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Effect of Feeding Synthetic Antioxidants and Prepartum Evaporative Cooling on Performance of Periparturient Holstein Cow...

Permanent Link: http://ufdc.ufl.edu/UFE0042227/00001

Material Information

Title: Effect of Feeding Synthetic Antioxidants and Prepartum Evaporative Cooling on Performance of Periparturient Holstein Cows During Summer in Florida
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Wang, Dan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antioxidants, dairy, evaporative, oxidative, transition
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this study was to evaluate the effect of supplementation with 0 or 250 mg of synthetic antioxidants (AO, Agrado Plus, Novus International, MO) per kg of dietary DM and prepartum evaporative cooling on periparturient Holstein cows (n = 35) from 21 days before through 49 days after parturition in a 2 by 2 factorial design. Uterine health was evaluated via metricheck at 7, 16, and 25 days in milk (DIM). Blood was collected at -15, 1, 8, 15, and 29 DIM for oxidative markers. Phagocytosis and oxidative burst of neutrophils were measured in whole blood collected at -15, 0, 7, and 14 DIM. Acute-phase proteins were measured in plasma collected three times weekly. A uterine horn was flushed at 40 plus or minus 2 DIM for diagnosis of subclinical endometritis. Rectal temperature of cooled cows was lower prepartum (39.2 vs. 39.6degreeC). Prepartum cooling resulted in greater mean concentration of milk fat during 7 wk (3.54 vs. 3.32%) and mean production of 3.5% FCM during the first 4 wk postpartum (26.5 vs. 23.0 kg/d). Cooling reduced the concentration of circulating WBC postpartum (7864 vs. 10,199 per microL of blood) and of circulating lymphocytes (3463 vs. 5432 per microL of blood) and increased proportion of neutrophils undergoing oxidative burst (83 vs. 77%) isolated from cows fed the control diet. Prepartum cooling of multiparous cows resulted in less oxidative stress as evidenced by lower activity of GPx in RBC (8,854 vs. 12,247 nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). Feeding AO increased concentration of milk true protein (3.07 vs. 2.94%) but decreased concentration of milk fat (3.25 vs. 3.61%) resulting in less production of milk fat (0.88 vs. 1.04 kg/d) and of 3.5% FCM (26.2 vs. 29.5 kg/d). In addition, cows fed AO had a greater incidence of endometritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs. 33%). Feeding AO to prepartum cooled cows reduced plasma concentration of TBARS (1.78 vs. 2.33 nmol/mL), proportion of neutrophils undergoing oxidative burst (77 vs. 83%), and mean florescence intestity of phagocytosis of primiparous cows postpartum (36 vs. 57%).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dan Wang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Staples, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042227:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042227/00001

Material Information

Title: Effect of Feeding Synthetic Antioxidants and Prepartum Evaporative Cooling on Performance of Periparturient Holstein Cows During Summer in Florida
Physical Description: 1 online resource (162 p.)
Language: english
Creator: Wang, Dan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: antioxidants, dairy, evaporative, oxidative, transition
Animal Sciences -- Dissertations, Academic -- UF
Genre: Animal Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The objective of this study was to evaluate the effect of supplementation with 0 or 250 mg of synthetic antioxidants (AO, Agrado Plus, Novus International, MO) per kg of dietary DM and prepartum evaporative cooling on periparturient Holstein cows (n = 35) from 21 days before through 49 days after parturition in a 2 by 2 factorial design. Uterine health was evaluated via metricheck at 7, 16, and 25 days in milk (DIM). Blood was collected at -15, 1, 8, 15, and 29 DIM for oxidative markers. Phagocytosis and oxidative burst of neutrophils were measured in whole blood collected at -15, 0, 7, and 14 DIM. Acute-phase proteins were measured in plasma collected three times weekly. A uterine horn was flushed at 40 plus or minus 2 DIM for diagnosis of subclinical endometritis. Rectal temperature of cooled cows was lower prepartum (39.2 vs. 39.6degreeC). Prepartum cooling resulted in greater mean concentration of milk fat during 7 wk (3.54 vs. 3.32%) and mean production of 3.5% FCM during the first 4 wk postpartum (26.5 vs. 23.0 kg/d). Cooling reduced the concentration of circulating WBC postpartum (7864 vs. 10,199 per microL of blood) and of circulating lymphocytes (3463 vs. 5432 per microL of blood) and increased proportion of neutrophils undergoing oxidative burst (83 vs. 77%) isolated from cows fed the control diet. Prepartum cooling of multiparous cows resulted in less oxidative stress as evidenced by lower activity of GPx in RBC (8,854 vs. 12,247 nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). Feeding AO increased concentration of milk true protein (3.07 vs. 2.94%) but decreased concentration of milk fat (3.25 vs. 3.61%) resulting in less production of milk fat (0.88 vs. 1.04 kg/d) and of 3.5% FCM (26.2 vs. 29.5 kg/d). In addition, cows fed AO had a greater incidence of endometritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs. 33%). Feeding AO to prepartum cooled cows reduced plasma concentration of TBARS (1.78 vs. 2.33 nmol/mL), proportion of neutrophils undergoing oxidative burst (77 vs. 83%), and mean florescence intestity of phagocytosis of primiparous cows postpartum (36 vs. 57%).
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Dan Wang.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Staples, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042227:00001


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EFFECT OF FEEDING SYNTHETIC ANTIOXIDANTS AND PREPARTUM
EVAPORATRIE COOLING ON PERFORMANCE OF PERIPARTURIENT HOLSTEIN
COWS DURING SUMMER IN FLORIDA




















By

DAN WANG


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA

2010































2010 Dan Wang






























To my husband, Zheng Fu who loves me, encourages me, and supports me









ACKNOWLEDGMENTS

First of all, I sincerely thank my dear Lord Jesus who has strengthened me all the

time since I put my faith in Him in 2007 so that I am able to do everything through Him.

1 Peter 5:10 states, "the God of all grace, He who has called you into His eternal glory

in Christ Jesus, after you have suffered a little while will Himself perfect, establish,

strengthen, and ground you". You make your home in my heart through faith.

I would like to thank my mentor Dr. Charles R. Staples for allowing me to pursue

the MS degree in the Department of Animal Sciences. He guided me in the academic

field and cared me for when I was lost in the American life. His warm heart has

supported me from the beginning to the end of my MS program. I am grateful for his

infinite patience to interpret every single question I ask and throughout the study.

I greatly thank Dr. Adegbola Adesogan for agreeing to be my committee member. I

deeply appreciate his prayers concerning how I could finish my MS while taking care of

my little baby. I would like to give thanks to Dr. Jose Eduardo Santos who collected

uterine flush samples from the experimental cows and built the statistical model for

analyzing the categorical data. Thanks Dr. Santos for inspiring me to deepen my

knowledge of ruminant nutrition, metabolic physiology, and statistics. Also thanks go to

his wife, Cristiana R.C. Santos, for sending those sweet messages to me when Eunice

was born and giving her a playing mat and baby clothes.

I deeply appreciate Dr. Lokenga Badinga for allowing me to voluntarily work with

his formal student, Dr. Cristina Caldari Torres, at the university dairy farm. I am grateful

that I was able to work with Cristina and to learn a lot of techniques from her.

I owe special thanks to all the biological scientists who at one time or another

technically supported my study. These include Sergei Sennikov who collected milk









samples every Tuesday during the trial and helped performed plasma analysis for

glucose, BUN, BHBA, and NEFA. Jan Kivipelto shared experiences about the 8-

isoprostane assay, tested it for bovine species with me for a long period of time, and

taught me how to operate the Elemantar and plate reader. Joyce Hayen updated milk

yield data every month. Jae-Hyeong Shin not only helped daily with the management of

the study but also was my good friend after work. Andrea Dunlap shared her

experiences with the haptoglobin assay. I also would like to thank Eric Diepersloot,

Grady Byers, Mary Russell, Jerry Langford, Molly Gleason, Patty Best, Sherry Hay, Jay

Lemmermen, Megan Manzie, Krista Seraydar, and all the Dairy Unit employees who

offered time and help to make this trial successful. I have great appreciation for Dr.

Bruno do Amaral and Dr. Lilian Oliveira for elucidating for me the working principles of

flow cytometry and how to analyze neutrophil function data.

I want to extend my appreciation to my research companions in the Department of

Animal Sciences that directly or indirectly helped in my research and cared for me:

Miriam Garcia Orellana who offered her help to the uttermost in this study and also

shared her deep knowledge of nutrition with me, Sha Tao who I knew before I entered

into the MS program, gave me encouragement when I was weak, Leandro Greco who

often says "We are on the same team so it's not necessary to always thank me", and

Rafael Bisinotto and Eduardo Ribeiro who helped me perform the progesterone assay

and professionally answered my many questions about uterine diseases. Special

thanks go to Milerky Perdomo and Kathy Arriola who shared their precious experiences

with me about being a good mom. I would also like to thank Oscar Queiroz and Fabio









Lima for being around the building and their good friendship. I am very grateful for what

all of you have done for me.

My special thanks to Dr. Joel Brendemuhl and Joann Fischer for their advice and

help with academic questions. Special thanks to Sabrina Robinson and Delores

Bradshaw for their hugs and help with all kinds of miscellaneous issues.

I give my deep appreciation to my husband (Gougou) and my parents. Thank you

all for shepherding me and supporting me immeasurably. Dear Gougou, I am grateful

that you make me stronger little by little. I thank you for your sweet love during the past

7 years. Without your support, I would not have finished this endeavor.

Praise you Lord unceasingly!









TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ........................ ........................................................................... 4

L IS T O F TA B L E S .......................... ............................................................ 9

L IS T O F F IG U R E S ......................................... ... ....................................... ...... ................. 1 0

L IS T O F A B B R E V IA T IO N S .............................................. .................................................. 15

A B S T R A C T ............ ........ .. ............ .. ......................................... ........... ...... 17

CHAPTER

1 IN T R O D U C T IO N ...... .............................................................. .................................. 1 9

Oxygen and O xidative Stress ...................................................................................... 19
Oxidative Status and Health of Dairy Cattle ...................................... ................. 20

2 LIT E R A T U R E R E V IE W .......................... .. ............................................. ....... ....... 22

Reactive Oxygen Species Formation ........................................................................ 22
Oxidative Stress and Oxidative Damage ........................................... ....................... 22
Lipid Peroxidation ............................................................................. .... ............ .................23
A antioxidant D defenses .............................. ............. ...................... 23
1) E nzym atic A ntioxida nts ......... ................. ................................... ............... 23
a) S uperoxide D ism utase ......... ................. .............................................. 23
b) G lutathio ne P eroxidase ..................................... .................... .. ............... 24
2 ) N o ne nzym a tic A ntioxidants ..................................... ..................................... 2 7
a) G lutathio ne (G S H ) ............................................................ ...... .... .... 2 7
b) V itam in A and B-carotene ......................................................................... 28
c ) V ita m in E .................................................................................. 3 0
d ) S e le n iu m ............................................................................................. 3 1
3) S ynthetic A ntioxida nts ........... ........................................................ .............. 33
O verview of Im m une Function .................................................................................... 34
Effect of Feeding Antioxidants on the Immune System ................ .................35
Effect of Feeding Synthetic Antioxidants on Performance ........................ ........... 37
Effect of Feeding Antioxidants on Oxidative Status and Stability................................. 39
Effect of Prepartum Heat Stress on Performance and Metabolites........................ 43
Effect of Prepartum Heat Stress on the Immune System .................................45
The Effect of Cooling System for Dairy Cows in Hot Environment.........................46
Shading. ......... ..... .. .................................46
Cooling. ................ .................. ........ ......... 47





7









3 EFFECT OF FEEDING ANTIOXIDANTS AND PREPARTUM EVAPORATR/E
COOLING ON PERFORMANCE OF TRANSITION HOLSTEIN COWS DURING
S U M M E R IN F LO R ID A ......... ............................................ .......................................... 5 0

Introd uctio n .......... ....................... ................. 50
M ateria l a nd M methods ... .. .. ...... ............. .... .... ... .... ........ .............. ......... .... 5 1
Animals, Treatments, and Management ..................................... ........ ......... 51
Sam ple Collection and Analysis ........ ......... .. ......................... ................. 52
Processing of Red Blood Cell (RBC)....................... ... ............................... 54
Thiobarbituric Acid Reactive Substances (TBARS) Assay .......................... 55
Superoxide Dism utase (SOD) Assay ........................................................... 55
Glutathione peroxidase (GPx) Assay ................. ............................................ 57
A cute P hase Protein A ssays...................................... ....................... ........... 58
Neutrophil Function, W BC and Lymphocyte ................................................. 59
O va Ib um i n C ha lle ng e ......... ................. ..................................................... 6 0
V agi noscopy ............ ..... ...... .... ......... ... .................... 6 1
Uteri ne C yto logy.............. ..... ................................ ... .. ............ .. 62
S tatistica l A na lyses ......... ..... ........ ...................................... .. .... .... ..... 62
Results and Discussion ............. ........................................ .. .. ............ 64
Body Temperature, BW, BCS, and DMI.................. .............................64
Milk Production and M ilk Composition ... ... .............................. ................ .. ................ 68
P la sm a M e ta bo lite s ........... ............ ............... ..... .. .............. .... ...... .......... 7 1
Postpartum Body Temperature and Oxidative Markers .......................................73
In B lo o d .............. ...... ... ........... .................. ..........................7 3
W white B lood C ells ............................................. ..........................................78
Function of Blood Neutrophils .............................................................................. 80
Ova Ibumin Challenge and Acute-phase Proteins........................................... 83
Progesterone ............ .... ..... ......... ........ 85
Vaginoscopy and Uterine cytology ......................................... 86
S um m ary ............................................................................. ....................... 87

L IS T O F R E F E R E N C E S ......... .... ........ ............................................................ .......... 14 9

B IO G R A P H IC A L S K E T C H ...................................................................... .................. ... ..... 16 2









LIST OF TABLES


Table page

3-1 Ingredient composition of diets fed to nonlactating and lactating Holstein
cow s. ................ .... ..... ..... ........................................... 90

3-2 Chemical composition of diets fed to nonlactating and lactating Holstein
cow s. ................ .... ..... ..... ........................................... 92

3-3 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on body
temperature, body weight, BCS, and DMI of nonlactating pregnant Holstein
cow s during sum m er in Florida. ............................ ... ..... ................................... 93

3-4 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on
performance of lactating pregnant Holstein cows during summer i n Florida. ......97

3-5 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on
plasma concentration of metabolites of lactating Holstein cows during
s um m e r in F lo rid a .................................................................. ................... 10 9

3-6 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on
plasma concentration of oxidative markers of lactating Holstein cows during
sum m er in Florida. ........... .... ... ..................................... ... ...... .....114

3-7 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on
concentration of white blood cells (WBC), lymphocytes, and neutrophils,
function of blood neutrophils, and plasma concentration of acid soluble
protein (ASP) and haptoglobin (Hp) of periparturient Holstein cows during
summer in Florida. ...... ........ ....... ............................... 123

3-8 Profile of plasma progesterone of postpartum dairy cows fed with or without
synthetic antioxidants (AO) and cooled or noncooled during the prepartum
period during the summer season in Florida. ................................... ................. 145

3-9 Incidence of postpartum health disorders of dairy cows fed with or without
synthetic antioxidants (AO) and cooled or noncooled during the prepartum
period during the summer season in Florida. ................................... ................. 148









LIST OF FIGURES


Figure page

3-1 Least squares means for mean dry matter intake of prepartum primiparous (n
= 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
w ith dietary antioxidants (A O ) .......................... ...... ......... .......... .................95

3-2 Least squares means for mean body weight of prepartum primiparous (n =
22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
w ith dietary antioxidants (A O ) .......................... ...... ......... .......... .................96

3-3 Least squares means for mean dry matter intake of postpartum primiparous
(n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
w ith dietary antioxidants (A O ) .......................... ...... ......... .......... .................99

3-4 Least squares means for mean body weight (BW) of postpartum primiparous
(n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
w ith dietary antioxidants (A O ) .......................... ...... ....... .. ....... .................100

3-5 Least squares means for weekly body weight (BW) of postpartum
primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows housed in
cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented
without (Control) or with dietary antioxidants (AO). ......................... ................. 01

3-6 Least squares means for weekly dry matter intake of postpartum Holstein
cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed
diets supplemented without (Control) or with dietary antioxidants (AO). ............ 102

3-7 Least squares means for weekly milk production of Holstein cows (n = 35)
housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets
supplemented without (Control) or with dietary antioxidants (AO)...................... 103

3-8 Least squares means for weekly concentration of milk fat of Holstein cows (n
= 35) housed in cooled (Cool) or noncooled (Hot) freestalls. .................................104

3-9 Least squares means for weekly milk fat production of Holstein cows (n = 35)
housed in cooled (Cool) or noncooled (Hot) freestalls. ......... ............................ 05

3-10 Least squares means for production of 3.5% fat-corrected milk by postpartum
Holstein cows (n = 35) housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot). .......... ............... .................................. 106









3-11 Least squares means for weekly concentration of milk protein of Holstein
cows (n = 35) fed diets supplemented without (Control) or with dietary
antioxidants (AO ). .................. .................................................. ....107

3-12 Least squares means for somatic cell counts of postpartum Holstein cows (n
= 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (H o t). ................................................................. ............ 108

3-13 Least squares means for weekly plasma concentrations of NEFA of
postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein
cows fed diets supplemented without (Control, C) or with synthetic
antioxidants (AO) and housed in shaded freestalls equipped with fans and
sprinklers (C ool) or just shade (Hot). ............................................................ ... 111

3-14 Least squares means for weekly energy balance of postpartum primiparous
(A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets supplemented
without (Control) or with synthetic antioxidants (AO) and housed in shaded
freestalls equipped with fans and sprinklers (Cool) or just shade (Hot)..............112

3-15 Least squares means for weekly plasma concentration of beta-hydroxyl
butyric acid of primiparous primii, n = 22) and multiparous (multi, n = 13)
Holstein cows fed diets supplemented without (Control) or with synthetic
antioxidants (AO). .................................... .............................. .......113

3-16 Least squares means for plasma thiobarbituric acid reactive substances
(TBARS) on -15, 1, 8, 15, 29 d relative to calving.. .............. ........ ............... 116

3-17 Least squares means for mean plasma concentration of thiobarbituric acid
reactive substances (TBARS) of Holstein cows (n = 35) fed diets
supplemented without (Control) or with dietary antioxidant (AO) and housed
in shaded freestalls equipped with fans and sprinklers (Cool) or just shade
(H o t) ................... ........................................................................... 1 1 7

3-18 Least squares means for weekly activity of glutathione peroxidase (GPx) per
mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented without
or with synthetic antioxidants and housed in shaded freestalls equipped with
fans and sprinklers or just shade on -15, 1, 8, 15, and 29 d relative to calving. 118

3-19 Least squares means for mean activity of glutathione peroxidase corrected
for pack cell volume of primiparous (n = 22) and multiparous (n = 13)
Holstein cows fed diets supplemented without (Control) or with synthetic
antioxidants (AO) and housed in shaded freestalls equipped with fans and
sprinklers (C ool) or just shade (Hot). ............................................................ ... 119

3-20 Least squares means for mean activity of glutathione peroxidase per mL of
erythrocyte of primiparous (n = 22) and multiparous (n = 13) Holstein cows
fed diets supplemented without (Control) or with synthetic antioxidants (AO)









and housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (H o t). ................................................................. ............ 12 0

3-21 Least squares means for activity of superoxide dismutase (SOD) corrected
for pack cell volume of Holstein cows (n = 35) fed diets supplemented
without or with synthetic antioxidants and housed in shaded freestalls
equipped with fans and sprinklers or just shade on -15, 1, 8, 15, and 29 d
relative to calving ........................................ ............ ................... 121

3-22 Least squares means for activity of superoxide dismutase (SOD) per mL of
erythrocyte of Holstein cows (n = 35) fed diets supplemented without
(Control) or with dietary antioxidants (AO) on -15, 1, 8, 15, and 29 d relative
to calvi ng ......... ................................. ........... 122

3-23 Least squares means for number of white blood cells (WBC), neutrophils,
and lymphocytes per pL of whole blood on -15, 0, 7, 14 d relative to calving....125

3-24 Least squares means for number of white blood cells (WBC) per pL of whole
blood of Holstein cows (n = 35) housed in shaded freestalls equipped with
fans and sprinklers (Cool) or just shade (Hot). ............................................... 126

3-25 Least squares means for number of neutrophils per pL of whole blood of
Holstein cows (n = 35) housed in shaded freestalls equipped with fans and
sprinklers (C ool) or just shade (Hot). ..................................................................... 127

3-26 Least squares means for number of lymphocytes per pL of whole blood of
Holstein cows (n = 35) housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot) ............................................................ .......... 128

3-27 Least squares means for percentage of neutrophils with phagocytic activity
(solid line) and neutrophil mean florescence intensity (MFI, indication of
number of bacteria phagocytised per neutrophil, dash line) of Holstein cows
(n = 35) on -15, 0, 7, 14 d relative to calving ............................... 129

3-28 Least squares means for percentage of neutrophils with oxidative burst
activity (solid line) and neutrophil mean florescence intensity (MFI, indication
of intensity of reactive oxygen species produced per neutrophil, dash line) of
Holstein cows (n = 35) on -15, 0, 7, 14 d relative to calving............................... 130

3-29 Least squares means for mean percentage of neutrophils with phagocytic
activity of primiparous primii, n = 22) and multiparous (multi, n = 13) Holstein
cows housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (H o t). .................................................................... ............. 13 1

3-30 Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows housed in









shaded freestalls equipped with fans and sprinklers (Cool) or just shade
(H o t) ................... ........................................................................... 1 3 2

3-31 Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO). ................33

3-32 Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO) on -15, 0,
7, 14 d relative to ca living ...... ................................ ........ .. .. .. 134

3-33 Least squares means for mean percentage of neutrophil with oxidative burst
activity of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed
diets supplemented without (Control) or with synthetic antioxidants (AO) and
housed in shaded freestalls equipped with fans and sprinklers (Cool) or just
s h a d e (H o t) ...................................... ................................................. 1 3 5

3-34 Least squares means for percentage of neutrophil with oxidative burst activity
of Holstein cows (n = 35) fed diets supplemented without (Control) or with
synthetic antioxidants (AO) and housed in shaded freestalls equipped with
fans and sprinklers (Cool) or just shade (Hot) on -15, 0, 7, 14 d relative to
calvi ng ............. ... ...... ............ ......................... 136

3-35 Least squares means for IgG response against ovalbumin of Holstein cows
(n = 35) fed diets supplemented without (Control) or with synthetic
antioxidants (AO) and housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot) on -4, -2, 0, 1, 2, 3, 4, and 7 wk relative
to c a lv in g ................. .... .... ......... ..... ............ ................................... 1 3 7

3-36 Least squares means for IgG response against ovalbumin of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO). ................38

3-37 Least squares means for concentrations of acid soluble protein (ASP) of
primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets
supplemented with or without dietary antioxidant (Agrado Plus) and housed
in shaded freestalls equipped with fans and sprinklers or just shade ................ 139

3-38 Least squares means for concentrations of acid soluble protein of Holstein
cows (n = 35) diagnosed as healthy (n = 14) or unhealthy metritiss, mastitis,
or retained fetal membranes, n = 21).................................... .......................... 140

3-39 Least squares means for concentrations of acid soluble protein (ASP) of
Holstein cows (n = 35) fed diets supplemented without (Control) or with
synthetic antioxidants (AO) ............................................................................................. 141









3-40 Least squares means for concentrations of haptoglobin of primiparous (n =
22) and multiparous (n = 13) Holstein cows fed diets supplemented with or
without dietary antioxidant (Agrado Plus) and housed in shaded freestalls
equipped with fans and sprinklers or just shade. ................................................. 142

3-41 Least squares means for concentrations of haptoglobin of Holstein cows (n =
35) diagnosed as healthy (n = 14) or unhealthy metritiss, mastitis, or retained
fetal m em branes, n = 21 ) ............. ......... ........... ...................... .................143

3-42 Least squares means for concentrations of haptoglobin of Holstein cows (n =
35) fed diets supplemented with or without dietary antioxidant (Agrado Plus)
and housed in shaded freestalls equipped with fans and sprinklers (cool) or
just shade (hot) ................. .............................................. .................... 144

3-43 Least squares means for mean peak concentration of progesterone (P4) of
the first cycle of primiparous primii, n = 22) and multiparous (multi, n = 13)
Holstein cows fed diets supplemented without (Control) or with synthetic
antioxidants (A O ) .................................................. ......... .. ...............147










LIST OF ABBREVIATIONS

ADF acid detergent fiber

AO antioxidants

ASP acid soluble protein

BCS body condition score

BHA butylated hydroxyanisole

BHBA beta-hydroxy butyric acid

BHT butylated hydroxytoluene

BUN blood urea nitrogen

Con A concanavalin A

DHR dihydrorhodamine 123

DIM days in milk

DMI dry matter intake

ELISA enzyme-linked immunosorbent assays

GPx glutathione peroxidase

GSH glutathione

GSSG glutathione disulfide

Hb hemaglobin

Hp haptoglobin

IgG immunoglobulin G

I.m. intramascularly

LCFA long chain fatty acids

MDA malondialdehyde

MFI mean fluorescence intensity

NDF neutral detergent fiber









NEFA non esterified fatty acid

NIR near infrared

PBMC peripheral blood mononuclear cell

PBS phosphate buffer solution

PCV packed cell volume

RBC red blood cell

ROS reactive oxygen species

SCC somatic cell count

SDS sodium dodecyl sulfate

SOD superoxide dismutase

TBA thiolbarbituric acid

TBARS thiolbarbituric acid reactive substances

THI temperature humidity index

WBC white blood cell









Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

EFFECTOF FEEDING SYNTHETIC ANTIOXIDANTS AND PREPARTUM
EVAPORATIVE COOLING ON PERFORMANCE OF PERIPARTURIENT HOLSTEIN
COWS DURING SUMMER IN FLORIDA

By

Dan Wang

August 2010

Chair: Charles R. Staples
Major: Animal Sciences

The objective of this study was to evaluate the effect of supplementation with 0 or

250 mg of synthetic antioxidants (AO, Agrado Plus, Novus International, MO) per kg of

dietary DM and prepartum evaporative cooling on periparturient Holstein cows (n = 35)

from 21 days before through 49 days after parturition in a 2 by 2 factorial design.

Uterine health was evaluated via metricheck at 7, 16, and 25 days in milk (DIM). Blood

was collected at -15, 1, 8, 15, and 29 DIM for oxidative markers. Phagocytosis and

oxidative burst of neutrophils were measured in whole blood collected at -15, 0, 7, and

14 DIM. Acute-phase proteins were measured in plasma collected three times weekly.

A uterine horn was flushed at 40 2 DIM for diagnosis of subclinical endometritis.

Rectal temperature of cooled cows was lower prepartum (39.2 vs. 39.60C). Prepartum

cooling resulted in greater mean concentration of milk fat during 7 wk (3.54 vs. 3.32%)

and mean production of 3.5% FCM during the first 4 wk postpartum (26.5 vs. 23.0 kg/d).

Cooling reduced the concentration of circulating WBC postpartum (7864 vs. 10,199 per

pL of blood) and of circulating lymphocytes (3463 vs. 5432 per pL of blood) and

increased proportion of neutrophils undergoing oxidative burst (83 vs. 77%) isolated









from cows fed the control diet. Prepartum cooling of multiparous cows resulted in less

oxidative stress as evidenced by lower activity of GPx in RBC (8,854 vs. 12,247

nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). Feeding AO increased

concentration of milk true protein (3.07 vs. 2.94%) but decreased concentration of milk

fat (3.25 vs. 3.61%) resulting in less production of milk fat (0.88 vs. 1.04 kg/d) and of

3.5% FCM (26.2 vs. 29.5 kg/d). In addition, cows fed AO had a greater incidence of

endometritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs.

33%). Feeding AO to prepartum cooled cows reduced plasma concentration of TBARS

(1.78 vs. 2.33 nmol/mL), proportion of neutrophils undergoing oxidative burst (77 vs.

83%), and mean florescence intestity of phagocytosis of primiparous cows postpartum

(36 vs. 57%).









CHAPTER 1
INTRODUCTION

Oxygen and Oxidative Stress

Animals do not use energy in feed directly for requirements of maintenance,

activity, pregnancy, and productive purposes. Carbohydrates, lipids, and amino acids

must undergo oxidative phosphorylation as the final stage to generate ATP. During

oxidative phosphorylation, molecular oxygen is reduced by accepting 4 electrons to

produce two molecules of water. The electrons which reduce oxygen to water are

derived from metabolism of feed. This process is called cellular respiration.

