either destroying or neutralizing

materials within the body that are foreign to the "normal self'. The immune

defense system provides protection against foreign and abnormal cells and

removes cellular debris. Pathogenic bacteria and viruses are the major targets

of the immune defense system. Leukocytes (white blood celIs) and their

derivatives (neutrophils, eosinophils, basophils, B lymphocytes, T

lymphocytes and monocytes) are the effector cells of the immune defense

system. Immune responses may he either nonspecific or specifie.

18

2.1. Nonspecific Immune Response

Nonspecific defenses include inflammation, interferon, neutrophils,

natural killer ceUs, macrophages, lysozyme and the complement system.

Inflammation refers to an innate, nonspecific series of highly interrelated

events that are set into motion in response to foreign invasion, tissue damage

or both. The ultimate goal of inflammation is to bring to the invaded or injured

area phagocytes and plasma proteins then can (1) isolate, destroy or inactivate

the invaders; (2) remove debris; and (3) prepare for subsequent healing and

repair.

Numerous drugs can suppress the inflammatory response, the Most

effective are the salicylates and glucocorticoids (drugs similar to the steroid

hormone cortisol, which is secreted by the adrenal cortex). Glucocorticoids,

which are potent anti-inflammatory drugs, suppress almost every aspect of the

inflammatory response. In addition, they destroy lymphocytes within

lymphoid tissue and reduce antibody production. By suppressing inflammatory

and other immune responses that localize and eliminate bacteria, such therapy

also reduces the body's ability to resist infection.

2.2. Specifie Immune Response

Specific immune defenses include lymphocytes, macrophages and

immunoglobulins. Specifie immunity can he divided into two types of

responses, antibody-mediated immunity accomplished by B lymphocyte

derivatives and cell-mediated immunity accomplished by T lymphocytes.

Lymphocytes, categorized under Band T lymphocytes, are responsible for

specifie recognition of the antigen but have different functions.

Both Band T ceUs must he able to specificaUy recognize unwanted

ceUs and other materials to he destroyed or neutralized as heing distinct from

the body's own normal cells. The presence ofantigens enables lymphocytes to

make this distinction. An antigen is a large, complex Molecule that triggers an

immune response against itself when it gains entry into the body. In general,

the more complex a Molecule results in greater antigenicity. Foreign proteins

are the MOst common antigens because oftheir size and structural complexity.

Bach B and T cell bas receptors on its surface for binding with one particular

type of the Many possible antigens. For B ceUs, binding with antigen induces

the cell to differentiate into a plasma ceU, which produces antibodies that are

19

able to combine with the specifie type of antigen that stimulated the

antibodies' production. Each individual must be able to produce thousands of

different antibody molecules in order to identify the large array of potential

antigens and pathogens. The functions of antibodies include their ability to

bind to antigen and to increase phagocytosis or killing by polymorphonuclear

ceUs and macrophages, complement activation, direct inactivation of virus or

toxin, and enhancement of antigen clearance.

2.2.1. B Lymphocytes: Antibody-Mediated Immunity

The B lymphocytes express surface Ig and are the precursors of plasma

ce Us that synthesize antibodies that indirectly lead to the destruction of foreign

material. B lymphocytes are responsible for antibody-mediated immunity

involving antibodies that amplify the inflammatory response to promote

destruction of the antigen that stimulated their production. Bach antigen

stimulates a different clone of B lymphocytes to produce antibodies. The

production of antibodies as a result of exposure to an antigen is referred to as

active immunity against the antigen. Another way to acquire antibodies is

passive immunity, which is achieved by direct transfer of antibodies actively

formed by another animal.

2.2.2. T Lymphocytes: Cell-Mediated Immunity

T lymphocytes are responsible for ceU~mediated immunity involving

direct destruction of virus-invaded ceUs and mutant ceUs through

nonphagocytic means. The T lymphocytes are divided into three discrete

subpopulations based on the expression of cell surface receptors.

Cytotoxic T ceUs (killer T ceUs or CD 8 ceUs) destroy host ceUs

bearing foreign antigen. Helper T cells (CD4 ceUs) enhance the development

of antigen-stimulated B ceUs into antibody-secreting ceUs, enhance the activity

of the appropriate cytotoxic and suppressor T cells, and activate macrophages.

Suppressor T ceUs suppress both B cell antibody production and cytotoxic and

helper T cell activity. These functions are mediated by the production of a

wide range of cytokines and enzymes.

Measures of lymphocyte activity, such as antibody and cytokine

production, cytotoxicity, and proliferation, have been used to indicate the

functional status of the immune system (MaUard et al., 1998). In the current

study, lymphocyte proliferation and antibody production in response to

20

chicken ovalbumin were examined during treatment with Flucort® and Prede~

2X administered at calving.

2.3. Changes in the immune system during the transition period

Around the time of calving, host defense is impaired and the dairy cow

is immunosuppressed (Detilleux et al., 1995). These are associated with

changes in hormone profiles and metabolic and physical stresses ofpregnancy,

calving and lactation and largely mediated through the neuroendocrine­

immune axis (perkins et a1.,200 1).

In dairy cows, the weeks before and after calving are periods with high

incidence of infections. Diseases may occur when the immune system is

unable to respond efficiently to invading pathogens. An efficient immune

response relies on the interaction and balance between different ceH types and

their products. In the periparturient period, large changes occur in hormonal

levels and metabolism, adapting the animal to a high metabolism and high

milk production (Holtenius et al., 1996; Bell and Bauman, 1997; Kehrli et al.,

1999). It has been suggested that negative energy balance in combination with

other factors involved in calving, such as increases in cortisol, may contribute

to periparturient immunosuppression and disease susceptibility (perkins et al.

(200 l, Preisler et al. 1999 and Goff and Kimura, 2002)

As calving approaches, the total number of white blood cells increases,

mainly as a consequence of higher numbers of neutrophils (Saad et al., 1989;

Gilbert et al., 1993). However, the functional capacities ofthe neutrophils are

impaired during this periode Neutrophils are the tirst line of host

immunological defense against bacterial infections. One of the key points in

the control and eradication of an infection is rapid migration and recruitment

of neutrophils to the site of infection (Heyneman et al., 1990; Burton and

Erskine, 2003). The tirst step in neutrophil migration depends on the

coordinated function of selectins and ~2-integrins (adhesion molecules on

leukocytes and endothelial ceIIs) (Kehrli et al., 1999). As a consequence,

migratinglmarginating cells stop their rolling and adhere tightly to the

endothelium, initiating diapedesis. However, down-regulation and shedding of

CD62L molecules from neutrophils have been reported around calving,

consequently less numbers of cells are able to migrate into peripheral tissue

(Lee and Kehrli, 1998; Paape et al., 2002). In addition, the phagocytic and

21

killing ability of neutrophils are also impaired around calving (Saad et al.,

1989; Hoeben et al., 2000; Mehrzad et al., 2001).

As calving approaches, the proportion of blood lymphocytes and their

functional activities, such as cloning expansion and antibody production

decrease (Ishikawa et al., 1994; Detilleux et al., 1995; Kimura et aL, 1999).

The decrease in lymphocyte numbers is due to a net depression in CD4+, CD8+

and 10+ T lymphocytes (Van Kampen and Mallard, 1997; Kimura et al., 1999).

ln addition, functions of certain subpopulations change. It has been observed

that blood CD4+ T-cells preferentially produce IL-4 and IL-I0 around calving,

while they shift to IFN-y and IL-2 production during mid to late lactation

(Shafer-Weaver et al., 1999). Moreover, CD8+ lymphocytes of the suppressor

type predomina te at this time, which may also contribute to higher levels IL-4

and IL-lO, setting a humoral immune response (Shafer-Weaver and Sordillo,

1997). The changes in leukocytes and cytokine production observed around

calving result in a suppressed activity of the cellular immune response; which

is necessary to deal with intracellular bacteria and viruses, thereby making the

animal more susceptible to infections.

Numerous investigations (Ishikawa, 1987; Kehrli et al., 1989b; Saad et

al., 1989) have reported diminished lymphocyte responsiveness around

calving. Studies by Kehrli et aL (1989b) utilizing Holstein heifers

demonstrated that peripheral blood lymphocyte response to pokeweed mitogen

declined steadily from 2 weeks pre-calving until the week of calving and then

began to increase again at week 2 post-calving. This diminishing response was

substantiated by Saad et al. (1989), who reported a steady decline in

lymphocyte response of Swedish Red and White cows to concanavalin A and

pokeweed mitogen from 3 weeks pre-calving through to calving and recovery

at about week 2 or 3 post-calving. Those groups (Kehrli et al., 1989b; Saad et

al., 1989) speculated on the ability of reproductive hormones and

glucocorticoids to modulate this response in vivo. Those observations provide

sorne support for the notion that altered lymphocyte responsiveness around

calving is linked to increased mastitis susceptibility and that transition period

hormone changes may be influential.

Mitogens are often used to assess the proliferation ability of

lymphocytes and mitogens such as concanavalin A (ConA), pokeweed

22

mitogen (PWM), and lipopolysaccharide (LPS) are often used in such studies.

Concanavalin A is known to stimulate T -celI proliferation, whereas pokeweed

mitogen and bacterial lipopolysaccharide selectively stimulate B-cell

proliferation (Li et al., 2000).

Decline of certain subsets of blood lymphocytes in both healthy and

diseased cows may explain the increased disease incidence at calving.

Changes in lymphocyte subsets, particularly the ratios of CD4 to CDS have

been associated with immunosuppressive diseases in various species. It has

been shown that the proportion of certain T -cell subsets decreased

dramatically around calving, but B cells did not. In addition, the proportions of

T lymphocytes pre-calving and at calving were also significantly lower than

those of non-pregnant, non-Iactating cows of the same breed (Glass et

al., 1990). The percentage of T ceUs was lowest in milk (16%) during the

transition period, but increased to 62% in late lactation, whereas the

percentage of B ceUs and macrophages were reported at 25 and 69%,

respectively, during the same period, but declined in late lactation to 7 and

21 %, respectively (Glass et al., 1990).

As calving approaches, there are changes in the different T -cells

subsets, however, the percentage of B-Iymphocytes seems to remain fairly

constant (Shafer-Weaver et al., 1996). In contrast, Van Kimpen and Mallard

(1997) reported a higher proportion of B-cells in blood before and at calving

than after calving. In regards to functional activity of B-cells, a diminished

antibody production during the time of calving has been observed (Nagahata et

al., 1992; Detilleux et al., 1995).

Lacetera et al. (2004) demonstrated that high concentrations of fatty

acids impaired cow lymphocyte function in vitro. Results suggest that intense

lipomobilization with increased plasma concentrations of NEF As might be

factors that explain the higher incidence of infections observed in cows

suffering from energy deficit. Further, the same authors reported that the

increase of plasma NEF A is likely to exert negative effects on lymphocyte

functions in cows and that a deficiency of IFN production occurred in cows

with fat mobilization syndrome (Lacetera et al., 2004). Wentink et al. (1997)

documented that hepatic lipidosis due to intense lipomobilization is associated

with impaired immunodepression. High plasma NEF A is hypothesized as

23

directly responsible for impairment of physiological functions of live stock

suffering from energy deficit.

During the transition period, sorne animaIs experience a low antibody

response (Franco et al., 1990). Franco et al. (1990) reported that antibody

responses are suppressed due to stress released glucocorticoids. Although it

has been reported that low serum antibodies in cattle at calving may be due to

sequestration of immunoglobulin into the mammary gland (Detilleux et al.,

1995), the study done by Mallard et al (1997) suggest that lower specifie

antibody response in serum does not necessarily relate to immunoglobulin

transport. Mallard et al. (1997) demonstrated that animais with high serum

antibody response also supply higher concentrations of specifie antibody to the

mammary gland and that not aIl cows experience depression of antibody

response during the transition period.

2.4. Major diseases during the transitional period

Elevation of NEF A, BHB, and liver TG may predispose cows to high

incidence of metabolic disorders (Baird, 1982). Negative energy balance and

the degree of fatty acid mobilization before calving, as indicated by plasma

NEF A concentrations, has been positively related to the incidence of dystocia,

retained placentas, ketosis, fatty liver, displaced abomasums, and mastitis in

the transition period (Grummer, 1993). The risk of displaced abomasums,

retained placenta and clinical mastitis is significantly increased when marked

energy deficits occur prior to calving (Duffield et a1.,2002). Furthermore,

over-conditioned cows had a higher incidence of health problems within 75 d

post-calving. Profits are affected by metabolic disorders as expenses for most

metabolic disorders have been reported to vary from $200 to $400 per incident

(Harris, 2000).

Many of the resulting disorders are associated and are consequential of

each other. Studies of periparturient diseases suggest that clinical ketosis

precedes displaced abomasum in addition to increasing its risk (Curtis et al.,

1985; Grohn et al., 1989). In addition, subclinical ketosis has been identified

as a risk factor for metritis (Doohoo & Martin, 1984, Grohn et al., 1989) and

mastitis (Doohoo & Martin, 1984) and bas been associated with cystic ovaries

(Andersson & Emanuelson, 1985; Doohoo & Martin, 1984).

24

2.4.1. Mastitis

The dairy cow is at increased risk of infectious disease during the

transition period (Smith et al., 1985). Selection of dairy cows with superior

milk production traits has resulted in a steady increase in the incidence of

clinical mastitis (Harmon, 1994). As parity increases, so too does production

yield and the occurrence ofdisease (Dunklee et al., 1994).

Given the positive genetic correlation between selection for increased

milk production and the increased rate of clinical mastitis (Owen et al., 2000),

one might hypothesize that superior production is associated with unfavorable

changes in host defense mechanisms that could result in an increased

occurrence of mastitis.

More than 95% of somatic celIs (SC) (cells in bovine milk) are

leucocytes, including neutrophils, macrophages and lymphocytes. The somatic

cell count (SCC) is the number of SC/mL of milk and is a useful

approximation for the concentration of leucocytes in milk. Somatic celI counts

in milk are used as indicators of mammary health on the basis that they reflect

an immune response and thus the presence of infection. Although a raised

SCC is an accepted indicator of an existing bacterial infection, a very low SCC

has been associated with an increased subsequent susceptibility to clinical

mastitis. This suggests that SC may provide protection from bacterial

colonisation as weIl as being a marker of infection.

Mastitis is a common problem in modem dairy cows and a major cause

of lost income in the dairy industry. Mastitis is a disease that leads to reduced

milk yield and an increased number of clinical treatments and early cow

culling (Beaudeau et al., 1993; Lescourret and Coulon, 1994). This

inflammation of the mammary gland, usually a response to invasive agents,

can be characterized by an increase in SCC. A logarithmic transformation

calIed somatic celI score has been used as an indicator of udder health for

management and selection purposes (Rodriguez-Zas et al., 2000). Somatic cell

count values higher than 283, 000 cells/mL indicate the presence of mastitis

(Guidry, 1985; Reneau, 1986). Therefore, values lower than 283,000 cells/mL

do not reflect the health of the udder, but rather are associated with milk yield

(Hortet et al., 1999). The established association between milk production and

SCC in dairy cattle is increasingly used to estimate lost production due to

25

mastitis because important management decisions regarding cost-effective

prevention and control of mastitis are based on this relationship (Bartlett et al.,

1990). Jones (1986) suggested that sec of 600, 000 to 1, 000, 000 cells/mL

were associated with an 8 to 12% reduction in herd milk production.

According to Harmon (1994), mastitis or elevated sec is associated with a

decrease in lactose, a-Iactalbumin, and fat in milk because of reduced

synthetic activity in the mammary tissue.

2.4.2. Udder Defense

Wagner (2003) demonstrated that cows in the negative energy balance

period show an impairment of udder defense mechanisms. Lacetera et al.

(2001) reported immunodepression in ketotic ewes and negative relationships

between immune functions and plasma NEF A or BHB. Hyperketonemia is

hypothesized as one of the Most important factors leading to reduced udder

defenses. There are possible explanations for these effects via each of the

mechanisms of defense. Firstly, the capacity for phagocytosis by

polymorphonuclear cells and macrophages May be reduced in a state of

negative energy balance, furthennore bacterial killing capacity is impaired in

the presence ofketone bodies (Leslie et al., 2000). Secondly, lower amounts of

cytokines produced by lymphocytes in ketotic cows May cause udder

leukocytes to induce ceIl recruitment in intra·mammary infection (Leslie et al.,

2000). Finally, the capacity for blood leukocytes to migrate into the infected

gland is reduced (Leslie et al., 2000).

It is hypothesized that high plasma NEF A are directly responsible for

impairment of physiological functions of livestock suffering from energy

deficit (Drackley et al., 2001; Gillund et al., 2001). ResuIts from Lacetera et al.

(2004) suggest that intense lipomobilization May he potentially responsible for

immunodepression and also that plasma NEF A might represent biochemical

indicators of the immune reactivity of cows.

2.4.3. Compromised Reproduction

Lipid metaboHsm is related to the reproductive status of high yielding

dairy cows, for example during early lactation the rate of adipose tissue

lipolysis is high, but the rate of lipogenesis is almost negligible (Bareille and

Faverdin, 1996). In addition, subclinical ketosis has been associated with

26

decreased milk yield, increased risk of clinical ketosis, metritis, cystic ovarian

disease, and impaired reproductive performance (Doohoo and Martin, 1984)

Decreased reproductive performance can he explained partly by

delayed uterine involution (Higgins and Anderson, 1983). The delayed

involution can be explained by an increased incidence, length, and severity of

endometritis (Heinonen et al., 1987; Sheldon et al., 2002), which can be

caused by delayed and decreased immune response in the uterus (Zerbe et al.,

2000). Delayed initiation of ovarian activity is caused by a severe negative

energy balance (Herdt, 1991). Additionally, decreased concentrations of

insulin and elevated concentrations of NEF A can impair normal ovarian

function (Comin et al., 2002; Jorritsma et al., 2003).