However electrons which fail to be incorporated into the terminal acceptor of the

transport chain may cause problems. About 1 to 2% of consumed oxygen is not

completely reduced due to the escape of electrons from the intermediate complex in the

respiratory chain (Levine, 1985). For example, the passage of electrons from reduced

ubiquinone to complex III involves the radical Q which could pass an electron to oxygen

to form a superoxide radical (02-). Another example is the generation of 02- during the

hydroxylation reactions catalyzed by Cytochrome P-450 (Nelson, 2008). Anything which

increases metabolic demands such as parturition, lactation, heat stress, and disease or

disorders could increase oxygen requirements, number of electrons transferred, and

production of 02- (Sordillo and Aitken, 2009).

Superoxide radicals can be reduced to hydrogen peroxide (H202) by acceptance

of a second electron. With acceptance of a third electron, oxygen can be reduced to

hydroxyl radical (OH-). The generation of OH- may cause two types of damage

depending on the location. On the one hand, OH- may attack the peroxidative chain

which can damage cellular and subcellular membranes (Gutteridge, 1994). On the other









hand, OH- is produced at the site where Fe is associated with a macromolecule such as

DNA or protein causing damage of DNA and protein (Casciola-Rosen et al., 1997). The

term "reactive oxygen species (ROS)" refers to oxygen-derived free radicals such as 02

,H202 and OH-.

Oxidative Status and Health of Dairy Cattle

Cells normally are protected against the harmful effects of ROS by antioxidant

defenses. But when the generation of ROS exceeds the capacity of defensive systems

to eliminate ROS, the oxidation-antioxidation system is imbalanced. This process is

defined as oxidative stress. Levine and Kidd (1985) elucidated that the progression from

oxidative stress to chronic disease can be divided into 4 stages (Levine, 1985). In the

first stage, the individual is healthy and able to deal with oxidative stress. In the second

stage, due to chronic deficiency of antioxidant nutrients or exposure to oxidants, the

individual adapts to oxidative stress. In the third stage, continued oxidative stress

depletes antioxidants so that the individual is subjected to oxidative damage. The third

stage can become more severe so that absorption of antioxidant nutrients is influenced.

In the fourth stage, the rate of deterioration of antioxidant defense exceeds the rate of

recovery resulting in subclinical or clinical diseases.

The effects of antioxidants on oxidative status and health of dairy cows have been

examined in recent years. Supplementation with vitamin E and Se usually reduced

incidence of retained fetal membranes and mastitis (Miller et al., 1993; Allison and

Laven, 2000). The supplementation of antioxidants also has been reported to improve

oxidative status (Brzezinska-Slebodzinska et al., 1994; Vazquez-Anon et al., 2008;

Sahoo et al., 2009).









The objectives of the literature review (Chapter 2) are the following: 1) to introduce

the classes of antioxidants namely enzymatic antioxidants including superoxide

dismutase (SOD) and glutathione peroxidase (GPx), nonenzymatic antioxidants

including glutathione (GSH), vitamin A, 3-carotene, vitamin E and Se; and synthetic

antioxidants including ethoxyquin, butylated hydroxyanisole (BHA), and butylated

hydroxytoluene (BHT), 2) to summarize the effectiveness of antioxidants on

performance, immune system, and oxidative status of animals. In addition the effect of

heat stress on performance and immune system of animals is reviewed. In the

subsequent research (Chapter 3), the effects of feeding synthetic antioxidants and

prepartum cooling on the performance of periparturient Holstein cows was investigated.









CHAPTER 2
LITERATURE REVIEW

Reactive Oxygen Species Formation

Free radicals are molecules within the animals' body that have at least an

unpaired electron in the outer orbit. They can accept or donate electrons from other

molecules to generate a more stable molecule through oxidation and reduction

reactions (Gitto et al., 2002; Halliwell, 2007a; Sordillo and Aitken, 2009). Reactive

oxygen species (ROS) is a collective term to classify oxygen-derived free radicals,

including superoxide radical (02-), hydrogen peroxide (H202), and hydroxyl radical (OH-)

(Gitto et al., 2002). Normally, ROS are produced either through oxidative

phosphorylation within the inner membrane of mitochondria or phagocytosis of

pathogens to stimulate NADPH oxidase in neutrophils (Paape et al., 2003; Sordillo and

Aitken, 2009). Animals and human beings may undergo metabolic and physiological

adaptations during the transition from pregnancy to lactation accompanied by elevated

requirements for oxygen. This increased oxygen demand augments the production and

accumulation of ROS in tissues.

Oxidative Stress and Oxidative Damage

Oxidative stress refers to an imbalance between production of free radicals and

antioxidant mechanisms (Halliwell, 2007a). It can result from dietary imbalances,

pregnancy, environmental pollutants, solar radiation, or heat stress (Gitto et al., 2002,

Miller et al., 1993). Oxidative stress can result in oxidative damage to molecules, cells,

and tissues which may subsequently develop to certain kinds of diseases or disorders

such as endometriosis, heart failure, diabetes and so on in different species (West,

2000; Sun et al., 2002; Jackson et al., 2005). For example, oxidative stress could









indirectly lead to Ca2+ overload which activates phospholipase A2 and C. These

enzymes lead to membrane phospholipid hydrolysis (Gitto et al., 2002; Halliwell,

2007a). Another example is that oxidative stress could lead to membrane lipid

peroxidation which gives rise to peroxyl radicals.

Lipid Peroxidation

Lipid peroxidation is a chain reaction (initiation, propagation, and termination). This

reaction is initiated by attack of reactive hydroxyl radicals on polyunsaturated fatty acids

in plasma membranes. Propagation of lipid peroxidation gives rise to a lipid radical (L-)

which interacts with oxygen under aerobic conditions to produce a peroxyl radical

(LOO'). The reaction between peroxyl radicals generates nonradical products which

terminates this chain reaction (Burton and Ingold, 1984, Halliwell, 2007a).

Antioxidant Defenses

Antioxidants can be defined as any substance that helps protect cells by delaying,

preventing or removing oxidative damage (Halliwell, 2007b). Antioxidants can be

classified based on their chemical and physical characteristics, namely enzymatic

antioxidants, such as superoxide dismutase (SOD) and glutathione peroxidase (GPx),

nonenzymatic antioxidants such as glutathione, ascorbic acid, vitamin E, 3-carotene,

and ubiquinone, and synthetic antioxidants such as ethoxyquin, butylated

hydroxyanisole (BHA), and butylated hydroxytoluene (BHT).

1) Enzymatic Antioxidants

Enzymatic antioxidants work most efficiently and directly reduce ROS.

a) Superoxide Dismutase

Superoxide dismutase catalyzes the dismutation of superoxide to H202 and 02

(Miller et al., 1993; Mates et al., 1999).









+ SOD
202 + 2H o H202 + 02

The three forms of SOD are the following: Cu/Zn-SOD in cytosols, Mn-SOD in

mitochondria, and Fe-SOD. Cu/Zn-SOD plays an important role in the first defense

against ROS (Mates and Sanchez-Jimenez, 1999; Bernabucci et al., 2005). The activity

of Cu/Zn-SOD in milk was similar to that in bovine erythrocytes (Przybylska et al.,

2007). Mn-SOD, which is a homotetramer, functions to remove superoxide produced in

the electron transport chain in mitochondria. It can be induced and depressed by

cytokines (Mates et al., 1999). Experiments using gene knock-out mice demonstrated

that Mn-SOD is essential for life, but that Cu/Zn-SOD is not (Mates and Sanchez-

Jimenez, 1999). The Fe-SOD are found only in prokaryotes such as Escherichia coli

(Fridovich, 1975). It seems to provide a defense against exogenous superoxide.

Superoxide dismutase activity has been used to evaluate oxidative status in

animals. Cows in the last 3 wk of pregnancy had an incremental increase in plasma

SOD activity and reached peak at 4 d after calving (Bernabucci et al., 2005). The same

laboratory reported in 2002 that cows giving birth during summer had greater

erythrocyte SOD activity compared with the cows calving in spring. The activity of SOD

in placental tissues was higher in cows with retained placental membranes than those

without retained placental membranes (Kankofer et al., 1996).

b) Glutathione Peroxidase

Glutathione peroxidase can be classified as Se-dependent and Se-independent.

The Se-dependent enzyme catalyzes the reduction of hydroperoxides to water,

accompanying the oxidation of glutathione (GSH) to glutathione disulfide (GSSG).

GPx
ROOH + 2GSH ROH + GSSG + H20









Glutathione can be regenerated by reducing equivalents from NADPH2 (Mates et

al., 1999, Miller et al., 1993).

Glutathione peroxides are composed of at least 5 isoenzymes (GPx 1 to 5) in

mammals (Przybylska et al., 2007). Glutathione peroxidase 1 (GPx1) which is

comprised of four identical SeCys-containing subunits is the most predominant. It is

found in the cytoplasm of erythrocytes, kidney, and liver. The preferred substrates are

hydrogen peroxide and a wide range of organic hydroperoxides. Glutathione peroxidase

4 (GPx4), also called phospholipid hydroperoxide glutathione peroxidase, is distributed

in both cytosol and the membrane fraction, whose preferred substrate is phospholipid

and cholesterol hydroperoxides (Thomas et al., 1990). Glutathione peroxidase 2 (GPx2)

is a cytosolic enzyme, whereas glutathione peroxidase 3 (GPx3) is extracellular,

especially abundant in plasma. The GPx3 metabolizes phospholipid hydroperoxides

and plays a direct role in protection of membranes (Takahashi et al., 1987). Glutathione

peroxidase 5 (GPx5), being Se-independent, is newly discovered in mouse epididymis

(Przybylska et al., 2007). Glutathione peroxidase has been detected in bovine milk and

its activity was strongly and positively correlated with Se concentration (Przybylska et

al., 2007).

Glutathione peroxidase activity can be used to evaluate oxidative status. Generally

speaking, the activity of this enzyme could be elevated during parturition and lactation,

by heat stress, by BW loss, by diseases, or by consumption of different types of

feedstuffs. When blood samples were collected from dairy cows at 21 7 d before

calving, calving, and 21 DIM, the GPx activity in peripheral blood mononuclear cells

(PBMC) increased at calving and at 21 DIM compared with the prepartum period









(Sordillo et al., 2007). Similarly, weekly blood samples collected from 30 d before

calving to 30 d after calving indicated that plasma GPx activity began to increase a

week before calving and was greater after calving than before calving (Bernabucci et

al., 2005). The same authors also reported that cows giving birth during summer had

erythrocytes of greater GPx activity at 21 d before calving compared to that of cows

giving birth during spring (Bernabucci et al., 2002). However, GPx activity in plasma did

not differ during the transition period between cows calving in the summer and spring

season. Burke et al. (2007) reported that PBMC of heifers had less GPx activity when

housed under high temperature-humidity indices (THI) compared to neutral THI.

Brennan conducted a study to determine the effect of BW loss of beef cows which could

increase the production of ROS through fat mobilization on the antioxidant activity of

GPx and antioxidant mRNA levels (Brennan et al., 2009). Total RNA was isolated from

skeletal muscle, glutathione peroxidase 1 and GPx4 target gene mRNA was measured

using real-time reverse transcription-PCR. Erythrocyte GPx activity was measured to

determine oxidative status. No differences were found in GPx1 activity between cows

losing or maintaining BW. Therefore BW loss did not influence the GPx activity of

erythrocytes. However, abundance of GPx4 mRNA in skeletal muscle was 1.4-fold

greater during BW loss. Activity of plasma GPx was greater in patients who were

diagnosed with ulcerative colitis or Crohn's disease compared with healthy individuals

who didn't have any sign, symptom or previous records of inflammatory bowel disease

(Tuzun et al., 2002). The activity of GPx in placental tissues was greater in cows with

retained placental membranes than those without retained placenta membranes

(Kankofer et al., 1996). Cows fed grain or a mixed diet had a greater muscle GPx









activity than cows consuming only pasture (Mercier et al., 2004; Descalzo and Sancho,

2008), probably due to the different concentrations of Se in different feedstuffs

(Ammerman, 1975). Lambs fed brown seaweed extract exhibited greater GPx activity in

erythrocyte and white blood cell compared with animals not fed the extract, indicating

that brown seaweed extract improved lamb antioxidant status by increasing antioxidant

capacity (Saker et al., 2004). 2) Nonenzymatic Antioxidants

a) Glutathione (GSH)

Glutathione, a low-molecular-weight thiol, is abundant in animal cells and plasma.

Majority of the cellular GSH (85 to 90%) is distributed in the cytosol, with the rest

located in many organelles including the mitochondria, nuclear matrix, and

peroxisomes. On average, the concentration of GSH ranges from 0.5 to 10 mmol/L, with

the exception of bile acid which contains more than 10 mmol/L. The roles of glutathione

include the following: 1) antioxidant defense including the scavenging of free radicals

and other reactive species, removal of hydrogen and lipid peroxides, and prevention of

oxidation of biomolecules; 2) metabolic roles such as serving as a substrate for

synthesis of leucotriene C4, a substrate to convert formaldehyde to format, and for

storage and transport of cysteine; 3) aids in regulation of cytokine production, immune

responses, and mitochondrial function and integrity (Wu et al., 2004). Usually the

concentration ratio of GSH: GSSG is used as an index of the cellular oxidative status,

which is greater than 10 under normal physiological conditions (Griffith, 1999). The

GSH:GSSG ratio in PBMC of heifers was less under heat stress, which indicated either

increased production of ROS or decreased antioxidant status (Burke et al., 2007). Cows

exposed to heat stress and fed endophyte-infected tall fescue had a lower whole-blood

GSH concentration compared with those under heat stress without endophyte-infected









tall fescue in the diet (Lakritz et al., 2002). The addition of GSH did not alter the

proliferative response of lymphocyte isolated from the blood of Holstein cows at 38.50C

(neutral) or 420C (heat stress) (Kamwanja et al., 1994).

b) Vitamin A and B-carotene

B-carotene is the major dietary precursor of vitamin A in dairy cattle (LeBlanc et

al., 2004; Przybylska et al., 2007). B-carotene that escapes from the rumen is absorbed

and converted to retinol in the intestinal mucosa and transported to the liver with fat

(LeBlanc et al., 2004). Experiments indicate that /-carotene is neither a peroxide-

decomposing antioxidant such as catalase and GPx nor a conventional chain-breaking

antioxidant such as vitamin E (Burton and Ingold, 1984). It functions as an unusual

antioxidant at low oxygen pressures (Burton and Ingold, 1984) and a scavenger of free

radicals produced from unsaturated long chain fatty acid (LCFA) peroxidation (Hino et

al., 1993).

The bioavailability of vitamin A for cattle is considerably limited due to its

destruction by ruminal microbes (Rode et al., 1990; Weiss et al., 1995). Hino et al.

(1993) did an in vitro study to examine whether the addition of /-carotene could

alleviate the inhibition of bacterial growth caused by LCFA. The results demonstrated

that the addition of p-carotene increased bacterial growth with the presence of LCFA.

The active form of vitamin A could be different for different functions (Hemken and

Bremel, 1982). Alosilla et al. (2007) reported that feeding different commercial vitamin A

sources to yearling beef cattle led to different retinol concentrations in liver, indicating

that some supplemental vitamin A sources had greater amounts of vitamin A reaching

the duodenum for absorption and storage than others.









Vitamin A plays an important role in resistance to infectious disease, especially

mastitis (NRC, 2001). Johnston and Chew (1984) studied the peripartum concentrations

of vitamin A and /-carotene in Holstein cows with or without mastitis. They reported that

plasma concentrations of vitamins A and 3-carotene decreased rapidly before calving

and reached their lowest point at calving (vitamin A) or on d 4 to d 6 postpartum (,3-

carotene). In addition, concentration of vitamin A in plasma was lower in mastitic cows

from d 0 to 7 and at wk 2 and 4 than nonmastitic cows, whereas /-carotene was greater

in mastitic cows from prepartum to d 7. When feeding lactating Holstein cows /-

carotene at a rate of 0 or 300 mg/d from 3 to 98 d postpartum, concentration of,3-

carotene in serum declined in both control and carotene-supplemented groups between

1 and 2 wk postpartum, but increased to 225 pg/dl after 3 wk in supplemented group,

whereas the concentration stayed the same in control group. Additionally, feeding 3-

carotene did not affect the length of first estrous cycle or peak concentration of

progesterone in the first estrous cycle. However, the incidence of mastitis was less for

the supplemented group (Wang et al., 1988). In a review paper Hemken and Bremel

(1982) indicated that a deficiency of vitamin A was associated with a number of

reproductive problems such as retained placenta and abortions. Consumption of vitamin

A to meet requirement could improve conception rate or reduce days open for cows.

Pregnant Holstein cows were given 0, 300, or 600 mg/d of p-carotene or 120,000

IU/d of vitamin A from 4 wk before expected calving date to 4 wk postpartum (Michal et

al., 1994). Blood lymphocyte proliferation in response to 5 pg/mL of concanavalin A

(Con A) was greater at 1 wk before calving and at 2 wk postpartum in cows fed 600

mg/d of, -carotene compared to unsupplemented group. The phagocytic ability of









Staphylococcus aureus by neutrophils was enhanced in cows fed 300 mg/d of 3-

carotene at 1 wk after calving compared with cows in other treatments. Tjoelker and co-

workers (1990) conducted a trial to evaluate the impact of supplementation of vitamin A

and 3-carotene on neutrophil and lymphocyte function of dairy cows in the nonlactating

period. Cows were assigned randomly to one of 3 treatments: 53,000 IU of vitamin A,

213,000 IU of vitamin A, or 53,000 IU vitamin A plus 400 mg of p-carotene per d from 6

wk before to 2 wk after dry-off. Phagocytosis and bacterial killing ability of S. aureus by

neutrophils were not different among treatments, but lymphocyte blastogenesis was

stimulated by 10 ipg/mL of Con A on wk 2 for cows fed 53,000 IU of vitamin A but did not

change in other treatments throughout the experiment.

Whether supplemental vitamin A exerts these effects through its role as an

antioxidant is unknown.

c) Vitamin E

Vitamin E is the term for a class of lipid-soluble tocopherols (a, 8/, y, 5) and

tocotrienols (a, 8, y, 5), of which a-tocopherol has the highest biological activity

(Brigelius-Flohe and Traber, 1999; NRC, 2001). Vitamin E acts as a chain-breaking

antioxidant that limits the propagation of peroxidation by trapping free radicals (Nockels

et al., 1996; Brigelius-Flohe and Traber, 1999; Goupy P., 2007; Gobert et al., 2009). It

also exerts other functions involving cellular signaling, immunity and reproductive

function. Vitamin E is absorbed in the small intestine and enters the circulation via the

lymphatic system. It is absorbed with lipids, transported from the small intestine to the

liver, packed in lipoproteins, and distributed via plasma to the rest of the body (Herdt

and Smith, 1996). Although found in feedstuffs, the concentration and activity of vitamin









E is easy to lose during processing and storage of feedstuffs. Therefore,

supplementation of vitamin E in the diet is necessary. The common form of

supplemental vitamin E fed to dairy cattle is DL-a-tocopheryl acetate.

Plasma concentration of a-tocopherol was reduced during the last month

prepartum and at 1 or 2 wk postpartum (LeBlanc et al., 2002; Rezamand et al., 2007) in

dairy cows. Goff et al. (2002) reported a similar pattern in that the concentration of

vitamin E declined from 1 wk before calving to 3 DIM. A steady vitamin E state may be

reached by supplementation of 3000 IU/d for 2 wk prepartum to pregnant heifers

(Bouwstra et al., 2008). Oxidative damage during lipid peroxidation and its prevention

by vitamin E can be analyzed by quantification of 8-isoprostane (F2-isoprostanes). 8-

isoprostane concentration was reduced when overweight patients received 800 IU/d of

vitamin E for 3 mon and 1200 IU/d for another 3 mon compared with a placebo group

(Sutherland et al., 2007), indicating that supplementation of vitamin E may improve

oxidative status in obese humans. Two studies (Rimm et al., 1993, Stampfer et al.,

1993) in human species reported that high intakes of vitamin E reduced the risk of

cardiovascular diseases. Supplementation or injection of vitamin E to animals/humans

has reduced oxidative damage in the body.

d) Selenium

Many selenoproteins have Se in their structures to participate in the antioxidant

defense system of cells (Cerri et al., 2009). Considerable evidence exists that Se

functions by a similar mechanism as vitamin E in lipid peroxidation (Hamilton and

Tappel, 1963). Selenium is an integral part of the enzyme GPx which functions to

prevent oxidative damage to tissues or cells.









Dietary supplementation of 2 mg/d of Se in the form of sodium selenite increased

concentration of Se and GPx activity in blood of dairy cows during the periparturient

period compared to cows not supplemented with Se (Grasso et al., 1990). However,

feeding Se in the form of sodium selenite at 0.3 mg/kg of dietary DM from 25 d before to

70 d after calving did not influence incidence of postpartum diseases metritiss, ketosis,

and mastitis) and ovarian responses compared with cows fed Se yeast at the same rate

(Cerri et al., 2009) due to the lack of effects of source of dietary Se on Se status. A

similar dietary supplementation of Se study was conducted by Silvestre (2006), in which

they reported Se yeast reduced the risk of some postpartum uterine problems

compared with that of a supplemental inorganic source of Se. The disparity of effects of

supplementation of Se on health was likely due to differences in Se status due to

location. Cows on the study of Silvestre (2006) were managed in Florida using forages

grown on Se-deficient soils whereas cows used by Cerri et al. (2009) were managed in

California on Se-adequate soils. This was reflected by different blood concentrations of

Se.

Erskine et al. (1987) reported that somatic cell count (SCC) obtained from 9 dairy

herds decreased as concentration of plasma Se increased in cows. The GPx activity

was positively correlated with Se intake but negatively with SCC. Injection of 1 mg/kg of

BWof Se at 21 d prior to estimated calving date for dairy cows reduced the incidence of

mastitis by 12% compared to cows not receiving Se injection (Smith et al., 1984). The

effect of supplementation or injection of vitamin E and Se on mammary health has been

reviewed by Smith et al. (1997) who pointed out that vitamin E and Se deficiency were









associated with greater incidence of mastitis and greater SCC. Therefore alleviating

deficiencies of vitamin E and Se can enhance mammary health.

3) Synthetic Antioxidants

Three commonly used synthetic antioxidants are ethoxyquin, butylated

hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). These synthetic

antioxidants are used mainly by the feed industry to delay the peroxidation of feed lipids

and to stabilize the formulation of vitamin A and vitamin D3 in premises and feeds. In

addition, Kahl (1984) summarized that synthetic antioxidants have been associated with

a wide variety of molecular, cellular, and organ activity, roughly divided into three

categories: 1) modulation of growth, macromolecule synthesis and differentiation; 2)

modulation of immune response; and 3) interference with 02 activation.

Ethoxyquin is used widely as an antioxidizing agent in food formulation for animals

including fish, livestock and pets. It functions as a scavenger of free radicals which are

formed during lipid peroxidition (Yamashita, 2009). The FDA approved feeding rate of

ethoxyquin for use in animal feeds is 150 ppm (FDA, 2010). It has been used effectively

to promote color retention and preserve fat-soluble vitamins. However, its toxic effect in

vitro on phagocytosis by leukocytes isolated from swim bladder of tilapia was reported

by Yamashita et al. (2009) when concentration of ethoxyquin in media was > 0.1 mg/L.

They also reported phagocytic activity by inflammatory leucocytes isolated from fish fed

ethoxyquin at a rate of 150 mg/kg for 30 d was reduced compared with the activity in

fish fed no ethoxyquin.

Two additional synthetic antioxidants, BHA and BHT, are used as food additives

by the feed industry. Butylated hydroxyanisole is effective to preserve animal fats but

not vegetable oils. It is approved for human and animal use. Butylated hydroxytoluene









has similar properties. Bjorkhem et al. (1991) reported that BHT had an antiatherogenic

effect in cholesterol-fed rabbits. However, organ proliferation and histopathological

changes were induced by BHT and BHA in rat liver (Kahl, 1984).

More studies of the effects and mechanism of action of synthetic antioxidants in

animals need to be investigated in the future.

Overview of Immune Function

The immune system can be divided simply into two systems: innate and adaptive

systems. In the innate system, foreign bodies are destroyed and/or neutralized by an

array of cells and molecules that initiate immediate responses without any "memory"

about that foreign body. In the adaptive system, immune responses are the result of a

previous memory obtained by exposure to a particular antigen. Therefore, adaptive

immunity provides life-long protection against reinfection by the same pathogens

(Janeway, 2004). Both innate immunity and adaptive immunity responses depend upon

the activities of WBC or leukocytes. Neutrophils, macrophages (the mature form of

monocytes), and eosinophils are the primary cells to arrive at the infection site and

phagocytize pathogens or parasites without requiring memory (innate immunity).

Phagocytosis by macrophages and neutrophils is triggered by the binding of ligand to

the receptors, and subsequent destruction of pathogens takes place by complement or

by the generation of toxic chemicals, such as superoxide radicals, hydrogen peroxide,

and nitric oxide (Calder, 2007). Adaptive immune responses rely upon lymphocytes, i.e.

B cells and T cells. Therefore, the components of the immune system communicate and

work with each other to help hosts defend against infectious agents from the

environment.









Acute-phase proteins, as their names imply, are synthesized by the liver and

secreted into the blood to protect the host from local inflammation or stress. These

proteins mimic the action of antibodies but are nonspecific. Acid-soluble protein is one

acute-phase protein which contains mainly al-acid glycoprotein. It is an anti-

inflammatory agent that controls inappropriate or extended activation of the immune

system (Jafari et al., 2006). Acid-soluble protein has a dual immune-modulatory effect in

that it causes immune activation of macrophages to secrete cytokines or immune

suppression to control immune response. Haptoglobin (Hp) is another acute-phase

protein which binds to hemoglobin and so inhibits bacterial proliferation by reducing the

availability of iron (Wassell, 2000; Huzzey, 2009) and functions as an antioxidant by

virtue of its ability to prevent hemoglobin-driven oxidative damage of tissues (Melamed-

Frank et al., 2001). Many studies (Hirvonen et al., 1996; Wittum et al., 1996; Huzzey,

2009) have reported that an increase in plasma concentration of acute-phase proteins,

especially haptoglobin, is an indicator of severity or chronicity of sickness in cattle.

Effect of Feeding Antioxidants on the Immune System

Numerous studies have been conducted to evaluate the effects of

supplementation of antioxidants on neutrophil functions, acute-phase proteins and

immune challenge. A review of effects of vitamin E supplementation on health and

fertility of dairy cattle (Allison and Laven, 2000) indicated that Holstein calves receiving

up to 500 IU/d of dietary vitamin E increased the blastogenic responses of T cells and B

cells (Reddy et al., 1987). Injection of 3000 IU of vitamin E at 10 to 5 d before calving

increased the killing ability of bacteria by neutrophils at calving (Hogan et al., 1992).

Weiss and Hogan (2005) reported that supplementing Se to provide 0.3 mg/kg of

dietary DM from either sodium selenate or Se-yeast with 500 IU of vitamin E did not









affect the percentage of neutrophils that phagocytized E. coli in Holstein heifers or

cows, but bacterial killing ability by neutrophils tended to be increased for cows fed

selenite. Similarly, supplementation of vitamin E (400 to 600 mg/d) or Se (0.3 mg/kg of

dietary DM) alone to 21 multiparous Holstein cows increased the proportion of bacteria

killed by neutrophils, but did not influence phagocytic ability (Hogan et al., 1990).

Grasso et al. (1990) reported that cows supplemented with 2 mg/d of sodium selenite

during the transition period had greater bacterial killing ability by neutrophils in milk and

increased viability of neutrophils when challenged with S. aureus compared with cows

not supplemented with Se. The percentage of neutrophils that phagocytized and killed

Candida albicans was greater for cows that received sufficient Se (0.1 ppm of dietary

DM) than cows given a deficient Se diet (Boyne and Arthur, 1979). However, the

percentage of neutrophils that phagocytized C. albicans did not differ between cows fed

the two dietary treatments. Ascorbic acid (vitamin C) is considered to be the most

abundant and important water-soluble antioxidant. Functions of neutrophils (the

proportion of neutrophils that phagocytized bacteria and number of intracellular bacteria

per neutrophil) isolated from whole blood were not influenced by supplementation with

either 0 or 30 g/d of vitamin C starting from 2 wk before calving through 7 DIM (Weiss

and Hogan, 2007). Yamashita et al. (2009) evaluated the effect of a synthetic

antioxidant, ethoxyquin, on immunity of tilapia. The phagocytic activity of leucocytes in

vitro was lower in leucocytes exposed to 0.1 mg/L of ethoxyquin than leucocytes not

exposed to ethoxyquin. In summary, supplementation of natural antioxidants to dairy

cows improved killing ability by neutrophils measured as oxidative burst.