The etiology of retained placenta is not completely understood,

however, impaired immune function may play an important role (Goff and

Horst, 1997). For example, after calving, reduced neutrophil chemoattraction

for fetal tissue has been observed in cows with retained placenta (Cai et al.,

1994). In addition, Gunnink (1984) observed that impaired leukocyte ability to

attack cotyledon material existed prior to calving in cows that subsequently

developed retained placenta.

Lucy et al. (1991) found that the number of small follicles «5 mm)

decreased while the number of large follicles (> 1 0 mm) increased between d 0

and d 25 post-calving, and energy balance was related to changes in follicular

populations. As the degree of negative energy balance increases during early

lactation, the interval from calving to first ovulation has been shown to be

lengthened (Butler & Smith, 1989). Butler and Smith (1989) suggested that

cows with a longer interval from calving to first ovulation experience a

decrease in pregnancy rate at frrst service because the conception rate is

related to the number of ovulatory cycles that occur before insemination

(Stevenson & Call, 1983). In addition, cows that express estrus before the frrst

post-calving ovulation have greater energy balance than cows that do not

express estrus (Spicer et al., 1990). Negative energy balance is, therefore, a

likely cause for poor reproductive efficiency in lactating dairy cows (Opsomer

et al., 1996).

Because plasma cholesterol (Carroll et al., 1990; Spicer et al., 1993),

insulin (Koprowski and Tucker; 1973) and IGF-I (Spicer et al., 1990, 1993)

27

increase, whereas plasma NEF A decrease (Staples et al., 1990) with increasing

week of lactation, those hormones and metabolites are primary candidates for

transmitting the metabolic status of a cow to its reproductive axis.

Concentrations of cholesterol and IOF-I in blood of cattle are modified by

variations in fat, prote in, and (or) energy intake, and increase as energy

balance increases (Grummer and Davis, 1984; Spicer et al., 1990). Moreover,

insulin and IOF-I stimulate mitogenesis and steroidogenesis of bovine ovarian

ce Us in vitro (Spicer and Echternkamp, 1995), and thus, negative energy

balance may affect ovarian activity by decreasing luteal progesterone (P4)

production (Orummer and Carroll, 1988; Spicer et al., 1993). Recent studies

also implicate leptin as a possible metabolic mediator of reproduction by

inhibiting steroidogenesis of bovine granulosa and theca ceUs (Spicer and

Francisco, 1997, 1998).

3. Adrenal Cortex

The adrenal cortex produces corticosteroids or a number of different

adrenocortical hormones, aIl of which are steroids derived from cholesterol.

Adrenal corticosteroids can he divided into three categories; (l)

mineralocorticoids; (2) glucocorticoids; and (3) sex hormones.

3.1. Corticosteroids

The adrenal glands are stimulated to produce corticosteroids by

adrenocorticotropic hormone (ACTH) released by the anterior pituitary gland

in the brain. Release of ACTH from the pituitary gland is influenced by factors

including exercise, stress, surgery, cold exposure, and hypoglycemia. The

major control of ACTH release is feedback inhibition by high blood levels of

corticosteroids. This occurs with the natural release of corticosteroids from the

adrenal glands, or with the administration of a corticosteroid drug formulation.

After release from the adrenal glands, corticosteroids circulate in the

bloodstream until they reach cellular targets. At the level of individual cells,

eortieosteroids enter the eell and bind to specifie protein receptors. The

corticosteroid-protein complex then enters the cell nucleus and alters the cell's

production of proteins. Changes in protein production result in altered cellular

functions, which can have a wide variety of effects in the body, depending on

the type of cell involved.

28

3.1.1. Mineralocorticoids

Mineralocorticoids (primarily aldosterone) have a major effect on

electrolyte (sodium and potassium) balance and blood pressure homeostasis.

Specifically aldosterone stimulates sodium reabsorption and potassium

secretion in the kidneys.

3.1.2. Glucocorticoids

Glucocorticoids have an important role in adaptation to stress, however

the natural function of glucocorticoids is to protect the supply ofblood glucose

critical for normal brain function. They increase blood glucose concentration

by counteracting the effect of insulin and by mobilizing fatty acids and amino

acids from body stores for additional glucose production by the liver.

Therefore, glucocorticoids have a breakdown (catabolic) effect on body

muscle and fat stores, but can cause excessive fat to accumulate in the liver.

The primary glucocorticoid, cortisol, plays an important role in carbohydrate,

prote in, and fat metabolism; exhibits significant permissive actions for other

hormonal activities; and helps resist stress.

3.1.2.1. Cortisol

The overall effect of cortisol is to metabolically increase the

concentration of blood glucose at the expense of prote in and fat stores.

Cortisol also plays a major role in glucose metabolism (carbohydrate, fat, and

protein) by stimulating hepatic gluconeogenesis, the conversion of non­

carbohydrate (amino acids) into carbohydrate sources within the liver during

periods of starvation, in other words it mobilizes the body's nutrient stores so

that metabolic fuel is readily available to keep pace with the body's energy

needs when feed intake is low and to maintain normal blood glucose levels.

Cortisol inhibits glucose uptake and use by many tissues, but not the brain,

therefore sparing glucose for use by the brain. Cortisol stimulates prote in

degradation in Many tissues, especially muscle. Muscle proteins are broken

down into amino acids, increasing the blood amino acid concentration, which

are available for use in gluconeogenesis. Cortisol facilitates lipolysis, releasing

fatty acids into the blood which are available as an altemate metabolic fuel for

tissues that can use this energy source, therefore sparing glucose for the brain.

Cortisol plays a key role in adaptation to stress. The release of

glucocorticoids in response to stress May in part serve to control the

29

magnitude of an immune response (Blalock, 1994; Derijk and Sternberg,

1994). For example, cytokines released during inflammation or infection can

stimulate the eventual release of glucocorticoids (Vassilopoulou-Sellin. 1994;

Spangelo and Gorospe, 1995). The glucocorticoid cortisol delivers its

hormonal message to ceUs via cytoplasmic glucocorticoid receptors (Preisler

et al., 2000), which may act in a feedback loop to control the magnitude of the

immune response (Vassilopoulou-Sellin. 1994; Spangelo and Gorospe, 1995).

o 4 (1

4. Syntbetic Glucocorticoids

21jH20H

2OC=O 12 18CH3 _. -OH

17

Synthetic glucocorticoids are potent anti-inflammatory drugs that

suppress immune response, delay wound healing, and depress numbers of

circulating lymphocytes (Griffin, 1989). Synthetic glucocorticoids have been

developed to maximize anti-inflammatory and immunosuppressive effects

while minimizing the metabolic effect, which is useful for treating undesirable

immune responses such as allergic reactions and inflammation. Anti­

inflammatory action has been attributed only to pharmacological levels of

glucocorticoids (administration of cortisol-like drugs that are higher in blood

concentrations than the nonnal physiological range). When phannacological

glucocorticoids are administered to yield higher than physiological

concentrations, the metabolic effects increase in magnitude in addition to

immunosuppressive and anti-inflammatory effects.

Glucocorticoid therapy has been used widely in dairy cattle for the

treatment of ketosis, however there is Httle current research documenting the

efficacy of these treatments, especially in the early stages of the condition.

Glucocorticoid concentrations are usually elevated in the serum of ketotic

cows. AlI of their actions except adipose lipolysis and increased ketogenesis

are favorable in terms ofketosis prevention (Morin, 2004).

30

Administration of large amounts of glucocorticoids inhibits almost

every step of the inflammatory response by acting in a negative-feedback

fashion to suppress the hypothalamus-pituitary axis that drives normal

glucocorticoid secretion and maintains the integrity of the adrenal cortex.

Glucocorticoids stimulate the cellular production of lipocortin, which inhibits

phospholipase A2, the enzyme responsible for cleaving arachidonic acid from

cell walls therefore inhibiting arachidonic acid release (Morin, 2004). The

corticosteroids inhibit both the lipoxygenase and cyclooxygenase pathways of

inflammation, blocking formation of leukotrienes as well as prostaglandins,

prostacyclin, thromboxan and in addition to blocking the inflammatory

Mediators, the corticosteroids suppress white blood cell functions and antibody

production, specifically destroying lymphocytes within lymphoid tissue which

are responsible for antibody production and destruction of foreign cells. This

is reflected in alterations in the numbers of white blood cells circulating in the

bloodstream and the white blood cell response to injured and infected tissues.

In acute inflammation, the corticosteroids maintain the integrity of the blood

vessels and reduce edema formation, and limit the movement of white blood

ceUs into injured tissues.

The use of glucocorticoids which are effective in the management of

anti-inflammatory disorders such as bovine mastitis is hampered by their

adverse effects on hormonal, metabolic and skeletal systems (Sigeal, 1985).

To overcome these drawbacks, research has been performed on the structural

modifications of glucocorticoids in an attempt to increase their potencies while

reducing their serious adverse systemic effects.

A hydroxyl group at carbon Il is essential for glucocorticoid and anti­

inflammatory activity. The 4, 5 double bond and a 3 ketone on ring A is

essential for both mineralocorticoid and glucocorticoid activity. Synthetic

analogues of cortisone and hydrocortisone contain a double bond between

carbon 1 and 2 of the corticosteroid nucleus and have a greatly decreased

effect on electrolyte metabolism and increased glucocorticoid activity. In

addition, unsaturation of hydrocortisone at the number one carbon

(prednisolone) bas been shown to enhance its glucocorticoid properties four to

five times in lactating normal and ketotic dairy cattle. Methylation at carbon 6

or 16 and hydroxylation at carbon 16, have led to further decrease in

31

electrolyte imbalance noted with the naturally occurring glucocorticoids

(package insert). Fluorination at carbon 6 and/or 9 has led to an increase in

anti-inflammatory activity. It has also been shown that when hydrocortisone

bears fluorine in the 9-alpha position (9-alpha-fluorohydrocortisne), it

becomes more potent than the parent compound. Fluorination and methylation

prolongs the half life of glucocorticoids (package insert). Fusion of the

heterocyclic rings onto the steroid nuclei and fluorination at the 90. position,

have been very effective in improving the pharmacological activity of

glucocorticoids.

There are many corticosteroid preparations available for veterinary use.

• Methylprednisolone sodium succinate (Solu-Medrol'Iil)

• Prednisolone sodium succinate (Solu-Delta-Corte~)

• Dexamethasone sodium phosphate (Azium SP~

• Dexamethasone (Azium~

• Flumethasone (Flucort®)

• Methylprednisolone acetate (Depo-Medrol~

• Triamcinolone acetonide (Vetalog®)

• Isoflupredone acetate (Predef!' 2X)

• Betamethasone dipropionate (Betasone ~

Differences in chemical structure of these drugs determine the potency

of anti-inflammatory activity, duration of effect, and duration of suppression

ACTH release from the brain, shown in Table 1. The corticosteroids can be

classified by comparing them to hydrocortisone, which is identical to cortisol,

the natural corticoid hormone.

32

Table 1. Comparison of Drug Duration and Action Potency*

(Dowling, 2004)

Drug Duration Potency

Hydrocortisone 8-12 hrs 1

Prednisone 12-36 hrs 4

Methylprednisone 12-36 hrs 5

Triamcinolone 12-36 hrs 5

Isoflupredone 12-36 hrs 50

Dexamethasone 32-48 hrs 30

Betamethasone 32-48 hrs 30

Flumethasone >48 hrs 120

* Potency is determined by comparison to a cortisol value of 1.0.

Corticosteroid therapy is directed at modifYing the body's response to

inflammation and not at treating the underlying disease process (Dowling,

2004). A veterinarian will use the smallest dose that achieves the desired effect

in order to limit adverse side effects. Generally, anti-inflammatory doses are

10 times the physiological levels, doses to suppress the immune system are

twice the anti-inflammatory dose, and doses to treat shock are 5 to 10 times

the immunosuppressive dose (Dowling, 2004). Product formulations have

differences in onset and duration of action.

Flucort® and Prede~ 2X are two corticoid steroids used in this study

and their characteristics will he further described.

Solutions of free steroid alcohols such as Flumethasone (Flucort®;

Syntex Animal Health, Inc) are administered intravenously or intramuscularly

and their use is usually limited to acute, but not immediately life·threatening

conditions such as chronic obstructive pulmonary disease (heaves) attacks,

snake bites, vaccine reactions, and insect bite hypersensitivity (Dowling,

2004).

33

4.1. Flucort®

F

Flucort® solution is made by Fort Dodge. It is a chemical modification

of prednisolone, which possesses greater anti-inflammatory and gluconeogenic

properties than the parent compound when compared on an equivalent basis

(package insert).

Chemically it is 6a-9a-difluoro-l6a methylprednisolone. The active

ingredient of Flucort® solution is flumethasone which occurs as a white to

creamy white, odorless, crystalline powder. The appearance of Flucort®

solution is a clear colorless to slightly yellowish mobile liquid. Each mL of the

injectable preparation contains

• 0.5 mg flumethasone

• 420 mg polyethylene glycol 400

• 9 mg benzyl alcohol (as a preservative)

• 8 mg sodium chloride

• 0.1 mg citric acid

• Water for injection USP q.s.

• When necessary, pH is adjusted with hydrochloric acid and/or

sodium hydroxide

The eosinophil depression test in normal dogs and blood glucose

elevation and eosinophil depression in normal cattle have been used as

parameters of drug activity to compare prednisone and dexamethasone.

According to the company package insert, the assays indicate that

34

flumetbasone possesses greater anti-inflammatory and gluconeogenic activity

than these compounds, on an equivalent basis (Flucort® package insert).

Acetate and acetonide esters of flumethasone are given

intramuscularly, subcutaneously or intra-articular (into the joint) for a

prolonged effect. Absorption of drug into the systemic bloodstream oceurs

slowly, over days to weeks. Examples of long-acting formulations include

Isoflupredone acetate (PredefP 2X; Pharmacia and Upjohn) and

Betamethasone dipropionate (Betasone®; Schering-Plough Animal Health)

(Dowling, 2004).

4.2. PredefP 2X

I~H c=o

CHs, .'.OH

o

Since the successful treatment of bovine ketosis with cortisone and

hydrocortisone in 1950 (Hatziolos and Shaw, ] 950), a number of synthetic

glucocorticoids have been recommended for the use (Butler and Elliot, 1970),

and research continues to develop more potent steroids to do more specifie

jobs. The biological activity of (9a.-fluoroprednisolone acetate was first

reported in 1955 by Stafford et al. Their work in the rat demonstrated that this

compound was about 50 times as potent as hydrocortisone as a gluconeogenic

agent (measured by liver glycogen deposition assay), and almost 20 times as

potent as desoxycorticosterone acetate in causing sodium retention in the

adrenalectomized rat. Data such as these prompted experiments leading to the

possible use ofthis compound in the treatment of bovine ketosis. Goetsch et al

(1959) reported 9a-fluoroprednisolone to be about four times as potent as

prednisolone or 9a-fluorohydrocortisone in its ability to elevate blood glucose

of normal dairy cows.

Prede~ 2X is an isoflupredone acetate sterile aqueous suspension

made for intramuscular or intrasynovial injection by Pharmacia & Upjohn to

35

treat bovine ketosis and other conditions that require a potent gluconeogenic

and anti-inflammatory agent. It is noted for its gluconeogenic and glycogen

deposition activity and is an effective and valuable treatment for the endocrine

and metabolic imbalance of bovine ketosis. In secondary bovine ketosis,

where the condition is complicated by pneumonia, mastitis, endometritis, and

traumatic gastritis, isoflupredone acetate exerts an inhibitory influence on the

mechanisms and the tissue changes associated with inflammation. Vascular

penneability is decreased, exudation diminished, and migration of the

inflammatory ceUs markedly inhibited. In addition, systemic manifestations

such as fever and signs of toxemia may also be suppressed. White certain

aspects of this alteration of the inflammatory reaction may be beneficial, the

suppression of inflammation may mask the signs of infection and tend to

facilitate spread of microorganisms. Predet«' 2X therapy should he used in

conjunction with appropriate antibacterial therapy. Without concurrent use of

an antibiotic, the use of the adrenal honnones in animaIs with infections can

be hazardous. No sodium retention or potassium depletion has been observed

at the doses recommended in animaIs receiving 9-fluoroprednisolone acetate

(package insert).

Each mL of Predet«' 2X contains 2 mg of isoflupredone acetate. Non-

medicinal ingredients include (Package insert):

• 4.5 mg sodium citrate hydrous

• 120 mg polyethylene glycol 3350

• 1 mg polyvinylpyrrolidone (povidone)

• 0.201 mg Myristyl-gamma-picolinium chloride (as preservative)

• Water for injection USP q.s.

• When necessary, pH is adjusted with hydrochloric acid and/or

sodium hydroxide

According to the package insert, Predef> 2X results in a more potent

corticoid whose activity is greater than the additive effects of unsaturation and

fluorination. Predet«' 2X has greater glucocorticoid activity than an equal

quantity of prednisolone. Isoflupredone acetate has less than half the anti­

inflammatory potency of dexamethasone (Langston, 1993). The glucocorticoid

36

activity of isoflupredone is approximately ] 0 times that of prednisolone, 50

times that of hydrocortisone, and 67 times that of cortisone as measured by

liver glycogen deposition in rats. Isoflupredone acetate is a less potent

glucocorticoid than dexamethasone, does not cause abortion, and presumably

has less risk of immunosuppression (Morin, 2004). Isoflupredone acetate has

less than half the anti-inflammatory potency of dexamethasone (Langston,

1993). However, isoflupredone acetate has more mineralocorticoid activity,

which can lead to hypokalemia and recumhency when repeated doses are used

in sick cows (Sielman et al.,1997; Sielman et al.,1997). Other sources state

that PredefP 2X is a safe substance at one 10mL dose (Radostits, et al. 2000).

And its gluconeogenic activity is based on its hyperglycemic effect in both

normal and ketotic cattle. A possible explanation for the increased potency of

PredefB> 2X over prednisolone is the difference in the rates of reduction of !:14_3

and C20 ketone groups of prednisolone and PredefP 2X by a liver enzyme

system. It has been shown that the ketone groups of prednisolone were

reduced to biologically inactive products more than twice as rapidly as those

of Predef> 2X. Consequently, Prede~ 2X remains present in a biologically

active form more than twice as long as prednisolone. This factor would

increase the length of time the active steroid remained in the circulatory

system and thus contributed to a longer elevated blood glucose level. Prede~

2X is long-lasting (48 h gluconeogenic activity). Isoflupredone is 10 times

more glucogenic than prednisolone (package insert), thus 10 mg of

isoflupredone acetate therapeutically equals 100 mg of prednisolone (package

insert). The usual intramuscular dose for cattle is 10 to 20 mg according to the

size of the animal and severity of the condition (package insert). This dose

may be repeated in 12 to 24 h if indicated (package insert). It is reported that

blood glucose levels return to normal or above within 8 to 24 h after injection,

following a reduction in blood and urine ketone levels by increasing its

gluconeogenic and glycogen disposition activity. It does not cause pregnancy

termination or induce calving. There is a decrease in the circulating amount

eosinophils. Usually the general attitude of the cow is much improved,

appetite retums, and milk production rises to previous levels within 3 to 5 d. In

secondary bovine ketosis, where the condition is complicated by pneumonia,

mastitis, endometritis, traumatic gastritis, etc, PredefB> 2X should he used

37

concurrently with proper local and parenteral antibacterial therapy, infusion

solutions, and other accepted treatments for the primary conditions (Package

insert). Milk from treated animais must not be used for food within 72 h after

the last treatment with the drug, in addition animaIs intended for human

consumption should not be slaughtered within 7 d of last treatment (package

insert).

In ruminants, the comparison and effects of Flucort® and Predefll 2X

on blood metabolites, immune function and milk composition has not been

investigated. The main objective of this study was therefore to determine

whether an intramuscular injection 10mg/mL of Flucort® or Prede~ 2X on the

day of calving improved the energy status of cows without depressing immune

function.

38

III. Hypothesis & Objectives

Hypothesis

1. We hypothesize that cows will respond to glucocorticoid treatments on

the day of calving by increasing gluconeogenic activities and reducing

production of ketone bodies, without compromising the immune

function.

Objectives

The objective of this research project was to evaluate specifie intervention

strategies for negative energy balance and observe its effects on immune

function. Specifically:

1. Determine the effects of glucocorticoid (Predet«' 2X and Flucort~

treatment on energy balance

2. Determine the effects of glucocorticoid (Predet«' 2X and Flucort®)

treatment on immune function.

3. Determine the effects of glucocorticoid (Predet«' 2X and Flucort®)

treatment on milk composition.

39

IV. Materials and Methods

1. Experimental design

Thirty Holstein cows (pre-calving first lactation heifers and

multiparous cows) were randomly selected and enrolled in the trial 10 d prior

to the expected calving date. Two different glucocorticoids were compared

with a placebo control group in a randomized double-blind design. Using

random number tables, animaIs were assigned to one of three treatment groups

to receive a 10 mg/mL intramuscular injection in the right hind leg of

Flucort®, Prede~ 2X or 10.5 mL of placebo on the day of calving. The

placebo solution contained 10 mL sterile water and 0.5 mL penicillin.

Approximately 33% cows were in flfSt lactation, 20% in second lactation, 17%

in third lactation, 17% in fourth lactation, and the remainder were fifth parity

or greater cows as shown in Table 2.

Table 2. Frequency of parities (1 to >5) within treatment

Parity Control Flueort'Ïl Prede~2X Total Total %

1 3 3 4 10 33.3

2 2 2 2 6 20.0

3 2 2 1 5 16.7

4 2 1 2 5 16.7

>5 1 2 1 4 13.3

For simplicity, parities 3 and greater were combined. Therefore

approximately 47% of the animaIs were in parity group ~3.

Twenty percent of the cows calved during the summer, over 70%

calved in the falI and approximately 7% calved in the winter as shown in

Table 3.

Table 3. Frequency of calving seasons within treatment

Prede~

Season of Calving Control Flutort@ 2X Total Total %

Summer 2 3 1 6 20.0

FaU 7 7 8 22 73.3

Winter 1 0 1 2 6.7

40

2. Feeding

AU cows were based in a tie-staU and fed total mixed rations. Daily

feed intakes were recorded from two weeks pre-calving until one month post­

calving. The herd utilized two dry cow feeding groups (far-off and close-up).

Cows were fed twice per day with the first feeding occurring after the morning

milking and the second feeding before the afternoon milking. Feed intake was

recorded daily from day lOto day 28.

2.1. Close-Up Dry Ration

Specifie diet formulations were changed frequently, however the

predominant forages used were dry hay, haylage and corn silage; the main

components of the concentrate mix were high moisture corn, cracked corn,

raw soybean, soybean meal and commercial supplements (Anion Tech).

Table 4. Diet Composition (% of DM) of Close-Up Ration

Chemical Composition %

Crude Protein (CP) 15.5

Acid-Detergent Fiber (ADF) 25.8

Neutral-Detergent Fiber (NDF) 41.2

Non-Fiber Carbohydrate (NFC) 32

NEL, McaVkg of DM 1.51

2.2. Fresh Cow Ration

Specifie diet formulations were changed frequently, however the

predominant forages used were dry hay, haylage, corn silage and alfalfa hay;

the main components of the concentrate mix were high moisture corn, cracked

corn, soybean meal and raw soybean, and commercial supplements (Ener-g-II,

Melass Sec, RTM Amino).

Table 5. Diet Composition (% of DM) of Fresh Cow Ration

Chemical Composition %

Crude Protein (CP) 16.2-18

Acid-Detergent Fiber (ADF) 18-21.7

Neutral-Detergent Fiber (NDF) 28-32.5

Non-Fiber Carbohydrate (NFC) 41-41.2

NEL, Meal/kg of DM 1.6-1.71

41

3. Blood Serum Collection

Blood was collected from the coccygeal vein into 10 mL vacuum tubes

without anticoagulant (Monoject® red stopper blood collection tubes;

Sherwood Medical, St. Louis, MO) d-l0, d-5, dO, d 1, d 7, d 14, d 21, and d

28 relative to calving. Pre-calving sampling dates were assigned

retrospectively and labeled according to closest d-l0 or d-5 in the original

sampling schedule. Blood samples were taken at approximately the same time

of day. Blood samples were stored in an insulated cooler before samples were

returned to the laboratory where blood samples were allowed to clot and went

through clot retraction by centrifugation at 3,000 x g for 10 min. The serum

was separated from the clotted sample by aspiration and stored in a -20°C

freezer until further serum analysis.

4. Milk Sampling

Composite aseptic milk samples were taken weekly from dito d 21

post-calving and submitted directly to PATLQ (Programme d'Analyze

Traitement Laitiers du Quebec; Ste-Anne-de-Bellevue, Quebec) for analysis of

SCC, fat (%), protein (%) and lactose (%). In addition, daily milk yields were

reeorded from dIto d 28 post-calving. Fat, prote in and lactose yield were

calculated based on milk yield and the respective percentages.

5. Milk Sodium and Potassium Analysis

An extra composite aseptic milk sample was taken on d 1 post-calving

and an aliquot of 5 mL was placed in a 100 mL volumetrie flask, 50 mL of

24% (w/v) TCA was added to the sample and then diluted with deionized

water to a volume of 100 mL. Milk proteins including casein were precipitated

using trichloroacetic acid (TCA). Samples were shaken at 5-min intervals for

30 min and then filtered. A 5 mL aliquot of the filtrate was transferred to a 50

mL volumetrie flask, 1 mL of 5% (w/v) lanthanum solution was added and

then diluted with deionized water to a volume of 50 mL. A mixed standard

was prepared containing 5.0 mg/L Ca, 0.6 mg/L Mg, 1.6 mg/L Na, 5.0 mg/L

K, 500 mgIL La and 1.2% (w/v) TCA. The filtra te was then analyzed for

sodium and potassium by atomic absorbance, all measurements were made

relative to a blank reagent containing 500 mgIL La and 1.2% TCA .

42

6. Body Condition Scoring

Cows were scored for body condition on a scale of 1 to 5 using

increments of 0.25 according to Edmonson et al. (1989) on d -10, d 0, d 14 and

d 28 relative to calving.

7. Serum Biocbemical Analysis

Frozen serum was submitted to the Animal Health Laboratory,

University of Guelph for the measurement of serum glucose, BHB, NEF A,

calcium, phosphorus, sodium, potassium, chloride and magne sium using a

serum autoanalyzer (Hitachi, model911, Roche, Laval, Quebec).

8. Antibody Production (ELISA)

To evaluate post-ealving immune response to Predef!> 2X and Flueort®,

each eow was immunized on d 0 with an intramuscular injection in the left

hind leg of 3mg/mL saline solution of Grade V chicken ovalbumin (minimum

98%; Sigma A 5503) ftom chicken egg albumin, an inert antigen to whieh

these animaIs had not been previously exposed. One ml of the chicken

ovalbumin solution was mixed immediately before injection with 1 mL of

Freund's incomplete adjuvant (PICA), therefore 2 mL of the mixed solution

was injected. Cows received a boost of the same chicken ovalbumin solution

on d 14.

Specifie antibody response to chicken ovalbumin was determined at d

1, d 7, d 14, d 21, and d 28 relative to calving. Frozen serum samples were

thawed at room temperature. Chicken ovalbumin IgG titer was determined by

ELISA. Flat-bottom 96-well plates (Immulon 2, Dynatech, Chantilly, VA)

were coated with 100 uL of chicken ovalbumin (l ug/mL) in carbonate buffer

(pH 9.6), at 4°C, ovemight. After eoating, the wells were blocked with 1%

gelatin (Sigma) in PBS containing 0.05% Tween 20 (Sigma), 200 uL per weU,

at 37°C for 1 h. After 1 wash with PBS-Tween 20 (0.05%), serum samples

were diluted 40 fold with PBS-Tween 20 (0.05%) eontaining 0.1% gelatin and

added to each weil (100 uL) in triplicate. Fetal bovine serum (FBS) was

diluted 20 fold with PBS-Tween 20 (0.05%) eontaining 0.1% gelatin and

added to 3 wells per plate as negative eontrols. The plates were then incubated

at room temperature to allow attachment of sera chieken ovalbumin

antibodies, for 1 h. Wells were washed 3 times with PBS-Tween 20 (0.05%).

Then, 100 uL/well of IgG (l :6000, HRP-goat and bovine IgG (Kirkgaard &

43

Perry Laboratories, Gaithersburg, MD» diluted in PBS Tween 20 (0.05%)

containing 0.1% gelatin was added to each weil (l00 uL) and incubated at

37°C, for 1 h. After three washings with PBS-Tween 20 (0.05%), peroxidase

activity was assayed by adding 100 uL of ABTS substrate solution (Kirkgaard

& Perry Laboratories, Gaithersburg, MD) and allowed for color development

in the dark for 30 min. Finally, the absorbance values of IgG titers were

evaluated and determined by a microplate reader at 400 nm in a Multiskan®

MCC 340 plate reader (Titertek, Alabama, USA).

9. ConA-Induced Lymphocyte Proliferation

In order to determine the proliferation of lymphocytes, blood was

collected from the coccygeal vein into 3 x 10 mL vacuum tubes (Monoject®

green heparin vacutainer stopper blood collection tubes; Sherwood Medical,

St. Louis, MO) d 0 and d 7 relative to calving. Blood samples were stored in

an insulated cooler before samples were retumed to the laboratory where fresh

blood samples were analyzed for lymphocyte proliferation.

Bovine lymphocytes were isolated from whole blood taken from

Holstein cows. Heparin-anticoagulated blood was mixed with an equal volume

of Ca2+-free Hank's balanced salt solution (HBSS, GIBCOIBRL,

Gaithersburg, MD) and added into 20 mL Ficoll-Paque solution (l.077 g/mL,

Amersham Biosciences, Piscataway, NJ) in 50 mL tubes. After centrifugation

at 3,000 x g for 40 min at 20°C, the buffy coat layer containing the

lymphocytes was removed by pipetting and placed into another 50 mL tube

and then filled with Ca2+-free Hank's balanced salt solution (HBSS,

GIBCOIBRL, Gaithersburg, MD). After centrifugation at 1,500 x g for 10 min

at 20°C, aIl remaining liquid was aspirated and discarded leaving a pellet

containing the lymphocytes. The pellet was washed twice with Ca2+ -free

Hank's balanced salt solution (HBSS, GIBCOIBRL, Gaithersburg, MD) and

centrifuged at 1,500 x g for 10 min at 20°C. The isolated lymphocytes were

resuspended (1 x 107 cells/mL) in the proliferated medium containing RPMI

(Sigma), 10% inactivated fetal bovine serum, 1 % L-glutamine, >5 mMol

HEPES, 0.05 mMol 2-mercapto-ethanol (Fischer, Pittsburgh, PA), 1%

antibiotics/antimycotic. In a 96 weIl-round bottom tissue culture plate, 100 uL

of the cell suspension were cultured in 100 uL of medium to measure the

background, unstimulated proliferation. In the same plate, 100 uL of cell

44

suspension was stimulated with a 100 uL of a 2 /lg/mL concanavalin A

solution containing ConA (Sigma) and a phosphate buffer solution. The plate

was placed in a humidified incubator with 5% CO2 for 54 h at 37°C. During

the last 18 h of culture, ceIls were pulsed with (20 /lCi/mL) eH]-thymidine

solution ([3H]-thymidine, RPMI, antibioticl antimycotic, L-glutamine and

added 20 uL in each weIl. After incubation, the ceUs were harvested with a

semi-automatic celI harvester (Skatron, Sterling, VA) and placed into 4 mL

scintillation cocktail (ICN, Montreal, Canada). The [3H]-thymidine

incorporation into ceUs (radioactivity) was measured with a liquid scintillation

counter (Beckman LS 60001C; Beckman Instruments, Inc., Columbia, MD).

Each sample was run in ten replicate wells and the values were averaged and

expressed as mean counts per minute (cpm). A stimulation index (SI) was

calculated as the mean cpm of tritium incorporated in ceUs treated with ConA

divided by the mean cpm of tritium incorporated in ceUs not treated with

ConA. Stimulation index is the ratio of mitogen-specific proliferation and

background proliferation with error of the mean (SEM). Con A is a

glycoprotein extracted from the jack bean that promotes mitosis and stimulates

T lymphocytes. The usefulness of ConA is the specifie binding action with

certain carbohydrate-containing receptors, a commonly occurring sugar, (1-

linked mannose. Since a wide variety of serum and membrane glycoproteins

have a "core oligosaccharide" structure which includes a-linked mannose

residues, many glycoproteins can be examined or purified with ConA and its

conjugates. Concanavalin A agglutinates red blood ceUs and complexes with

blood group substance (Clark and Denborough, 1971) and immunoglobulin

glycopeptides (Komfeld and Ferris, 1975). Con A is one of the most widely

used and weIl characterized lectins that is also a lymphocyte mitogen (Ruscetti

and Chervenick, 1975).

10. Serum Insulin Analysis

Frozen serum samples were analyzed for insulin by a radio-immuno

assay (RIA) procedure using a commercial kit (KTSP-l1002, Medicorp,

Montreal, Canada).

11. Statistical Analyses

This study followed a randomized double-blind placebo-controIled

design. AIl statistical analyses on serum metabolites, immune function

45

parameters, and milk composition parameters were conducted with SAS

version 8.0 for Windows using PROC MIXED. The mixed-model

methodology was used according to a repeated-measures covariance structure,

Autoregressive(l) or AR(l), as weIl as the restricted maximum likelihood

method (REML) available in the MIXED software of SAS (1995).

The mixed model applied to ail data sets included a random cow effect

and fixed effects of treatment group, parity, calving season, sampling date,

treatment-by-sampling date interaction, treatment-by-parity interaction,

treatment-by-calving season interaction, parity-by-calving season interaction,

parity-by-sampling date interaction, and calving season-by-sampling date

interaction.

Differences among treatments were compared by Least Squares Means

(SAS, 1999) and the Scheffé multiple comparison test. Statistical significance

was considered at P < 0.05. The statistical models were described as

following.

Yykl(ykm) = p. + T,.+ Pj+ CSk + SDl+ COWykm+ T;*SD1+ Pj*SD1+ CSk*SD, +

eijkl(ijkm)

Where, Yijk1(ijlan) = dependent observation

p. = overall mean

T;= fixed effect of the th treatment {i;;;:: l(control), 2 (Flucort®) or 3 (prede~

2X)}

Pr fixed effect ofthelh parity {j = 1,2,3 (or >3)}

CSk = fixed effect of the J(h calving season {k = 1 (June 22 to September 21-

summer),2 (September 22 to December 21-fall), 3 (December 22 to

March 20-winter)}

SDI ;: fixed effect of the th sampling date {l = 1 (D-10), 6 (D-5), Il (DO), 12

(Dl), 18 (D7), 25 (D14), 32 (D21), 39 (D28)}

COWij/rm = random effect of the mth animal nested in the lh treatment and in the

/h parity and in the IIh calving season <ijkm == 1, ... , 30)

T;*SDF fixed effect of interaction the th treatment and the t' sampling date

Pj*SDF fixed effect of interaction thelh parity and the lth sampling date

eSk *SDF fixed effect of interaction the ft' calving season and the th sampling

date

eijk/(ijkm) = random residual error associated with the observation from the ijkmth

46

cow on the th treatment ofthelh parity in the Iéh calving season and

ofthe th sampling date eijkl(ijkm) - N (0, R); if = random residual

variance

AlI other interactions were tested for significance (P<O.05), and were

eliminated from the model since they were not significant.