Bull calves were assigned randomly to one of three supplementation rates of

vitamin E (285, 570, and 1140 IU/d, respectively) for 21 d. They were vaccinated with 4

mL of ovalbumin (2 mg/mL of PBS). A linear increase in IgG concentration with

increased dietary supplementation of vitamin E was detected at 21 d after ovalbumin

ingestion (Rivera et al., 2002). Similar to this finding, calves vaccinated with 125 IU of

vitamin E at 7 wk of age had greater IgG values compared with those receiving no

vitamin E (Reddy et al., 1987). The enhancement of serum IgG to Pasteurella

haemolytica was detected in steers injected i.m. with 25 mg of Se and 340 IU of vitamin

E (Droke and Loerch, 1989).

Heifer calves were fed 2000 IU of vitamin E for 0, 7, 14, or 28 d (Carter et al.,

2002). No differences were detected in Hp concentrations in plasma among treatments

on any sampling day. However plasma concentrations of acid-soluble protein were

lower in calves fed 7, 14, or 28 d of vitamin E on d 7 of experimental period compared to

concentration of calves not receiving vitamin E. The effects of supplementation of

vitamin E on responses of antibody and acute-phase proteins were not consistent

possibly due to differing initial concentrations of vitamin E that may have made the

animals more or less susceptible to respond to additional vitamin E supplementation.

Effect of Feeding Synthetic Antioxidants on Performance

Few experiments have been conducted to examine the effects of supplementation

of synthetic antioxidants on the performance of dairy cattle. Feeding lactating Holstein

cows a basal diet containing distillers grains (15% of dietary DM) supplemented without

or with (0 or 0.02% of dietary DM) a blend of ethoxyquin, BHA and BHT (Agrado Plus,

Novus International, St. Louis, MO) did not affect DMI (26.6 0.5 kg/d) or milk yield

(49.8 1.7 kg/d) (Preseault, 2008). Although milk fat depression occurred in both control









and Agrado Plus supplemented treatment groups, the extent of milk fat depression

was less for cows fed Agrado Plus (3.22 vs. 3.32%). In another abstracted study (He,

2008), milk yield of dairy cows was not influenced by adding Agrado Plus (0 or 0.025%

of dietary DM) to six diets containing 1 of 5 vegetable oils (control, palm, high-oleic

safflower, high-linoleic safflower, linseed, or corn oil) at 5% of dietary DM.

However Bowman et al. (2008) reported that dairy cows fed Agrado Plus at 250

mg/kg of dietary DM increased DMI (22.2 vs. 22.7 kg/d) and tended to increase milk

yield (P < 0.10) (Bowman, 2008). Vazquez-An6n et al. (2008) also detected increased

DMI (20.2 vs. 20.9 kg/d, SEM = 0.22) and increased production of 3.5% FCM (27.3 vs.

28.3 kg/d, SEM = 0.36) when dairy cows (171 10 DIM) were fed Agrado Plus at 200

mg/kg of dietary DM.

An in vitro study using continuous cultures (Vazquez-Anon et al., 2008) was

conducted to investigate the effect of presence or absence of Agrado Plus on nutrient

digestibility, microbial N and fatty acid metabolism. This study was a 2 x 2 factorial

design in which two types of oil (oxidized vs. unoxidized mixture of unsaturated oil) and

supplementation or not of Agrado Plus at 200 mg/kg of dietary DM were combined.

Feeding Agrado Plus increased NDF and ADF digestion, and increased conversion of

feed N to microbial N, but tended to reduce the outflow of 18:3 in the effluent.

Few experiments have been conducted to investigate the effect of supplementary

synthetic antioxidants on performance of species other than dairy cattle. In poultry,

feeding laying hens ethoxyquin at 250 mg/kg of food from 32- to 88-wk of age did not

affect weight gain and egg production compared with hens fed control diet which

contained 5 mg of vitamin E and 125 mg of ethoxyquin/kg of food (Bartov et al., 1991).









Similar results have been reported for chicks in that dietary ethoxyquin fed at 0, 125,

500, and 1000 ppm did not affect body weight and weights of liver, spleen, kidney or

heart. Average carcass weights of broilers were greater when supplementing BHA at

the rate of 12.5 mg/d/bird from 3- to 7-wk of age and BHT at the same rate for the last 5

d of the trial compared with those only fed unoxidized sunflower oil at 55 g/kg of dietary

DM (Lin et al., 1989). Rainbow trout were utilized in a 2 x 2 x 2 factorial design with 2

feeding rates of oxidized fish oil (peroxide value of 5 and 120 meq/kg of oil), 2 feeding

rates of a-tocopheryl acetate (0 and 33 mg/kg of food), and 2 feeding rates of

ethoxyquin supplementation (0 and 125 mg/kg of food). Live weight gains and carcass

composition were not different among treatments (Hung et al., 1981). In summary

feeding ethoxyquin did not affect performance of chicks and fish.

Effect of Feeding Antioxidants on Oxidative Status and Stability

Dunkley et al. (1967) conducted a trial to evaluate the effects of supplementation

of either tocopherol at 0.0025% of dietary DM or ethoxyquin at 0.0125% of dietary DM

on oxidative stability of milk from dairy cows. Oxidative stability of the milk fat was

increased by the supplementation of tocopherol but not by ethoxyquin. A second

experiment was conducted to study the effect of feeding increasing dietary

concentrations of ethoxyquin (0.015 and 0.15% of dietary DM) on milk quality. They

reported an increase in endogenous tocopherol concentration and oxidative stability of

the milk when feeding either concentration of ethoxyquin but the improvement was

greater when more ethoxyquin was fed. The same research group also reported that

supplemental ethoxyquin in the diet was transferred to milk and accompanied by

appearance of an unidentified compound using a fluorimetric method (Dunkley et al.,

1968).









Oxidative status of mammals can be monitored by several markers such as SOD,

GPx, and TBARS. The activity of erythrocyte SOD was decreased compared with the

value before treatment after 3 injections of Vitamin E (i.m., 500 IU/injection) and Se

(i.m., 15 mg/injection) on alternate days up to the 5th day as a therapy for subclinical

ketosis. Comparing the activities of erythrocyte SOD after treatments, cows receiving

additional vitamin E and Se had the lowest SOD activity followed by those receiving 5

injections of 25% dextrose daily at 540 mL plus 1 injection of 4 mg/mL of

dexamethasone at 2.5mL, whereas cows receiving no treatment had the highest SOD

activity (Sahoo et al., 2009). Vazquez-Ah6n et al. (2008) reported that the activity of

plasma SOD was decreased (22.02 vs. 19.34 U/g of protein) for cows with vs. without

supplementation of 200 mg ofAgrado Plus /kg of dietary DM when an unoxidized blend

of unsaturated oil was fed, whereas SOD activity was increased (23.74 vs. 26.35 U/g of

protein) by feeding Agrado Plus with oxidized oil compared to cows without Agrado

Plus. The varied responses of activity of SOD may due to differences in degrees of

oxidative stress among animals used in the studies.

Usually supplementation of Se is correlated positively with GPx activity in RBC or

plasma. Hafeman et al. (1974) conducted a study of increasing concentrations of Se

supplementation (0, 0.005, 0.1, 0.5, or 1.0 ppm) on erythrocyte GPx of the rat. Activity

of GPx was increased markedly with increased concentration of Se in the diet. The

difference among these groups became greater as the diets were fed over a longer

period of time. Activity of plasma GPx was not different between cows fed an inorganic

vs. organic source of Se (0.3 mg/kg of dietary DM) from 25 d before calving to 70 DIM

(Cerri et al., 2009). The GPx activity in RBC of horses was increased with increasing the









distance of endurance race up to 80 km for horses under vitamin E (5000 IU/d of a-

tocopheryl acetate) supplementation and horses supplemented with 5000 IU/d of

vitamin E plus 7 g/d of ascorbic acid (Williams et al., 2004).

The supplementation of vitamin A (5000 IU/d), vitamin E (100 IU/d), and vitamin C

(50 mg/d) resulted in decreased concentration of plasma TBARS in HIV-infected

patients compared to the patients who received a placebo (Jaruga et al., 2002),

indicating the reduced production of lipid peroxides in liver. In another human study

involving 24 participants who were nonsmokers and not supplemented with vitamin

(Jialal and Grundy, 1993), concentrations of plasma TBARS did not differ between

volunteers who received 1.0 g/d of vitamin C, 800 IU/d of vitamin E, and 30 mg/d of

vitamin A and those receiving placebo capsules. Two amounts of vitamin E (0 or 1000

IU/d) and of Se (0 or 3 mg/kg of dietary DM) were studied for 6 wk using multiparous

dairy cows. Concentration of TBARS in erythrocytes was decreased by supplementation

of vitamin E regardless of Se supplementation (Brzezinska-Slebodzinska et al., 1994).

Holstein steers were fed supplemental vitamin E at 4 concentrations (0, 250, 500, or

2000 mg/d) for either 42 or 126 d. Concentrations of TBARS in meat increased with

increased length of display of retail cuts but accumulation of TBARS was less in beef

from vitamin E supplemented steers than from controls (Liu et al., 1996). Descalzo

and Sancho (2008) reported that meat from pasture-fed steers had lower TBARS

concentration than that from grain-fed steers. They suggested that cattle grazed on

good quality pasture incorporated enough vitamin E in their tissues to prevent lipid

peroxidation in meat. A 2 x 2 factorial design was arranged with two types of corn oil

(oxidized vs. unoxidized) and with or without Agrado Plus to test the effect of synthetic









antioxidants on shelf-life of pork after pigs were or were not given antioxidants. After 21

d in display case, the concentrations of TBARS in loin chop were lowest from pigs fed

fresh oil with Agrado, whereas TBARS were greatest in loin chop from pigs fed oxidized

oil without Agrado (D. M. Fernandez-Dueias, 2009). Supplementation of lipid-soluble

antioxidant effectively reduced the lipid peroxidation by scavenging peroxyl radicals

generated through the chain-reaction.

Limited studies have been published on the effects of synthetic antioxidants on

oxidative stability. Bartov et al. (1991) reported that initial oxidation of uterine tissue

from laying hens after 30 d of frozen storage as measured by thiobarbituric acid (TBA)

was reduced by supplementing ethoxyquin at 250 mg/kg of food compared with those

given 5 and 125 mg/kg of vitamin E and ethoxyquin, respectively. Lin et al. (1989)

reported that adding BHA at the rate of 12.5 mg/d/bird for 7 wk plus BHT at the same

rate for the last 5 d before slaughter with unoxidized sunflower oil increased the

oxidative stability of dark and while meat from boilers after 9 d of refrigerated storage in

contrast with meat from boilers given only unoxidized sunflower oil. In a study by Bailey

et al. (1996), 4 feeding rates of ethoxyquin (0, 125, 500, and 1000 ppm) were given to

chicks from 30-d to 6-wk of age. Both 500 and 1000 ppm ethoxyquin rates resulted in

lower TBARS concentration in liver and spleen tissues but not in kidney compared to 0

ppm, indicating that ethoxyquin is effective to reduce tissue peroxidation.

Murai and Andrews (1974) reported that channel catfish fed oxidized menhaden oil

had reduced lipid content of liver and lipid peroxidation of pelleted diets when

supplemented with 125 mg/kg of ethoxyquin.









Effect of Prepartum Heat Stress on Performance and Metabolites

Commonly, temperature-humidity index (THI) is used to indicate the degree of

heat stress on cattle (Armstrong, 1994; West, 2003). A THI above 72 indicates heat

stress such that performance and physiological status will be altered. Typically, major

responses to heat stress by dairy cattle include the following: reduced DMI and activity,

increased sweating, water intake, respiratory rate, body temperature and maintenance

requirement, and changes in metabolic and hormonal status (Fuquay, 1981; Armstrong,

1994; West, 2003). Ultimately, heat stress results in loss of milk production (Collier et

al., 1982, Rhoads et al., 2009).

Avendaho-Reyes et al. (2006) reported that non-cool cows which were only under

shades tended to have a greater respiratory rate (95.7 vs. 89.5 breaths/min) than

cooled cows which were cooled by soaking the entire body with a hose that delivered

approximately 25 L of 270C water/cow/cooling event for every 2 min each day from

1130 to 1430 h for a 60-d nonlactating period, but rectal temperatures and BWwere not

different between treatment groups. After calving, cows were moved to a common pen

with only shade. Milk production was numerically greater for cows exposed to the

cooled environment relative to cows exposed to heat stress (22.3 vs. 20.2 kg/d, but

proportion of milk fat and milk fat yield were not affected by treatment. Amaral et al.

(2009) reported that rectal temperatures in the afternoon were greater for cows offered

shade alone from dry off (60 d) until calving than cows offered fans and sprinklers. Milk

yield, milk fat content, and milk fat yield were greater for cows under prepartum

evaporative cooling even though all cows were cooled after calving. The DMI (as % of

BW) of cooled vs. noncooled cows were not different during the nonlactating period, but

was greater for cows under prepartum heat stress compared to those given evaporative









cooling from calving to 14 DIM. Pregnant dairy cows were assigned randomly to either a

shaded or a non-shaded group during the last 60 d of pregnancy (Collier et al., 1982).

After calving, all cows were cooled with fans and sprinklers. The shaded cows yielded

13.6% more milk for a 305-d lactation. Wolfenson et al. (1988) reported that prepartum

cows under shade alone had greater rectal temperature in the afternoon period than

cows under shade with fans and sprinklers (39.2 vs. 38.70C) but BCS did not differ

between treatments. Milk yield corrected for fat was increased by prepartum cooling

compared with noncooled cows during 150 d of lactation. A 2 x 2 factorial trial was

conducted using 112 growing replacement heifers in which animals were assigned to

one of 4 treatments: no shade or misting, only misting, only shade, or shade and

misting. Misted heifers had lower rectal temperatures and respiratory rates than

unmisted heifers. After 131 d on treatment, BWwas greater (520 vs. 547 kg) for shaded

than for the unshaded heifers (Mitlohner et al., 2001).

Amaral et al. (2009) reported that prepartum cows under shade alone had lower

NEFA in plasma at parturition and for the following 28 DIM compared with cows

exposed to shade plus fans and sprinklers. This result was due to lower DMI and

greater BCS for the cooled cows. They also detected lower BHBA in plasma from 14 to

28 DIM for prepartum cows only under shade than cows under shade with fans and

sprinklers. Pregnant multiparous Holstein cows were assigned randomly to 2 study pens

where cows were provided with either sprinklers over the feed bunk or sprinklers, fans,

and shade over the feed bunk around 30 d before calving during summer. After

parturition, cows were housed under identical conditions. Body condition scores and

serum NEFA were not different between treatment groups (Urdaz et al., 2006).









The somatic cell count (SCC) response of cows responding to heat stress is

consistent. Wegner et al. (1976) reported that cows under mild to severe heat stress

from June to November had increased SCC during the hot summer season (August to

October) than the rest of the study period. Mohammed and Johnson (1985) also

reported that the number of somatic cells increased 56% when cows were exposed to

heat stress (28.90C and 55% RH).

Effect of Prepartum Heat Stress on the Immune System

Nardone et al. (1997) conducted a trial in which primiparous Holsteins were

housed either in a cool (THI = 65) or hot environment (THI = 82 from 0900 to 2000 h,

THI = 76 from 2100 to 0800 h) from 3 wk before calving to 36 h after calving.

Concentration of IgG in colostrum was lower for cows housed in the hot vs. cool

prepartum environment. Secretion of IgM by PBMC isolated from cows calving in

summer (THI = 79) was greater than those from cows calving in spring (Lacetera et al.,

2005).

Several studies have been carried out to evaluate the effect of heat stress on

immune cell function in the bovine. However, the results of these studies are

inconsistent regarding lymphocyte function in cows exposed to a hot environment.

Soper et al. (1978) tested PBMC immunostimulation on lactating dairy cows every 2 wk

for 1 yr. The greatest PBMC response to mitogens was shown for 4- to 6- yr old cows in

August when heat stress occurred, whereas the least response was shown for 7- to 9- yr

old cows in Feburary. Contrary to this result, after in vivo heat stress of lactating

Holstein cows, the responses of polymorphonuclear leukocytes in vivo was not

influenced by heat stress, but the decrease in proliferation of lymphocytes isolated from

cows exposed to heat stress was less at 420C (Elvinger et al., 1991). The same lab









(Kamwanja et al., 1994) also reported that proliferation of phytohemagglutinin-

stimulated lymphocytes isolated from 3 Holstein cows was decreased when cells were

exposed to 420C compared with 38.50C. Lymphocytes were isolated from Holstein cows

and stimulated by Con A. Cows provided only shade prepartum had less proliferation

compared with those provided shade plus fans and sprinklers (Amaral et al., 2009). Yet

Lacetera et al. (2002) reported that the response of Con A-treated PBMC isolated from

calves exposed to a constantly hot environment (350C) was not different from those of

calves exposed to thermoneutral conditions. These variations may be due to the

duration of the exposure, intensity of heat stress, and immune function variables

measured (Kelley et al., 1982).

The Effect of Cooling Systems for Dairy Cows in Hot Environments

The characteristic climate in the southeastern United States is the high ambient

temperature and relative humidity. Hot and humid conditions are associated with

numerous physiological changes that occur in the digestive system, acid-base

chemistry, and blood hormones (West, 2003). In order to improve cow performance in

such conditions, alterations of the cow's environment have been developed.

Shading.

Firstly, shade structures should be used to reduce the direct and indirect solar

radiation reaching the animals during the day (Ryan and Boland, 1992; West, 2003).

They have little effect on changing ambient temperature and humidity (Buffington et al.,

1981).

Shading during the prepartum period. Reducing the negative effect of heat

stress by shading for nonlactating and pregnant cows has shown benefits. Two studies

evaluated the effect of heat stress relief by shading during the last 80 d of pregnancy on









prepartum and postpartum responses of Holstein cows (Collier et al., 1982; Lewis et al.,

1984). Upon calving, all cows were managed uniformly. Cows with shade during the

last 80 d of gestation had numerically greater milk yields in the subsequent lactation,

lower rectal temperatures and respiratory rates, greater thryoxine and lower NEFA

concentration in plasma, and gave birth to heavier calves (the difference about 3 kg)

compared with cows offered no shade (Collier et al., 1982). Prepartum shading reduced

postpartum plasma concentrations of prostaglandin F2a, but increased rectal

temperatures compared with non-shaded cows (Lewis et al., 1984).

Shading during the postpartum period. Providing shade to cows in midlactation

for 11 wk during summer in Florida resulted in lower rectal temperatures (38.9 vs.

39.40C), reduced respiratory rates (54 vs. 82 breaths/min), and about 10% more milk

yield (Roman-Ponce et al., 1977). Similarly, mid-lactating cows with shade for 102d had

lower rectal temperatures (38.7 vs. 39.60C) and respiratory rates (79 vs. 115

breaths/min), increased ruminal contractions (2.3 vs. 1.6 times/min), and greater milk

yield corrected for stage of lactation (15.1 vs. 12.7 kg/d) compared with cows with no

shade indicating that environmental modification altered physiological responses of the

cows (Collier et al., 1981).

Mechanical Cooling.

Air movement (fans), wetting the cow (sprinklers and sprayers), evaporation to

cool the air (misting), and their combinations are effective to enhance heat dissipation.

Mist precools the air by evaporation before it reaches the hair coat and respiratory

system of the cows, whereas sprinklers and sprayers dampen the hair and skin of the

cows, increase the rate of evaporation and subsequent heat removed from the skin The

main disadvantage of sprinklers is that they create an environment saturated with water









which markedly reduces the capability of animals to dissipate heat by evaporation

(Flamenbaum et al., 1986).

Cooling during the prepartum period. Cooling with sprinklers and fans during

the last 60 d of gestation reduced rectal temperatures (38.7 vs. 39.0C), increased 150-

d lactation mean milk yield by 3.5 kg/d, and increased calf birth weights by 3.3 kg

compared to cows managed under shade only. Prepartum cooling may alleviate any

detrimental effects of heat stress on the development of mammary parenchyma or its

lactogenic capacity. The effect of adding shade and fans to a sprinkler system for

periparturient cows was evaluated in California (Urdaz et al., 2006). Shade with

sprinklers and fans during the last 3 wk of gestation did not affect either the incidence of

postparturient disorders/diseases or serum concentration of NEFA in the prepartum

period compared with cows managed with sprinklers only but milk yield was increased

by 1.4 kg/d.

Cooling during the postpartum period. Two evaporative cooling systems (Korral

Kool vs. fans and sprinklers) were evaluated for lactating dairy cows for a 142-d period

from the end of May through the middle of October (Ryan and Boland, 1992). The mean

rectal temperature (39.0 vs. 38.80C) and milk production (27.7 vs. 26.8 L/d) of cows

managed with Korral Kool compared with cows managed with fans and sprinklers.

Correa-Calderon et al. (2004) also reported that using a Korral Kool cooling system for

lactating Holstein cows for 18 wk reduced rectal temperature by 0.90C compared with

cows under shade only. Flamenbaum et al. (1986) reported that mean rectal

temperature of Israeli-Holstein lactating cows was reduced by 0.60C by cooling

(sprinklers and forced ventilation) for 5 cooling periods of 30 min each during the day









compared with cows under no cooling system. Shading and cooling cows during late

gestation and postpartum will improve subsequent lactation performance and may result

in heavier calves at birth.









CHAPTER 3
EFFECT OF FEEDING ANTIOXIDANTS AND PREPARTUM EVAPORATIVE
COOLING ON PERFORMANCE OF TRANSITION HOLSTEIN COWS DURING
SUMMER IN FLORIDA

Introduction

Dairy cattle undergo tremendous physiological and nutritional changes during the

periparturient period. As a result they might experience a variety of metabolic disorders

ketosiss, hepatic lipidosis, displaced abomasum, and hypocalcemia) and infectious

diseases (retained fetal membranes, mastitis, endometritis/metritis) (Goff and Horst,

1997; Bernabucci et al., 2005; Sordillo et al., 2007; Sordillo and Aitken, 2009). Many

disorders and diseases during this special period are associated with suppressed host

defense mechanisms (Sordillo, 2005; Sordillo et al., 2009) and oxidative stress.

Oxidative stress results from excess production of reactive oxygen species (ROS) and

insufficient antioxidant production to remove these ROS (Miller et al., 1993; Bernabucci

et al., 2002, 2005; Castillo et al., 2005; Sordillo et al., 2007).

Recently, clinical medicine has given increased attention to the detection and

protection of ROS (Castillo et al., 2003). Oxidative status can be monitored by several

biomarkers, such as superoxide dismutase (SOD) which is the enzyme that catalyzes

the dismutation of superoxide radicals to hydrogen peroxide and oxygen, glutathione

peroxidase (GPx) which is a Se-dependent enzyme that decomposes hydrogen

peroxide, thiobarbituric acid reactive substances (TBARS) which present a composite

number of lipid oxidative end products including malondialdehyde and indicates the

level of lipid peroxidation (Trevisan et al., 2001; Bernabucci et al., 2005), and total

antioxidant status (TAS) which provides more overall evaluation of oxidative status.









The relationship between heat stress and oxidative status in dairy cattle has been

examined only minimally. Activities of SOD, GPx and concentration of TBARS in

erythrocytes as indicators of increased oxidative challenge were increased during heat

stress (Bernabucci et al., 2002; Saker et al., 2004), whereas a decrease in GPx activity

by peripheral blood mononuclear cells (PBMC) was detected in spite of heat stress

(Burke et al., 2007).

Supplementation with 200 mg/kg of dietary synthetic antioxidant (Agrado Plus,

Novus International, St. Charles, MO) increased plasma SOD activity when cows were

fed oxidized rather than fresh oil and increased plasma GPx activity across both types

of oil fed (Vazquez-Ai6n et al., 2008). The effect of supplementation of synthetic

antioxidants on oxidative status and performance of dairy cattle during the transition

period is limited.

The aim of this study was to evaluate whether the supplementation with a blend of

synthetic antioxidants (Agrado Plus) would ameliorate the expected negative effect of

heat stress on performance and oxidative status of Holstein cows.

Material and Methods

Animals, Treatments, and Management

The experiment was conducted at the University of Florida Dairy Research Unit

(Hague, FL) during the months of July through December 2008. All experimental

animals were managed according to the guidelines approved by the University of

Florida Animal Research Committee. Periparturient Holstein primiparous (n = 22) and

multiparous (n = 13) cows were blocked by parity and were assigned to treatments at 29

7 days prior to their calving date. Four treatments were arranged in a 2 x 2 factorial

design including 2 dietary concentrations of antioxidants (0 vs. 250 mg/kg of dietary DM,









AO) and 2 environmental housing conditions for pregnant cows. The AO is a liquid

mixture of ethoxyquin and tertiary-butyl-hydroquinone which was added to corn oil by

the manufacturer and shipped to the research site. The corn oil was mixed with ground

corn just prior to preparing the concentrate portion of the diet in 909 kg batches. The

corn oil was devoid of commercial antioxidants except AO. Pregnant cows were housed

in an open-sided free-stall barn with sand bedding equipped with or without fans (J & D

Manufacturing Eau Claire, WI) and sprinklers (Rainbird Manufacturing, Glendale, CA) in

cooled vs. noncooled treatments, respectively. Sprinklers were intermittently operated

every 6 min for 1.5 min. Lights were operational from 0600 h to 2000 h. Calan gates

(American Calan Inc., Northwood, NH) were used to measure DMI of individual cows.

Pregnant cows were fed twice daily ad libitum amounts of a bermudagrass silage-corn

silage-based TMR (Table 3-1). Refusals of TMR were measured daily. Rectal body

temperatures were recorded daily between 1430 and 1530 h using a GLA M700 digital

thermometer (GLA Agriculture Electronics, San Luis Obispo, CA). After calving, all

animals were moved to a sand-bedded, open-sided, free-stall barn equipped with fans

and sprinklers and Calan gates. Cows were milked twice daily at 0700 and 2000 h and

fed in ad libitum amounts a corn silage-alfalfa hay-based TMR twice daily at 0800 and

1300 h for 7 weeks. Dry matter intake was recorded daily. Prepartum and postpartum

cows were weighed on the same day each week before the morning feeding. Rectal

body temperature was measured on 4, 7, and 12 DIM using a GLA M700 digital

thermometer.

Sample Collection and Analysis

Representative samples of corn silage, bermudagrass silage, alfalfa hay and grain

mixes were collected weekly, composite monthly and ground through a 1-mm Wiley









mill screen (A. H. Thomas, Philadelphia, PA). Silage and hay samples were ground

before compositing whereas grain mix samples were composite before grinding.

Composited feed samples were analyzed for CP using a macro elemental analyzer

vario MAX CN (Elementar Analysensystene GmbH, Hanau, Germany), NDF (Mertens,

2002), ADF (AOAC, 1995), ether extract (AOAC, 2003), and minerals (Dairy One,

Ithaca, NY). The chemical composition of diets are shown in Table 3-2.

Milk samples were collected from two consecutive milkings weekly using bronopol-

B-14 as a preservative, and analyzed for true protein, fat, and SCC by Southeast Milk

laboratory (Belleview, FL) using a Bently 2000 NIR analyzer. Final concentrations of fat

and protein were calculated after adjusting for milk production during those 2 milk

collections.

Blood samples were collected at 0900 h on Monday, Wednesday and Friday

weekly from the coccygeal vessels into sodium heparinized tubes (Vacutainer, Becton

Dickinson, Franklin Lakes, NJ) from calving until 49 DIM. Blood samples were placed on

ice immediately after collection until centrifuged at 1200 x g at 40C for 15 min (Allegra

X-15R Centrifuge, Beckman Coulter). Plasma was separated after centrifugation and

stored at -200C for subsequent metabolite and hormone analyses.

Plasma concentrations of NEFA (NEFA-C kit; Wako Diagnostics, Inc., Richmond,

VA; as modified by Johnson, 1993) and BHBA (Wako Autokit 3-HB; Wako Diagnostics,

Inc., Richmond, VA) were determined weekly for 7 wk. A Technicon Autoanalyzer

(Technicon Instruments Corp., Chauncey, NY) was used to determine weekly

concentrations of plasma BUN (a modification of Coulombe and Favreau, 1963 and

Marsh et al., 1965) and plasma glucose (a modification of Gochman and Schmitz,









1972). Concentrations of progesterone were determined on all plasma samples

collected using Coat-A-Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA)

solid phase 1251 RIA. The sensitivity of the assay was 0.02 ng/mL and the intra-assay

CV was 3.8%.

Processing of Red Blood Cell (RBC)

Approximately 7 mL of blood was collected on -15, 1, 8, 15, and 29 DIM from the

coccygeal vessels into evacuated tubes containing 17.55 mg of K2 EDTA (Vacutainer,

Becton Dickinson, Franklin Lakes, NJ, USA). The blood samples were placed on ice

immediately following collection and transported to the laboratory within 3 h. To

measure haematocrit, blood was drawn into a haematocrit capillary tube (Fisher

Scientific, Cat. No. 22-362-566) and centrifuged for 2.5 min. Plasma and RBC were

separated using a refrigerated centrifuge operating at 1200 x g for 10 mins at 4 C

(Allegra X-15R Centrifuge, Beckman Coulter). Plasma was stored at -800C until

analyzed for TBARS. The RBCs were processed as follows. The buffy coat leukocytess)

was discarded via pipette. The RBC (2 mL) were transferred using a positive

displacement pipette to a 13 x 100 mm polypropylene tube. Cold (40C) physiological

saline (4 mL) was added to the RBC, capped, and gently mixed. The mixture was

centrifuged at 1200 x g for 10 min, and saline pippetted off. This washing process was

repeated 2 more times for a total of 3 saline washes. After the third saline wash, the

washed RBC were lysed by adding 2 mL of cold (40C) UltraPure water (Cayman

Chemical, Catalog No. 400000, Ann Arbor, MI) at which time a dark maroon color was

achieved upon vortexing. Samples (400 uL) were stored in triplicate vials at -800C for

analysis for superoxide dismutase (SOD) and glutathione peroxidase (GPx),

respectively.









Thiobarbituric Acid Reactive Substances (TBARS) Assay

Plasma concentrations of TBARS were determined by the method modified by

Armstrong et al. (1998). Briefly, fresh thiobarbituric acid (TBA) buffer was made (200

mL) by adding 40 mL of glacial acetic acid and 1.060 g of TBA to 160 mL distilled water.

Sodium dodecyl sulfate (SDS) reagent (8.1%) was prepared by mixing 8.1 g of SDS

with 100 mL of distilled water. Malondialdehyde (MDA) stock solution (100 nmol/mL)

was made by mixing 82 uL of MDA (Sigma-Aldrich Inc., cat no. 108383, St. Louis, MO)

and 1 mL of concentrated HCI with 100 mL of distilled water. One portion of stock MDA

and 9 portions of distilled water were combined to make the MDA working solution (10

nmol/mL). Working MDA standards of 0, 1, 2, 3 and 4 nmol/mL were prepared by

adding 0, 10, 20, 30, and 40 uL of MDA working solution respectively to 100, 90, 80, 70,

and 60 uL of distilled water. In each labeled test tube, 100 uL of diluted sample (1:100)

or working MDA standards, 100 uL of SDS reagent, and 2.5 mL of TBA buffer were

added by force in order to mix them well. Tubes were covered with a marble and

incubated at 95C for 1 h. After incubation, tubes were placed in an ice water bath for

10 min. Lastly, fluorescent readings were obtained using a florescence

spectrophotometer (RF-1501, Shimadzu International, Columbia).

Superoxide Dismutase (SOD) Assay

The SOD activities of RBC were measured using the Superoxide Dismutase

Assay Kit (Cayman Chemical, Catalog No. 706002, Ann Arbor, MI). This assay utilizes a

tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and

hypoxanthine. All 3 types of SOD (Cu/ZnSOD, MnSOD and FeSOD) are measured in

this 96-well microplate assay. Briefly, 2.5 mL of concentrated assay buffer was diluted

with 22.5 mL of HPLC-grade water. Concentrated sample buffer (2.5 mL) was diluted









with 22.5 mL of HPLC-grade water. Diluted assay buffer and sample buffer was kept at

room temperature while performing the assay. Erythrocyte lysate samples (10 iiL) from

15 d before expected calving date, 1, 8, 15, and 29 DIM were thawed and diluted 5000

times with diluted sample buffer. The SOD Stock standard was prepared by diluting 20

piL of concentrated SOD standard with 1.98 mL of diluted sample buffer. Working

standards of 0, 0.025, 0.05, 0.1, 0.15, 0.2, and 0.25 U/mL were made by adding 0, 20,

40, 80, 120, 160, and 200 piL respectively of SOD standard stock to 1000, 980, 960,

920, 880, 840, and 800 piL of diluted sample buffer. Diluted samples and working

standards were kept on ice. Diluted radical detector was obtained by diluting 50 piL of

concentrated radical detector with 19.95 mL of diluted assay buffer. The wells on a 96-

well plate were categorized as SOD standard wells and sample wells. Each

standard/sample was measured in duplicate. For the SOD standard wells, 200 uL of the

diluted radical detector and 10 uL of working SOD standard (7 standards) were added

to each corresponding well. For sample wells, 200 piL of the diluted radical detector and

10 piL of the diluted sample were added to each corresponding well. After this step,

xanthine oxidase was prepared by adding 50 piL of the supplied enzyme to 1.95 mL of

sample buffer. As quickly as possible 20 piL of xanthine oxidase was added to each well

to initiate the reaction. The plate was covered, incubated on a shaker for 1 h, and read

at 450 nm using a plate reader (SpectraMax 340PC384, Molecular Devices, CA).

Superoxide dismutase activity was calculated based on the standard curve. Enzyme

activity was expressed as units per milliliter of packed cell volume (PCV). Plates

contained samples from each treatment and all the samples from the same cow were









analyzed in the same plate. This method was used for all variables measured using the

microplate reader.

Glutathione peroxidase (GPx) Assay

The GPx activities of erythrocytes were measured using the assay kit supplied by

Cayman Chemical Company (catalog no. 703102, Ann Arbor, MI). This assay measures

GPx activity indirectly by a coupled reaction with glutathione reductase (GR).

GPx
ROOH + 2GSH ROH + GSSG + H20

+ GR
GSSG + NADPH + H o 2GSH + NADP

The procedure of this assay is as below. The assay buffer and sample buffer were

diluted 10 fold and kept at room temperature. Erythrocyte lysate samples from 15 d

before expected calving date, 1,8, 15, and 29 DIM were thawed and diluted 500 times

with diluted sample buffer. Ten iil of supplied glutathione peroxidase was diluted with

490 uL of diluted sample buffer. Cayman sells a 96-well kit and a 480-well kit. The 96-

well kit contains 3 vials of co-substrate mixture. Each vial was reconstituted by adding 2

mL of HPLC-grade water. The 480-well kit contains 5 vials of co-substrate mixture.

Each vial was reconstituted by adding 6 mL of HPLC-grade water. The diluted samples,

enzyme, and co-substrate mixture were kept on ice at all times. The wells in a 96-well

plate were categorized as background wells, positive control wells, and sample wells.

For background wells, 50 piL of co-substrate mixture and 120 piL of assay buffer were

added to each well. For positive control wells, 20 piL of diluted enzyme, 50 piL of co-

substrate mixture and 100 uL of assay buffer were added to each well. For sample

wells, 20 piL of diluted RBC sample, 50 piL of co-substrate mixture, and 100 uL of assay









buffer were added to each well. The reaction was initiated by adding 20 piL of cumene

hydroperoxide to all wells as quickly as possible. The plate was covered and shaken for

a few seconds. Lastly, plate was read at 340 nm once every minute for at least 6 min.

GPx activity was calculated using the formula below:

GPx activity = AA34/min .19 ml x sample dilution = nmol/min/ml
0.00373 0.02 ml

AA340/min was calculated by subtracting the change in absorbance per minute for

the background from the change in absorbance per minute for the sample. Enzyme

activity was expressed as nmol/min/mL, which is the amount of enzyme used to oxidize

1.0 nmol of NADPH to NADP+ per minute at 25 C. Enzyme activity was expressed as

nmol per min per mL of PCV.

Acute Phase Protein Assays

Plasma samples collected on Monday-Wednesday-Friday were used to determine

the concentrations of acid soluble protein (ASP) and haptoglobin (Hp). Acid soluble

protein was extracted from 50 piL of plasma with 1 mL of 0.6 M perchloric acid after 20

min of incubation at room temperature in duplicate. Tubes were centrifuged at 1200 x g

for 30 min at room temperature. Supernatant was analyzed with the bicinchoninic acid

kit (Sigma-Aldrich, Saint Louis, MO; Cat No. 096k9802). Concentrations of unknowns

were obtained from the standard curve. The inter-assay variation was 9.2%. If CV%

between replicates was greater than 10%, samples were re-analyzed.

Concentrations of plasma Hp were determined by measuring the differences of

hydrogen peroxide activity with haptoglobin-hemoglobin (Hb) complex (modified by

Tarukoski 1966). Briefly, O-dianisidine solution (4 L) was prepared by adding 2.4 g of O-

dianisidine, 2.0 g of Na2EDTA, and 55.2 g of NaH2PO4 to 4 L of distilled water and









adjusted to pH of 4.1. Hemoglobin stock solution (25 uL) was added to each tube to

form a Hb-Hp complex with Hp from 5 uL of plasma sample. O-dianisidine solution (7.5

mL) was added to each tube. Tubes were incubated in a water bath for 45 min at 370C.

Then 100 uL of hydrogen peroxide were added to react with Hb-Hp complex to liberate

oxygen which oxidized O-dianisidine to a yellow color compound. Samples from the

tubes were transferred to the 96-well plate and read at 450 nm using the microplate

reader.

Neutrophil Function, WBC and Lymphocyte

Blood (6 mL) was collected from coccygeal vessels at- 21, 0, 7, and 14 DIM in

vacutainer (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) tubes containing acid

citrate dextrose. Tubes were not put on ice but were gently mixed by hand

approximately every 15 min. Counts of WBC, lymphocytes, and neutrophils were done

using a Bayer Advia 120 cell counter (Fisher Diagnostic, Middletown, VA) within 3 h of

collection. Phagocytosis and oxidative burst by neutrophils were assessed within 3 h

after blood collection. Neutrophil, WBC and lymphocyte concentrations were estimated

by a demacytometer. Whole blood (100 uL) was pipeted into each of 3 tubes. Then 10

pL of 50 pM dihydrorhodamine 123 (DHR) (Sigma-Aldrich, Saint Louis, MO) was added

to all tubes. Tubes were vortexed and incubated in an oven at 370C for 10 min with

constant rotation using the Clay Adams nutator (BD Bioscience, San Jose, CA). Ten pL

of 20 pg/mL solution of phorbol 12-myristate, 13-acetate (PMA) (Sigma-Aldrich, St.

Louis, MO) were added to the second tube only. An Escherichia col bacterial

suspension (109 cells/pL) labeled with propidium iodide (Sigma-Aldrich) was added to

the third tube to establish bacteria to neutrophil ratio of 40:1 (Escherichia col strain

were isolated from a dairy cow with mastitis and grown in vitro for labeling). Tubes were









vortexed and incubated in oven at 370C for 30 min with constant rotation using the Clay

Adams nutator (BD Bioscience, San Jose, CA). Then all tubes were removed and

placed immediately on ice to stop phagocytosis and oxidative burst activity. Tubes were

processed in a Q-Prep Epics immunology workstation on the 35 cycle. Cold distilled

water (500 pL) and 0.4% tryphan blue (10 pL) were added to each tube. Then tubes

were vortexed, kept on ice, and 10,000 neutrophil cells were read using the Facsort flow

cytometer (BD Biosciences, San Jose, CA). The percentage of total neutrophils able to

phagocytize E. coli, undergo oxidative burst, and efficiency of bacteria killing (mean

fluorescence intensity, MFI) were measured using the flow cytometer.

Ovalbumin Challenge

All cows were injected i.m. with 1 mg of ovalbumin (Sigma-Aldrich, Saint Louis,

MO) diluted in 1 mL of sterile Quil A adjuvant (0.5 mg of Quil A/ mL of PBS) (Accurate

Chemical & Scientific Corp. Westbury, NY) at -4 and -2 wk relative to expected calving

date and at calving. Blood samples (8 mL) for measurement of anti-ovabumin IgG were

collected at -4, -2, 0, 1, 2, 3, 4, and 7 wk relative to calving. Samples were taken in

vacutainer tubes containing no anticoagulant before the ovalbumin injection. Serum

concentration of anti-ovalbumin IgG was measured by an Enzyme Linked

ImmunoSorbent Assay (ELISA) as described by Mallard et al. (1997). Briefly, flat bottom

96-well polystyrene plates (Immulon 2, Dynex Tech., Chantilly, VA) were coated with a

solution of ovalbumin dissolved in carbonate-bicarbonate coating buffer (1.4 mg OVA/

mL of carbonate-bicarbonate buffer). Plates were incubated at 40C for 48 h, then

washed with PBS and 0.05% Tween-20 solution (pH = 7.4). Plates were blocked with a

PBS-3% Tween-20 and bovine serum albumin (Sigma Chemical, St. Louis, MO)

solution and incubated at room temperature for 1 h. Plates were washed and serum









samples and control sera diluted at 1/50 and 1/200 were added in duplicate using a

quadrant system (Wright, 1987). Positive and negative control sera to anti-ovalbumin

IgG were obtained from a pool of sera of known high (21 d after the third injection of

ovalbumin) and low (no ovalbumin injection) concentrations, respectively. All samples

from the same cows were analyzed in the same plate and plates contained balanced

number of animals from each diet group. Plates were incubated at 240C for 2 h and

washed with the previously described buffer solution. Subsequently, alkaline

phosphatase conjugate rabbit antibovine IgG whole molecule (Sigma Chemical, St.

Louis, MO) was dissolved in Tris/HCI Buffer, added to the plates and incubated for 1 h

at room temperature. After incubation, plates were washed 4 times and substrate

solution [P-nitrophenyl phosphate disodium (Sigma Chemical, St. Louis, MO)] was

added and the plate was incubated at room temperature for 30 min. Plates were read

on an automatic ELISA plate reader (MRX Revelation; Dynex Technologies Inc.,

Chantilly, VA) and the optical density was recorded at 405 nm and the reference at 650

nm.

Vaginoscopy

Cows were evaluated for cervical discharge on 7, 16, and 25 DIM using the

metricheck (Metricheck, Simcro, New Zealand) tool. The vulva was cleaned using a

povidone-iodine scrub (0.75% titratable iodine and 1% povidone solution, Agripharm,

Memphis, TN, USA) and dried with clean gauze. The metricheck was inserted in the

vagina close to the cervix. The floor of the vagina was scraped, the discharge collected

in a 50-ml conical tube (Fisher Diagnostics, Middletown, VA), and scored according to

Sheldon et al. (2006). Scoring system was as follows: 0 = translucent or clear, 1 =

flecks of white or off-white pus, 2 = discharge containing < 50% white or off-white









mucopurulent material, 3 = discharge containing > 50% white or yellow mucopurulent

material, 4 = discharge containing > 50% sanguineous mucopurulent material.

Uterine Cytology

At 40 2 days postpartum, one of the uterine horns was flushed with saline. After

thorough sanitation of the vulva and entrance of the vagina using chlorhexidine

diacetate (Nolvasan, Fort Dodge, Overland Park, KS), an 18 French, 30 mL, 56 cm, 2-

way Foley catheter was placed randomly in 1 uterine horn at approximately 2 cm past

the bifurcation of the uterus. The cuff of the catheter was inflated with 7 to 10 mL of air

according to the diameter of the uterine horn, and 20 mL of sterile isotonic saline

solution was infused into the uterine horn and then recovered using a 35 mL sterile

syringe. The aspirated fluid was transferred to a 50 mL sterile conical tube, placed in ice

and transported to the laboratory within 4 h of collection. In the laboratory, the aspirated

fluid was centrifuged at 750 x g for 10 min and the supernatant discarded. The pellet

was resuspended with 2 mL of saline, and an aliquot of 20 uL was pipetted onto glass

slides and smeared in triplicate. Smears were air-dried and stained using a Diff-Quick

(Fisher Diagnostics, Middletown, VA) stain. Slides were examined under a microscope

and the number of total leukocytes, epithelial endometrial cells, and neutrophils were

counted to complete a 100 cell-count per slide, and percentage neutrophiles was

calculated. Subclinical endometritis was diagnosed when the proportion of neutrophils

exceeded 5% after 30 DIM (Gilbert et al., 2005).

Statistical Analyses

Treatments were arranged in a 2 x 2 factorial completely randomized design.

Repeated measurements were made on nearly all variables and were analyzed using

the PROC MIXED procedure of SAS (Release 9.2) according to the following model:









Yjkl = p + ai + (j + (a3)ij + Yk + (ca)ik + (PY)jk + (C3Y)ijk + CI(ijk) + Wm + (aW)im +

(3WV)jm + (apW)ijm + (YW)km + (ayW)ikm + (3yW)jkm + (a3YW)jkm + Eijklm

where Yjkl is the observation, p is overall mean, ai is the fixed effect of diet (i = 1,

2), P3j is the fixed effect of environment (j = 1, 2), (ap)ij is the interaction of diet and

environment, Yk is the fixed effect of parity (k = 1, 2), (ay)ik is the interaction of diet and

parity, (3p)jk is the interaction of environment and parity, (apy)ijk is the interaction of diet,

environment, and parity, Cl(ijk) is the random effect of cow within diet and environment

and parity (I = 1, 2, ... n) + WI is the fixed effect of week (I = 1, 2, 3, 4, 5, 6, 7), (aW)il is

the interaction of diet and week, (3W)jl is the interaction of environment and week,

(aP3W)ij is the interaction of diet, environment, and week, (yW)ki is the interaction of

parity and week, (ayW)iki is the interaction of diet, parity, and week, (P3yW)j is the

interaction of environment, parity, and week, (apyW)ijkl is the interaction of diet,

environment, parity, and week, and Eijkm is the residual error. Different temporal

responses to treatments were further examined using the SLICE option of the MIXED

procedure. Mean treatment, parity, and time (week or day relative to calving) effects are

presented as least square means. A covariate representing the initial measurement for

IgG against ovalbumin and BCS of individual cows was included in the model.

Data were tested to determine the structure of best fit, namely AR(1), ARH(1), CS,

or CSH, as indicated by a lower Schwartz Baesian information criterion value (Littell et

al., 1996). If repeated measures were taken on unequally spaced intervals, the sp(pow)

covariance structure was used.

Measures of blood cell numbers, neutrophil function, acute-phase proteins,

oxidative markers, and IgG against ovalbumin were tested for normality before and after









transformation (log or square root transformation) by using the PROC UNIVARIATE

procedure of SAS (SAS Institute, 2007). Probability values > 0.05 using the Shapiro-

Wilk test for variables were considered normal. After data were transformed, the PROC

MIXED procedure with the same model described above was used. Progesterone data

(DIM of first ovulation, number of ovulations, peak concentration of progesterone in the

first ovulation, the length of the first cycle, and accumulated progesterone over 49 DIM)

were analyzed using Proc MIXED of SAS with the accumulated progesterone analysis

requiring the repeated measures function. Metricheck scores were analyzed using

logistic regression with the Odds-Ratio option. If cervical discharge of cows was scored

as 0 or 1, cows were classified as healthy whereas scores of 2, 3, or 4 resulted in a

diagnosis of metritis/endometritis. These reclassified scores were analyzed as binomial

data. Uterine cytology data were used to classify cows as either clean or with

subclinical endometritis. This binary data also was analyzed using logistic regression

techniques. Differences discussed in the text were significant at P < 0.05 and tended to

be significant at 0.05 < P < 0.15.

Results and Discussion

Body Temperature, BW, BCS, and DMI

In the prepartum period, cows stayed in the assigned freestall barn area for 29 7

d before calving. However the data for body temperature, BW, and DMI are reported

only for the last 21 d prepartum because most cows contributed data during this time

period. Mean rectal temperature over the last 21 d prior to calving was less for cooled

vs. noncooled cows (39.2 vs. 39.60C, P < 0.001, Table 3-3) as expected. This difference

was consistent across prepartum days as tests of all interactions of week with

treatments were not significant. Rectal temperatures above 39.20C may represent









reduced ability of lactating dairy cows to adapt to thermal stress resulting in reduced

performance (Staples and Thatcher, 2003). Therefore providing fans and sprinklers to

shaded freestalls helped reduce heat stress and established the planned differences of

prepartum environments for cows assigned to the study.

Prepartum intake of DM did not differ among the treatment groups, averaging 10

kg/cow per day (Table 3-3). However effect of treatment on DM intake when expressed

as a percentage of BW differed according to parity. Feed intake (% of BW) by

primiparous cows was unaffected by treatments but DMI by multiparous cows fed the

control diet was decreased by cooling whereas intake was increased by cooling when

cows were AO (diet by cooling by parity interaction, P = 0.03, Figure 3-1). Typically

cows under significant heat stress reduce DMI. Multiparous cows fed the AO diet

demonstrated this response. The DMI of multiparous cows fed the control diet and

evaporatively cooled responded unexpectedly as mean DMI was only 1.09% of BW.

Multiparous cows on this treatment were much heavier than those in the other groups,

averaging 843 kg, about 147 kg heavier than multiparous cows on the other treatments

(Table 3-3). Mean prepartum BW of primiparous cows did not differ across treatments

resulting in a diet by environment by parity interaction (P = 0.001, Figure 3-2). This

disparity in BW across treatments likely occurred due to the removal of some lighter BW

cows from this treatment (control diet-cooled environment) because of poor health

postpartum. Overconditioned cows may consume less DM postpartum (Jones and

Garnsworthy, 1989) and both multiparous cows on the control diet-cool treatment in this

study were poor eaters and had a mean prepartum BCS of 3.75 whereas other

treatment groups were 5 3.4 (Table 3-3). Cows maintained under evaporative cooling









had greater body condition throughout the prepartum period (3.48 vs. 3.18, Table 3-3, P

= 0.05). This difference was a result of removing cows assigned to this treatment from

the experiment due to health reasons. Evaporative cooling of prepartum cows did not

affect BW change although BW gain between 3 and 1 wk before calving was

numerically greater for cooled vs noncooled cows (1.17 vs. 0.25 kg/d, P = 0.24, data not

shown).

As expected, mean DMI during the first 7 wk postpartum was greater (P < 0.001)

for multiparous compared with primiparous cows (16.8 vs. 13.8 kg/d, Table 3-4).

However no difference in DMI was detected between parities when DMI was expressed

as a percentage of BW(2.67 vs. 2.69%) thus indicating that more DM can be consumed

by larger animals (BW of 643 vs. 516 kg for multiparous and primiparous cows,

respectively). Effect of parity by diet by environment interaction on DMI was the same

whether expressed as amount (P < 0.01) or as a proportion of BW (P = 0.001, Table 3-

4). Postpartum DMI was greater when primiparous cows were evaporatively cooled

prepartum and fed the control diet compared to the uncooled cows fed the control diet

(2.98 vs. 2.62% of BW) but prepartum cooling had no effect on postpartum DMI of

primiparous cows fed AO (2.49 vs. 2.68% of BW). However prepartum evaporative

cooling of multiparous cows fed the control diet resulted in less DMI compared to those

not cooled (1.99 vs. 3.24% of BW) whereas prepartum cooling had no effect on

postpartum DMI of multiparous cows fed AO (2.83 vs. 2.64% of BW, Figure 3-3, diet by

environment by parity interaction, P = 0.001). The cooled multiparous cows fed the

control diet were the heaviest (Figure 3-4) and the heaviest conditioned at calving which

they maintained throughout the study (Figure 3-5). Cows on this treatment had a BCS









of 3.75 at calving whereas the BCS of the other treatment groups ranged from 3.06 to

3.37 at calving (data not shown). These cows were the poorest eaters (DMI at 1.99% of

BW), continuing their prepartum pattern of poor DMI (Table 3-3). Therefore the

interaction effect of treatment with parity on postpartum DMI was likely largely due to

greater body condition for the one group of animals at calving. Amaral et al. (2009)

reported lower DMI by multiparous cows during the first 14 DIM when they were

evaporatively cooled prepartum compared to cows provided shade only. Similarly,

cows fed the control diet in the current study consumed less DM postpartum when

cooled prepartum compared to those not cooled (2.57 vs. 2.79% of BW). However

postpartum DMI of cows fed AO was not affected by prepartum cooling (2.66 vs. 2.66%

of BW, diet by environment interaction, P = 0.05, Table 3-4). Mean DMI postpartum

was not affected by diet although DMI was lower during the first week postpartum for

cows fed AO compared to control cows (1.36 vs. 1.72% of BW, diet by week interaction,

P = 0.10, Figure 3-6). This difference in DMI at wk 1 postpartum may have been due to

a greater incidence of serious health disorders mastitiss, metritis, retained fetal

membranes, milk fever, or displaced abomasum) for cows fed AO (n = 11/20) vs. the

control diet (n = 3/15) (Table 3-9). In contrast to the current study, Vazquez-A~in et al.

(2008) reported that feeding AO to lactating dairy cows increased DMI.

Neither mean BCS (3.21) nor change in BCS over 7 wk postpartum were

influenced by treatments (Table 3-4). This result is consistent with a study in which the

feeding of AO did not influence BCS of lactating dairy cows (Vazquez-A~in et al.,

2008).









Milk Production and Milk Composition

As expected multiparous cows produced more milk (Table 3-4) than primiparous

cows (32.8 vs. 24.4 kg/d, P = 0.001) likely due to greater DMI by multiparous cows.

Mean and pattern of milk yield measured over the first 7 wk were not different among

treatments (Table 3-4 and Figure 3-7). This is consistent with 3 studies in which milk

yield was not influenced by dietary supplementation with AO (Bowman et al., 2008; He

et al., 2008; Preseault et al., 2008). However, lactating cows did respond to

supplemental AO by increasing milk yield adjusted for fat (3.5% FCM) (Vazquez-Ao6n

et al., 2008). Smith et al. (2002) is the only group to report improvements in

uncorrected milk yield (38.5 vs. 32.3 kg/d, P < 0.05) despite a 2.6 kg/d decrease in DMI

when adding synthetic antioxidants (50 ppm) in the form of ethoxyquin to the diet for 2

wk. Possibly differences in concentration of natural dietary antioxidants such as

vitamins A and E and Se in the control diets may account for differences in milk yield

responses to AO across these studies. In the current study, mean prepartum and

postpartum intakes were 174,853 and 83,781 IU/d for vitamin A, 848 and 446 IU/d for

vitamin E, and 4.6 and 4.7 mg/d for Se, respectively.

Mean concentration (3.25 vs. 3.61%, P = 0.03) and yield (0.87 vs. 1.04 kg/d, P =

0.04) of milk fat were less for cows fed AO compared to control cows (Table 3-4). Smith

et al. (2002) reported numerical decreases in milk fat concentration when ethoxyquin

was fed at 0, 50, 100, or 150 ppm (3.6, 3.2, 3.5, and 3.4%, respectively) although milk

fat production was unchanged. Milk fat depression is usually associated with changes

in biohydrogenation of long chain polyunsaturated fatty acids to conjugated linoleic fatty

acids by ruminal bacteria in cows fed high concentrate/low forage diets and/or diets rich

in plant-oil supplementation (Bauman and Griinari, 2001; Zebeli and Aetaj, 2009).









Vazquez-Ai6n et al. (2007) reported a trend for reduced escape of linolenic acid from

continuous cultures fed AO indicating that AO may stimulate some of the steps in the

biohydrogenation process, although the outflow of cis-9, trans-11 CLAwas not changed

by adding AO. Likewise concentration of trans- 0 C18:1 and the 3 CLA isomers (cis-9,

trans-11 CLA, trans-10, cis-12 CLA, and trans-9, trans-11 CLA) in milk fat were not

changed by feeding AO (Vazquez-Ai6n et al.,2008). Indeed milk fat yield was increased

(0.95 vs. 1.00 kg/d) in dairy cows fed AO in that study. Their reported improvements in

milk fat yield might be associated with positive effects of AO on efficiency of ruminal

microbes (Vazquez-Ai6n et al., 2007). The AO appears to act as a microbial modifier in

the rumen because feeding AO improved fiber digestibility and conversion of dietary N

to microbial N in continuous culture systems (Vazquez-Anon et al., 2007), Cows in the

current study were fed diets of low NDF and ADF concentrations (Table 3-2; NRC,

2001) and supplemented with polyunsaturated fat in the form of corn oil. A more acidic

ruminal environment in the presence of additional polyunsaturated fatty acids may be a

situation in which a microbial modifier such as AO can shift fatty acid metabolism toward

CLA and result in reduce milk fat production.

Although the effect of prepartum cooling did not affect mean concentration or yield

of milk fat, the pattern of milk fat concentration and yield over weeks postpartum

differed; cows not evaporatively cooled prepartum experienced decreased milk fat % at

wk 3 and 4 postpartum compared to cows evaporatively cooled prepartum (environment

by week interaction, P = 0.01, Figure 3-8). Production of milk fat over time responded in

a similar fashion (environment by week interaction, P = 0.02, Figure 3-9). Production of

3.5% FCM was reduced at wk 3 and 4 postpartum due to lack of prepartum cooling









(environment by week interaction, P= 0.07, Figure 3-10). Dairy cows under similar

management conditions (Amaral et al., 2009) also tended to produce milk of less fat

concentration due to lack of prepartum cooling (3.5 vs. 3.9% milk fat) and yielded less

milk fat (0.9 vs. 1.3 kg/d). Avendaho-Reyes et al. (2006) reported that cows cooled with

water spray and fans during pregnancy had an increase in milk fat yield in the

subsequent lactation compared with cows managed under heat stress. It is unknown

why lack of evaporative cooling prepartum would depress milk fat postpartum.