47

v. Results and discussion

This study comprised of a trial on 30 Holstein primiparous and

multiparous dairy cows, in which two veterinary drugs, Flucort® and Predefl'

2X were evaluated on blood metabolites, immune function and milk

composition in the weeks immediately pre-calving and post-calving. This is

the first work that compared Flucort® and Prede~ 2X and their effects on

immune function. The data collected from cows are particularly interesting

because there is limited literature published on the influence of Flucort® and

Prede~ 2X for this important time period after ealving.

F values of aIl effects tested in the model are represented in Table 6.

Table 6. F value of effecfs from mixed models for ail ~arameters in cows that were either treated day of calving with a Flucort ,Predef' 2X or

• 1 served as nee:atIve con trois Fvalue

Parameter T P CS SD T*P T*CS T*SD p*cs P*SD CS*SD

Glucose 0.5 0.75 0.86 <0.05 0.92 0.69 0.92 0.88 0.71 0.32

NEFA 0.35 <0.05 0.53 <0.05 0.75 0.19 0.29 0.26 0.09 0.21

BHB 0.11 0.24 0.19 <0.05 0.32 0.57 0.56 0.26 0.87 0.35

Calcium 0.65 0.07 0.92 <0.05 0.76 0.99 <0.05 0.72 <0.05 <0.05

Phosphorus 0.91 0.02 0.82 <0.05 0.71 0.51 0.26 0.81 <0.05 <0.05

Sodium 0.99 0.45 0.54 <0.05 0.27 0.39 0.51 0.85 0.34 0.99

Potassium 0.23 0.46 <0.05 0.27 0.53 0.43 0.29 0.39 0.23 0.05

Chloride 0.75 0.9 0.27 <0.05 0.28 0.28 0.75 0.31 0.11 <0.05

Magnesium 0.9 0.31 0.24 <0.05 0.99 0.93 0.049 0.87 0.71 0.11

Insulin 0.99 0.22 0.83 <0.05 0.38 0.91 0.01 0.42 <0.05 0.23

Antibody 0.83 0.14 0.59 <0.05 0.72 0.64 0.54 0.12 0.49 0.94

Lymphocyte 0.7 0.39 0.l3 0 0.42 0.67 0.53 0.96 0.5 0.03

Fat (%) 0.98 0.18 0.85 0.63 0.64 0.5 0.85 0.73 0.37 0.43

Fat (kg) 0.61 0.01 0.48 <0.05 0.32 0.41 0.73 0.24 0.l8 0.69

Protein (%) 0.4 0.l9 0.09 <0.05 0.23 0.25 <0.05 0.7 0.09 0.02

Protein (kg) 0.84 0.15 0.24 0.03 0.36 0.29 0.43 0.19 0.31 0.05

Lactose (%) 0.77 <0.05 0.08 <0.05 0.41 0.19 0.32 0.37 0.69 0.19

Lactose (kg) 0.72 0.51 0.64 <0.05 0.62 0.9 0.3 0.75 0.25 0.54

MilkNa 0.002 0.06 0.59 0.59 0.28 0.47

MilkK 0.85 0.69 0.06 0.5 0.71 0.52

SCC 0.52 0.28 0.59 <0.05 0.7 0.84 0.11 0.92 0.03 0.58

AvgMilk 0.34 <0.05 <0.05 <0.05 0.71 0.58 0.72 0.46 0.04 0.56 Yield

1 . Models usmg proc mlxed (SAS) mcluded panty, calvmg season, sample date

and the random effeet ofeow.

48

1. Flucort®

1.1. Energy Status

Blood energy metabolites included glucose, insulin, NEF A and BHB.

Ali the blood energy metaboHtes were evaluated on d-IO, d-5, dO, d 1, d 7, d

14, d 21 and d 28 relative to calving. Among the ketone bodies (BHB,

acetoacetate and acetone), only BHB was evaluated because most other

cowside tests lack sensitivity as compared to serum BHB. Testing for BHB

remains the gold standard for studying ketosis (Duffield, 1997).

1.1.1. Glucose

Statistical analysis of serum glucose levels revealed no effect of

treatment (P>0.05), however there was an effect of sampling date (P<0.05).

Furthermore, there was no significant differences (p>0.05) in serum glucose

levels among treatments. Results for overall least square means and least

square means for individual days are shown in Table 7 and 8, respectively.

49

Table 7. Overall least squares metabolites, immune function parameters in cows tbat were

means from mixed models for blood parameters and milk compositional eitber treated day of calving witb a

FI ®p df'Y2X 1 b ucort , re e or pl ace 0 contro Il

Parameter Trt OverallLSM S.E.M. Glucose (mmollL) C 3.2019 0.1690

F 2.9591 0.1690 P 3.0869 0.1733

NEFA (mmollL) C 0.4755 0.04830 F 0.4311 0.04830 P 0.5443 0.04958

BHB (IlmollL) C 700.90 234.26 F 1139.00 234.26 P 1513.69 240.22

Calcium (mmollL) C 2.5156 0.04900 F 2.5108 0.04900 P 2.4441 0.05024

Phosphorus C 2.0917 0.08423 (mmollL) F 2.0713 0.08423

P 2.0329 0.08639 Sodium (mmollL) C 143.16 1.1717

F 143.18 1.1681 P 143.38 1.1987

Potassium (mmollL) C 4.5964 0.1108 F 4.7745 0.1108 P 4.4768 0.1139

Chloride (mmollL) C 100.99 1.2121 F 99.8916 1.2091 P 100.19 1.2407

Magnesium C 1.0076 0.03493 (mmollL) F 0.9995 0.03493

P 1.0219 0.03583 Insulin (uIU/mL) C 15.0863 1.3706

F 15.0698 1.3821 P 15.2290 1.4153

Antibody (OD) C 0.8657 0.1020 F 0.8142 0.1020 P 0.7722 0.1046

Lymphocyte (SI) C 11.6433 5.8275 F 11.6078 5.9168 P 12.6123 5.6497

MilkFat(%) C 4.5035 0.4983 F 4.6083 0.4983 P 4.5023 0.5106

Milk Fat (kg) C 1.5617 0.1831 F 1.6013 0.1831 P 1.3137 0.1790

Milk Protein (%) C 3.3905 0.2482 F 3.3889 0.2482 P 3.8890 0.2543

Milk Protein (kg) C 1.1748 0.1620 F 1.1490 0.1620

50

P 1.0248 0.1584 Milk Lactose (%) C 4.2874 0.08765

F 4.3562 0.08765 p 4.2814 0.08981

Milk Lactose (kg) C 1.4566 0.08851 F l.4017 0.08851 P l.3460 0.08654

Milk sec (,000) c 108.00 50.9855 F 132.25 50.1947 p 203.90 5l.5055

Milk Sodium (mglL) C 34.3840 1.9029 F 24.4662 1.8073 p 21.6297 1.9771

Milk Potassium C 8l.5700 3.8217 (mgIL) F 79.9409 3.6296

p 78.5493 3.9708 Milk Yield (kg) C 36.8114 0.7521

F 35.4448 0.7399 p 35.5731 0.7199

1 . Models usmg proe mlxed (SAS) meluded panty, ealvmg season, sample date

and the random effeet of eow.

51

Table 8. Least squares means from mixed models for serum energy parameters in cows on D-10, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort@, P d ~2X 1 b 1 re e or place 0

Serum Energy Parameter (LSM ± S.E.M.)

Day Trt Glucose NEFA BHB Insulin (mmollL) (mmollL) (mmollL) (uIU/mL)

D-10 C 3.325 0.1413a 320.15 22.23488

F 3.40723 0.0841 3 467.33 22.6638

P 3.46518 0.21593 1076.668 25.82528

D-S C 3.6357a 0.2657a 373.94 20.41998

F 3.42798 0.1875° 415.898 21.40448

P 3.41428 0.3066° 935.96° 22.86188

DO C 3.65728 0.6943b,c 669.91 20.3482A.8

F 3.38948 0.8121b,d,e 523.268 15.1735c

P 3.5239c 0.9044b,d,e 1074.328 13.0722B,b

Dl C 2.899611 0.9305A.II,a,e 758.21 10.6079A.II

F 3.0818° 0.6873B,b,d,g 566.768 18.3892B,e

p 3.1712 0.7221 b,d,g 1450.81 14.5857b

D7 C 2.9916b 0.6264b,t;g 858.65 9.4076b

F 2.4138b,d 0.5782b,d,t:i 1444.1b,c 9.4989b,d,f

P 2.624b,d 0.6411 b,d,f 1846.49d 10.5376b

D14 C 3.1528 0.541 b,t;g 759.09 11.799b

F 2.745b 0.4208b,d,f,h 1237.15c 10.4086b,f

P 2.9251d 0.5297b,f 1623.19 12.2096b

D21 C 2.896911 0.3235a,t,h 785.59A 11.8335b

F 2.5791b 0.3483b,f,h,j 1865.24B,b,c 11.8039b,f

P 2.6776b,d 0.554Sb,d,f 2098.9SB,b,d 10.0026b

D28 C 3.056811 0.281rt,h 1081.65A 14.039411

F 2.629b 0.3305b.f,h,j 2592.3B,b.d 11.2168b,f

P 2.893r 0.4799b,f,h 2003.13d 12.7372b

S.E. C 0.2541 0.08818 364.11 2.2141

F 0.2541 0.08818 364.11 2.2704

P 0.2586 0.08955 370.29 2.2938 1 .

Models UStng proe mlxed (SAS) tncluded panty, ealvtng season, sample date and the random effeet of cow. A and B, C and D differ significantly among treatments within the same day a and h, c and d, e and f, g and h, i and j differ significantly among days within the same treatment

52

The only day that Flucort® treated cows had better energy status than

control cows as indicated by the numerically higher serum glucose (P>0.05)

was on d 1. AIl cows experienced decreases in glucose levels after d 0,

however, serum glucose values for Flucort® treated cows experienced an even

further significant decrease by d 7 (P<0.05). Furthennore, glucose

concentrations on d 14, d 21 and d 28 were significantly lower than d -10, d-5

and d 0 values (P<0.05), and this is comparable to the trend observed in

control cows.

When a glucocorticoid is administered, the concentration of glucose in

the blood should increase through the synthesis of glucose from amino acids

(gluconeogenesis), a decrease in the synthesis of prote in from amino acids,

and altered lipid metabolism, thereby satisfying the systemic demand for

glucose and helping to prevent the metabolism of fats and production of

ketones. Also, peripheral utilization of glucose is reduced and liver storage of

glycogen is increased.

Butler and Elliot (1970) observed an increase (P<O.OI) in blood

glucose concentrations from the administration of Flucort®. An inhibition of

glucose utilization as a result of Flucort® administration may account for sorne

of the increase in blood glucose levels. There is evidence that when a

glucocorticoid honnone 1s administered to sheep (Butler and Elliot, 1970),

blood glucose is increased as a result of impaired glucose utilization rather

than by an increase in the rate of gluconeogenesis. It has also been reported

(Butler and Elliot, 1970) that cortisol administration to sheep markedly

inhibited peripheral glucose utilization. and had an antagonistic effect on

insulin action. Butler and Elliot (1970) further suggested that the increase in

blood glucose concentration in the lactating cow may be due to the depression

in milk production (P<0.05) and consequent reduction in lactose secretion.

However in the present study, milk yield and lactose yield were not

significantly reduced with the administration of Flucort® which may explain

the lack of increase in glucose levels. A larger dose than 10 mg/mL of

Flucort® is probably needed to induce more significant effects on milk yield,

lactose yield and glucose concentrations. However, despite the dosage, effects

of glucose would only be temporary and would have to be measured on an

53

hourly basis. Butler and Elliot (1970) demonstrated blood glucose

concentrations reached maximum levels at about 18 h after treatment of 0.7

mg of Flucort® per 100 kg body weigh and this dosage is routinely used for

treating ketotic dairy cows.

1.1.2. Insulin

Statistical analysis of insulin levels revealed no effect of treatment

(P>0.05), however there was an effect of sampling date, interaction of

treatrnent and sampling date, and an interaction of parity and sample dating

(P<0.05). Results for overall least square means and least square means for

individual days are shown in Table 7 and 8, respectively.

The only significant difference observed in serum insulin levels was on

d 1 where Flucort® treated cows experienced significantly higher (P<0.05)

levels than control cows, indicating better energy status. The only other day

that Flucort® treated cows had better energy Status than control cows as

indicated by the slightly numerically higher serum insulin (P>0.05) was on d

7. In contrast, Flucort® treated cows had numerically (P>0.05) lower insulin

values from d 14 to d 28 than control cows.

Flucort® treated cows experienced a significant decrease in serum

insulin concentration by d O. From dIto d 7, insulin levels decreased

significantly (P<0.05) to almost 50%. Insulin levels increased by the end of

the trial (P>0.05), however, post--calving values remained significantly lower

than pre-calving values (P<0.05).

The higher insulin values experienced on d 1 should have stimulated

the uptake, utilization and storage of glucose. In addition, insulin should have

facilitated the entry of glucose into muscle, adipose tissue and other tissues. If

the chain of events listed had occurred upon Flucort® administration, the

mobilization of fat would have been avoided.

1.1.3. Non-esterified fatty acids

Statistical analysis of serum NEF A levels revealed no effect of

treatment (P>O.05), however there was an effect of parity and sampling date

(P<O.05). Results for overall least square means and least square means for

individual days are shown in Table 7 and 8, respectively.

The only significant difference observed in serum NEF A levels was on

d 1 where, Flucort® treated cows experienced significantly lower (P<O.05)

54

levels than control cows, indicating a better energy status. Other days that

Flucort® treated cows experienced numerically lower NEF A values and a

better energy status than control cows were on d 0, d 7 and d 14 (P>0.05).

Furthermore, from d 0 to d l, NEFA values for Flucort® treated cows

decreased numerically, whereas NEF A values for control cows increased

significantly. NEFA values for Flucort® treated cows continued to decrease

until d 28.

The better energy status experienced by Flucort® treated cows indicate

that there is less fat mobilization in these cows (Grummer, ] 993) and therefore

less NEF A transported to the liver. Plasma NEF A concentration and

triglyceride concentration in the liver (Bertics et al., 1992) are positively

correlated. As fatty liver and ketogenesis are closely linked (Dann et al.,

1999), a reduction in plasma NEF A concentration should result in a decreased

risk of the cow developing fatty liver and ketosis which supports the

hypothesis that Flucort® increased gluconeogenic activities by inhibiting fat

mobilization.

An analysis of the frequency of cows with high NEFA (>0.7 mmol/L)

values indicative of a state of negative energy balance revealed that less cows

had higher NEF A levels than control cows after a Flucort® treatment in ail

cows (both primiparous and multiparous cows) as shown in Tables 9,10 and

Il. In this respect, treatment of Flucort® may provide potential benefits in the

reduction of fat mobilization. However, due to limited numbers of cows used

in this study, a concrete conclusion cannot he made and further experiments

are warranted.

Table 9. Frequency of cows with high NEFA levels at D-10, D-5, DO, Dl, D7 D14 D21 d D28 b t t t , , an 'Y rea men· 2roup

Ali cows - % Hi2h NEFA (NEFA Value >0.7 mmollL) Trt D-I0 D-5 DO Dl D7 D14 D21 D28

C 0 10 70 90 50 50 20 10 F 0 10 60 50 40 20 10 0 p 0 10 44.44 50 10 10 20 20 Total 0 10 58.62 63.33 33.33 26.67 16.67 10

55

Table 10 Frequency of multiparous cows with high NEFA levels at D-10, D 5 DO Dl D7 D14 D21 d D28 b t t t - , , , ' , , an >y rea men group

MULTIPAROUS - % High NEFA (NEFA Value >0.7 mmol/L) Trt D-I0 D-5 DO Dl D7 D14 D21 D28

C 0 14.29 85.71 100 57.14 57.14 28.57 14.29 F 0 14.29 71.43 71.43 57.14 28.57 14.29 0 p 0 14.29 42.86 71.43 14.29 14.29 28.57 28.57 Total 0 14.29 66.67 80.95 42.86 33.33 23.81 14.29

Table 11. Frequency of primiparous cows with high NEFA levels at D-10, D 5 DO Dl D7 D14 D21 d D28 b t t t - , , , , , an )y rea men group

PRIMIPAROUS - % High NEFA (NEFA Value >0.7 mmol/L) Trt D-I0 D-5 DO Dl D7 D14 D21 D28

C 0 0 33.33 66.67 33.33 33.33 0 0 F 0 0 33.33 0 0 0 0 0 p 0 0 33.33 0 0 0 0 0 Total 0 0 33.33 20.00 10.00 10.00 0.00 0

Analysis of the frequency of body condition score (BCS) within

treatment and within day, demonstrate that approximately 73% of the animaIs

were classified as being in fair body condition prior to calving, and 63% in

thin body condition post-calving as shown in Table 12. This indicates that fat

was mobilized from adipose tissue during the transition from late gestation to

early lactation.

T bl 12 F a e . requency 0 fDes . h' wIt ID treatment an d . h' d wIt ID ay

D-I0 DO D14 Trt Thin Fair Fat Thin Fair Fat Thin Fair Fat Thin

C 0 9 1 2 7 1 6 3 1 8

F 2 6 2 3 7 0 5 5 0 6 p 1 7 2 1 8 1 3 7 0 5 Total (%) 10 73.3 16.7 20 73.3 6.7 46.7 50 3.3 63.3 .

Thm=BCS <3.25, Falr=BCS 3.25 to 3.75, Fat=BCS >3.75

1.1.4. Il-hydroxybutyrate

D28 Fair Fat

2 0

4 0

5 0

36.7 0

Statistical analysis of serum BHB Ievels revealed no effect of treatment

(P>O.05), however there was an effect of sampling date (P<O.05). Results for

overall least square means and least square means for individual days are

shown in Table 7 and 8, respectively.