Concentration of milk fat often is reduced when lactating cows undergo moderate

serious heat stress, thought to be due to respiratory alkalosis. Collier et al. (1982)

indicated that the effects of heat stress during the last trimester of pregnancy reduced

placental and maternal hormone concentration, which in turn reduced mammary gland

growth and function that may have led to the reduction in milk fat concentration.

Mean concentration of milk true protein tended to be greater for cows fed AO (3.07

vs. 2.94%, P = 0.06, Table 3-4). Much of this increase was due to a difference detected

at wk 1 postpartum (diet by week interaction, P = 0.04, Figure 3-11) therefore AO did

not improve milk protein long-termWhen feeding AO to lactating cows, no dietary effect

on milk protein content and yield were reported (Vazquez-A~in et al., 2008).

Prepartum cooling did not affect concentration or yield of milk protein. Milk protein

concentration dropped acutely when cows were housed under heat stress for 5 d

compared with the same cows housed under thermoneutral conditions for 5 d (Ominski

et al., 2002).

Somatic cell count was greater in milk produced by cows without prepartum

evaporative cooling at wk 5 and 6 after calving (environment by week interaction, P =









0.03, Figure 3-12). This was largely driven by a cow diagnosed with mastitis during this

time postpartum although 5 additional animals had elevated SCC during this same time

as well. The SCC was numerically greater at 5 of the 7 wk postpartum for cows not

cooled prepartum. Partially relieving heat stress during the immediate prepartum period

may reduce the susceptibility of cows to mammary gland infections postpartum.

Wegner et al. (1976) reported that 64 cows housed in mild to severe heat stress starting

in June, through the hot summer months of July and August, and into the cooler month

of November had increased SCC from August to October. The number of somatic cells

in milk from cows exposed to heat stress for 5 d was elevated compared with that from

cows exposed to thermo-neutral conditions for the same period (Mohammed and

Johnson, 1985).

Plasma Metabolites

The mean concentration of plasma NEFA tended to be greater for cows fed the

control diet compared to that of cows fed AO (325 vs. 246 piEq/L, P = 0.07). However

this dietary difference in plasma NEFA concentration was for multiparous cows only.

Mean plasma concentrations of NEFA tended to be greater for multiparous cows fed the

control diet (469 vs. 305 ipEq/L) but the mean plasma concentrations of NEFA for

primiparous cows were not affected by diet (182 vs. 187 piEq/L, diet by parity

interaction, P = 0.06). These greater concentrations of plasma NEFA for multiparous

cows in control cool treatment group occurred only during the first 3 wk postpartum

(diet by environment by parity by week interaction, P = 0.03; Figure 3-13). Therefore

feeding AO to multiparous cows reduced plasma concentrations of NEFA during the

early postpartum period. Elevated concentrations of plasma NEFA during the first 3 wk









postpartum were likely due to greater mobilization of adipose tissue in support of milk

and maintenance requirements. Loss of BW and feed efficiency as well as energy

balance (figure 3-14, diet by environment by parity by week interaction, P = 0.03) during

the first 3 wk postpartum for these 4 treatment groups followed rather closely the same

ranking as the plasma concentrations of NEFA; namely 2.05, 1.95, 1.69, 0.86 kg/d for

BWloss; 2.55, 2.12, 1.90 1.73 kg of 3.5% FCM/kg of DMI, and 802, 492, 259, and 270

piEq/L of NEFA for multiparous-control diet, multiparous-AO diet, primiparous-control

diet, and primiparous-AO diet groups, respectively.

The weekly postpartum pattern of concentration of plasma BHBA was similar to

that of plasma NEFA; that is, plasma concentrations of BHBA were greater in

multiparous cows fed the control diet during the first 3 wk postpartum (diet by parity by

week interaction, P < 0.01, Figure 3-15). Greater loss of BW during this time for this

group of cows likely accounted for the elevated BHBA concentrations. Thus feeding AO

to multiparous cows reduced plasma concentrations of BHBA but not those of

primiparous cows. Urine of all cows were checked for ketosis using a ketostik at 4, 7,

and 12 DIM. Ketosis was diagnosed for 22 of the 35 cows at least once during the 3 d.

A greater proportion of multiparous cows were diagnosed with ketonemia than

primiparous cows (10/13 vs. 12/22) and mean concentration of BHBA was greater for

multiparous cows (8.9 vs. 6.1 mg/dL, P < 0.01, Table 3-5). Incidence of ketosis was not

different between the 4 treatment groups; namely 4/7 for control-cool, 5/8 for control-

noncool, 6/10 for AO-cool, and 7/10 for AO-noncool.

Mean plasma concentration of glucose was greater for cows fed AO compared

with that of control cows (65.7 vs. 62.7 mg/100 mL, P = 0.03, Table 3-5). This









increased glucose concentration may be due to greater glucose synthesis or less

glucose utilization. Glucose production from propionate was likely similar between the 2

dietary groups because DMI did not differ. However the inclusion of AO in the diet may

have caused a shift towardsbacterial species that produce propionic acid, such that

glucose synthesis was increased. The reduced production of milk fat by cows fed AO

may suggest a bacterial shift in the rumen against fiber digesters. On the other hand,

glucose utilization may have been less for cows fed AO. There was a weak tendency

detected (P = 0.11) for cows fed AO to produce 3.3 kg/d less 3.5% FCM compared with

controls (26.2 vs. 29.5 kg/d, Table 3-4). If glucose production was similar between the 2

groups but glucose utilization was less due to lowered milk production, concentrations

of plasma glucose could be elevated. The cost of energy to maintain the immune

system should be taken into account in energy expense equations (Lochmiller and

Deerenberg, 2000). If feeding AO improved the energy efficiency of the cow's immune

cells, glucose utilization would be less and concentrations of plasma glucose could rise.

Neither mean nor weekly pattern of concentration of plasma BUN differed among

treatment groups (10.9 mg/dL, Table 3-5). The overall mean concentrations of BUN did

change with week postpartum (P < 0.001), increasing from 9.9 mg/dL at wk 1 to 12.2

mg/100 mL at wk 7 postpartum. This probably reflected increasing intake of CP with

increasing intake of DM. Blood urea nitrogen is a major end product of N metabolism

by ruminal microorganisms and can be an indicator of efficiency of utilization of dietary

N (Nousiainen et al., 2004).

Postpartum Body Temperature and Oxidative Markers in Blood

Mean rectal temperatures of postpartum cows (taken at 4, 7, and 12 DIM) were

greater for primiparous than multiparous cows (39.0 vs. 38.7C, P = 0.01, Table 3-6).









This elevated temperature may reflect greater stress typically experienced by

primiparous cows from the new experiences of calving and lactating for the first time.

Cows without evaporative cooling prepartum had reduced mean body temperature

postpartum compared with cows provided evaporative cooling (38.6 vs. 39.0C, P <

0.01). This may result from cow's adaption to heat stress before calving. McDowell et

al. (1969) reported that cows exposed to heat stress for 2 wk experienced improved

surface evaporation compared to cows exposed for only 1 wk. After calving, all cows

were under the same environmental conditions with fans and sprinklers. Assuming that

heat production by the 2 groups of cows were similar postpartum, those that lacked

cooling prepartum had greater evaporative heat loss than cooled cows which resulted in

lower body temperature postpartum.

Plasma TBARS represented a composite number of lipid peroxidative end

products (Bernabucci et al., 2005), but it should perhaps be considered as an index of

oxidative stress (Armstrong and Browne, 1994). Plasma concentration of TBARS was

greatest at -15 d prepartum and at 1 DIM and then decreased through 29 DIM (effect of

week, P < 0.001, Figure 3-16). This pattern was followed by both parities and all

treatment groups (tests of interactions of week with other independent variables were

not significant). Data suggest that these postpartum cows were under less oxidative

stress than prepartum cows. Bernabucci et al. (2005) collected 5 prepartum blood

samples (30 to 4 d prepartum) and 5 postpartum blood samples (4 to 30 DIM) from 24

Holstein cows for analysis of oxidative markers. Plasma concentrations of TBARS were

not different between prepartum and postpartum cows with the exception that cows

exceeding a BCS of 3.0 had greater plasma concentrations of TBARS post vs.









prepartum (2.1 vs.1.5 nmol/mL). Cows calving in the summer in Italy had numerically

greater plasma concentrations of TBARS in the prepartum (21 and 3 d prepartum)

compared to the postpartum period (1, 3, and 35 DIM, Bernabucci et al., 2002). Cows

in the current study may have been consuming more antioxidants than those in Italy and

thus responded differently.

Prepartum cooling did not reduce oxidative stress as measured with TBARS (2.05

vs. 1.79 nmol/mL for cooled and hot respectively, Table 3-6). Likewise, Bernabucci et

al. (2002) reported that moderate heat stress (summer vs. spring calving cows) had no

effect on concentration of plasma TBARS in cows during the transition period.

Feeding AO tended to reduce mean plasma concentration of TBARS when cows

were evaporatively cooled prepartum (2.33 vs. 1.78 nmol/mL) but diet had no effect

when prepartum cows were offered shade alone (1.74 vs. 1.83 nmol/mL, diet by

environment interaction, P = 0.07, Figure 3-17). Bernabucci et al. (2005) suggested that

plasma concentrations of TBARS were positively correlated with NEFA and BHBA

values. In the current study, when the NEFA values for the first 4 wk postpartum were

examined (same time period of measurement for TBARS), the mean concentration for

the cooled cows fed the control diet were only numerically greater compared with that of

the other 3 treatments (503 vs. 321, 390, and 325 nmol/mL for control-cool, AO-cool,

control-hot, and AO-hot, respectively, P = 0.26 for diet by environment interaction). The

same treatment group (control-cool) had the greatest 4-wk mean concentration of

plasma BHBA (8.4 vs. 6.8, 7.2, and 5.7 mg/100 mL) but the test of diet by environment

interaction was P = 0.95). Therefore newly calved cows that are mobilizing more









adipose tissue may be under greater oxidative stress but plasma concentrations of

NEFA and BHBA are imperfect indicators of that stress.

Activity of GPx per mL of erythrocyte decreased from 15 d prepartum to 8 DIM by

7% and then plateaued (effect of week, P = 0.02; Figure 3-18). Bernabucci et al. (2005)

also reported GSH-Px activity of erythrocytes to drop between -4 d and +11 d of calving.

This periparturient pattern followed that of TBARS.

The effect of prepartum cooling on mean activity of erythrocyte GPx uncorrected

and corrected for PCV was influenced by the feeding of AO. Heat stress of multiparous

cows fed the control diet elevated GPx corrected for PCV (8,854 vs.12,247

nmol/min/mL). Including AO in the diet of multiparous cows reversed this effect of heat

stress by reducing GPx activity (10,720 vs. 8,697 nmol/min/mL, Table 3-6) whereas the

activity of erythrocyte GPx corrected for PCV of primiparous cows was not affected by

diet or environment (diet by environment by parity interaction, P = 0.01, Figure 3-19).

The same 3-way interaction for GPx activity per mL of erythrocyte was detected. Lack

of evaporative cooling of multiparous cows fed the control diet elevated GPx (34,960

vs.40,505 nmol/min/mL of RBC). Including AO in the diet of multiparous cows reversed

this effect of heat stress by reducing GPx activity (35,716 vs. 30,203 nmol/min/mL of

RBC) whereas the activity of erythrocyte GPx per mL of erthrocyte of primiparous cows

was not affected by diet or environment (diet by environment by parity interaction, P =

0.05, Figure 3-20). The mechanism by which AO alleviated the negative effect of heat

stress by reducing the GPx activity for multiparous cows is unclear. It is possible that

AO scavenged ROS molecules and reduced the load of peroxides generated from

increased panting under lack of evaporative cooling, as a result, GPx activity was









suppressed. Vazquez-Anon et al. (2008) reported that feeding AO at 200 mg/kg

increased the activity of plasma GPx (0.39 vs. 0.55 U/mg of protein for the absence and

presence of AO, respectively) for mid- to late-lactation dairy cows regardless of the type

of soybean oil (oxidized vs. unoxidized) fed. Hafeman et al. (1974) reported that activity

of erythrocyte GPx was increased when increasing Se supplementation, indicating that

there is a correlation between the concentration of Se in diet and the activity of GPx. In

current study, the concentration of Se that multiparous cows consumed postpartum

followed the same pattern as the activity of erythrocyte GPx corrected for PCV (4.3, 5.8,

5.4, and 5.1 mg/d for control cool, control hot, AO cool, and AO hot,

respectively).

Concentration of erythrocyte SOD corrected for PCV increased 22%, from 2,412

U/mL on d 15 prepartum to 2937 U/mL on 8 DIM (effect of week, P = 0.02, Figure 3-21).

Bernabucci et al. (2005) reported this same dependent variable to increase about the

same percentage as the current study from -17 d to -4 d prepartum but then decreased

thereafter until 30 DIM. The periparturient pattern of SOD followed the opposite pattern

of TBARS and GPx.

Mean concentration of SOD activity corrected for packed cell volume was 2955

and 2495 U/mL for multiparous and primiparous cows, respectively (P = 0.05). High

producing dairy cows were more likely to suffer from oxidative stress (Castillo et al.,

2005; Lohrke et al., 2005) which can be monitored by elevated activities of antioxidant

enzymes. Parity affected SOD activity in the opposite way compared to that of plasma

TBARS.









Feeding AO resulted in an increase in SOD per mL of erythrocytes at 8 DIM,

reversing the trend for cows not fed AO (diet by week interaction, P = 0.03, Figure 3-

22). Contrary to current study, Sahoo et al. (2009) reported that the activity of

erythrocyte SOD was decreased compared with the value before treatment after 3

injections of Vitamin E (i.m., 500 IU/injection) and Se (i.m., 15 mg/injection) on alternate

days up to the 5th day as a therapy for subclinical ketosis. Vazquez-Ai6n et al. (2008)

reported that the activity of plasma SOD was decreased (22.02 vs. 19.34 U/g of protein)

for cows with vs. without supplementation of 200 mg of Agrado Plus per kg of dietary

DM when an unoxidized blend of unsaturated oil was fed, whereas SOD activity was

increased (23.74 vs. 26.35 U/g of protein) by feeding Agrado Plus with oxidized oil

compared to cows without Agrado Plus.

The different responses of SOD and GPx to feeding of AO may be because AO

might do a better job of scavenging H202 than 02- radicals along the sequence of

reactions of reduction of oxygen in the electron transport chain.

White Blood Cells

The predominant cells comprising white blood cells (WBC) are lymphocytes and

neutrophils, making up from 86 to 91% of the WBC in the current study (Table 3-7).

The lymphocytes are mononuclear cells that are part of the adaptive immune system in

that they produce antibodies against antigens to help the host organism resist infection

by foreign pathogens. The neutrophils are polynuclear cells that are part of the innate

immune system that are the first cells to arrive at sites of infection. The concentration of

WBC decreased from 10,887 per pL of blood at 15 d prepartum to 7711 per pL of blood

at 7 DIM (effect of DIM, P < 0.001, Figure 3-23). The type of WBC responsible for this

decrease was the neutrophil. Neutroophils migrate to the mammary gland and the









uterus in response to the presence of pathogenic microorganisms after calving. The

concentration of blood neutrophils was greatest at 15 d prior to calving (4617/pL) and

decreased to 2272/pL by 7 DIM (effect of DIM, P < 0.001, Figure 3-22). Concentration

of lymphocytes did not decrease over this same time period (Figure 3-22).

Prepartum cooling of cows tended to reduce mean concentration of WBC for the

periparturient cow (7900 vs. 10,176 per pL of whole blood, P = 0.09, Table 3-7). The

reduction was detected only at 7 and 14 DIM (environment by DIM interaction, P = 0.05,

Figure 3-24) in which the concentration dropped 32% from 9577 to 6524 per pL of

whole blood as cows transitioned from the prepartum to postpartum period. The WBC

concentration of noncooled cows remained the same throughout the periparturient

period (mean of 10,220 per pL of whole blood. The blood cell type that was responsible

for this postpartum drop in WBC of cooled cows was the neutrophil. Concentration of

blood neutrophils decreased from 0 to 7 DIM by 57% when cows were cooled

prepartum but only by 24% when cows were not evaporatively cooled (environment by

DIM interaction, P = 0.11, Figure 3-25). On the other hand, the concentration of

lymphocytes did not change (mean of 3455 per pL of whole blood) during the transition

period for cows cooled prepartum (Figure 3-26). Therefore the innate immune system

of bovine appeared to be more sensitive than the adaptive system to parturition. Lack

of prepartum cooling appeared to minimize the typical decrease in immuno-suppression

reported for cows in the first 2 wk postpartum. Possibly the prepartum exposure of

cows to hotter environmental conditions prepared their immune system for the stress of

parturition. The cows with the elevated WBC count postpartum had a lower mean rectal

temperature at 4, 7, and 12 DIM (Table 3-6) suggesting that elevated body temperature









postpartum may reflect a degree of suppressed immune response. Cows not offered

evaporative cooling prepartum had a 53% greater mean concentration of lymphocytes

throughout the study (5411 vs. 3455 per pL of whole blood, P = 0.03, Table 3-7). The

concentration of blood lymphocytes tended to decrease on the day of calving and then

rebound for noncooled cows whereas that of the cooled cows did not change over time

(environment by DIM interaction, P = 0.09, Figure 3-25). Again, hotter environmental

conditions resulted in elevated blood concentration of immune cells.

Function of Blood Neutrophils

Effect of calving. Measures of neutrophil numbers and activities were lowest at 7

or 14 DIM compared with -15 d or day of calving indicating a partially suppressed

immune system within a week or 2 after calving (effect of day, P < 0.001, Figure 3-22).

The proportion of neutrophils carrying out phagocytosis against labeled E. coli was less

at 7 compared to 0 DIM (72.6 vs. 83.3%, effect of day, P < 0.001) and the MFI for

phagocytosis also decreased from 60.4 to 33.5 on these same days (effect of day, P <

0.001, Figure 3-27). Proportion of neutrophils undergoing oxidative burst was less at 7

compared to 0 DIM (84 vs. 78%, effect of day, P < 0.01, Figure 3-28) and the MFI for

oxidative burst also decreased progressively over time from 1687 at -15 DIM to 868 at

14 DIM (effect of day, P < 0.01, Figure 3-27). This decrease after calving has been

reported by other investigators as indicators of suppressed immune response

immediately postpartum.

Effect of parity. In general, the immune system of multiparous cows appeared to

be more suppressed than that of primiparous cows. The concentration of neutrophils

(2894 vs. 3545/pL of blood, P = 0.11), the MFI of phagocytosis (36 vs. 62, P < 0.001),

the percentage of neutrophils conducting oxidative burst (76 vs. 82, P = 0.03), and the









MFI of neutrophils conducting oxidative burst (1041 vs. 1431, P = 0.03) were lower for

multiparous cows (Table 3-7). This effect of parity for these dependant variables was

consistent across all days of measure with the exception of percentage of neutrophils

conducting oxidative burst. Multiparous cows had a reduced percentage only at 7 DIM

compared with primiparous cows (71 vs. 86%, parity by week interaction, P = 0.05,

Table 3-7).

Effect of treatments. The concentration of blood neutrophils (number per pL)

throughout the study was unaffected by diet or prepartum evaporative cooling.

Prepartum cooling increased phagocytosis of E. coli by blood neutrophils of

multiparous cows (77.1 vs. 71.6%) but the reverse occurred for primiparous cows (77.1

vs. 71.6%, cooling by parity interaction, P = 0.03, Figure 3-29). This decrease in %

neutrophils undergoing phagocytosis due to the cooling of primiparous cows was

matched with a decrease in MFI for phagocytosis (69 vs. 55) but MFI for phagocytosis

by neutrophils of multiparous cows was not affected by prepartum cooling (37 vs. 34,

cooling by parity interaction, P = 0.05, Figure 3-30). Feeding AO did not influence the

proportion of neutrophils that phagocytized E. coli. However the MFI for phagocytosis

was affected by diet. The MFI for phagocytosis was reduced by feeding AO to

primiparous cows (69 vs. 55) but was unchanged by feeding AO to multiparous cows

(33 vs. 39, diet by parity interaction, P = 0.02, Figure 3-31). This reduction by AO on

MFI of phagocytosis as cows transitioned from prepartum to 7 and 14 DIM was greater

for primiparous than for multiparous cows (diet by parity by time interaction, P = 0.04,

Figure 3-32).









The consumption of oxygen during the generation of ROS is a critical process

termed oxidative burst to kill bacteria after phagocytosis. Killing ability by neutrophils

oxidativee burst) tended to be compromised by feeding AO when prepartum cows were

cooled (83 vs. 77%) vs. noncooled (77 vs. 80%, diet by environment interaction, P =

0.10, Figure 3-33). This effect tended to occur at 7 and 14 DIM (diet by environment by

day interaction, P = 0.08, Figure 3-34). Including AO in the diet of hotter cows

prepartum resulted in a similar pattern of oxidative burst by neutrophils as cows fed the

control diet and kept with evaporative cooling. Feeding ethoxyquin at 150 ppm inhibited

phagocytosis of leucocytes of tilapia (Yamashita et al., 2009) indicating that to some

extent, the synthetic antioxidants can suppress partially neutrophil activity.

Several studies have examined the impact of feeding vitamin antioxidants on

neutrophil function. When cows were injected with 3000 IU of vitamin E at 10 and 5 d

before calving, intracellular kill of bacteria by neutrophils was increased at calving

(Hogan et al., 1990). The same research group (1992) also reported that

supplementation of vitamin E (400 to 600 mg/d) or Se (0.3 mg/kg of dietary DM)

increased the proportion of bacteria killed by neutrophils. Weiss and Hogan (2005) also

reported that bacterial killing ability by neutrophils tended to be increased for cows fed

selenite at 0.3 mg/kg of dietary DM with 500 IU/d of vitamin E compared with cows fed

an organic source of Se. Boyne and Arthur (1979) concluded that the percentage of

neutrophils that phagocytized and killed bacteria was greater for cows that received

sufficient Se (0.1 ppm of dietary DM) than cows given a Se-deficient diet. Grasso et al.

(1990) reported that cows supplemented with 2 mg/d of sodium selenite during the

transition period had greater bacterial killing ability by neutrophils in milk compared with









cows not supplemented with Se. However function of neutrophils (the proportion of

neutrophils that phagocytized bacteria and number of intracellular bacteria per

neutrophil) isolated from whole blood were not influenced by supplementation with

either 0 or 30 g/d of vitamin C starting from 2 wk before calving through 7 DIM (Weiss

and Hogan, 2007). No published study has reported on the effect of feeding synthetic

antioxidants on neutrophil function.

Ovalbumin Challenge and Acute-phase Proteins

Cows were given 3 separate injections of ovalbumin at approximately 4 and 2 wk

before calving and on the day of calving. Plasma concentrations of IgG for ovalbumin

increased after each injection as expected (Figure 3-35). The IgG concentration at wk 4

before calving was used as a covariate in the statistical analysis because analysis of wk

4 alone in a reduced model resulted in a P = 0.12 for the test of diet by environment

interaction. The mean circulating concentrations of IgG against injected ovalbumin was

less for multiparous cows fed AO vs. the control diet (0.80 vs. 0.63 optical density)

whereas the response to diet was unchanged for primiparous cows (0.68 vs. 0.73 OD,

diet by parity interaction, P = 0.05). This difference between multiparous cows occurred

primarily from calving to 7 wk postpartum (Figure 3-36). From my knowledge, this is the

first study to report the effect of feeding synthetic antioxidants on IgG responses to

ovalbumin injections of bovine. Under the current conditions of this trial, supplementing

synthetic antioxidants appeared to suppress adaptive immunity responses of

multiparous cows. A linear increase in IgG concentration with increased dietary

supplementation of vitamin E (285, 570, and 1140 IU/d, respectively) for bull calves was

detected at 21 d after ovalbumin ingection (Rivera et al., 2002). Similar to this finding,









calves injected with 125 IU of vitamin E at 7 wk of age had greater IgG values

compared with those receiving no vitamin E (Reddy et al., 1987).

Primiparous cows had greater mean plasma concentration of ASP than

multiparous cows (49.5 vs. 39.8 pg/mL, P = 0.02, Table 3-7) with the greater values

occurring during the first 2 wk after calving (parity by week interaction, P < 0.001, Figure

3-37). This result is in agreement with our previous studies (Amaral et al., 2008).

Primiparous cows had a greater mean rectal temperature than multiparous cows

on 4, 7, and 12 DIM (Table 3-6) suggesting that primiparous cows were experiencing

more stress and responding with greater circulation of acid soluble protein. When

plasma ASP values were plotted for cows diagnosed as healthy (n = 18) vs. unhealthy

mastitiss, metritis, or retained fetal membranes in the first 14 DIM, n = 17), unhealthy

cows had greater mean concentrations of plasma ASP (51.2 vs. 40.1 pg/mL, P < 0.01)

and had greater peak concentrations of plasma ASP between 6 to 22 DIM (78.0 vs.

50.9 pg/mL, healthy vs. unhealthy by time interaction, P = 0.001, Figure 3-38). Lastly,

cows supplemented with AO had greater plasma concentrations of ASP from 6 to 15

DIM compared with the control group (diet by DIM interaction, P = 0.03, Figure 3-39).

This dietary effect on plasma ASP during the first 2 wk postpartum may have resulted

from a greater incidence of endometritis (10/20 vs. 3/15) and retained fetal membranes

(2/10 vs. 0/15) for cows fed AO vs. control, respectively (Table 3-9).

Similarly to ASP, plasma concentrations of Hp in early in lactation tended to be

greater for primiparous compared to multiparous cows (parity by day interaction, P =

0.09, Figure 3-40). Also in agreement with ASP responses to disease was the effect of

mastitis and metritis on plasma Hp. Plasma concentrations of Hp were greater between









6 and 15 DIM in unhealthy vs. healthy cows (healthy vs. unhealthy by time interaction, P

< 0.01, Figure 3-41). Plasma concentrations of Hp may not be as sensitive an acute

phase protein to reflect disease as plasma concentrations of ASP (6 vs. 4 significant

time points). Diet did not affect mean or weekly pattern of plasma Hp. Although

prepartum cooling did influence plasma concentrations of Hp over DIM, the influence

was mixed (environment by DIM interaction, P = 0.03, Figure 3-42); namely that cooled

cows had greater plasma Hp at 6 DIM but less at 13 DIM.

Progesterone

The reproductive system of multiparous cows appeared to respond better than

primiparous cows in the first 7 wk postpartum. Multiparous cows had a longer first

estrous cycle (19.3 vs. 15.1 d, P = 0.06) and a greater peak concentration of plasma

progesterone in the first cycle (8.2 vs. 5.8 ng/mL, P = 0.04) although the number of

ovulations were fewer (1.3 vs. 1.9, P = 0.04, Table 3-8). Thirty-two of the 35 cows

ovulated within the first 49 DIM. When considering only the 32 cows, treatment did not

influence the day of first ovulation which averaged 19.7 2.6 DIM (Table 3-8). However

when the 3 cows that did not ovulate were assigned 49 DIM as their day of first

ovulation, the day of first ovulation was affected by treatment. Providing evaporative

cooling to cows fed the control diet tended to increase the number of days to first

ovulation compared to noncooled cows (26.0 vs. 19.8 DIM) whereas cooling of cows fed

AO tended to decrease time to first ovulation (20.5 vs. 28.9 DIM, diet by environment

interaction, P = 0.08). This pattern was repeated for length of first estrous cycle in that

providing evaporative cooling to cows fed the control diet resulted in a reduced length of

cycle compared to noncooled cows (14.6 vs. 19.1 DIM) whereas cooling of cows fed AO

increased length of first cycle (20.1 vs. 15.0 DIM, diet by environment interaction, P =









0.04). Likewise the plasma progesterone values accumulated over all 21 d of sampling

times tended to be greater for these same 2 groups of cows (P = 0.10). Therefore

noncooled cows fed the control diet and cooled cows fed AO had an earlier ovulation, a

longer first estrous cycle, and produced more progesterone over 7 wk than the other 2

treatment groups. This effect can not be explained adequately by treatment differences

in plasma concentrations of NEFA or in feed efficiency, nor by lower incidence of health

disorders (Table 3-10). Multiparous cows fed the control vs. the AO diet had greater

peak concentrations of plasma progesterone in the first estrous cycle (10.6 vs. 5.8

ng/mL) whereas that of primiparous cows did not differ between diets (6.0 vs. 5.7

ng/mL, parity by diet interaction, P = 0.05, Figure 3-43).

Vaginoscopy and Uterine cytology

Cervical discharge was scored based upon extent of presence of pus on 7, 16,

and 25 DIM. Cows having clear discharge or only specks of pus were classified as

healthy whereas significant amounts of pus resulted in diagnosis of metritis (7 and 16

DIM) or endometritis (25 DIM) (Sheldon et al., 2006), creating a binomial set of data.

Compared with multiparous cows, primiparous cows had a greater incidence of uterine

disease at 7 DIM (96 vs. 62%), at 16 DIM (91 vs. 42%), and at 25 DIM (59 vs. 23%).

Primiparous cows were 18.2 (P = 0.02), 12.8 (P = 0.01), and 11.5 (P = 0.01) times more

likely to have a uterine infection on 7, 16, and 25 DIM than multiparous cows based on

odds ratio analysis. At 25 DIM, cows fed AO had a greater incidence of endometritis

than cows not fed AO (60 vs. 27%, odds ratio of 9.6, P = 0.02).

At 40 2 DIM, one uterine horn was flushed and the proportion of neutrophils

calculated after counting 100 stained cells identified using a microscope. Neutrophils

comprised a greater proportion of cells when cows were fed AO (21.9 vs. 6.8%, P =









0.02). Cows were diagnosed as having subclinical endometritis when the proportion of

neutrophils exceeded 5% (Gilbert et al., 2005) thus creating a binominal data set. The

incidence of subclinical endometritis was greater in cows fed AO (80 vs. 33%, odds ratio

of 3.4, P < 0.01). This negative effect of AO on increasing uterine subclincial

endometritis at 40 DIM agrees with AO's negative effect on increasing endometritis at

25 DIM as reported in the previous paragraph. Primiparous cows tended to have more

cases of endometritis than multiparous cows (68 vs. 46%, odds ratio = 9.3, P = 0.06), a

pattern that agrees with the greater incidence of metritis for primiparous vs. multiparous

cows. Lastly, cows lacking prepartum evaporative cooling tended to have a greater

incidence of subclinical endometritis (72 vs. 47%, P = 0.08).

Summary

Parity. Primiparous cows appeared to be under greater stress postpartum than

multiparous cows as evidenced by greater or tendency for greater mean body

temperature at 4, 7, and 12 DIM (39.0 vs. 38.7C), greater metricheck score which is

greater than 1 at 7 DIM (95 vs. 62%), 16 DIM (91 vs. 42%), and 25 DIM (59 vs. 23%),

greater incidence of subclinical endometritis around 40 DIM (68 vs. 46%), and

increasing concentrations of BHBA with increasing DIM. As a consequence of a greater

incidence of uterine diseases, the immune responses were stimulated; namely, an

increase in the concentration of blood neutrophils (3545 vs. 2894 per pL), in MFI for

phagocytosis (62 vs. 36) and for oxidative burst (38 vs.32) by neutrophils, in % of

neutrophils undergoing oxidative burst (82 vs. 76%), and in plasma concentration of

acute phase proteins (acid soluble protein and haptoglobin) in the first 2 wk postpartum.

Also as a result of increased metritis and endometritis, ovarian activity was affected

negatively; namely, a shorted length of the first estrous cycle (15.1 vs. 19.3 d) and a









lowerpeak concentration of progesterone in the first cycle (5.8 vs. 8.2 ng/mL). Lastly,

oxidative markers in the blood (TBARS, SOD, and GPx) reacted differently. Mean

plasma concentrations of TBARS were greater (2.12 vs. 1.71 nmol/mL) whereas mean

RBC concentrations of SOD corrected for packed cell volume were lower (2495 vs.

2955 U/mL) for primiparous vs. multiparous cows. Activity of GPX per mL of RBC were

lower at 15 d before parturition (34,601 vs. 40,096 nmol/min/mL) but greater at 29 DIM

(36,629 vs. 32,440 nmol/min/mL) for primiparous vs. multiparous cows.

Cooling. Prepartum evaporative cooling during the 3 wk prior to calving had or

tended to have several positive benefits. Prepartum cooling resulted in a lower mean

rectal temperature during the prepartum period (39.2 vs. 39.6C) but a greater mean

rectal temperature at 4, 7, and 12 DIM (39.0 vs. 38.7C). This slightly greater mean

rectal temperature was not increased enough to negatively affect production or health.

Indeed prepartum cooling resulted in greater mean concentration of milk fat during 7 wk

(3.54 vs. 3.32%) and mean production of 3.5% FCM during the first 4 wk postpartum

(26.5 vs. 23.0 kg/d). Additionally, incidences of mastitis (1/17 vs. 6/18), displaced

abomasum (0/17 vs. 3/18), and subclinical endometritis (47 vs. 72%) were reduced due

to prepartum cooling. Associated with the reduction in health disorders, the number and

activity of select immunity cells were affected as evidenced by lower concentration of

circulating WBC postpartum (2049 vs. 2804 per pL of blood) and of circulating

lymphocytes (3455 vs. 5411 per pL of blood) and increased proportion of neutrophils

undergoing oxidative burst of E. coli of cows fed the control diet (83 vs. 77%).

Assuming that an elevated concentration of oxidative markers in blood is indicative of

greater oxidative stress, prepartum cooling of multiparous cows resulted in less









oxidative stress as evidenced by lower activity of GPx in RBC (8,854 vs. 12,247

nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). The only negative effects of

prepartum cooling were a greater plasma concentration of TBARS for control-fed cows

(2.33 vs. 1.74 nmol/mL), a decreased length of the first estrous cycle of control-fed

cows (14.6 vs. 19.1 d), and a reduction in phagocytosis by neutrophils (74 vs. 81% and

55 vs. 67 MFI) in primiparous cows.

Synthetic antioxidants. Feeding AO did not influence DM intake or uncorrected

milk production. However supplemental AO had mixed effects on milk composition.

Concentration of milk true protein was increased (3.07 vs. 2.94%) but concentration of

milk fat was decreased (3.25 vs. 3.61%) resulting in less production of milk fat (0.88 vs.

1.04 kg/d) and of 3.5% FCM (26.2 vs. 29.5 kg/d). Therefore AO seems to influence the

microbial population in the rumen. In addition, cows fed AO had a greater incidence of

endometritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs.

33%). These reproductive disorders may have contributed to a lower peak

concentration of plasma progesterone in the first estrous cycle of multiparous cows (5.8

vs. 10.6 ng/mL).

A greater peak concentration of plasma ASP postpartum in cows fed AO vs.

control diet may reflect greater stress.

The effect of AO on oxidative markers or immune cells was not consistent. Under

4 different scenarios of more stressful (primiparous or prepartum noncooling) or less

stressful multiparouss or prepartum cooling) scenarios, cows fed AO responded

differently. Results are the following:









Nonstressful situation improved. Feeding AO to cows managed under

prepartum evaporative cooling conditions resulted in

1. a longer first estrous cycle (20.2 vs. 14.6 d).

2. A decrease in plasma concentration of TBARS (1.78 vs. 2.33 nmol/mL).

Nonstressful situation aggravated. Feeding AO to cows managed under

prepartum evaporative cooling conditions resulted in

1. a decrease proportion of neutrophils undergoing oxidative burst (77 vs. 83%).

Stressful situation improved. Feeding AO to multiparous cows managed without

prepartum evaporative cooling resulted ina reduced activity of GPX in RBC corrected

for packed cell volume (8,697 vs. 10,720 nmol/min/mL).

Stressful situation aggravated: Feeding AO to primiparous cows

1. reduced MFI of phagocytosis by neutrophils (36 vs. 57%)

2. Feeding AO at 250 mg per kg of dietary DM to periparturient Holstein cows fed
diets of minimal fiber density resulted in reduced production of fat-corrected milk,
increased incidence of uterine infections, reduced neutrophil activity, and mixed
effects on oxidative markers in blood.










Table 3-1. Ingredient composition of diets fed to nonlactating and lactating Holstein
cows.
Ingredient, % of DM Nonlactating Lactating
Corn silage 30.0 39.5
Bermudagrass silage 35.0 .
Alfalfa hay ... 12.5
Ground corn 9.5 18.9
Soybean meal 9.1 7.8
Soy Plus1 1.2 7.3
Citrus pulp 6.7 4.0
Corn gluten feed ... 4.4
Corn oil 2.0 2.0
Mineral/vitamin mix 6.52 3.7j
SWest Central Soy, Ralston, IA.
2Contained 18.3% CP, 20.5% Ca, 0.3% P, 3.1% Mg, 9.6% CI, 0.3% K, 1.6% Na, 2.4% S, 11 ppm Co, 176
ppm Cu, 8 ppm I, 158 ppm Fe, 142 ppm Mn, 7 ppm Se, 268,00 IU vitamin A, 40 IU vitamin D, and 1300
IU of vitamin E (DM basis).
3 Contained 24% CP, 9% Ca, 1% P, 4% K, 3% Mg, 10% Na, 1.1% S, 1200 ppm Mn, 158 ppm Fe, 500
ppm Cu, 1500 ppm Zn, 8.25 ppm Se, 2% CI, 20 ppm I, 147,756 IU/kg of vitamin A, 43,750 IU/kg of
vitamin D, and 787 IU/kg of vitamin E (DM basis).










Table 3-2. Chemical composition of diets fed to nonlactating and lactating Holstein
cows.
% DM


CP
NDF
ADF
Ether extract
NEL, Mcal/kg
Ca
P
Mg
K


Nonlactating
Control
15.1
37.6
23.5
3.9
1.31
1.86
0.32
0.43
1.25
0.15
0.34
0.99
29
256
107
46


cows
Antioxidant'
15.3
37.7
23.6
4.0
1.34
1.85
0.33
0.44
1.25
0.15
0.33
0.93
28
252
100
48


Contro
17.9
26.0
15.8
4.5
1.75
0.83
0.40
0.33
1.46
0.31
0.23
0.38
25
18
78
69


1 Agrado Plus (Novus International Inc., St. Charles, MO).


Lactating cows
Antioxidant
17.7
25.8
16.1
4.7
1.75
0.95
0.39
0.35
1.45
0.31
0.24
0.45
30
18
87
59


Chemical










Table 3-3. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on body temperature, body weight, BCS,
and DMI of nonlactating pregnant Holstein cows during summer in Florida.
Treatment
Control diet AO diet'
Measure Cooled Non-cooled Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous
Number of animals 5 2 6 2 5 5 6 4
Body temperature, C 39.2 0.1 39.4 0.2 39.5 0.1 39.8 0.2 39.2 0.1 39.2 0.1 39.6 0.1 39.6 0.1
DMI, kg/d 10.6 0.6 9.2 0.9 10.4 0.5 9.4 0.9 10.3 0.7 11.7 0.6 9.7 0.5 9.0 0.6
BW, kg 568 18 843 28 585 16 680 28 596 18 691 18 587 16 720 20
DMI, % of BW 1.86 0.10 1.09 0.16 1.77 0.09 1.41 0.16 1.69 0.12 1.70 0.10 1.62 0.09 1.25 0.11
BCS2 3.37 0.17 3.75 0.27 3.21 0.16 3.06 0.27 3.37 0.16 3.41 0.19 3.35 0.16 3.09 0.19
SAgrado Plus (Novus Internat. Inc., St. Charles, MO).
2 Least squares mean of BCS at the time of enrollment in the study and at calving.
3N/A = Not available.










Table 3-3. Continued
P values



0 O oO O
o> E "- o E



Body temperature, C 0.38 0.001 0.77 0.19 0.15 0.85 0.76 0.53 0.48 0.35 0.39 0.81 0.98 0.44 0.34
DMI, kg/d 0.56 0.09 0.10 0.33 0.12 0.39 0.18 <0.001 0.23 0.49 0.22 0.85 0.73 0.96 0.47
BW, kg 0.20 0.04 0.01 <0.001 0.02 0.02 0.001 0.21 0.14 0.40 0.43 0.47 0.36 0.95 0.97
DMI, % of BW 0.73 0.42 0.03 <0.001 0.03 0.91 0.03 <0.001 0.36 0.64 0.18 0.56 0.85 0.97 0.75
BCS 0.77 0.05 0.37 1.00 0.43 0.16 0.69 0.03 0.39 1.00 0.34 0.03 0.77 0.56 0.70










* Control Cool E Control Hot E AO Cool F AO Hot


2.1 -

1.8 -

1.5 -

1.2 -

0.9 -

0.6 -

0.3 -


Primiparous Multiparous
Parity

Figure 3-1. Least squares means for mean dry matter intake of prepartum primiparous
(n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
with dietary antioxidants (AO). Effect of diet by environment by parity
interaction (P = 0.03).










C Control-Hot E AO Cool


800 -
700
600
tXO 500 -
400
g 300
A 200
100
0
Primiparous Multiparous

Parity

Figure 3-2. Least squares means for mean body weight of prepartum primiparous (n =
22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
with dietary antioxidants (AO). Effect of diet by environment by parity
interaction (P = 0.001).


* Control Cool


F9 AO Hot











Table 3-4. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on performance of lactating pregnant
Holstein cows during summer in Florida.
Treatment


Measure


Number of animals
BW, kg
BCS
DMI, kg/d
DMI, % of BW
Milk, kg/d
Milk fat, %
Milk protein, %
Milk fat, kg/d
Milk protein, kg/d
Milk SCC, x1000
3.5% FCM, kg/d
Feed efficiency
'Agrado-Plus (Novus
2 N/A = Not Available.


Control diet
Cooled Non-cooled


Primiparous
6
518 15
3.12 0.12
13.5 0.8
2.62 0.12
24.5 2.8
3.21 0.17
3.02 0.07
0.7 0.1
0.7 0.1
89 20
22.5 2.2
1.72 0.15


Primiparous Multiparous
5 2
504 16 724 26
3.23 0.13 3.46 0.20
15.0 0.91 14.1 1.4
2.98 0.14 1.99 0.21
27.7 2.8 31.1 2.5
3.35 0.19 4.21 0.31
2.97 0.08 2.76 0.14
0.9 0.1 1.3 0.1
0.8 0.1 0.9 0.1
174 21 182 28
26.8 2.4 33.8 3.8
1.81 0.16 2.48 0.25
Internat. Inc., St. Charles, MO).


Multiparous
2
590 26
3.08 0.20
18.9 1.4
3.24 0.21
34.4 4.4
3.67 0.30
3.00 0.14
1.2 0.1
0.9 0.1
300 28
34.8 3.8
2.00 0.25


AO diet'
Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous
5 5 6 4


516 16
3.19 0.12
12.7 0.9
2.49 0.14
23.6 2.5
3.22 0.19
2.98 0.08
0.7 0.1
0.7 0.1
105 21
22.0 2.4
1.79 0.16


625 16
3.33 0.14
17.7 0.8
2.83 0.14
36.0 4.4
3.38 0.19
3.10 0.08
1.2 0.1
1.1 0.1
47 21
34.3 2.4
1.97 0.16


526 15
3.19 0.12
14.1 0.8
2.68 0.12
22.0 2.8
3.12 0.17
3.09 0.07
0.7 0.1
0.7 0.1
190 20
20.1 2.2
1.45 0.14


631 18
3.08 0.14
16.6 1.0
2.64 0.15
29.8 3.1
3.29 0.21
3.09 0.09
1.0 0.1
0.9 0.1
177 22
28.4 2.7
1.79 0.18












Table 3-4. Contiuned
P values


-0o 0 0
Measure |o > 8 o o
Ui 0 0 C 0 0
0> 0 Q -

Number of animals N/A2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A
BW, kg 0.48 0.06 0.02 < 0.001 0.16 < 0.01 0.01 < 0.001 0.12 0.56 0.29 0.52 0.80 0.16 0.41
BCS 0.83 0.10 0.59 0.62 0.72 0.23 0.97 0.001 0.59 0.25 0.18 0.58 0.32 0.82 0.53
DMI, kg/d 0.88 0.25 0.34 < 0.001 0.33 0.22 < 0.01 < 0.001 0.04 0.14 0.10 0.02 0.38 0.65 0.07
DMI, % of BW 0.69 0.06 0.05 0.87 0.14 0.01 0.001 < 0.001 0.10 0.21 0.26 0.28 0.60 0.72 0.30
Milk, kg/d 0.50 0.41 0.41 0.001 0.46 0.84 0.24 < 0.001 0.39 0.36 0.20 < 0.001 0.13 0.20 0.32
Milk fat, % 0.03 0.18 0.43 0.01 0.13 0.55 0.51 < 0.001 0.10 0.01 0.95 0.02 0.36 0.47 0.94
Milk protein, % 0.06 0.17 0.49 0.70 0.19 0.78 0.27 < 0.001 0.04 0.15 0.41 0.94 0.21 0.33 0.53
Milk fat, kg/d 0.04 0.11 0.86 < 0.001 0.72 0.96 0.40 < 0.001 0.17 0.02 0.83 < 0.01 0.48 0.33 0.55
Milk protein, kg/d 0.69 0.38 0.36 < 0.001 0.27 0.97 0.13 < 0.001 0.84 0.79 0.54 0.10 0.27 0.27 0.64
Milk SCC, x1000 0.35 0.31 0.23 0.82 0.22 0.27 0.80 < 0.001 0.15 0.03 0.68 0.27 0.26 0.68 0.40
3.5% FCM, kg/d 0.11 0.18 0.58 < 0.001 0.86 0.88 0.26 < 0.001 0.19 0.07 0.65 < 0.001 0.61 0.22 0.39
Feed efficiency 0.06 0.05 0.90 0.01 0.42 0.66 0.30 < 0.001 0.37 0.08 0.05 0.15 0.68 0.52 0.03













3.5

3

2.5

2

1.5

1


* Control-Cool M Control-Hot E AO-Cool 9 AO-Hot


T


0.5


Primiparous Multiparous
Parity

Figure 3-3. Least squares means for mean dry matter intake of postpartum primiparous
(n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or
noncooled (Hot) freestalls and fed diets supplemented without (Control) or
with dietary antioxidants (AO). Effect of diet by environment by parity
interaction (P = 0.001).











E Control Hot 0 AO Cool


800 -

700 -

600 -

0 500 -
-^

400 -

e 300 -

200 -

100 -

0-


Primiparous Multiparous

Parity


Figure 3-4. Least squares means for mean body weight (BW) of postpartum
primiparous (n = 22) and multiparous (n = 13) Holstein cows housed in cooled
(Cool) or noncooled (Hot) freestalls and fed diets supplemented without
(Control) or with dietary antioxidants (AO). Effect of diet by environment by
parity interaction (P = 0.01).


100


* Control Cool


9 AO Hot










-4-Control Cool
580

560

540

520

500

480

460


--E- Control Hot


-- AO- Cool -0- AO- Hot


0 1 2 3 4 5 6 7


--Control Cool
820

770-

720

670

620

570

520


Week postpartum


--- Control Hot


-- AO- Cool -0- AO- Hot


0 1 2 3 4 5 6 7

Week postpartum
Figure 3-5. Least squares means for weekly body weight (BW) of postpartum
primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows housed in
cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented
without (Control) or with dietary antioxidants (AO). Effect of parity by diet by
environment by week (P = 0.41).


101










---Control Cool
4.5
4
3.5
3
2.5
2 O
1.5
1
0.5
0 -


- Control Hot -A- AO- Cool


--AO- Hot


1 2 3 4 5 6 7

Week postpartum



Figure 3-6. Least squares means for weekly dry matter intake of postpartum Holstein
cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed
diets supplemented without (Control) or with dietary antioxidants (AO). Effect
of diet by week interaction (P = 0.10). Week with asterisk indicates that diets
differed (P < 0.05).


102











45
40
35
30
25
20
15
10
5


--$-Control Cool -i- Control Hot --- AO Cool --AO Hot


1 2 3 4 5 6 7
1 2 3 4 5 6 7


Week postpartum

Figure 3-7. Least squares means for weekly milk production of Holstein cows (n = 35)
housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets
supplemented without (Control) or with dietary antioxidants (AO). Effect of
diet by environment by week (P = 0.20). Week with dagger indicates that
means differed (P < 0.10).


103












4.5

4
aNN'

M~ 3.5




2.5

2
1 2 3 4 5 6 7
Week postpartum


Figure 3-8. Least squares means for weekly concentration of milk fat of Holstein cows
(n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls. Effect of
environment by week interaction (P = 0.01). Week with asterisk indicates that
means differed using slice command (P < 0.05).


104









1.4 --Cool -[- Hot
1.3
"o 1.2 -




> 0.9
"- 0.8
| 0.7 -
0.6
0.5
1 2 3 4 5 6 7

Week postpartum

Figure 3-9. Least squares means for weekly milk fat production of Holstein cows (n =
35) housed in cooled (Cool) or noncooled (Hot) freestalls. Effect of
environment by week interaction (P = 0.02). Week with asterisk indicates that
means differed using slice command (P < 0.05).


105










-Cool -- Hot
40

35 t

L.30

25

2 20



10
1 2 3 4 5 6 7
Week postpartum


Figure 3-10. Least squares means for production of 3.5% fat-corrected milk by
postpartum Holstein cows (n = 35) housed in shaded freestalls equipped with
fans and sprinklers (Cool) or just shade (Hot). Effect of cooling by week
interaction (P = 0.07). Week with dagger indicates that means differed using
slice command (P < 0.10).


106









4.75 Control -AO


S4.25 \


3 3.75


S3.25


2.75


2.25 ..
1 2 3 4 5 6 7
Week postpartum


Figure 3-11. Least squares means for weekly concentration of milk protein of Holstein
cows (n = 35) fed diets supplemented without (Control) or with dietary
antioxidants (AO). Effect of diet by week interaction (P = 0.04). Week with
asterisk indicates that means differed using slice command (P < 0.05).


107










1000 Cool Hot
0 900
0
S800 \
x
700
600 -
O 500
400 -
U
0 300 *
S200 -
E
0 100 -
Cn
0 -
1 2 3 4 5 6 7

Week postpartum

Figure 3-12. Least squares means for somatic cell counts of postpartum Holstein cows
(n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool)
or just shade (Hot). Effect of cooling by week interaction (P = 0.03). Week
with asterisk indicates that means differed using slice command (P < 0.05),
with dagger differed (P < 0.10).


108










Table 3-5. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on postpartum plasma concentration of
metabolites of lactating Holstein cows during summer in Florida.
Treatment
Control diet AO diet'
MeasCooled Non-cooled Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous
NEFA, peq/L 195 47 557 74 168 44 381 74 222 48 288 47 152 43 321 52
BHBA, mg/dL 7.22 0.97 10.46 1.54 5.17 0.94 9.28 1.54 6.53 1.00 8.62 0.97 5.55 0.91 7.40 1.09
Glucose, mg/dL 64.2 1.6 58.2 2.6 68.5 1.5 59.9 2.6 69.9 1.6 60.6 1.6 70.8 1.5 61.5 1.8
BUN, mg/dL 11.8 0.8 11.1 1.2 10.4 0.7 9.5 1.2 11.5 0.8 10.9 0.8 10.7 0.7 11.0 0.9
'Agrado-Plus (Novus Internat. Inc., St. Charles, MO).


109










Table 3-5. Continued
P values



Measure -8 < O o
A ) 0 .
0 0 5 0
NEFA, peq/L 0.07 0.16 0.31 < 0.001 0.06 0.78 0.14 <0.001 <0.01 <0.01 <0.01 <0.001 0.03 < 0.01 0.02
BHBA, mg/dL 0.22 0.11 0.75 < 0.01 0.30 0.85 0.73 <0.01 0.03 0.87 0.15 0.01 <0.01 0.61 0.22
Glucose, mg/dL 0.03 0.16 0.45 < 0.001 0.45 0.63 0.63 0.08 0.63 0.49 0.67 0.12 0.37 0.70 0.18
BUN, mg/dL 0.61 0.15 0.37 0.46 0.59 0.79 0.70 <0.001 0.38 0.52 0.08 0.59 0.94 0.98 0.19


110










A ---C-Cool -[0-C-Hot -r-AO-Cool --AO Hot
800 -
700
S600 -
( 500 -
1 400 \
300
Z 200
100
0
0 I I I I I I
0 1 2 3 4 5 6 7
Week postpartum
B
C Cool -- C- Hot --r AO -Cool GAO Hot
1400

1200 -

S1000

u. 800

600
Ui-
Z 400

200 -

0I I I I I I
0 1 2 3 4 5 6 7
Week postpartum

Figure 3-13. Least squares means for weekly plasma concentrations of NEFA of
postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows
fed diets supplemented without (Control, C) or with synthetic antioxidants
(AO) and housed in shaded freestalls equipped with fans and sprinklers
(Cool) or just shade (Hot). Effect of parity by diet by environment by week
interaction (P = 0.02). Week with one asterisk indicates that means for the 8
treatments differed for that week using slice command (P < 0.05); with two
asterisks differed at P < 0.001.


111











-- Control Cool --- Control Hot AO Cool
10

6*



-2
-4 -
-6
-8
-10


-0- AO- Hot
**


1 2 3 4 5 6 7


Week postpartum


---Control Cool -0- Control Hot
10
7
4
1
-2
-s5
-8
-11 -
-14
-17


---AO Cool -0- AO Hot


L`I


2 3 4 5 6 7


Week postpartum
Figure 3-14. Least squares means for weekly energy balance of postpartum
primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO) and
housed in shaded freestalls equipped with fans and sprinklers (Cool) or just
shade (Hot). Effect of parity by diet by environment by week interaction (P =
0.03). Week with one asterisk indicates that 8 treatment means differed for
that week using slice command (P < 0.05); with two asterisks differed at P <
0.01; dagger differed at P< 0.10.


112










--D- Primi AO -A- Multi Control


16
14
12
10 -
8
6
4-


0 **


- -4


U I I I I I I I
0 1 2 3 4 5 6 7
Week postpartum


Figure 3-15. Least squares means for weekly plasma concentration of beta-hydroxyl
butyric acid of primiparous primii, n = 22) and multiparous (multi, n = 13)
Holstein cows fed diets supplemented without (Control) or with synthetic
antioxidants (AO). Effect of diet by parity by week interaction (P < 0.01).
Week with two asterisks indicates that means differed for that week using
slice command (P < 0.01).


113


Primi Control


SMulti AO










Table 3-6. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on postpartum body temperature and
plasma concentration of oxidative markers of lactating Holstein cows during summer in Florida.
Treatment


Measure


Body temp, C
TBARS2,
nmol/mL
GPxj,
nmol/min/mL
GPx4,
nmol/min/mL
SOD3, U/mL
SOD6, U/mL


Control diet


Cooled
Primiparous Multiparous
39.0 0.1 39.0 0.2


Non-cooled
Primiparous Multiparous
38.7 0.1 38.4 0.2


2.32 0.19 2.34 0.29 2.08 0.17 1.39 0.31


10,302 720
35,985
2181
2635 + 272


8854 1133
34,960
3444
2503 427


9247 1078 9709 1701


12,247 +
9384 659 1 7
1157
35,802 + 40,505 +
1993 3464
2486 248 3111 439
10,634 +
9635 985 10,634
1713


PCV', % of
PCV % of 28.6 1.1 25.5 1.8 26.0 1.0 30.3 1.8
blood
'Agrado-Plus (Novus Internat. Inc., St. Charles, MO).
2 TBARS = thiobarbituric acid reactive substances.
3GPx = glutathione peroxidase expressed as per ml of blood corrected for PCV.
4GPx = glutathione peroxidase expressed per ml of red blood cell.
5SOD = superoxide dismutase expressed as per ml of blood corrected for PCV.
6 SOD = superoxide dismutase expressed per ml of red blood cell.
7 Packed cell volume.


AO diet'
Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous
39.0 0.1 38.9 0.1 39.0 0.1 38.4 0.2


1.95 0.19 1.61 0.19
10,720 +
9305 723 10,72
726


33,456
2184
2405 271
8806 +1079


35,716
2187
2759 275
9120 + 1081


2.15 0.17 1.52 0.21

9733 659 8697 801


37,214
1993
2454 248
9484 + 985


30,203
2435
3448 302
11,839
1203


27.4 1.1 29.8 1.1 26.2 1.0 29.1 1.2


114










Table 3-6. Continued
P values
0) 00) > 0) 0)
S- .- 0 -
.- -a_ -$
>0 >, 0 T i o
Measure o- O > o i n
0 < 1 0 g a a

Body temp, C 0.70 < 0.01 0.51 0.01 0.26 0.12 0.52 0.56 0.79 0.87 0.55 0.88 0.68 0.95 0.15
TBARS 0.19 0.13 0.07 0.02 0.68 0.15 0.52 <0.001 0.51 0.54 0.70 0.71 0.77 0.25 0.62
GPx 0.34 0.72 0.10 0.46 0.67 0.44 0.01 0.45 0.83 0.46 0.91 0.24 0.24 0.83 0.76
GPx 0.15 0.62 0.33 0.88 0.25 0.63 0.05 0.02 0.72 0.37 0.69 <0.001 0.35 0.89 0.98
SOD 0.72 0.20 0.76 0.05 0.35 0.13 0.90 0.02 0.39 0.73 0.71 0.83 0.10 0.73 0.92
SOD 0.99 0.20 0.56 0.26 0.74 0.48 0.68 0.82 0.03 0.91 0.22 0.17 0.09 0.69 0.25
PCV 0.57 0.94 0.28 0.10 0.27 0.04 0.07 <0.01 0.91 0.43 0.82 0.78 0.22 0.42 0.67


115










2.3

2.2

2.1

E 2
0 1.9
E
1.8

W 1.7

S1.6

1.5
-15 -10 -5 0 5 10 15 20 25 30

Days relative to calving

Figure 3-16. Least squares means for plasma thiobarbituric acid reactive substances
(TBARS) on -15, 1, 8, 15, 29 d relative to calving. Effect of time (P < 0.0001).