AlI cows experienced a graduai increase in BHB levels throughout the

trial. The days that Flucort® treated cows had a better energy status than

control cows were d 0 and d 1 (p>O.05). However, from dIto d 7, Flucort®

56

treated cows experienced a significant increase of serum BHB. Flucort®

treated cows had a worse energy status on d 7 and d 14 as indicated by the

numerically higher BHB values than control cows (P>0.05). The only

significant differences observed in serum BHB levels was on d 21 and d 28

where Flucort® treated cows experienced significantly higher (P<0.05) BHB

levels than control cows, indicating a worsening energy status.

The high levels of BHB indicate higher ketone bodies concentrations

post-calving in cows receiving Flucort® and an increased risk of ketosis. An

analysis of the frequency of cows with high BHB values (BHB > 1400

mmollL) (Duffield, 2000) indicative of subclinical ketosis revealed that more

cows had higher BHB levels (> 1400 JUllollL) than control cows after Flucort®

treatment in an cows (both primiparous and multiparous cows) as shown in

Tables 13, 14 and 15. In this respect, treatment of cows with Flucort® may

contribute to problems ofketosis. However, due to the limited number of cows

used in the CUITent study, a concrete conclusion cannot be made and further

experiments are warranted.

Table 13. Distribution of subclinical ketosis at D-10, D-5, DO, Dl, D7, D14, D21 d D28 b t t t an )y rea men group

Ail cows - % Ketotic (BHB Value >1400 pmollL) Trt 0-10 D-5 DO Dl D7 D14 D21 D28 C 0 0 20 10 20 10 10 40 F 0 0 0 10 40 50 50 50 p 0 10 11.11 10 30 50 40 30 Total 0 3.33 10.34 10 30 36.67 33.33 40

Table 14. Distribution of subclinical ketosis in multiparous cows at D-10, D 5 DO Dl D7 D14 D21 d D28 b t t t - , , , , , an 'Y rea men 2rOUp

MULTIPAROUS - % Ketotic (BHB Value >1400 p.mollL) Trt D-I0 O-S DO Dl D7 D14 D21 D28 C 0 0 28.57 14.29 28.57 14.29 14.29 42.86 F 0 0 0 14.29 42.86 57.14 42.86 42.86 P 0 0 14.29 14.29 42.86 42.86 42.86 28.57 Total 0 0 14.29 14.29 38.10 38.10 33.33 38.095

Table 15. Distribution of subclinical ketosis in primiparous cows at D-l0, D 5 DO Dl D7 D14 D21 d D28 b t t t - , , , , , an )y rea meoRroup

PRIMIPAROUS - % Ketotic BHB Value >1400 ~mollL) Trt D-I0 D-S DO Dl D7 D14 D21 D28 C 0 0 0 0 0 0 0 33.33 F 0 0 0 0 33.33 33.33 66.67 66.67 P 0 25 0 0 0 50 25 25 Total 0 10 0 0 10 30 30 40

57

The majority of metabolic problems associated with the transition

period occurs by d 1 post-calving. Cows that experience difficult transitions,

may be predisposed to undesirable metabolic outcomes, such as ketosis, that

occur by d 1 post-calving or will eventually take place in the following weeks

(Veenhuizen et al., 1991). If displaced abomasum is related to rumen fill, then

the extent of pre-calving DMI decrease may be an important factor

determining whether the cow develops this disorder. In the study done by

Butler and Elliot (1970), the Flucort® treatment caused a small increase

(P<O.OI) in blood ketone concentrations. This response bas also been observed

in sheep (Butler and Elliot, 1970). The response in blood ketone levels is

presumably the result of amino acid breakdown or fat mobilization.

To be sure that dry matter intake (DMI) and rumen fill was not affected

by treatment, DMI was recorded daily. Tables 4 and 5 specity the diet

composition (% of DM) of close-up rations and fresh cow ration. Statistical

analysis of DM! revealed no effect of treatment (p>0.05), and no differences

in DMI were observed among treatments (P>O.05) as shown in Table 16.

Table 16. Least squares means from mixed models for average dry matter intake in cows on D-I0, D-5, DO, Dl, week 1, week 2, week 3, and week 4 post-calving that were either treated day of calving with a Flucort®, P d f® 2X d ti t 1 1 re e or serve as nega vecon ro s

Average DMI (kg) (LSM Trt ±S.E.M.)

C F P

D-I0 23.8±1.28 21.4±1.37 21.4±O.81

D-5 22.6±1.57 21.07±1.50 20.4±1.71

DO 22.5±2.24 26.4±3.69 21.3±2.60

Dl 30.2±4.47 34.0±1.83 31.8±3.96

D7 35.0±1.45 30.8±2.00 37.4±2.75

D14 36.4±2.45 36.0±1.60 37.0±1.71

D21 37.2±2.81 36.6±1.76 37.5±1.51

D28 38.3±1.61 36.4±2.43 36.4±2.64 1 . .

Modeis usmg proc mlxed (SAS) mcluded panty, calvmg season, samphng date and the random effect of cow.

In this study, it appeared that after Flucort® treatment, less cows had

higher NEFA (>0.7 mmoIIL) Ieveis and more animaIs had higher BHB

(>1400, J..I.IllollL) leveis. High levels of BHB would be the result of fat

58

mobilization and a high concentration of NEF As released into bloodstream

causing high NEF A levels in the liver, and the eventual production of ketone

bodies. Therefore, this trend is difficult to explain and may be due to small

cow numbers. A larger sample size is needed to make a concrete conclusion.

Shpigel et al. (1996) compared the relative efficacy of 40 mg of

dexamethasone and 5 mg flumethasone alone or in combination with rapid IV

infusion of 500 mL of 50% glucose solution for treatment of ketosis in cattle.

Treatment success was defined as recovery after a single treatment without

relapse during the same lactation. Flumethasone in combination with glucose

and dexamethasone in combination with glucose were significantly more

effective than dexamethasone and flumethasone administered alone.

Flumethasone administered alone did not differ significantly from

dexamethasone administered alone. Flumethasone is approved and marketed

for the treatment of clinical ketosis in dairy cows. In this regard, the fmdings

of the current experiment of Flucort® administration are not consistent with

other studies.

1.2. Mineral status

Blood mineraI metabolites included calcium, phosphorus, potassium,

sodium, chloride and magnesium. Ail the blood minerai metabolites were

evaluated on d -10, d -5, dO, d 1, d 7, d 14, d 21 and d 28 relative to calving.

Due to the potential upset of the electrolyte balance the effects of Flucort® and

Prede~ 2X were evaluated on serum minerai parameters.

1.2.1. Calcium

Statistical analysis of serum calcium levels revealed no effect of

treatment (p>0.05), however there was an effect of sampling date, interaction

of treatment and sampling date, interaction of parity and sampling date, and

interaction of calving season and sampling date (P<0.05). Results for overall

least square means and least square means for individual days are shown in

Table 7 and 17, respectively.

59

Table 17. Least squares means from mixed models for serum minerai parameters in cows on D-lO, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort®, Predetll> 2X or placebo·

Serum Mineral Parameter (LSM ± S.E.M.)

Day Trt Calcium Phosphorus Potassium (mmollL) (mmollL) (mmollL)

D-I0 C 2.588 2.35638 4.92488

F 2.54938 2.27968 5.02418

P 2.47928 2.22068 4.5316

D-S C 2.5868c 2.28768 4. 1656A ,b,c

F 2.57318 2.3439c 5.0249B,8

P 2.50288 2.26188 4.6429

DO C 2. 165D,a,e 2.0233D,c 4.7137

F 2.2153b,c 2.0246d,e 4.9338

p 2.0909b,c 1.7802b,c 4.5896

Dl C 2.1024 A,b,a,g 1.6834A,b,a,e 4.8686A ,a

F 2.3907B,b,d,e 1.3337B,b,d,t;g 5. 1 079A,c

P 2.18A,b,c 1.5372b,e 4.1278B

D7 C 2.6662t;b 1. 7571 b,a,e 4.5352

F 2.5095d,g 1.9764b,d,b,i 4.4345d

P 2.5567d 1.901 b,f,g 4.3977

D14 C 2.6493I)l 2.11011 4.5894

F 2.6386d,f,h 2. 1374h 4.7887

P 2.5761d 2.0699d,f 4.5308

D21 C 2.6632t;b 2.23571 4.4815

F 2.6115d,f 2.226h 4.7008

P 2.5759d 2.2531 d,t;h 4.6894

D28 C 2.7116b,t;b 2.27971 4.4928

F 2.5989d,f 2.24~ 4.1821b,d

P 2.5915d 2.2394d,f,b 4.3046

S.E. C 0.0646 0.1203 0.2423

F 0.0646 0.1203 0.2423

P 0.06584 0.1224 0.2457 1 . . .

Models usmg proc mlxed (SAS) mc1uded parlty, calvmg season, sample date and the random effect of cow. A and B, C and D differ significantly among treatments within the same day a and b, c and d, e and f, g and h, i and j differ significantly among days within the same treatment

60

The only significant difference observed in serum calcium levels was

on d 1 where Flucort® treated cows experienced significantly higher (P<0.05)

levels than Predef!> 2X and control cows. There were no significant

differences between pre-calving and post-calving serum calcium

concentrations (d 7, d 14, d 21 and d 28). A higher serum calcium status may

provide a beneficial effect as most fresh-cow health problems such as

displaced abomasums, metritis and clinical and subclinical milk fever are part

of one disease complex greatly dependent on the cow's calcium status (Acre,

1998).

The interaction between parity and sampling date indicated that on

sorne days older cows presented lower calcium concentrations than younger

animaIs. The relationship between age and hypocalcemia has also been weil

documented (Goff and Horst, 1997). In the present study, there were 4 first

parity heifers and 6 multiparous cows in the Predef!> 2X treatment group and

this might explain the lower serum calcium levels on d 1 compared to the

Flucort® treatment group.

1.2.2. Phosphorus

Statistical analysis of serum phosphorus levels revealed no effect of

treatment (P>0.05), however there was an effect of parity, sampling date,

interaction of parity and sampling date, and interaction of calving season and

sampling date (P<0.05). Results for overallleast square means and least square

means for individual days are shown in Table 7 and 17, respectively.

The only significant difference observed in serum phosphorus levels

was on d 1 where Flucort® treated cows experienced significantly lower

(P<0.05) levels than control cows. This was not surprising as sorne

glucocorticoids have a suppressive effect on phosphorus levels. In comparison

to control cows phosphorus levels in this experiment were relatively normal

and not affected by treatment and probably were the response to an adequate

use of anionic salts. There were no differences observed between pre-calving

(d -10, d -5) and post-calving (d 14, d 21, d 28) phosphorous levels (p>O.05).

1.2.3. Potassium

Statistical analysis of serum potassium levels revealed no effect of

treatment (P>O.05), however there was an effect of calving season (P<0.05).

No significant differences were observed in serum potassium levels between

61

Flucort® treated cows and control cows. In the Flucort® treatment cows, cows

that calved in the summer season had significantly lower potassium levels than

cows that calved in the winter season (P<O.05). In both the control and

Flucort® treatments, cows that calved in the fall season had significantly lower

potassium levels than cows that calved in the winter season (P<O.05). These

differences are probably due to the different feeds available in each season.

Results for overall least square means and least square means for individual

days are shown in Table 7 and 17, respectively.

1.2.4. Sodium

Statistical analysis of serum sodium levels revealed no effect of

treatment (P>O.05). however there was an effect of sampling date (P<O.05).

There were no differences (P>O.05) in serum sodium levels among treatments.

In comparison to control cows sodium levels in this experiment were relatively

normal and not affected by treatment and probably were the response to an

adequate use of anionic salts. Serum sodium post-calving values (d 21 and d

28) were significantly lower than pre-calving values in Flucort® treated cows

(P<O.05). which was comparable to control cows. Results for overall least

square means and least square means for individual days are shown in Table 7

and 18, respectively.

62

Table 18. Least squares means from mixed models for serum minerai parameters in cows on D-10, D-5, DO, Dl, D7, D14, D21 and D28 relative to calving that were either treated on day of calving with a Flucort®, P d ~2X 1 b 1 re e or place 0

Serum Mineral Parameter (LSM :i: S.E.M.)

Day Trt Sodium Chloride Magnesium (mmol/L) (mmol/L) (mmol/L)

D-lO C 144.808 105.068 1.07118

F 144.178 105.328 1.0192

P 145.068 105.618 1.03248

D-5 C 147.128 107.058 0.9988

F 145.92c 106.93c 1.06698

P 143.61 104.728 0.958c

DO C 147.298 107.058 1.0243

F 146.06c 104.628 1.0224

P 144.388 102.55c 1.0745d

Dl C 145.958 105.178 0.9929

F 145.198 102.90d,e 1.0009

P 147.34c 104.608 1.06518,d

D7 C 140.81 11 99.70 1411,c 0.9443 11,c

F 143.498 99.8668b,d,e 0.9424b

P 142.32d 99.3861b,e 0.9441b,e

Dl4 C 140.8611 97.415911,e 1.0381e

F 141.73d 96.2813b,d,:t;g 0.9762b

P 141.50d 96.7195b,d 1.0035

D2l C 139.2211 94.514711,11 0.948311,lI,t,g

F 139.70b,d 92.1801 b,d,:t;h 0.9864

P 142.40d 94.80 12b,d,f 1.0275

D28 C 139.2411 91.96701l,<I,t 1.043211,b

F 139.21b,d 91.0324b,d,:t;h 0.9813

P 140.39b,d 93.1059b,d,f 1.0697d,f

S.E. C 1.73 1.73 0.04467

F 1.72 1.71 0.04467

P 1.75 1.76 0.04587 . Models usmg proc mlxed (SAS) mcluded panty, calvmg season, sample date

and the random effect of cow. A and B, C and D differ significantly among treatments within the same day a and b, c and d, e and f, g and h, i and j differ significantly among days within the same treatment

1.2.5. Chloride

Statistical analysis of chloride levels revealed no effect of treatment

(P>O.05), however there was an effect of sampling date and an interaction of

calving season and sampling date (P<O.05). There were no differences

63

(P>0.05) in serum chloride levels among treatments. Results for overall least

square means and least square means for individual days are shown in Table 7

and 18, respectively.

In comparison to control cows, chloride levels in this experiment were

relatively normal and not affected by treatment and probably were the

response to an adequate use of anionic salts.

1.2.6. Magnesium

Statistical analysis of magne sium levels revealed no effect of treatment

(p>0.05), however there was an effect of sampling date and an interaction of

treatment and sampling date (P<0.05). There were no differences (p>0.05) in

serum Magnesium levels among treatments. Results for overall least square

means and least square means for individual days are shown in Table 7 and

18, respectively.

1.3. Immune Function

Cows were challenged to evaluate the effect of Flucort® and Prede~

2X on the immune system. The cows were immunized with chicken

ovalbumin on the day of calving and given a boost 2 weeks post-calving.

Immune function parameters included antibody parameters which were

evaluated on d 0, d 7, d 14, d 21 and d 28 relative to calving and analyzed by

an ELISA assay measuring the concentration of chicken ovalbumin specific

IgG (antibody) in serum; and lymphocyte proliferation which was evaluated

on d 0 and d 7 relative to calving and analyzed by an in vitro assay

determining if bovine ConA-induced lymphocyte proliferation is altered in

response to treatments.

1.3.1. Antibody production

Statistical analysis of antibody levels revealed no effect of treatment

(p>0.05), however there was an effect of sampling date (P<0.05). No

statistically significant differences (P>0.05) were observed between Flucort®

treated cows and control cows for antibody production values. Results for

overall least square means and least square means for individual days are

shown in Table 7 and 19, respectively.

64

Table 19. Least squares means from mixed models for antibody production values in eows DO, D7, D14, D21 and D28 rela.tive to ealving that were either treated day of calving with a Flucort®, Predef!> 2X or

d t , t 1 1 serve a.s nega IVe con ro s LSM±S.E.M.

Parameter Trt DO D7 Dt4 D2t D2S

S.E.M. Antibody Cl 0.4271 3 0.58803 0.8674D,c 1.1708D

,Q 1.275D,Q 0.1246

(OD-400 F 0.34553 0.41143 0.9755b,c 1.1841b,d 1.1548b 0.1246 nm)

P 0.29653 0.4608& 0.8562b,c 1.0824b,d 1.1652b,d 0.1271

1 Models usmg proe mixed (SAS) included panty, ealvmg season, sample date and the random effeet of eow. a and b, c and d differ significantly among days within the same treatment

1.3.2. Lymphocyte proliferation

Statistieal analysis of lymphocyte proliferation levels revealed no

effect oftreatment (P>O.05), there was however an effect of sampling date and

an interaction of calving season and sampling date (P<O.05). Furthermore, in

the present study, a single injection of Flucort® on the day of calving did not

affect ConA-induced lymphocyte proliferation compared to the control group

at d 0 or d 7. Differences may not have been statistieally significant due to a

large variation in lymphocyte proliferation in response to ConA among

animaIs and among replicates. However, the administration of a Flucort® to

dairy cows on the day of calving resulted in a significantly reduced d 7 blood

lymphocyte proliferation values in response to ConA compared to the

respective d 0 blood lymphocyte proliferation values and the control cows

receiving the placebo treatment (P<O.05). The decreased celI proliferation

observed in Flucort® treated cows on d 7 could also be a result of cell death.

Results for overall least square means and least square means for individual

days are shown in Table 7 and 20, respectively.

65

Table 20. Least squares means from mixed models for lymphocyte proliferation values in cows DO and D7 relative to calving that were either treated day of calving with a Flucort®, Predefi> 2X or served as negative controls1

Parameter Trt LSM±S.E.M.

DO D7 Lymphocyte proliferation C 18.22 ± 5.11 7.71 ± 3.47 (stimulation index)

F 24.10 ± 5.36a 6.32 ± 3.6b

P 19.72 ± 5.98 11.78 ± 6.08

Models usmg proc mlxed (SAS) mcluded panty, calvmg season, sample date and the random effect of cow. a and b differ significantly among days within the same treatment

This indicates that Flucort® did not induce more of an

immunosuppressive effect in cows receiving treatment on day of calving

compared to control, however Flucort® treated cows did cause more of a

significant decrease in circulating lymphocytes.

Thanasak et al. (2004) also found that dexamethasone treatment did not

induce a significant decrease in the number of circulating lymphocytes or

changes in lymphocyte subsets, considering the population of lymphocytes in

the study. Our fmdings are in accordance with the investigations of Burton and

Kehrli (1996) who demonstrated that the percentages of lymphocyte subsets

were not altered by 0.04 mglkgl d dexamethasone administered to young bulls

on three consecutive days. However, in other studies glucocorticosteroids have

been shown to cause lymphopenia and alterations of lymphocyte subsets

(Anderson et al., 1999; Doherty et al., 1995; Winnicka et al., 2000). Thus, the

differences between the results of previous studies and the present one cao

mainly be explained by the differences in the sort of corticosteroids

administered and the dosage regimen.

Nagahata et al. (1992) examined B lymphocyte populations to evaluate

host defense in dairy cows during the transition period and found no

significant changes in the number of B lymphocytes of cows from 2 weeks

before until 2 weeks after calving. Other studies of glucocorticosteroids such

as single injections with high doses or repeated treatments of dexamethasone

resulted in decreased lymphocyte proliferation and have been shown to cause

66

lymphopenia and alterations of lymphocyte subsets (Anderson et al., 1999).

On possible mechanism underlying the reduced lymphocyte proliferative

responses is the down modulation of CD 18 expression which has been shown

to act as a co-stimulatory Molecule in the process of lymphocyte proliferation

(Thanasak et al., 2004). Thanasak et al. (2004) found reduced CDl8

expression on lymphocytes in dexamethasone treated cows, however their

proliferative responses remained unaffected.

A depression in lymphocyte proliferation has been shown to be related

to increased blood glucose concentrations (Franklin et al., 1991; Thanasak et

al., 2004). In the study by Thanasak et al. (2004), although increases in blood

glucose and insulin concentration in the dexamethasone treated group

indicated that hypoglycemia was overcome, they did not affect lymphocyte

proliferation. In this study neither lymphocyte proliferation or blood glucose

concentrations was affected by Plucort® administration, however Plucort®

treated cows had significantly higher insulin concentrations on d 1. Perhaps

the therapeutic dosage of 10 mg of Flucort® is not an effective dosage to

induce significant depression in lymphocyte proliferation, and a significant

increase in blood glucose and insulin concentrations allowing cows to

overcome hypoglycemia.

1.4. Milk Status

Milk composition parameters included fat (% and yield), protein (%

and yield), lactose (% and yield), SCC, sodium and potassium. Milk

composition parameters were evaluated on d 1, d 7, d 14 and d 21 relative to

calving, with the exception of sodium and potassium which were evaluated on

d 1 by digestion and atomic absorption.

1.4.1. Milk Yield (kg)

Statistical analysis of milk yield revealed no effect of treatment

(P>0.05), however there was an effect of parity, calving season, sampling date

and an interaction of parity and sample dating (P<O.05) on milk yield. There

were no differences (P>O.05) in milk yield among treatments. Results for

overall least square means and least square means for weekly average milk

yield are shown in Table 7 and 21, respectively.

67

Table 21. Least squares means from mixed models for average milk yield in eows on week 1, week 2, week 3, and week 4 post-ealving that were either treated day of calving with a Flucort®, Prede~ 2X or served as

. 1 1 nel!atIve contro s Trt Average Milk Yield (kg) (LSM::l:: S.E.M.) S.E.M.

Weekl Week2 Week3 Week4 C 28.9072 37.8828 39.595 40.8606 1.445

F 28.6201 36.0701 38.519 38.5701 1.4387

P 27.4726 37.3306 38.8986 38.5906 1.3742 J Models usmg proc mlxed (SAS) mcluded panty, calvmg season, sample date and the random effect of cow

1.4.2. Protein

1.4.2.1. Protein (%)

Statistical analysis of protein (%) revealed no effect of treatment

(P>O.05), however there was an effect of sampling date, interaction of

treatment and sampling date, and an interaction of calving season and

sampling date (P<O.OS) on prote in %. Results for overallleast square means

and least square means for individual days are shown in Table 7 and 22,

respectively.

68

Table 22. Least squares means from mixed models for milk component parameters in cows Dl, D7, D14 and D21 relative to calving tbat were eitber treated day of calving witb a Flucort®, Prede~ 2X or served as

. 1 1 negatlve contro s Milk Parameters (LSM ± S.E.M.)

Trt Dl D7 D14 D21

S.E.M. Pa ra meter MilkFat C 4.6431a 4.1751° 4.89940 4.2963b 0.7864 (%)

F 4.9832" 3.9832b 5.4195b 4.0474b 0.7864

P 4.6032& 4.5920b 4.2354b 4.5786b 0.7997

MilkFat C 0.8852a 1.5660 1.959fD 1.8363b 0.2920 (kg)

F 0.8499& 1.3585 2.2760b 1.921 Ob 0.2920

P 0.4406a 1.4478b 1.5699b 1.7964b 0.2828

Milk C 4.4474A,a 3.3832° 2.9286° 2.8029D 0.3616 Protein

F 4.2938A.a 3.3417b 2.9820b 2.9383b 0.3616 (%)

P 6.3307B,a 3.2693b 3. 1346b 2.8213b 0.3681

Milk C 0.7809a 1.3621° 1.2680b 1.2882 0.2121 Protein

F 0.7831a 1.221 Ob 1.2346 1.3571b 0.2121 (kg)

P 0.9695 1.0083 1.1169 1.0045 0.2060

Milk C 3.7292a 4.3148b 4.5662° 4.5396° 0.1271 Lactose

F 3.9858a 4.3693b 4.4628b 4.6071 b 0.1271 (%)

P 3.6076a 4.3942b 4.6174b 4.5065b 0.1294

Milk C 0.5929a 1.5952b 1.7765b 1. 8620b 0.1171 Lactose

F 0.6418 a 1.4419 b,c 1.6288 b,e 1.8943 0.1171 (kg) b,d,f

P 0.4209 a 1.4671 b,c 1.7745 1.7215 b 0.1137 b,d

MilkSCC C 169.97A 96.3405 41.5749 124.11 67.6351

F 236.258 117.32 110.14 65.3144b 65.9659

P 437.45B,a 105.30b 141.29b 131.56b 67.1982

Milk C 34.3840A 1.9029 Sodium

F 24.4662B 1.8073 (mg/L)

P 32.62A 1.9771

Milk C 81.5700 3.8217 Potassium

F 79.9409 3.6296 (mg/L)

p 78.5493 3.9708

1 . Models usmg proc ffilxed (SAS) mcluded panty, calvmg season, sample date

and the random effect of cow. A and B differ significantly among treatments within the same day a and b, c and d, e and f, g and h, i and j differ significantly among days within the same treatment

69

AIl cows experienced a decline in milk prote in (%) with advancing

lactation, furthermore a significant decrease was observed on d 7 for aIl

treatments (P<O.05), and d 14 and d 21 values were significantly lower than d

1 values (P<O.05).

1.4.2.2. Protein Yield (kg)

Statistical analysis of protein yield (kg) revealed no effect of treatment

(P>O.05), however there was an effect of sampling date (P<0.05) on prote in

yield. There were no difIerences (p>0.05) in protein yield among treatments.

Results for overall least square means and least square means for individual

days are shown in Table 7 and 22, respectively.

Milk protein yield levels increased numerically from dIto d 7

(P>0.05) and milk protein yield on d 7 and d 21 values were significantly

higher than d 1 levels in Flucort® treated cows (P<0.05).

In a study done by Shamay et al. (2000), a single intramuscular dose of

40 mg of dexamethasone was injected into dairy cows in order to get a better

insight into the effects of corticosteroids on milk secretion and composition.

Dexamethasone caused a 45% reduction in milk yield after 24 h, full recovery

took 5 d. The concentration of total prote in increased then decreased in direct

proportion to the changes in milk yield. In the present study, as milk yield

increased, protein (%) decreased, however protein yield increased throughout

the trial, particularly between d 1 and d 7 (P<0.05). Protein yield on d 7 and d

21 were significantly higher than d 1 levels. In the study by Shamay et al.

(2000), the secretion of total protein decreased as a result of the

dexamethasone injection. In the present study, treatment had no effect on

prote in (%) or protein yield, however there was an interaction between

treatment and sampling date for protein (%) which may explain the

discrepancies.

1.4.3. Fat

1.4.3.1. Fat (%)

Statistical analysis of fat (%) revealed no effect of treatment (p>0.05)

and no significant differences (p>0.05) were observed among treatments.

Results for overall least square means and least square means for individual

days are shown in Table 7 and 22, respectively.

70

1.4.3.2. Fat Yield (kg)

Statistical analysis of fat yield revealed no effect oftreatment (p>O.05),

however there was an effect of parity and sampling date (P<O.05). There were

no differences (p>O.05) in fat yield among treatments. Results for overallleast

square means and least square means for individual days are shown in Table 7

and 22, respectively.

AH cows experienced an increase in milk fat yield with advancing

lactation and Milk fat levels on d 14 and d 21 for aH treatments were

significantly higher than their respective d 1 milk fat levels (P<0.05)

Flucort® treated cows with parity >3 had significantly higher levels of

milk fat (kg) than the frrst parity cows and cows that calved in the winter had

higher milk fat (kg) levels than cows that calved in the fall season (P<0.05).

These differences between increasing milk protein yield and increasing parity

are due to the different feeds and their respective protein content which are

available in different seasons.

In the study done by Shamay et al. (2000), the concentration of fat also

increased then decreased in direct proportion to the changes in milk yield. In

the present study fat (%) fluctuated, however fat yield (kg) increased until d 14

and then decreased by d 21 which further substantiates this observation.

1.4.4. Lactose

1.4.4.1. Lactose (%)

Statistical analysis of lactose (%) revealed no effect of treatment

(P>0.05), however there was an effect of parity and sampling date (P<0.05) on

lactose %. There were no differences (P>O.05) in lactose (%) among

treatments. Results for overall least square means and least square means for

individual days are shown in Table 7 and 22, respectively.

AlI cows experienced an increase in the percentage of milk lactose and,

d 7, d 14, and d 21 values were significantly higher than d 1 values (P<0.05).

A more increase in lactose % was observed between d 0 and d 7 for all

treatment groups (P<0.05).

1.4.4.2. Lactose Yield (kg)

Statistical analysis of lactose yield (kg) revealed no effect of treatment

(P>0.05), however there was an effect of sampling date (P<0.05) on lactose

yield. There were no differences (p>0.05) in lactose yield among treatments.

71

Results for overall least square means and least square means for individual

days are shown in Table 7 and 22, respectively.

Lactose yield from Flucort® treated cows inereased with milk yield

throughout the trial. The increase in lactose yield was particularly more

between d 14 to d 21. As expected, lactose yields for d 7, d 14, and d 21 were

significantly higher than their respective d 1 values for Flueort® treated cows

(P<O.05). The simultaneous increase in lactose and milk yield is expected as it

plays a major role in milk synthesis. It is the major osmole in milk and the

process of synthesis of lactose is responsible for drawing water into the milk

as it is being formed in the mammary gland.

1.4.5. Milk potassium (mgIL)

Sodium and potassium in milk were analyzed to see if ion transport

aeross the mammary epithelial cell was affected by treatment. Results for

overall least square means and least square means for individual days are

shown in Table 7 and 22, respectively.

Statistical analysis of milk potassium levels revealed no effect of

treatment (p>0.05). There were no differenees (p>0.05) in potassium levels

among treatments on d 1.

In the study done by Shamay et al. (2000), the concentration of lactose

and monovalent ions (sodium and potassium) was unaffeeted. AIl other results

in the study done by Shamay et al. (2000) were similar to the present study

except that of milk potassium levels (P>0.05).

1.4.6. Milk sodium (mgIL)

Statistical analysis of milk sodium levels revealed an effect of

treatment (P<0.05). Flueort® treated cows had significantly lower milk sodium

levels in comparison to the control cows (P<0.05) on d 1. Results for overall

least square means and least square means for individual days are shown in

Table 7 and 22, respeetively.

In the study done by Shamay et al. (2000), the concentration of lactose

and monovalent ions (sodium and potassium) was unaffeeted. Furthermore,

milk Na was the only parameter that responded to the treatment effects. This

would imply that the administration of Flucort® prevented the release of Na

into the mammary epithelial eeU thereby affeeting the ion transport across the

mammary epithelial celI.

72

Opening of mammary tight junctions is associated with increase in

milk sodium and chlorine, due to their leakage from the blood into the lumen,

and decrease in potassium concentration, due to its leakage from milk to blood

(Stelwagen et al., 1995). Data by Shamay et al. (2000) on monovalent ion

concentration in the milk suggest that depressed milk yield following

dexamethasone treatment cows was not associated with the disruption of the

integrity of the mammary cell tight junctions. Furthermore, dexamethasone

decreased tight junction permeability in vitro and cortisol appears to be

associated with closure of tight junction in the late pregnant mammary gland

of goats (Nguyen et al., 1998). One of the most basic principles of milk

secretion is that the total osmotic pressure of milk remains approximately

constant and equal to the blood (Holt, 1993). The output of osmotic

components, of which 60% are contributed by lactose and 40% by the

monovalent ions, determines the volume of milk (Peaker, 1977). Thus, the

reduction in milk volume following dexamethasone treatment can he fully

explained by reduction in the secretion of osmotic components. As lactose is

the main osmotic component, it is suggested that reduction in lactose secretion

induced a coordinated reduction in the output of sodium, potassium and

chlorine. However, in this study lactose percentage and yield experienced no

significant losses.

1.4.7. Somatic cell count

Statistical analysis of sec revealed no effect of treatment (p>0.05),

however there was an effect of sampling date and an interaction of parity and

sampling date (P<0.05). No significant differences (p>0.05) were observed

among treatments. Results for overall least square means and least square

means for individual days are shown in Table 7 and 22, respectively.

Numericallyon dl, Flucort® treated cows had higher sec than control

cows. As sec decreased slightly, lactose (%) increased slightly and this is

supported by Harmon (1994) who observed that elevated sec are associated

with a decrease in lactose because of reduced synthetic activity of the

mammary tissue. An analysis of the frequency of mastitis, as denoted by sec > 283,000 celIs/mL (Guidry, 1985; Reneau, 1986), indicative of cows with

subclinical mastitis, revealed greater mastitis in the flumethasone-treated cows

73

on d 1 and d 14 as shown in Table 23. However, due to limited cow numbers,

a concrete conclusion cannot be made and further studies are warranted.

Table 23. Frequency of mastitis, as denoted by sec > 283,000 cells/mL (Guidry, 1985; Reneau, 1986 , percentages of cows by treatment 2roup Trt Dl D7 D14 D21 C 14 22 0 20 F 30 20 13 10 P 78 11 20 20

2. PredefP 2X

2.1. Energy Status

Blood energy metabolites included glucose, BHB, NEF A and insulin.

AlI the blood energy metabolites were evaluated on d -10, d -5, d 0, d 1, d 7, d

14, d 21 and d 28 relative to calving. Among the ketone bodies (BHB,

acetoacetate and acetone), only BHB was evaluated because most other

cowside tests lack sensitivity as compared to serum BHB. Testing for BHB

remains the gold standard for studying ketosis (Duffield, 1997).

2.1.1. Glucose

Statistical analysis of serum glucose levels revealed no effect of

treatment (p>0.05), however there was an effect of sampling date (P<0.05).

Furthermore, there were no significant differences (P>0.05) in serum glucose

levels among treatments. Results for overall least square means and least

square means for individual days are shown in Tables 7 and 8, respectively.

The only day that Prede~ 2X treated cows had better energy status

than control cows as indicated by numerically higher serum glucose (p>0.05)

was on d 1. However, serum glucose values for Predef!> 2X treated cows

experienced a significant decrease by d 7 (P<0.05). Furthermore, glucose

concentrations on d 14, d 21 and d 28 were significantly lower than d -10, d-5

and d 0 values (P<0.05) and a similar trend was observed in control cows.

The numerical increase in glucose on d 1 has also been reported by

Convey et al (1970), in which lactating Holstein cows receiving 5 or 10 mg of

9a-fluoroprednisolone acetate (also called isoflupredone acetate or Predef!>

2X) immediately following moming milking experienced a linear increase

(P>0.05) in plasma glucose levels with time. The earliest significant increase

occurred 3 h after treatment and this agrees with the findings of Neff et al.

74

(1960), who also observed increased blood glucose 3 to 6 hr following

intramuscular administration of 10 mg of 9a-fluoroprednisolone acetate and

suggests equal availability of this steroid by intramuscular or intravenous

route. Philip et al. (1991), observed a significant increase in plasma glucose

concentration which persisted until d 5 in cows given a single intramuscular

injection of dexamethasone-21-isonicotinate of (2mg/l OOmg bodyweight).

When a glucocorticoid is administered, the concentration of glucose in

the blood should increase through the synthesis of glucose from amino acids

(gluconeogenesis), a decrease in the synthesis of proteins from amino acids,

and an altered lipid metabolism, thereby satisfying the systemic demand for

glucose and helping to prevent the metabolism of fats and production of

ketone bodies. AIso, peripheral utilization of glucose is reduced and liver

storage of glycogen is increased.

Despite other studies using the same dosage of Prede~ 2X and

observing significant increases in blood glucose, a larger dose than 10 mg/mL

of Prede~ 2X is probably needed to induce more significant effects on glucose

concentrations. Furthermore, the significant inereases that were observed in

other studies were found only within 24 h of administration. The effects on

blood glucose levels would only be temporary and would therefore have to be

measured on an hourly basis in future studies.