116











2.5

E
2
E
S1.5




0.5

0
Control Cool Control Hot AO Cool AO Hot
Treatment


Figure 3-17. Least squares means for mean plasma concentration of thiobarbituric acid
reactive substances (TBARS) of Holstein cows (n = 35) fed diets
supplemented without (Control) or with dietary antioxidant (AO) and housed in
shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot).
Diet by environment interaction (P = 0.07).


117










39000


Z~ 38000
E
rc 37000

36000
E
-C 35000

(D 34000

33000
-15 -10 -5 0 5 10 15 20 25 30

Days relative to calving

Figure 3-18. Least squares means for weekly activity of glutathione peroxidase (GPx)
per mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented
without or with synthetic antioxidants and housed in shaded freestalls
equipped with fans and sprinklers or just shade on -15, 1, 8, 15, and 29 d
relative to calving. Effect of time (P = 0.02).


118











* Control Cool I Control Hot 0 AO Cool l AO Hot


0 -


Primiparous Multiparous


Parity

Figure 3-19. Least squares means for mean activity of glutathione peroxidase corrected
for pack cell volume of primiparous (n = 22) and multiparous (n = 13) Holstein
cows fed diets supplemented without (Control) or with synthetic antioxidants
(AO) and housed in shaded freestalls equipped with fans and sprinklers
(Cool) or just shade (Hot). Effect of diet by environment by parity interaction
(P = 0.01).


119


12000


9000


6000


3000










45000
Control Cool U Control Hot E AO Cool B AO Hot
43000

41000

E 39000

E 37000

35000
O
E 33000

X 31000

29000

27000

25000
Primiparous Multiparous

Parity

Figure 3-20. Least squares means for mean activity of glutathione peroxidase per mL
of erythrocyte of primiparous (n = 22) and multiparous (n = 13) Holstein cows
fed diets supplemented without (Control) or with synthetic antioxidants (AO)
and housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (Hot). Effect of diet by environment by parity interaction (P = 0.05).


120









3200


3000

-1 2800
E
) 2600

0 2400

2200

2000 I I I I I I I I I
-15 -10 -5 0 5 10 15 20 25 30

Days relative to calving


Figure 3-21. Least squares means for activity of superoxide dismutase (SOD) corrected
for pack cell volume of Holstein cows (n = 35) fed diets supplemented without
or with synthetic antioxidants and housed in shaded freestalls equipped with
fans and sprinklers or just shade on -15, 1, 8, 15, and 29 d relative to calving.
Effect of time (P = 0.02).


121











-- Control --[ AO


12000
11500
11000
-j

~ 10000
O 9500 ------
"~ 9000
8500
8000
-15 -10 -5 0 5 10 15 20 25 30

DIM

Figure 3-22. Least squares means for activity of superoxide dismutase (SOD) per mL
of erythrocyte of Holstein cows (n = 35) fed diets supplemented without
(Control) or with dietary antioxidants (AO) on -15, 1, 8, 15, and 29 d relative to
calving. Effect of diet by time (P = 0.03).


122










Table 3-7. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on concentration of white blood cells
(WBC), lymphocytes, and neutrophils, function of blood neutrophils, and plasma concentration of acid soluble
protein (ASP) and haptoglobin (Hp) of periparturient Holstein cows during summer in Florida.
Treatment


Measure


WBC, per pL

Lymphocytes, per pL
Neutrophils, per pL
Phagocytosis, %
Phagocytosis, MFIl
Oxidative burst, %
Oxidative burst, MFI
Acute phase proteins


Primiparous
9,273 +
1665
4272 +
1081
3,728 563
77.0 3.3
63.3 5.9
84.1 3.0
1374 + 211


Control diet
Cooled Non-cooled


Multiparous
5,998 +
1762
2208 944
2,983 + 730
80.4 5.2
34.5 5.1
81.2 4.6
1098 + 321


Primiparous
10,698
1766
4911 +
1131
4,571 661
80.3 3.2
75.3 6.8
84.7 2.8
1645 +219


Multiparous
12,917 +
3795


8145 3484
3,010 + 737
70.4 5.0
31.2 4.7
70.5 4.3
1094 + 297


ASP, pg/mL 45.1 4.6 39.9 7.3 48.6 4.2 35.3 7.3
0.015 0.015 0.013 0.012
Hp, arbitrary units
0.002 0.002 0.002 0.002
Agrado-Plus (Novus Internat. Inc., St. Charles, MO).
2MFI = Mean florescence intensity, to test the phagocytic ability of neutrophils.


Primiparous
8,488
1547
3912 996
3,031 489
71.1 3.0
48.5 4.4
77.1 2.7
1146 183

53.5 4.6
0.012
0.003


AO diet'
Cooled Non-cooled


Multiparous
8,249 +
1481


3862 978
2,819 + 426
73.8 3.6
40.4 4.2
76.1 3.2
950 196

41.5 4.6
0.011
0.003


Primiparous
8,969 +
1470
4700 +
1080
3,056 + 429
81.4 3.3
62.7 6.0
81.8 2.9
1589 222

50.7 4.2
0.011 +
0.002


Multiparous
8,652
1746
4559 1304
2,772 470
72.8 3.6
36.7 4.0
77.4 3.3
1025 209

42.6 5.1
0.013
0.002


123










Table 3-7. Continued
P values
C 0 >- 0
_o0 0 o 5 0- n 0
Measure o .- = 0 _
0- 0 L
0- 0 C o

WBC, per pL 0.56 0.09 0.18 0.60 0.76 0.30 0.29 <0.001 0.67 0.05 0.41 0.67 .042 0.97 0.83
Lymphocytes per pL 0.85 0.03 0.18 0.81 0.89 0.16 0.15 0.07 0.85 0.09 0.73 0.14 0.96 0.94 0.57
Neutrophils, per uL 0.14 0.68 0.66 0.11 0.35 0.66 0.74 <0.001 0.47 0.11 0.24 0.50 0.54 0.98 0.97
Phagocytosis, % 0.42 0.83 0.15 0.27 0.93 0.03 0.86 0.001 0.41 0.97 0.34 0.29 0.60 0.65 0.15
Phagocytosis, MFI 0.68 0.16 0.79 <0.001 0.02 0.05 0.79 <0.001 0.93 0.93 0.61 0.40 0.04 0.11 0.17
Oxidative burst, % 0.46 0.64 0.10 0.03 0.24 0.13 0.40 <0.01 0.36 0.73 0.08 0.05 0.86 0.96 0.31
Oxidative burst, MFI 0.46 0.29 0.70 0.03 0.96 0.40 0.90 <0.01 0.81 0.47 0.77 0.22 0.23 0.96 0.99
Acute phase proteins
ASP, pg/mL 0.22 0.85 0.97 0.02 0.92 0.78 0.43 <0.001 0.03 0.72 0.47 <0.001 0.74 0.89 0.86
Hp, arbitrary units 0.65 0.99 0.73 0.20 0.36 0.68 0.65 <0.001 0.24 0.03 0.72 0.09 0.97 0.60 0.35


124











--WBC (per IAL) -0- Neutrophil (per IAL) lymphocyte (per IL)
14017 -


CO
0
" 12017

c 10017
u
C
o 8017
u

u 6017

"5 4017

E 2017
I


-i


1. I I I I I I I
I/ -I ---- I----I----I---------------
-15 -10 -5 0 5 10 15

Days relative to calving


Figure 3-23. Least squares means for number of white blood cells (WBC), neutrophils,
and lymphocytes per pL of whole blood on -15, 0, 7, 14 d relative to calving.
Effect of time for WBC (P < 0.001), for neutrophil (P < 0.001), and for
lymphocyte (P = 0.07).


125


m i-----5


-

-










-*--Cool -I3- Hot


14017

12017

10017

8017

6017

4017

2017


- -


-15 -10 -5 0 5 10 15
Days relative to calving


Figure 3-24. Least squares means for number of white blood cells (WBC) per pL of
whole blood of Holstein cows (n = 35) housed in shaded freestalls equipped
with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by
week interaction (P = 0.05). Days relative to calving with asterisk indicates
that means differed (P < 0.05) using slice command.


126










-* Cool -I- Hot


6017
0
0
.o 5017

1 4017

0 3017

2017
0
E 1017
z
17
-15 -10 -5 0 5 10 15
Days relative to calving


Figure 3-25. Least squares means for number of neutrophils per pL of whole blood of
Holstein cows (n = 35) housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot). Effect of environment by week
interaction (P = 0.11).


127









-* Cool -I- Hot


7017
6017 -
5017
4017 -
3017
2017
1017
17


- -


Days relative to calving


Figure 3-26. Least squares means for number of lymphocytes per pL of whole blood of
Holstein cows (n = 35) housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot). Effect of environment by week
interaction (P = 0.09). Days relative to calving with asterisk indicates that
means differed (P < 0.05) using slice command.


128


$-











Phagocytosis % --- Phagocytosis MFI
90 80
80- 70
? 70 ..-60 |
60 0
60 >
S 50 u
S50 0bO
4- % r -40 c
>7 40 -- M.
0 30 -
S30 0
S2 20 M
2 20
10 10
0 I I I I 0
-15 -10 -5 0 5 10 15

Days relative to calving

Figure 3-27. Least squares means for percentage of neutrophils with phagocytic activity
(solid line) and neutrophil mean florescence intensity (MFI, indication of
number of bacteria phagocytised per neutrophil, dash line) of Holstein cows
(n = 35) on -15, 0, 7, 14 d relative to calving. Effect of time (P = 0.001) for
percentage of neutrophils with phagocytic activity, effect of time (P < 0.001)
for neutrophil MFI.


129










- oxidative burst % --- oxidative burst MFI


- 2000
- 1800
- 1600
- 1400
- 1200
- 1000
- 800
- 600
- 400
- 200
0


Days relative to calving


Figure 3-28. Least squares means for percentage of neutrophils with oxidative burst
activity (solid line) and neutrophil mean florescence intensity (MFI, indication
of intensity of reactive oxygen species produced per neutrophil, dash line) of
Holstein cows (n = 35) on -15, 0, 7, 14 d relative to calving. Effect of time (P <
0.01).


130


100
90
80
70
60
50
40
30
20
10
0


+


~

5
5~_ T


f f









90
80 T
70 -
60 -
O 50
U40 -
0
30,-
.. 20
10 -
0
Primi Cool Primi Hot Multi Cool Multi Hot
Treatment

Figure 3-29. Least squares means for mean percentage of neutrophils with phagocytic
activity of primiparous primii, n = 22) and multiparous (multi, n = 13) Holstein
cows housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (Hot). Effect of environment by parity interaction (P = 0.03).


131










80 -


.L 70 -
0
w60
U
0 50
tO
4 40

30
LL 20

10 -

0 I I I I
Primi Cool Primi Hot Multi Cool Multi Hot
Treatment


Figure 3-30. Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows housed in
shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot).
Effect of environment by parity interaction (P = 0.05).


132









80


70

o
U 60 -
0
o
O.

4I-
0 40

30 -

20

10 -

0
Primi Control Primi AO Multi Control Multi AO
Treatment

Figure 3-31. Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO). Effect of
diet by parity interaction (P = 0.02).


133










Primi Control --- Primi AO -A- Multi Control -- Multi AO


120

100

80

60

40

20


-15 -10 -5 0 5 10 15
Days relative to calving


Figure 3-32. Least squares means for neutrophil mean fluorescence intensity (MFI,
indication of number of bacteria phagocytised per neutrophil) of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO) on -15, 0,
7, 14 d relative to calving. Effect of diet by parity interaction (P = 0.04). Days
relative to calving with two asterisks indicates that means differed for that day
using slice command (P < 0.01), with three asterisks differed (P < 0.001), with
dagger differed (P <0.10).


134









86
84
o 82 -
80O
78 -
76
4 74
x 72
0
70 -
68
Control cool Control hot AO cool AO hot
Treatment

Figure 3-33. Least squares means for mean percentage of neutrophil with oxidative
burst activity of primiparous (n = 22) and multiparous (n = 13) Holstein cows
fed diets supplemented without (Control) or with synthetic antioxidants (AO)
and housed in shaded freestalls equipped with fans and sprinklers (Cool) or
just shade (Hot). Effect of diet by environment interaction (P = 0.10).


135










-- Control Cool --- Control Hot -A- AO- Cool -- AO- Hot
95

o0 90

85 -

G80



X 70


-15 -10 -5 0 5 10 15
Days relative to calving

Figure 3-34. Least squares means for percentage of neutrophil with oxidative burst
activity of Holstein cows (n = 35) fed diets supplemented without (Control) or
with synthetic antioxidants (AO) and housed in shaded freestalls equipped
with fans and sprinklers (Cool) or just shade (Hot) on -15, 0, 7, 14 d relative to
calving. Effect of diet by environment by time interaction (P = 0.08). Days
relative to calving with dagger indicates that means differed for that day using
slice command (P < 0.10).


136










--- Control Cool -I- Control Hot -A- AO Cool --- AO Hot
1.4

> 1.2

1,2

S0.8
S0.6

o 0.4


0
-3 -2 -1 0 1 2 3 4 5 6 7

Week relative to calving


Figure 3-35. Least squares means for IgG response against ovalbumin of Holstein
cows (n = 35) fed diets supplemented without (Control) or with synthetic
antioxidants (AO) and housed in shaded freestalls equipped with fans and
sprinklers (Cool) or just shade (Hot) on -4, -2, 0, 1, 2, 3, 4, and 7 wk relative
to calving. Arrows on the top of week indicate the week of ovalbumin
injection. Effect of diet by environment by week interaction (P = 0.69).


137










-* Prim-Control -0-- Prim-AO -- Multi-Control Multi-AO
1.4

> 1.2

Q. 1

0.8
U
0.6

.0 0.4 -
0.2

0
-4 -3 -2 -1 0 1 2 3 4 5 6 7

Week relative to calving

Figure 3-36. Least squares means for IgG response against ovalbumin of primiparous
primii, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets
supplemented without (Control) or with synthetic antioxidants (AO). Effect of
diet by parity interaction (P = 0.05). Week with two asterisks indicates that
means differed for that week (P < 0.01) using slice command.


138









-- Primiparous -&- Multiparous


80 -
70 -












0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
E
o50










DIM
40
S30

20
10


0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
DIM

Figure 3-37. Least squares means for concentrations of acid soluble protein (ASP) of
primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets
supplemented with or without dietary antioxidant (Agrado Plus) and housed in
shaded freestalls equipped with fans and sprinklers or just shade. Parity by
DIM interaction (P < 0.001). Days in milk with asterisks indicates that means
differed for that day using slice command (P < 0.05).


139











80

70 -

60
-j
E 50-

40-

S30 -

20

10


**


A4:


-4-Healthy -e- Unhealthy
1"
**

c~ "*


0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48

DIM

Figure 3-38. Least squares means for concentrations of acid soluble protein of Holstein
cows (n = 35) diagnosed as healthy (n = 14) or unhealthy metritiss, mastitis,
or retained fetal membranes, n = 21). Treatment by DIM interaction (P =
0.04). Days in milk with one asterisk indicates that means differed for that day
using slice command (P < 0.05), with two asterisks differed (P < 0.001).


140


IIIIIIIIIIIIIIIII


I i I I


I I I I









control -0- AO
**
80 **
70 *t
,60 6
E 50o1
?40
30 -
20
10 -
0

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
DIM


Figure 3-39. Least squares means for concentrations of acid soluble protein (ASP) of
Holstein cows (n = 35) fed diets supplemented without (Control) or with
synthetic antioxidants (AO). Diet by days interaction (P = 0.03). Days in milk
with dagger indicates that means differed for that day using slice command (P
< 0.10), with one asterisk differed (P < 0.05), with two asterisks differed (P <
0.01).


141










0.045 --Primiparous -- Multiparous
0.04
.E 0.035 t
C 0.03


U 0.02 -
o 0.015 \
o. 0.01
0.005

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
DIM


Figure 3-40. Least squares means for concentrations of haptoglobin of primiparous (n =
22) and multiparous (n = 13) Holstein cows fed diets supplemented with or
without dietary antioxidant (Agrado Plus) and housed in shaded freestalls
equipped with fans and sprinklers or just shade. Parity by DIM interaction (P =
0.09). Days in milk with asterisk indicates that means differed for that day
using slice command (P < 0.05), with dagger differed (P < 0.10).


142











0.05
0.045
S0.04
.1-a
S0.035
C-
o 0.03
'F 0.025
U
.1--
'* 0.02
. 0.015
e-
I 0.01

0.005
0


-- Healthy -0- Unhealthy


t /


I .... ....


0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48

DIM


Figure 3-41. Least squares means for concentrations of haptoglobin of Holstein cows
(n = 35) diagnosed as healthy (n = 14) or unhealthy metritiss, mastitis, or
retained fetal membranes, n = 21). Treatment by DIM interaction (P= 0.03).
Days in milk with one asterisk indicates that means differed for that day using
slice command (P < 0.05), with three asterisks differed (P < 0.001), with
dagger differed (P <0.10).


143


I I I I


I I


i l l i i l l i










0.04 Cool -- Hot

0.035 -

S0.03 -
0.025 -
'7 0.02
U
'. 0.015
o
0.01 -
0.005 -


0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48
DIM


Figure 3-42. Least squares means for concentrations of haptoglobin of Holstein cows
(n = 35) fed diets supplemented with or without dietary antioxidant (Agrado
Plus) and housed in shaded freestalls equipped with fans and sprinklers
(cool) or just shade (hot). Environment by DIM interaction (P = 0.03). Days in
milk with dagger indicates that means differed for that day using slice
command (P < 0.10).


144










Table 3-8. Profile of plasma progesterone of postpartum dairy cows fed with or without synthetic antioxidants (AO) and
cooled or noncooled during the prepartum period during the summer season in Florida.
Treatment
Control diet AO diet'
Measure Cooled Non-cooled Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous
First ovulation, DIM' 21.6 3.8 12.0 8.6 18.7 3.5 21.0 6.1 24.0 3.8 17.0 3.8 23.0 3.5 20.5 6.1
Length of first cycle, d 12.2 2.2 17.0 5.0 15.2 2.0 23.0 3.5 19.5 2.5 20.8 2.2 13.5 2.0 16.5 3.5
Peak progesterone in first
Peak progesterone in f 5.0 1.1 10.5 2.5 6.9 1.0 10.6 1.8 5.6 1.3 5.5 1.1 5.7 1.0 6.0 1.8
cycle, ng/mL
Number of ovulations 2.0 0.3 1.5 0.5 2.0 0.3 1.5 0.5 1.6 0.3 1.6 0.3 2.0 0.3 0.7 0.4
Accumulated
progesterone from 1 to 49 26.1 8.0 32.0 12.6 35.1 7.3 51.4 12.6 26.8 8.0 41.3 8.0 33.0 7.3 19.0 8.9
DIM, ng/ml
SAgrado-Plus (Novus Internat. Inc., St. Charles, MO).
2 Based on 32 of 35 cows ovulating in the first 49 DIM.


145










Table 3-8. Continued
P values
Diet by
Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P cooling P
cooling by P
First ovulation 0.45 0.57 0.81 0.26 0.88 0.27 0.62
Length of first cycle, d 0.74 0.88 0.04 0.06 0.35 0.59 0.88
Peak progesterone in first
0.03 0.54 0.75 0.04 0.05 0.74 0.60
cycle, ng/mL
Number of ovulations 0.33 0.68 0.68 0.04 0.81 0.25 0.25
Accumulatedprogesterone 0.36 0.64 0.10 0.40 0.42 0.50 0.15
from 1 to 49 DIM, ng/ml


146










14 -


12










2
10

c









Primi Control Primi AO Multi Control Multi AO
Treatment

Figure 3-43. Least squares means for mean peak concentration of progesterone (P4)
of the first cycle of primiparous primii, n = 22) and multiparous (multi, n = 13)
Holstein cows fed diets supplemented without (Control) or with synthetic
antioxidants (AO). Effect of diet by parity interaction (P = 0.05).


147










Table 3-9. Incidence of postpartum health disorders of dairy cows fed with or without synthetic antioxidants (AO) and
cooled or noncooled during the prepartum period during the summer season in Florida.
Control diet AO diet'
Measure Cooled Non-cooled Cooled Non-cooled
Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous
Number of animals 5 2 6 2 5 5 6 4
Endometritis 1 2 3 2 3 2
Retained fetal membranes 1 1
Mastitis 2 1 1 2 1
Ketosis 2 2 4 1 3 3 3 4
Displaced abomasum 1 1 1
Milk fever 1
' Agrado-Plus (Novus Internat. Inc., St. Charles, MO).


148









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BIOGRAPHICAL SKETCH

Dan Wang was born in Beijing, China on 1983. She is the daughter of Ruilin Wang

and Jinhua Fei. Dan Wang started her Bachelor of Science degree at Capital Normal

University in 2002 and graduated in 2006. Later she pursued her Master of Science

majoring in microbiology at the same university. But one year later, she married with

Zheng (Alex) Fu and moved to Gainesville with her husband in 2007. Dan Wang did a

volunteering work in the Department of Animal Sciences in University of Florida from

March to June in 2008. Then she started her Master of Science at University of Florida,

Department of Animal Science, under the guidance of Dr. Charles R. Staples. Her

research focused on the oxidative status of dairy cows during the transition period. After

graduation, she is going to stay on her Ph.D. under Dr. Charles R. Staples. Dan Wang

has a lovely daughter named Eunice and she really enjoys the life with this baby girl.


162





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1 EFFECT OF FEEDING SYNTHETIC ANTIOXIDANTS AND PREPARTUM EVAPORATIVE COOLING ON PERFORMANCE OF PERIPARTURIENT HOLSTEIN COWS DURING SUMMER IN FLORIDA By DAN WANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 2010 Dan Wang

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3 To my husband Zheng Fu who loves me, encourag es me, and support s me

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4 ACKNOWLEDGMENTS First of all, I sincerely thank my dear Lord Jesus who has strengthened me all the time since I put my faith in Hi m in 2007 so that I am able to do everything through Him. 1 Peter 5:10 s tate into His eternal glory in Christ Jesus, after you have suffered a little while will Himself perfect, establish, You make your home in my heart through faith. I would like to thank my mentor Dr. Charles R. Staples for allowing me to pursue the MS degree in the D epartment of A nimal S ciences. He guided me in the academic field and cared me for when I was lost in the American life. His warm heart has support ed me from the beginning to the end of my MS program. I am grateful for his infinite patience to interpret every single question I ask and throughout the study. I greatly thank Dr. Adegbola Adesogan for agreeing to be my committee member. I deeply app reciate his prayer s concerning how I could finish my MS while taking care of my little baby. I would like to give thanks to Dr. Jose Eduardo Santos who collect ed uterine flush samples from the experimental cows and buil t the statistical model for analyzing the categori cal data Thanks Dr. Santos for inspiring m e to deepen my knowledge o f rumina nt nutrition, metabolic physiology and statistics. Also thanks go to his wife, Cristiana R.C. Santos for sending those sweet messages to me when Eunice was born and giving her a playing mat and baby clothes. I deep ly appreciat e Dr. Lok enga Badinga for a llowing me to volunt a ri ly work with his formal student Dr. Cristina Caldari Torres at the university dairy farm. I am grateful that I was able to work with Cristina and to learn a lot of techniques from her. I owe special thanks to all the biological scientists who at one time or another technically supported my study. These include Sergei Sennikov who collected milk

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5 samples every Tuesday during the trial and helped performed plasma analysis for g lucose, BUN, BHBA, and NEFA. Jan Kivipe lto shared experiences about the 8 isoprostane assay, tested it for bovine species with me for a long period of time and taught me how to operate the Elemantar and plate reader. Joyce Hayen updated milk yield data every month Jae Hyeong Shin n ot only helped daily with the management of the study but also wa s my good friend after work Andrea Dunlap shared her experience s with the haptoglobin assay I also would like to thank Eric Diepersloot, Grady Byers, Mary Russell, Jerry Langford, Molly Gleason, P atty Best, Sherry Hay, Jay Lemmermen, Megan Manzie, Krista Seraydar and all the Dairy Unit employees who offered time and help to make this trial successful. I have great appreciation for Dr. Bruno do Amaral and Dr. Lilian Oliveira for elucidating for me the working principle s of flow cytometry and how to analyze neutrophil function data. I want to extend my appreciation to my research companion s in the D epartment of Animal Sciences that directly or indirectly help ed in my research and care d for me : Miria m Garcia Orellana who offered her help to the uttermost in this study and also shared her deep knowledge of nutrition with me Sha Tao who I knew before I entered into the MS program gave me encouragement when I was weak Leandro Greco who W e are o and Rafael Bisinotto and Eduardo Ribeiro who helped me perform the progesterone assay and professionally answered m y many questions about uterine diseases. Special thanks go to Milerky Perd omo and Kathy Arriola who shared their precious experiences with me about being a good mom I would also like to thank Oscar Queiroz and Fabio

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6 Lima for being around the building and their good friendship. I am very grateful for what all of you have done for me. M y special thanks to Dr. Joel Brendemuhl and Joann Fischer for their advice and help with academic questions. Special thanks to Sabrina Robinson and Delores Bradshaw for their hugs and help with all kinds of miscellaneous issue s. I give my deep ap preciation to my husband ( G ougou) and my parents. Thank you all for shepherding me and supporting me immeasurably. Dear G ougou, I am grat eful that you make me stronger little by little. I thank you for your sweet love during the past 7 years. Without yo ur support, I would not have finish ed this endeavor Praise you Lord unceasingly!