2.1.2. Insulin

Statistical analysis of insulin levels revealed no effect of treatment

(P>O.05), however there was an effeet of sampling date, interaction of

treatment and sampling date, and an interaction of parity and sampling date

(P<0.05). Results for overall least square means and least square means for

individual days are shown in Tables 7 and 8, respectively.

The only significant difference observed in serum insulin levels was on

d 0 where Prede~ 2X treated cows experienced significantly lower (P<0.05)

levels than control cows indicating a worse energy status. In contrast, Prede~

2X treated cows had numerically (P>O.05) higher insulin levels from dIto d

14, indieating a better energy status than control cows.

From dito d 7, insulin levels decreased significantly (P<O.05) to

almost 50% but increased towards the end of the trial (p>O.05). Post-calving

75

insulin levels however remained significantly lower than pre-calving levels

(P<0.05).

The higher insulin values experienced on d 1, d 7 and d 14 should have

stimulated the uptake, utilization and storage of glucose. In addition, insulin

should have facilitated the entry of glucose into muscle, adipose tissue and

other tissues. If the chain of events listed above had occurred upon Prede~ 2X

administration, the mobilization of fat would have been avoided.

2.1.3. Non-esterified fatty acids

Statistical analysis of serum NEF A levels revealed no effect of

treatment (p>0.05), however there was an effect of parity and sampling date

(P<0.05). Results for overall least square means and least square means for

individual days are shown in Tables 7 and 8, respectively.

There were no significant differences (p>0.05) in serum NEFA levels

between Prede~ 2X treated cows and control cows (p>0.05). Days at which

Prede~ 2X treated cows experienced numerically lower NEF A values and a

better energy status than control cows were d 1 and d 14 (P>0.05).

Furthermore, from d 0 to d 1, NEF A levels for Prede~ 2X treated cows

decreased numerically, whereas NEF A levels for control cows increased

significantly. NEF A values for Prede~ 2X treated cows continued to decrease

until d 28.

The better energy status experienced by Prede~ 2X treated cows

indicate less fat mobilization in these cows (Grummer, 1993) and therefore

less NEFA transported to the liver. Plasma NEFA concentration and

triglyceride concentration in the liver (Bertics et al., 1992) are positively

correlated. As fatty liver and ketogenesis are closely linked (Dann et al.,

1999), a reduction in plasma NEF A concentration should result in a decreased

risk of the cow developing fatty liver and ketosis. Our current findings

supports the hypothesis that, Prede~ 2X increases gluconeogenic activities

and therefore inhibits fat mobilization from the adipose tissue. FUrll and Jackel

(2005), investigated the effects of 0.02 mglkg body weight of dexamethasone

(in the form of Voren suspension) on d 7 and d 11 post-calving when high

levels of lipolysis normally occur and observed an increase in glucose and

insulin concentrations and a concomitant decrease free fatty acid

concentrations. Contrary to this fmdings, Seifi et al (2006) observed

76

significantly higher (P<0.02) NEFA concentrations 1 week after intramuscular

injection with 20 mg of isoflupredone acetate (Prede~ 2X) compared to the

control cows. In the present study, by d 21 Prede~ 2X treated cows had

numerically higher serum NEF A (P>0.05) levels and by d 28 the cows had a

worse energy status as indicated by higher serum NEF A (P>0.05)

concentrations.

An analysis of the frequency ofcows with high NEFA (>0.7 mmollL)

values indicative of a state of negative energy balance revealed that less

Prede~ 2X treatment cows had higher NEF A levels than the control cows in

both, primiparous and multiparous cows as shown in Tables 9,10 and 11. In

this respect, a treatment with Prede~ 2X may provide potential benefits in the

reduction of fat mobilization. However, due to limited numbers of cows used

in tbis study, a concrete conclusion cannot he made and further experiments

are warranted.

An analysis of the frequency of body condition score (BeS) within

treatment and within day, demonstrate that approximately 73% of the animaIs

were classified as being in fair body condition prior to calving, and 63% in

thin body condition post-calving as shown in Table 12. This indicates fat

mobilization during the transition from late gestation to early lactation.

2.1.4. p-hydroxybutyrate

Statistical analysis of serum BHB levels revealed no effect of treatment

(P>0.05), however there was an effect of sampling date (P<0.05). Results for

overall least square means and least square means for individuai days are

shown in Tables 7 and 8, respectively.

AlI cows experienced a graduaI increase in BHB levels throughout the

trial. From d 0 to d 28, Prede~ 2X treated cows had higher BHB values than

control cows but the only significant difference between the two groups was at

d 21 (P<0.05). This indicates that, Prede~ 2X cows were in a worse energy

status and that more fat was mobilized, resulting in an increase of serum

NEF A concentrations, which underwent more metabolism in the liver

resulting in more acetyl-CoA and its divergence into ketone body production

causing the eventual higher ketone concentrations post-calving in cows and

causing an increased risk of ketosis. This also may suggest that liver damage

may have occurred and that Prede~ 2X may have a lipolytic effect in vivo and

77

may aggravate or accelerate fatty degeneration of the liver. Seifi et al (2006)

also observed significantly higher (P<0.02) BHB concentrations 1 week after

intramuscular injection of cows with 20 mg of isoflupredone acetate (Prede~

2X) compared to control cows. In contrast, a study done by Philip et al.

(1991), cows given a single intramuscular injection of dexamethasone-21-

isonicotinate of (2mg/100mg bodyweight) increased plasma BHB

concentration insignificantly (P<0.05) on d 1 after treatment then showed a

graduaI and significant (p>0.05) decline until the end of the study.

An analysis of the frequency of cows with high BHB values (BHB >

1400 mmollL) (Duffield~ 2000) indicative of subclinical ketosis revealed that

more Prede~ 2X treated cows had higher BHB levels (> 1400 f.llllollL) than

control cows in both primiparous and multiparous cows as shown in Tables

13, 14 and 15. In tbis respect, treatment with Predef!> 2X may contribute to

problems of ketosis. However, due to limited numbers of cows used in the

current study, a concrete conclusion cannot he made and furtber experiments

are warranted.

To be sure that dry matter intake (DMI) was not affected by treatment,

and that DMI rumen fin, DMI was recorded on a daily basis. Tables 4 and 5

specify the diet composition (% of DM) of close-up rations and fresh cow

ration. Statistical analysis of DMI revealed no effect of treatment (p>0.05),

and no differences in DMI were observed among treatments (p>0.05) as,

shown in Table 16.

Seifi et al. (2006) observed that a treatment of 20 mg ofPrede~ 2X did

not resolve subclinical ketosis (SCK; ::: 1400 f.llllollL) among 190 cows that

were ketotic before treatment relative to control animaIs. Among 972 cows

that were not ketotic at enrollment, cows that received Prede~ 2X were 1.6

times more likely to develop subclinical ketosis 1 week after treatment.

Prede~ 2X is approved and marketed for the treatment of clinical ketosis in

dairy cows and in this regard, the findings of the current experiment are not

consistent with the approval.

In this study, it appeared that after a Prede~ 2X treatment, less cows

had higher NEFA (>0.7 mmollL) and more animaIs had higher BHB (>1400,

f.llllollL). High levels of BHB would he the result of fat mobilization and a

high concentration of NEF As released into bloodstream causing high NEF A

78

levels in the liver, and the eventual production of ketone bodies. Therefore,

this trend is difficult to explain and may be due to small cow numbers. A

larger sample size is needed to make a concrete conclusion.

2.2. Serum Mineral Status

Blood mineraI metabolites included calcium, phosphorus, potassium,

sodium, chloride and magnesium. AH the blood mineraI metabolites were

evaluated on d -10, d -5, d 0, d 1, d 7, d 14, d 21 and d 28 relative to calving

and due to the potential upset of the electrolyte balance, effects of Flucort®

and Predeflll2X were evaluated on serum mineraI parameters.

2.2.1. Calcium

Statistical analysis of serum calcium levels revealed no effect of

treatment (p>0.05), however there was an effect of sampling date, interaction

of treatment and sampling date, interaction of parity and sampling date, and

interaction of calving season and sampling date (P<0.05). Results for overall

least square means and least square means for individual days are shown in

Tables 7 and 17, respectively.

The only significant difference observed in serum calcium levels was

on d 1 where, Predef> 2X treated cows experienced signiticantly lower

calcium (P<0.05) levels than Flucort® treated cows. In a study done by Schafer

et al. (1983), 300 mg of prednisolone acetate injected intramuscularly caused a

decrease in blood plasma calcium sharply within 8 h. Seifi et al (2006)

observed significantly lower (p<O.Ol) calcium concentrations 1 week

following intramuscular injection with 20 mg of isoflupredone acetate

(prede~ 2X). AlI these observations contirm what was observed in the present

study. Furthermore, calcium concentrations of PredefJ 2X treated cows

remained lower than control cows until d 28 in the present study. The

numerically higher serum calcium concentrations observed on d 1 in Predef>

2X treated cows may provide a beneticial effect as most fresh-cow health

problems such as displaced abomasums, metritis, and clinical and subclinical

milk fever are part of one disease complex greatly dependent on the cow's

calcium status (Acre, 1998).

2.2.2. Phosphorus

Statistical analysis of serum phosphorus levels revealed no effect of

treatment (p>0.05), however there was an effect of parity, sampling date,

79

interaction of parity and sampling date, and interaction of calving season and

sampling date (P<0.05). No significant differences were observed among

treatments (p>0.05). Results for overall least square means and least square

means for individual days are shown in Tables 7 and 17, respectively.

Phosphorus levels in this experiment were relatively normal and not

affected by treatment and probably were the response to an adequate use of

anionic salts. No significant differences were observed between pre-calving (d

-10, d -5) and post-calving (d 14, d 21, d 28) levels (p>0.05). Sorne approved

glucocorticoid drugs have a suppressive effect on serum phosphorus levels. In

clinical safety studies on Prede~ 2X done for product registration, serum

phosphorus levels dropped to 60 to 70% of pretreatment levels and then rose

to 80% of those levels by 24 h. When treatment was terminated, phosphorus

levels rebounded to 40% above pretreatment levels (Dairy, Utah State

University Extension, July 1996). In the present study, the opposite was

observed and after d 1, phosphorus levels in Prede~ 2X treated cows

remained higher than control cows (not statistically significant).

2.2.3. Potassium

Statistical analysis of serum potassium levels revealed no effect of

treatment (P>O.05), however there was an effect of calving season (P<0.05).

There was a difference (P<0.05) in serum potassium levels on d 1 where,

Prede~ 2X treated cows had significantly lower potassium levels than

Flucort® treated cows and control cows (P<0.05). Results for overall least

square means and least square means for individual days are shown in Tables

7 and 17, respectively.

Sorne approved glucocorticoid drugs have decreasing effects on

potassium. Repeated or large, off-label doses of isoflupredone acetate can

upset electrolyte balance in the cow causing very low blood potassium levels

and down cows (Goff, 2001). Furthermore, in clinical safety studies on

Predet4!> 2X done for product registration. serum potassium values changed

very little with a single 10 mg injection, but dropped to 70 to 80% of

pretreatment levels with a second or successive injections. In the clinical

safety studies. doses of 100 mg of Predef!> 2X alone did not exhibit toxic signs

of hypokalemia (Dairy, Utah State University Extension, July 1996). In the

present study, potassium levels did change very little with the exception of d 1.

80

This observation was also observed by Schafer et al. (1983) where a decrease

in blood plasma potassium sharply occurred within 8 h following an

intramuscular injection of 300 mg of prednisolone acetate. In the present study

after d 1, potassium concentrations in the Predefl 2X treated cows were not

significantly different from those of the control cows. This is also confrrmed

by Seifi et al (2006), who observed that treatment had no influence on

potassium concentrations 1 week following an intramuscular injection of 20

mg of isoflupredone acetate (Predef!> 2X).

2.2.4. Sodium

Statistical analysis of serum sodium levels revealed no effect of

treatment (p>0.05), however there was an effect of sampling date (P<0.05) on

sodium levels. In comparison to control cows, sodium levels in this

experiment were relatively normal and not affected by treatment and probably

were the response to an adequate use of anionic salts. Serum sodium post~

calving values (d 21 and d 28) were significantly lower than pre-calving

values in Predefl2X treated cows (P<0.05), which was comparable to control

cows. Results for overall least square means and least square means for

individual days are shown in Tables 7 and 18, respectively.

2.2.5. Chloride

Statistical analysis of chloride levels revealed no effect of treatment

(P>0.05), however there was an effect of sampling date and an interaction of

calving season and sampling date (P<0.05) on serum chloride levels. There

were no differences (P>O.05) in serum chloride levels among treatments.

Results for overall least square means and least square means for individual

days are shown in Tables 7 and 18, respectively.

In comparison to control cows, chloride levels in this experiment were

relatively normal and not affected by treatment and probably were the

response to an adequate use of anionic salts. This is also confirmed by Seifi et

al (2006), who observed that treatment had no influence on sodium and

chloride concentrations 1 week following an intramuscular injection of 20 mg

of isoflupredone acetate (prede:t«> 2X). Prede~ 2X treated cows experienced a

graduaI decline in chloride levels and d 28 chloride values were significantly

lower than d -10 values (P<0.05). This trend was observed in aIl cows.

81

2.2.6. Magnesium

Statistical analysis of magnesium levels revealed no effect of treatment

(p>O.05), however there was an effect of sampling date and an interaction of

treatment and sampling date (P<O.05) on serum magne sium levels. There were

no differences (P>O.05) in serum magne sium levels among treatments. Results

for overall least square means and least square means for individual days are

shown in Tables 7 and 18, respectively.

Predefb 2X treated cows experienced slight fluctuations in magnesium

levels which was comparable to control cows. In a study done by Schafer et al.

(1983), 300 mg of prednisolone acetate injected intramuscularly caused a

decrease in blood plasma magnesium sharply within 8 h. This result was not

observed in the present study where d 0 and d 1 magnesium concentrations

values was much higher in PredefID 2X treated cows than control cows,

although not statistically different. There was a significant decrease (P<0.05)

in serum magnesium levels until d 7, which then significantly increased in

Predefb 2X treated cows and a similar trend was also observed in control

cows. Furthermore Goff and Horst (1998) reported a similar pattern of plasma

magnesium.

2.3. Immune Function

Cows were challenged to evaluate the effect of Flucort® and PredefID

2X on the immune system. The cows were immunized with chicken

ovalbumin on the day of calving and given a boost 2 weeks post-calving.

Immune function parameters included antibody parameters which were

evaluated on d 0, d 7, d 14, d 21 and d 28 relative to calving and analyzed by

an ELISA assay measuring the concentration of chicken ovalbumin specifie

IgG (antibody) in serum; and lymphocyte proliferation which was evaluated

on d 0 and d 7 relative to calving and analyzed by an in vitro assay

determining if bovine ConA-induced lymphocyte proliferation is altered in

response to treatments.

2.3.1. Antibody production

Statistical analysis of antibody levels revealed no effect of treatment

(p>0.05), however there was an effect of sampling date (P<0.05) antibody

levels. No statistically significant differences (P>0.05) were observed between

PredefID 2X treated cows and control cows for antibody production values.

82

Results for overall least square means and least square means for individual

days are shown in Tables 7 and 19, respectively.

2.3.2. Lymphocyte proliferation

Statistical analysis of lymphocyte proliferation levels revealed no

effect oftreatment (P>0.05), however there was an effect ofsampling date and

an interaction of calving season and sampling date (P<0.05). In addition, there

were no statistically significant differences (P>0.05) observed for ConA­

induced lymphocyte proliferation values for d 0 or d 7. Results for overall

least square means and least square means for individual days are shown in

Tables 7 and 20, respectively. However this may be due to the large variation

in lymphocyte proliferation among animais and among replicates. Nagahata et

al. (1992) examined B lymphocyte populations to evaluate host defense in

dairy cows during the transition period and found no significant changes in the

number of B lymphocytes of cows from 2 weeks before until 2 weeks after

calving. Thanasak et al. (2004) demonstrated that a single injection with

dexamethasone did not affect Con A-induced lymphocyte proliferation. In

previous studies, however, single injections with high doses or repeated

treatments resulted in decreased lymphocyte proliferation (Pruett et al., 1987).

Perhaps high dosages or repeated treatments of Prede~ 2X would cause

decreased lymphocyte proliferation, but the single therapeutic dosage does not

cause decreased lymphocyte proliferation. Thanasak et al. (2004) suggested

one possible mechanism underlying the reduced lymphocyte proliferative

responses is the down modulation of CD 18 expression which has been shown

to act as a co-stimulatory molecule in the process of lymphocyte proliferation.

Studies by Thanasak et al. (2004) found no detrimental effects of

glucocorticoid injection in the post-calving period on measures on immune

function. In contrast, FürU and Jackel (2005) found that two doses of Voren

resulted in typical glucocorticoid-related changes in the differential leukocyte

count.

A depression in lymphocyte proliferation bas been shown to be related

to increased blood glucose concentrations (Franklin et al., 1991; Thanasak et

aL, 2004). In the study by Thanasak et al. (2004), although increases in blood

glucose and insulin concentration in the dexamethasone treated group

indicated that hypoglycemia was overcome, they did not affect lymphocyte

83

proliferation. In this study neither lymphocyte proliferation, blood glucose nor

blood insulin concentrations were affected by Prede~ 2X administration.

Perhaps the therapeutic dosage of 10 mg of Prede~ 2X is not an effective

dosage to induce a significant depression in lymphocyte proliferation, and a

significant increase in blood glucose and insulin concentrations allowing cows

to overcome hypoglycemia.

2.4. Milk Status

Milk composition parameters included fat (% and yield), protein (%

and yield), lactose (% and yield), sec, sodium and potassium levels in milk.

Milk composition parameters were evaluated on d 1, d 7, d 14 and d 21

relative to calving, with the exception of sodium and potassium which were

evaluated on d 1 by digestion and atomic absorption.

2.4.1. Milk Yield (kg)

Statistical analysis of milk yield revealed no effeet of treatment

(P>0.05), however there was an etfect ofparity, calving season, sampling date

and an interaction of parity and sample dating (P<0.05) on milk yield. There

were no ditferences (p>0.05) in milk yield among treatments. Results for

overall least square means and least square means for weekly average milk

yield are shown in Tables 7 and 21, respectively.

Milk yield for Predef> 2X treated cows increased throughout the trial

but remained slightly lower than control cows although not significantly

ditferent from each other. This is supported by Wagner and Apley (2003) who

reported that isoflupredone acetate (20 mg, IV) did not atfect milk production

in mastitic or healthy cows. Wagner and Apley (2003) concluded that drug

treatment effects would have to be very strong or study power (cow numbers)

would have to be quite high to detect an effeet of drug treatment on milk

production. FürU and Jackel (2005) noted only very slight changes in daily

milkyield.

2.4.2. Protein

2.4.2.1. Protein (%)

Statistical analysis of protein % revealed no etfect of treatment

(P>0.05), however there was an effeet of sampling date, interaction of

treatment and sampling date, and an interaction of calving season and

sampling date (P<0.05) on prote in %. Results for overall least square means

84

and least square means for individual days are shown in Tables 7 and 22,

respectively.

Ail cows experienced a decline in milk prote in percentage and a

significant decrease was observed on d 7 for ail treatments (P<0.05). Day 14

and d 21 values were significantly lower than d 1 values (P<0.05). Prede~ 2X

treated cows had significantly higher milk protein % than Flucort® and control

cows on d 1 (P<0.05).

Predef> 2X treated cows in parity >3 had significantly higher milk

protein % levels than cows in parity l, in addition cows that calved in the

winter season had higher milk protein % levels than cows that calved in the

summer or faH season (P<0.05). These differences are due to increasing milk

protein yields with increasing parities and the different feeds available at each

season.

2.4.2.2. Protein Yield (kg)

Statistical analysis of protein yield (kg) revealed no effect of treatment

(P>0.05), however there was an effect of sampling date (P<0.05) on prote in

yield. There were no differences (p>0.05) in protein yield among treatments.

Results for overall least square means and least square means for individual

days are shown in Tables 7 and 22, respectively.

Milk protein yield levels increased numerically from dito d 7 for

Prede~ 2X treated cows (p>0.05).

In a study done by Shamay et al. (2000), a single intramuscular dose of

40 mg of dexamethasone was injected into dairy cows in order to get a better

insight into the effects of corticosteroids on milk secretion and composition.

Dexamethasone caused a 45% reduction in milk yield after 24 h and full

recovery took 5 d. The concentration of total protein increased then decreased

in direct proportion to the changes in milk yield. In the present study, as milk

yield increased, protein % decreased. On d l, protein % of Prede:r 2X treated

cows was significantly higher than control cows and d 7, d 14, and d 21,

furthermore Predet«> 2X treated cows experienced the biggest decrease in

protein (%) by d 7 The higher protein percentage observed in Prede:r 2X

treated cows May provide a beneficial effect considering that during the fIfSt

few weeks of lactation the cow is typically in negative energy and prote in

balance and these May both impact and limit milk protein synthesis (Green et

85

al., 1999). However, the higher protein percentage may also be due to the high

protein content of colostrum and the high sec experienced on d 1. Prote in

(%) on d 7, d 14 and d 21 for Prede~ 2X treated cows were not statistically

different from control cows. In the study by Shamay et al. (2000), the

secretion of total protein decreased as a result of treatment. In the present

study, protein yield) changed slightly throughout the trial and the difference

from control cows and the difference between days were not statistically

different. Treatment had no effect on protein % or protein yield, however there

was an interaction effect between treatment and sampling date for prote in %

which may be an explanation.

2.4.3. Fat

2.4.3.1. Fat (Ofo)

Statistical analysis of fat % revealed no effect of treatment (p>0.05)

and no significant (p>0.05) differences were observed among treatments.

Results for overall least square means and least square means for individual

days are shown in Tables 7 and 22, respectively.

2.4.3.2. Fat Yield (kg)

Statistical analysis of fat yield (kg) revealed no effect of treatment

(P>0.05), however there was an effect of parity and sampling date (P<0.05) on

fat yield. There were no differences (p>0.05) in fat (% and yield) among

treatments. Results for overall least square means and least square means for

individual days are shown in Tables 7 and 22, respectively.

AIl cows experienced an increase in milk fat yield. Prede~ 2X treated

cows experienced a significant increase on d 7 (P<0.05). Milk fat levels on d

14 and d 21 for aIl treatments were significantly higher than their respective d

1 milk fat levels (P<0.05)

In the present study, fat % of Prede~ 2X treated cows tluctuated and

was not statistically different from control cows. On d l, fat % was

significantly higher than d 7, d 14, and d 21. However, fat yield increased

throughout the trial but not statistically different from control cows. On dl, fat

yield was significantly lower than d 7, d 14, and d 21. Therefore, in the first

week post-calving (d 7), Predefi> 2X treated cows experienced a significant

increase in milk fat yield. In the study done by Shamay et al. (2000), the

concentration of fat increased then decreased in direct proportion to the

86

changes in milk yield. In the present study fat % fluctuated but not in

proportion ta changes in milk yield.

2.4.4. Lactose

2.4.4.1. Lactose (%)

Statistical analysis of lactose % revealed no effect of treatment

(P>0.05), however there was an effect of parity and sampling date (P<0.05).

There was no difference (P>0.05) in lactose % among treatments. Results for

overall least square means and least square means for individual days are

shown in Tables 7 and 22, respectively.

AlI cows experienced an increase in percentage of milk lactose %, and

d 7, d 14, and d 21 values were significantly higher than d 1 values (P<0.05).

A significant increase was observed on d 7 for aIl treatments (P<0.05). First

parity Predefl 2X treated cows experienced a significantly higher lactose (%)

than cows in parity >3 and, in addition cows that calved in the summer or fall

season had significantly higher lactose (%) than cows that calved in the winter

season (P<O.05). These differences may be due ta increasing lactose yield with

increasing parities, and the different feeds available in each season.

2.4.4.2. Lactose Yield (kg)

Statistical analysis of lactose yield revealed no effect of treatment

(p>0.05), however there was an effect of sampling date (P<0.05) on lactose

yield. There were no differences (P>0.05) in lactose yield among treatments.

Results for overall least square means and least square means for individual

days are shawn in Tables 7 and 22, respectively.

Lactose (%) and lactose yield from Predefl2X treated cows increased

with milk yield throughout the trial but was not statistically different from

control cows. The increase was particularly significant for lactose yield and

lactose % between d 7 ta d 21. As expected, lactose yield for d 7, d 14, and d

21 were significantly higher than their respective d 1 values for Predefl 2X

treated cows (P<O.05). The simultaneous increase in lactose and milk yield is

expected as it plays a major raIe in milk synthesis. It is the major osmole in

milk and the process of synthesis of lactose is responsible for drawing water

into the milk as it is being formed in the mammary gland. In a study done by

Shamay et al. (2000), the concentration of lactose and monovalent ions

(sodium and potassium) was unaffected. However, in a study done by Schafer

87

et al. (1983), 300 mg of prednisolone acetate injected intramuscularly caused

an increase in Nalkg milk solids, but no significant changes in Klkg milk

solids within 8 h. In the present study, milk sodium and potassium levels in

Prede~ 2X treated cows were unaffected by treatment (P<0.05).

2.4.5. Milk potassium (mgIL)

Sodium and potassium in milk were analyzed to see if ion transport

across the mammary epithelial cell was affected by treatment. Results for

overall least square means and least square means for individual days are

shown in Tables 7 and 22, respectively.

Statistical analysis of milk potassium levels revealed no effect of

treatment (p>0.05). There were no differences (p>O.05) in potassium levels

among treatments on d 1.

2.4.6. Milk sodium (mgIL)

Statistical analysis of milk sodium levels revealed an effect of

treatment (P<0.05). PredefP 2X treated cows had significantly lower milk

sodium levels in comparison to Flucort® treated cows on d 1 (P<O.05). This

would imply that the administration ofPrede~ 2X prevented the release of Na

into the mammary epithelial cell thereby affecting the ion transport across the

mammary epithelial cell. Results for overall least square means and least

square means for individual days are shown in Tables 7 and 22, respectively.

2.4.7. Somatic cell count

Statistical analysis of sec revealed no effect of treatment (P>O.05),

however there was an efIect of sampling date and an interaction of parity and

sampling date (P<0.05) on sec. A significant difference (p>0.05) in sec was

observed on d 1 where Prede~ 2X treated cows had significantly higher

(P<0.05) sec than control cows. Results for overallleast square means and

least square means for individual days are shown in Tables 7 and 22,

respectively.

Cows treated with Predet4l> 2X experienced fluctuations in milk SCC

throughout the trial. Prede~ 2X treated cows had sec significantly higher

(P<O.05) than control cows, and numerically higher (P>O.05) than Flucort®

treated cows on d 1. Furthermore, sec on d 1 was significantly higher than d

7, d 14, and d 21. Prede~ 2X treated cows experienced a significant decrease

in milk sec by d 7, a numerical increase by d 14 and a numerical decrease by

88

the d 21. As sec decreased, lactose % increased and this is supported by

Hannon (1994) who found that elevated sec are associated with a decrease in

lactose because of reduced synthetic activity of the mammary tissue. An

analysis of the frequency of mastitis, as denoted by sec> 283,000 cells/ml

(Guidry, 1985; Reneau, 1986) indicative of cows with subclinical mastitis

revealed greater mastitis in the Prede:f 2X treated cows on d 1 and d 14, as

shown in Table 23. However, due to limited cow numbers used in the current

study, a concrete conclusion cannot be made and further studies are warranted.

3. Flucort® versus Prede~ 2X

3.1. Energy Status

There were no statistically significant differences between Prede:f 2X

and Flucort® treated cows in tenns of energy status, Le. values for glucose,

insu lin, NEF A and BHB between Prede:f 2X and Flucort® treated cows were

not significantly different. However, serum glucose values of Prede:f 2X were

numerically higher than Flucort® treated cows on aIl days throughout the trial.

Serum insulin values for Flucort® treated cows were numerically higher than

Prede:f 2X treated cows on d 1 and d 21, whereas Prede:f 2X treated cows

had numerically higher insulin values on d 1, d 14, and d 28. Serum NEFA

values for Prede:f 2X treated cows were numerically lower than Flucort®

treated cows from d 0 to d 14. Serum BHB values for Prede:f 2X treated cows

were numerically lower than Flucort® treated cows.

3.2. Serum Mineral Status

There were no statistically significant differences between Prede:f 2X

and Flucort® treated cows in tenns of serum minerai status. Phosphorus,

sodium, chloride, and magnesium values were not statistically different with

the exception of calcium and potassium on d 1.

Serum calcium values for Flucort® treated cows were numerically

higher than Prede:f 2X treated cows on d 0, dl, d 14, d 21 and d 28. Flucort®

treated cows had significantly higher calcium values than Predet«' 2X treated

cows on d 1 (P<0.05). Serum phosphorus values for Prede:f 2X treated cows

were numerically higher than Flucort® treated cows from dIto d 28. Serum

sodium values for Prede:f 2X treated cows were numerically higher than

Flucort® treated cows on d 1, d 21, and d 28. Serum sodium values for

Flucort® treated cows were numerically higher than Prede~ 2X treated cows

89

on d 0, d 7, and d 14. Serum potassium values for Flucort® treated cows were

always numerically higher than Predet«' 2X treated cows except on d 7, d 21

and d 28. PredefID 2X treated cows had statistically lower potassium values

than Flucort® treated cows on d 1. This decrease in serum potassium May be

due to the hypokalemic effect of Predet«' 2X. Serum chloride values for

Prede~ 2X treated cows were numerically higher than Flucort® treated cows

from d 1, d 21 and d 28. Serum chloride values for Flucort®treated cows were

numerically higher than Prede~ 2X treated cows from d 0, d 7 and d 14.

Serum Magnesium values for Prede~ 2X treated cows were numerically

higher than Flucort® treated cows from d 0 to d 28.

3.3. Immune Function

There were no statistical differences between Prede~ 2X and Flucort®

treated cows for parameters of immune function. Numerically, Flucort® treated

cows experienced a higher ConA~induced lymphocyte proliferation on d 0

compared to Prede~ 2X treated cows, however numerically Prede~ 2X

treated cows experienced a higher ConA·induced lymphocyte proliferation on

d 7 compared to Flucort® treated cows. Flucort® treated cows experienced the

largest decrease in ConA .. induced lymphocyte proliferation.

Numerically, Predet«> 2X treated cows experienced a higher antibody

production from d 0 to d 28 compared to Flucort® treated cows.

3.4. Milk Status

There were no statistical differences between Predef' 2X and Flucort®

treated cows. Milk fat (% and yield), milk protein yield (kg), milk lactose (%

and yield), milk somatic cell count and milk yield, with the exception of milk

protein (%) on d 1.

Statistically, milk prote in % for Prede~ 2X treated cows was higher

than Flucort® treated cows on d 1. Numerically, milk protein % for Prede~ 2X

treated cows was higher than Flucort® treated cows on d 14. Numerically, milk

protein % for Flucort® treated cows was higher than Predet«' 2X treated cows

on d 7 and d 21. The higher prote in percentage observed in Predet«' 2X treated

cows May provide a beneficial effect considering that during the first few

weeks of lactation the cow is typically in negative energy and prote in balance

and these May both impact and limit milk protein synthesis (Green et al.,

90

1999). Numerically, milk protein yield for Prede-r 2X was higher than

Flucort® treated cows on d 1, d 7 and d 14.

Numerically, milk fat % for Flucort® treated cows was higher than

Predef\' 2X treated cows on d 1 and d 14. Numerically, milk fat yield (kg) for

Flucort® treated cows was higher than Predef!> 2X treated cows on d 1, d 14

and d 21. Numerically, milk lactose (% and yield) for Flucort® treated cows

was higher than Prede-r 2X treated cows on d 1, d 14 and d 21. Numerically,

milk sec for Flucort® treated cows was lower than Predefll 2X treated cows

on dl, d 14 and d 21. .

Numerically, average milk yield for Flucort® treated cows was higher

than Predefll 2X treated cows for the fust week and fourth week post-calving,

whereas average milk yield for Predefll 2X treated cows was higher than

Flucort® treated cows for the second and third week post-calving.

Numerically, Prede~ 2X treated cows experienced higher milk sodium

and milk potassium values than Flucort® treated cows on d 1. In the present

study, there was no statistically significant difference between treatments for

milk potassium levels (P>O.05). However, Prede-r 2X treated cows had

significantly lower (P<O.05) milk sodium levels than Flucort® treated cows

91

VI. Conclusion

In conclusion, the following new discoveries were found in the present

study: (1) There were no effect oftreatments on any of the parameters except

for milk sodium on d 1. (2) There were very few statistical differences

between treatments in tenns of energy parameters. Predef!' 2X treated cows

had significantly lower insulin values on d 0 and significantly higher BHB

values on d 21. Flucort® treated cows had significantly lower NEF A values on

d l, significantly greater insulin values on d l, significantly greater BHB

values on d 21 and d 28. (3) There were very few statistical differences

between treatments in tenns of serum mineraI parameters. Predef!' 2X treated

cows had significantly higher calcium values on d 1 and significantly lower

potassium values than control and Flucort® treated cows on d 1. Flucort®

treated cows had significantly lower calcium and phosphorus levels on d 1. (4)

The administration of Flucort® or Predefl> 2X on d 0 did not cause

immunosuppression. (5) There were very few statistical differences between

treatments in tenns of milk parameters. Predef!' 2X treated cows had

significantly higher milk protein % than both control and Flucort® treated

cows. Predef!' 2X treated cows had significantly higher milk sec than control

cows.

Based on the statistically significant data in this study, the use of

glucocorticoids Flucort® and Predef!' 2X in a single intramuscular injection on

d 1 for the treatment of ketosis is not warranted. In this study there is not

enough evidence to suggest such a treatment. Glucocorticoids, although

approved for the therapy of ketosis, may not be the best treatment option for

post-calving dairy cattle. From this study, dairy producers and veterinarians

may want to rethink the way that transition dairy cattle are treated in order to

prevent such metabolic diseases as ketosis and negative energy balance.

Future experiments for the treatment of ketosis could include the

combination therapy of Flucort® and intravenous glucose; FJucort® and

endogenous dextrose; Flucort® and exogenous insulin; Predef!' 2X and

intravenous glucose; Predef!' 2X and endogenous dextrose; Predef!' 2X and

exogenous insulin; larger dosage than 10mg of Flucort® or Predef!' 2X; second

or successive doses Flucort® or Predef!' 2X; administration of treatments on

92

other days in the fresh cow period rather than the day of calving; and the effect

of treatment on ion transport across mammary epithelial ceUs. The effects of

Flucort® and Predefl> 2X is not well documented on immune function and milk

composition. It would be interesting to continue to evaluate these experimental

designs on blood metabolites, immunologie, and milk composition parameters

with a larger sample size.

93

VII. References

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Anderson, B.H., D.L. Watson, and I.G. Colditz. 1999. The effect of dexamethasone on sorne immunological parameters in cattle. Veto Res. Commun. 23:399-413.

Andersson, L., and U. Emanuelson. 1985. An epidemiological study of hyperketonaemia in Swedish dairy cows; determinants and the relation to fertility. Prev. Veto Med. 3:449-462.

Andersson. L. 1988. Subelinical ketosis in dairy eows. Vet. Clin. N. Amer.­Food Animal Practice. 4:233-251.

Arieli, A, J. E. Vallimont, Y. Aharoni, and G. A. Varga. 2001. Monensin and growth hormone effeets on glucose metabolism in the pre-calving eow. J. Dairy Sci. 84:2770-2776.

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Appendices

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