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7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .............. 4 LIST OF TABLES ................................ ................................ ................................ ......................... 9 LIST OF FIGURES ................................ ................................ ................................ ..................... 10 LIST OF ABBREVIATIONS ................................ ................................ ................................ ...... 15 ABSTRACT ................................ ................................ ................................ ................................ 17 CHAPTER 1 INTRODUCTI ON ................................ ................................ ................................ ................. 19 Oxygen and Oxidative Stress ................................ ................................ ............................ 19 Oxidative Status and Health of Dairy Cattle ................................ ................................ ... 20 2 LITERATURE REVIEW ................................ ................................ ................................ ...... 22 Reactive O xygen S pecies F ormation ................................ ................................ .............. 22 Oxidative S tress and O xidative D amage ................................ ................................ ........ 22 Lipid P eroxidation ................................ ................................ ................................ ................ 23 Antioxidant D efenses ................................ ................................ ................................ .......... 23 1) Enzymatic Ant ioxidants ................................ ................................ .......................... 23 a) Superoxide Dismutase ................................ ................................ .................... 23 b) Glutathione Peroxidase ................................ ................................ ................... 24 2) Nonenzymatic Antioxidants ................................ ................................ ................... 27 a) Glutathione (GSH) ................................ ................................ ............................ 27 b) Vitamin A and carotene ................................ ................................ ............... 28 c) Vitamin E ................................ ................................ ................................ ............ 30 d) Selenium ................................ ................................ ................................ ............ 31 3) Synthetic Antioxidants ................................ ................................ ............................ 33 Overview of Immune Function ................................ ................................ .......................... 34 Effect of Feeding Antioxidants on the Immune System ................................ ................ 35 Effect of Feeding Synthetic Antioxidants on Performance ................................ ........... 37 Effect of Feeding Antioxidants on Oxidative St atus and Stability ............................... 39 Effect of Prepartum Heat Stress on Performance and Metabolites ............................ 43 Effect of Prepartum Heat Stress on the Immune System ................................ ............. 45 The Effect of Cooling System for Dairy Cows in Hot Environment ............................. 46 Shading. ................................ ................................ ................................ ........................ 46 Cooling. ................................ ................................ ................................ ......................... 47

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8 3 EFFECT OF FEEDING ANTIOXIDANTS AND PREPARTUM EVAPORATIVE COOLING ON PERFORMANCE OF TRANSITION HOLSTEIN COWS DURING SUMMER IN FLORIDA ................................ ................................ ................................ ...... 50 Introduction ................................ ................................ ................................ .......................... 50 Material and Met hods ................................ ................................ ................................ ......... 51 Animals, Treatments, and Management ................................ ................................ .. 51 Sample Collection and Analysis ................................ ................................ ................ 52 Processing of Red Blood Cell (RBC) ................................ ................................ 54 Thiobarbituric Acid Reactive Substances (TBARS) Assay ............................ 55 Superoxide Dismutase (SOD) Assay ................................ ................................ 55 Glutathione peroxidase (GPx) Assay ................................ ................................ 57 Acute Phase Protein Assays ................................ ................................ ............... 58 Neutrophil Function, WBC and Lymphocyte ................................ .................... 59 Ovalbumin Challenge ................................ ................................ ........................... 60 Vagi noscopy ................................ ................................ ................................ .......... 61 Uterine Cytology ................................ ................................ ................................ .... 62 Statistical Analyses ................................ ................................ ................................ ...... 62 Results and Discussion ................................ ................................ ................................ ...... 64 Body Temperature, BW, BCS, and DMI ................................ ................................ ... 64 Milk Production and Milk Composition ................................ ................................ ..... 68 Plasma Metabolites ................................ ................................ ................................ ..... 71 Postpartum Body Temperature and Oxidative Markers ................................ ........ 73 In Blood ................................ ................................ ................................ ......................... 73 White Blood Cells ................................ ................................ ................................ ......... 78 Function of Blood Neutrophils ................................ ................................ .................... 80 Ovalbumin Challenge and Acute phase Proteins ................................ ................... 83 Progesterone ................................ ................................ ................................ ................ 85 Vaginoscopy and Uterine cytology ................................ ................................ ............ 86 Summary ................................ ................................ ................................ .............................. 87 LIST OF REFERENCES ................................ ................................ ................................ ......... 149 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ..... 162

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9 LIST OF TABLES Table page 3 1 Ingredient composition of diets fed to nonlactating and lactating Holstein cows. ................................ ................................ ................................ ................................ 90 3 2 Chemical composition of diets fed to nonlactating and lactating Holstein cows. ................................ ................................ ................................ ................................ 92 3 3 Effect of fe eding synthetic antioxidants (AO) and prepartum cooling on body temperature, body weight, BCS, and DMI of nonlactating pregnant Holstein cows during summer in Florida. ................................ ................................ ................... 93 3 4 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on performance of lactating pregnant Holstein cows during summer in Florida. ...... 97 3 5 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on plasma concentration of metabolites of lactating Holstein cows during summer in Flo rida. ................................ ................................ ................................ ....... 109 3 6 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on plasma concentration of oxidative markers of lactating Holstein cows during summer in Florida. ................................ ................................ ................................ ....... 114 3 7 Effect of feeding synthetic antioxidants (AO) and prepartum cooling on c oncentration of white blood cells (WBC), lymphocytes, and neutrophils, function of blood neutrophils, and plasma concentration of acid soluble protein (ASP) and haptoglobin (Hp) of periparturient Holstein cows during summer in Florida. ................................ ................................ ................................ ....... 123 3 8 Profile of plasma progesterone of postpartum dairy cows fed with or without synthetic antioxidants (AO) and cooled or noncooled during the prepartum period during the summer season in Florida. ................................ .......................... 145 3 9 Incidence of postpartum health disorders of dairy cows fed with or w ithout synthetic antioxidants (AO) and cooled or noncooled during the prepartum period during the summer season in Florida. ................................ .......................... 148

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10 LIST OF FIGURES Figure page 3 1 Least squares means for mean dry matter intake of prepartum primiparous (n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ................................ ................................ ..................... 95 3 2 Least squares means for mean body weight of prepar tum primiparous (n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ................................ ................................ ..................... 96 3 3 Least squares means for mean dry matter intake of postpartum primiparous (n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and f ed diets supplemented without (Control) or with dietary antioxidants (AO). ................................ ................................ ..................... 99 3 4 Least squares means for mean body weight (BW) of postpartum primiparous (n = 22) and multiparous (n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ................................ ................................ ................... 100 3 5 Least squares means for weekly body weight (BW) of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ................................ ................ 101 3 6 Least squares means for weekly dry matter intake of postpartum Holstein cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ............ 102 3 7 Least squares means for weekly milk production of Holstein cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented without (Control) or with dietary antioxidants (AO). ...................... 103 3 8 Least squares means for weekly concentration of milk fat of Holstein cows (n = 35) housed in cooled (C ool) or noncooled (Hot) freestalls. ................................ 104 3 9 Least squares means for weekly milk fat production of Holstein cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls. ................................ .......... 105 3 10 Least squares means for production of 3.5% fat corrected milk by postpartum Holstein cows (n = 35) housed in shaded freestalls equipped with fa ns and sprinklers (Cool) or just shade (Hot). ................................ ................................ ........ 106

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11 3 11 Least squares means for weekly concentration of milk protein of Holstein co ws (n = 35) fed diets supplemented without (Control) or with dietary antioxidants (AO).. ................................ ................................ ................................ ....... 107 3 12 Least squares means for somatic cell counts of postpartum Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinkle rs (Cool) or just shade (Hot). ................................ ................................ ................................ ........... 108 3 13 Least squares means for weekly plasma concentrations of NEFA of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets supplemented without (Control, C) or with synthetic antioxidants (AO) and housed in sh aded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ........ 111 3 14 Least squares means for weekly energy balan ce of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ............. 112 3 15 Least squares means for weekly plasma concentration of beta hydroxyl butyric acid of primiparous (primi, n = 22) and multip arous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO).. ................................ ................................ ................................ ....... 113 3 16 Least squares means for plasma thiobarbituric acid reactive substances (TBARS) on 15, 1, 8, 15, 29 d relative to calving.. ................................ ................ 116 3 17 L east squares means for mean plasma concentration of thiobarbituric acid reactive substances (TBARS) of Holstein cows (n = 35) fed diets supplemented without (Control) or with dietary antioxidant (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ................................ ............................... 117 3 18 Least squares means for weekly activity of glutathione peroxidase (GPx) per mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented without or with synthetic antioxidants and housed in shaded freestalls equipped with fans and sprinklers or just shade on 15, 1, 8, 15, and 29 d relative to calving. 118 3 19 Least squares means for mean activity of glutathione peroxidase corrected for pack cell volume of primiparous (n = 22) and multiparous (n = 13) Hol stein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ........ 119 3 20 Least squares means for mean activity of glutathione peroxidase per mL of erythrocyte of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO)

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12 and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot) ................................ ................................ ................................ ........... 120 3 21 Least squares means for activity of superoxide dismutase (SOD) corrected for pack cell volume of Holstein cows (n = 35) fed diets supplemented without or with synthetic antioxidants and housed in shaded freestalls equipped with fans and sprinklers or just shade on 15, 1, 8, 15, and 29 d relative to calving. ................................ ................................ ................................ ......... 121 3 22 Least squares means for activity of superoxide dismutase (SOD) per mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented without (Control) or with dietary antioxidants (AO) on 15, 1, 8, 15, and 29 d relative to calving. ................................ ................................ ................................ ....................... 122 3 23 Least squares means for number of white blood cells (WBC), neutrophils, 15, 0, 7, 14 d relative to calving. ... 125 3 24 blood of Holstein cows (n = 35) housed in shade d freestalls equipped with fans and sprinkle rs (Cool) or just shade (Hot). ................................ ........................ 126 3 25 Least squares means for number of neutrophils Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ........ 127 3 26 Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot) ................................ ................................ ........ 128 3 27 Least squares means for percentage of neutrophils with phagocytic activity (solid line) and neutrophil mean florescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil, dash line) of Holstein cows (n = 35) on 15, 0, 7, 14 d relative to calving. ................................ .......................... 129 3 28 Least squares m eans for percentage of neutrophils with oxidative burst activity (solid line) and neutrophil mean florescence intensity (MFI, indication of intensity of reactive oxygen species produced per neutrophil, dash line) of Holstein cows (n = 35) on 15, 0, 7, 14 d relative to calving ......................... 130 3 29 Least squares means for mean percentage of neutrophils with phagocytic activity of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ................................ ........... 1 31 3 30 Least squares means for neutrophil mean fluorescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil) of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows housed in

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13 sh aded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ................................ ............................... 132 3 31 Least squares means for neutrophil mean flu orescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil) of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). .................. 133 3 32 Least squares means for neutrophil mean fluorescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil) of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) on 15, 0, 7, 14 d relative to calving.. ................................ ................................ .......................... 134 3 33 Least squares means for mean percentage of neutrophil with oxidative burst activity of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented without (Cont rol) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). ................................ ................................ ................................ ................... 135 3 34 Least squares means for percentage of neutrophil with oxidative burst activity of Holstein cows (n = 35) fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipp ed with fans and sprinklers (Cool) or just shade (Hot) on 15, 0, 7, 14 d relative to calving... ................................ ................................ ................................ ......................... 136 3 35 Least squares means for IgG response against ovalbumin of Holstein cows (n = 35) fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot) on 4, 2, 0, 1, 2, 3, 4, an d 7 wk relative to calving. ................................ ................................ ................................ ....................... 137 3 36 Least squares means for IgG response against ovalbumin of primiparous (primi, n = 22) and mul tiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). .................. 138 3 37 Lea st squares means for concentrations of acid soluble protein (ASP) of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fans and sprinklers or just shade. ................ 139 3 38 Least squares means for concentrations of acid soluble protein of Holstein cows (n = 35) diagnosed as healthy (n = 14) or unhealthy (metritis, mastitis, or retainted fetal membranes, n = 21). ................................ ................................ ...... 140 3 39 Least squares means for concentrations of acid soluble protein (ASP) of Holstein cows (n = 35) fed diets supplemented without (Control) or with synthetic antioxidants (AO). ................................ ................................ ........................ 141

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14 3 40 Least squares means for conce ntrations of haptoglobin of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fans and sprinklers or just shade. ................................ .................... 142 3 41 Least squares means for concentrations of haptoglobin of Holstein cows (n = 35) diagnosed as healthy (n = 14) or unhealthy (metritis, mastitis, or retainted fetal membranes, n = 21). ................................ ................................ ........................... 143 3 42 Least squares means for concentrations of haptoglobin of Holstein cows (n = 35) fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fans and sprinklers (cool) or just shade (hot). ................................ ................................ ................................ ............ 144 3 43 Least squares means for mean peak concentration of progesterone (P4) of the first cycle of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Cont rol) or with synthetic antioxidants (AO). ................................ ................................ ................................ ......... 147

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15 LIST OF ABBREVIATION S ADF acid detergent fiber AO antioxidants ASP acid soluble protein BCS body condition score BHA butylated hydroxyanisole BHBA beta hydroxy butyric acid BHT butylated hydroxytoluene BUN blood urea nitrogen Con A concanavalin A DHR dihydrorhodamine 123 DIM days in milk DMI dry matter intake ELISA enzyme linked immunosorbent assays GPx glutathione peroxidase GSH glutathione GSSG glutathione disulfide Hb hemaglobin Hp haptoglobin IgG immunoglobulin G I.m. intramascu larly LCFA long chain fatty acids MDA m alondialdehyde MFI mean fluorescence intensity NDF neutral detergent fiber

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16 NEFA non esterified fatty acid NIR near infrared PBMC peripheral blood mononuclear cell PBS phosphate buffer solution PCV pack ed cell volume R BC red blood cell ROS reactive oxygen species SCC somatic cell count SDS s odium dodecyl sulfate SOD superoxide dismutase TBA thiolbarbituric acid TBARS thiolbarbituric acid reactive substances THI temperature humidity index WBC white blood cell

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17 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECT OF FEEDING SYNTHETIC ANTIOXIDANTS AND PREPARTUM EVAPORATIVE COOLING ON PERFORMANCE OF PERIPARTURIENT HOLSTEIN COWS DURING SUMMER IN FLORIDA By Dan Wang August 2010 Chair: Charles R. Staples Major: Animal Sciences The objective of this study was t o evaluate the effect of supplementation with 0 or 250 mg of synthetic antioxidants ( AO Agrado Plus, Novus International MO ) per kg of dietary DM and prepartum evaporative cooling on p eriparturient Holstein cows (n = 35) from 21 days before through 49 days after parturition in a 2 by 2 factorial design. Uterine h ealth was evaluated via metricheck at 7, 16, and 25 days in milk (DIM). Blood was collected at 15, 1, 8, 15, and 29 DIM for oxidative markers. Phagocytosis and oxidative burst of neutrophils were measured in whole blood collected at 15, 0, 7, and 14 DIM Acute phase proteins were measured in plasma collected three times weekly. A uterine horn was flushed at 40 2 DIM for diagnosis of subclinical endometritis. Rectal temperature of cooled cows was lower prepartum (39.2 vs. 39.6 C). Prepartum cooling r esulted in greater mean concentration of milk fat during 7 wk (3.54 vs. 3.32%) and mean production of 3.5% FCM during the first 4 wk postpartum (26.5 vs. 23.0 kg/d). Cooling reduced the concentration of circulating WBC postpartum ( 7864 vs. 10,199 per L o f blood) and of circulating lymphocytes (34 63 vs. 54 32 per L of blood) and increased proportion of neutrophils undergoing oxidative burst (83 vs. 77%) i solated

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18 from cows fed the control diet. Prepartum cooling of multiparous cows resulted in less oxidat ive stress as evidenced by lower activity of GPx in RBC (8,854 vs. 12,247 nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). Feeding AO increased concentration of milk true protein (3.07 vs. 2.94%) but decreased concentration of milk fat (3.25 vs. 3.61% ) resulting in less production of milk fat (0.88 vs. 1.04 kg/d) and of 3.5% FCM (26.2 vs. 29.5 kg/d). In addition, cows fed AO had a greater incidence of endo metritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs. 33%). Feeding AO to prepartum cooled cows reduced plasma concentration of TBARS (1.78 vs. 2.33 nmol/mL), proportion of neutrophils undergoing oxidative burst (77 vs. 83%), and mean florescence intestity of phagocytosis of primiparous cows postpartum (36 vs. 57%).

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19 CHAPTER 1 INTRODUCTION Oxygen and Oxidative Stress Animals do not use energy in feed directly for requirements of maintenance, activity, pregnancy, and productive purposes. C arbohydrates, lipids, and amino acids must undergo oxidative phosphorylation as the fina l stage to generate ATP. During oxi dative phosphorylation, molecular oxygen is reduced by accepting 4 electrons to produce two molecules of water. The electrons which reduce oxygen to water are derived from metabolism of feed. This process is call ed cellular respiration. However electrons which fail to be incorporated into the terminal acceptor of the transport chain may cause problems. About 1 to 2% of consumed oxygen is not completely reduced due to the escape of electrons from the intermediate complex in the respiratory chain (Levine, 1985) For example, the passage of electrons from red uced ubiquinone to complex III involves the radica l Q which could pass an electron to oxygen to form a superoxide radical (O 2 ). Another example is the generation of O 2 during the hydroxylation reactions catalyzed by Cytochrome P 450 (Ne lson, 2008) Anything which increases metabolic demands such as parturition lact ation, heat stress, and disease or disorders could increase oxygen requirements, number of electrons transferred, and production of O 2 (Sordillo and Aitken, 2009) Superoxide radicals can be reduced to hydrogen peroxide (H 2 O 2 ) by acceptance of a second electron. With acceptance of a third electron, oxygen can be reduced to hydroxyl radical (OH ). The generation of OH may cause two types of damage depending on the lo cation. On the one hand, OH may attack the peroxidative chain which can damage cellular and subcellular membranes (Gutteridge, 1994) On the oth er

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20 hand, OH is produced at the site where Fe is associated with a macromolecule such as DNA or protein causing damage of DNA and protein (Casciola Rosen et al., 1997) The derived free radicals such as O 2 H 2 O 2 and OH Oxidative Status and Health of Dairy Cattle Cells normally are protected against the harmful effect s of ROS by antioxidant defense s But when the generation of ROS exceeds the capacity of defensive system s to eliminate ROS, the oxidation antioxidation system is imbalanced. This process is defined as oxida tive stress. Levine and Kidd (1985) elucidated that the progress ion from oxidative stress to chronic disease can be divided into 4 stages (Levine, 1 985) In the first stage, the individual is healthy and able to deal with oxidative stress. In the second stage, due to chronic deficiency of antioxidant nutrients or exposure to oxidants, the individual adapts to oxidative stress. In the third stage, c ontinued oxidative stress depletes antioxidants so that the individual is subjected to oxidative damage. The third stage can become more severe so that absorption of antioxidant nutrients is influenced. In the fourth stage, the rate of deterioration of ant ioxidant defense exceeds the rate of recovery resulting in subclinical or clinical disease s The effects of antioxidants on oxidative status and health of dairy cows have been examined in recent years. Supplementation with vitamin E and Se usually reduced incidence of retained fetal membranes and mastitis ( Miller et al., 1993; Allison and Laven, 2000). The supplementation of antioxidants al so has been reported to improve oxidative status (Brzez inska Slebodzinska et al., 1994; Vazquez Anon et al., 2008 ; Sahoo et al., 2009 )

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21 The object ives of the literature review (C hapter 2) are the following: 1) to introduce the classes of antioxidant s namely enzymatic antioxidants including superoxide dismutase ( SOD ) a nd glutathione peroxidase (GPx), nonenzymatic antiox idants carotene, vitamin E and Se ; and synthetic antioxidants including ethoxyquin, butylated hydroxyanisole ( BHA ), and butylated hydroxytoluene ( BHT ) 2) to summarize the e ffectiveness of antioxidants on performan ce, immune system and oxidative status of animals. In addition the effect of heat stress on performance and immune system of animal s is reviewe d. In the subsequent research (C hapter 3), the effects of feeding synthetic antioxidants and prepartum cooling o n the performance of periparturient Holstein cows was investigated.

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22 CHAPTER 2 LITERATURE REVIEW Reactive O xygen S pecies F ormation Free radicals are molecules that have at least an unpaired electron in the outer orbit. They can accept or donate electrons from other molecules to generate a more stable molecule through oxidation and reduction reactions (Gitto et al., 2002 ; Halliwell, 2007a; Sordillo and Aitken, 20 09) Reactive oxygen species ( ROS ) is a collective term to classify oxygen derived free radicals, including superoxide radical (O 2 ), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH ) (Gitto et al., 2002) Normally, ROS are produced either through oxidative phosphorylation within the in ner membrane of mitochondria or pha gocytosis of pathogens to stimulate NADPH oxidase in neutrophils (Paape et al., 2003; Sordillo and Aitken, 2009) Animals an d human beings may undergo metabolic and physiological adaptations during the transition from pregnancy to lactation accompanied by elevated requirements for oxygen. This increased oxygen demand augments the production and accumulation of ROS in tissues. Oxidative S tress and O xidative D amage Oxidative stress refers to an imbalance between production of free radicals and antioxidant mechanisms (Halliwell, 2007a) It can result from dietary imbalances, pregnacy, environmental pollutants, solar radiation or heat stress (Gitto et al., 2002, Miller et al., 1993) Oxidative stress can result in oxidative damage to molecules, cells, and tissues which may subsequently develop to certain kinds of diseases or disorders such as endometriosis, heart failure, diabetes and so on in different species ( West, 2000 ; Sun et al., 2002; Jackson et al., 2005 ) For example, oxidative stress could

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23 ind irectly lead to Ca 2+ overload which activates phospholipase A 2 and C. These enzymes lead to membrane phospholipid hydrolysis (Gitto et al., 2002; Halliwell, 2007a). Another example is that oxidative stress could lead to membrane lipid peroxidation which gi ves rise to peroxyl radicals. Lipid P eroxidation Lipid peroxidation is a chain reaction (initiation, propagation, and termination). This reaction is initiated by attack of reactive hydroxyl radicals on polyunsaturated fatty acid s in plasma membranes. Propagation of lipid peroxidation gives rise to a lipid radical (L ) which interacts with oxygen under aerobic conditions to produce a peroxyl radical (LOO ). The reaction between peroxyl radicals generates nonradical products which terminates this chain r eaction (Burton and Ingold, 1984, Halliwell, 2007a) Antioxidant D efenses Antioxidants can be defined as any substance that helps protect cells by delaying, preventing or removing oxidative damage (Halliwell, 2007b) A ntioxidants can be classified based on their chemic al and physical characteristics, namely e nzymatic antioxidants, such as s uperoxide dismutase ( SOD ) and glutathione peroxidase ( GPx ) nonenzymatic antioxidants such as glutathione, ascorbic acid carotene, and ubiquinone and synthetic antioxidants such as ethoxyquin, butylated hydroxyanisole ( BHA ), and butylated hydroxytoluene ( BHT ) 1) Enzymatic A ntioxidants E nzymatic ant ioxidants work most efficiently and directly reduce ROS. a) Superoxide Dismutase Superoxide dismutase catalyzes the dismutation of superoxide to H 2 O 2 and O 2 ( Miller et al., 1993 ; Mates et al., 1999 )

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24 The three forms of SOD are the fo llowing: Cu/Zn SOD in cytosols, Mn SOD in mitochondria, and Fe SOD. Cu/Zn SOD plays an important role in the first defense against ROS (Mates and Sanchez Jimenez, 1999 ; Bernabucci et al., 2005 ) The activity of Cu/Zn SOD in milk was similar to that in bovine erythrocyte s (Przybylska et al., 2007) Mn SOD, which is a homotetramer, functions to remove superoxide produced in the electron transport chain in mitochondria. It can be induced and depressed by cytokines (Mates e t al., 1999) E xperiments using g ene knock out mice demonstrated that Mn SOD is essential for life, but that Cu/Zn SOD is not (Mates and Sanchez Jimenez, 1999) The Fe SOD are found only in prokaryotes such as Escherichia coli (Fridovich, 1975) It seems to provide a defense against exogenous superoxide. Superoxide dismutase activity has been used to evaluate oxidative status in animals. Cows in the last 3 wk of pregnancy had an incremental increase in plasma SOD activity and reached peak at 4 d after calving (Bernabucci et al., 2005) The same laboratory reported in 2002 that cows giving birth during summer had greater e rythrocyte SOD activity compared with the cows calving in spring. The activity of SOD in placental tissues was higher in cows with retained placenta l membranes than those without retained placenta l membranes (Kankofer et al., 1996) b) Glutathione Peroxidase Glutathione peroxidase can be classified as Se dependent and Se independent. The Se dependent enzyme catalyzes the reduction of hydroperoxides to water, accompanying the oxidation of glutathione (GSH) to glutathione disulfide (GSSG).

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25 Glutathione can be regenerated by reducing equivalents from NADPH 2 (Mates et al., 1999, Miller et al., 1993) Glutathione peroxides are composed of at least 5 isoenzymes (GPx 1 to 5) in mammals (Przybylska et al., 2007) Glutathione peroxidase 1 (GPx1) which is comprised of four identical SeCys containing subunits is the most predominant It is found in the cytoplasm of erythrocytes kidney, and liver. The preferred substrate s are hydrogen peroxide and a wide range of organic hydroperoxides. Glutathione peroxidase 4 (GPx4) also called phospholipid hydroperoxide glutathione peroxidase, is distributed in both cytosol and the membrane fraction, whose preferred substrate is phospholipid and cholesterol hydroperoxides (Thomas et al., 1990) Glutathione peroxidase 2 ( GPx2 ) is a cytosolic enzyme, whereas glutathione peroxidase 3 (GPx3) is extracellular, especially abundant in plasma The GPx3 metaboliz es phospholipid hydroperoxides and plays a direct role in protec tion of membranes (Takahashi et al., 1987) Glutathione peroxidase 5 (GPx5), being Se independent, is newly discovered in mouse epididymis (Przybylska et al., 2007) Glutathione peroxidase has been detected in bovine milk and its activity was strongly and positively correlated with S e concentration (Przybylska et al., 2007) Glutathione peroxidase activity can be used to evaluate oxidative status. Generally speaking, the activity of this enzyme could be elevated during parturition and lactation by heat stress, by BW loss, by diseases, or by co nsumption of different types of feedstuffs. When b lood samples were collected from dairy cows at 21 7 d befor e calving, calving, and 21 DIM, t he GPx activity in peripheral blood mononuclear cell s ( PBMC ) increased at calvin g and at 21 DIM compared with the prepartum period

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26 (Sordillo et al., 2007) Similarly, weekly blood samples collected from 30 d before calving to 30 d after cal ving indicated that plasma GPx activity began to increase a week before calving and was greater after calv ing than before calving (Bernabucci et al., 2005) The same authors also reported that cows giving birth during summer had erythrocytes of greater GPx activity at 21 d before calving compared to that of cows giving birth during spring (Bernabucci et al., 2002) However, GPx activity in plasma did not differ during the transition period between cows calving in the summer and spring season. Burke et al. (2007) reported that PBMC of heifers had less GPx activity when housed under high temperature humidity indices (THI) compared to neutral THI. Brennan co nducted a study to determine the effect of BW loss of beef cows which could increase the production of ROS through fat mobilization on the antioxidant activity of GPx and antioxidant mRNA levels (Brennan et al., 2009) Total RNA was isolated from skeletal muscle, glutathi one peroxidase 1 and GPx4 target gene mRNA was measured using real time reverse transcription PCR. Erythrocyte GPx activity was measured to determine oxidative status. No differences were found in GPx1 activity between cows losing or maintaining BW. Theref ore BW loss did not influence the G Px activity of erythrocytes. However abundance of GPx 4 mRNA in skeletal muscle was 1 4 fold greater during BW loss. Activity of plasma GPx was greater in patients who were diagnosed with u disease compared with healthy individuals who d previous records of inflammatory bowel disease (Tuzun et al., 2002) The activity of GPx in placental tissues was greater in cows with retained placenta l membranes than those without retained placenta membranes (Kankofer et al., 1996) Cows fed grain or a mixed diet had a greater muscle GPx

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27 activity than cows consuming only pasture ( Mercier et al., 2004 ; Descalzo and Sancho, 2008 ) probably due to the different concentrations of Se in different feedstuffs (Ammerman, 1975) Lambs fed brown seaweed extract exhibited greater GPx activity in erythrocyte and white blood cell compared with animals not fed the extract indicating that brown seaweed extract improved lamb antioxi dant status by increasing antioxidant capacity (Saker et al., 2004) 2) Nonenzymatic A ntioxidants a) Glutathione (GSH) Glutathio ne a low molecular weight thiol is abundant in animal cells and plasma Majority of the cellular GSH (85 to 90%) is distributed in the cytosol, with the rest located in many organelles including the mitochondria, nuclear matrix, and peroxisomes. On avera ge, the concentration of GSH ranges from 0.5 to 10 mmol/L, with the exception of bile acid which contains more than 10 mmol/L. The roles of glutathione include the following: 1) antioxidan t defense including the scavenging of free radicals and other reacti ve species, removal of hydrogen and lipid peroxides, and prevention of oxidatio n of biomolecules; 2) m etabolic roles such as serving as a substrate for synthesis of leucotriene C4, a substrate to convert formaldehyde to formate, and for storage and transpo rt of cysteine; 3) a ids in r egulation of c ytokine production, immune responses, and mitochondrial function and integrity (Wu et al., 2004) Usually the concentration ratio of GSH: GSSG is used as an index of the cellular oxidative status, which is greater than 10 under normal physiological conditions (Griffith, 1999) The GSH:GSSG ratio in PBMC of heifer s was less under heat stress, which indicated either increased production of ROS or decreased antioxidant status (Burke et al., 2007) Cows exposed to heat stress and fed endophyte infected tall fescue had a lower whole blood GSH concentration compared with those under heat stress without endophyte in fected

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28 tall fescue in the diet (Lakritz et al., 2002) The addition of GSH did not alter the proliferative response of lymphocyte isolated from the blood of Holstein cows at 38.5 C (neutral) or 42 C (heat stress) ( Kamwanja et al., 1994) b) Vitamin A and carotene carotene is the major dietary precurs or of vitamin A in dairy cattle (LeBlanc et al., 2004; Przybylska et al., 2007) carotene that escapes from the rumen is absorbed and converted to retinol in the intestinal mucosa and transported to the liver with fat (LeBlanc et al., 2004) Experiments indicate that carotene is neither a peroxide decomposing antioxidant such as catalase and GPx nor a conventional chain breaking antioxidant such as vitamin E (Burton and Ingold, 1984) It functions as an unusual antioxidant at l ow oxygen pressures (Burton and Ingold, 1984) and a scavenger of free radicals produced from unsaturated long chain fatty acid ( LC FA ) peroxidation (Hino et al., 1993) The bioavailability of vitamin A for cattle is considerably limited due to its destruction by ruminal microbes (Rode et al., 1990; Weiss et al., 1995) Hino et al. (1993) did an in vitro study to examine whether the addition of carotene could alleviate the inhibition of bacterial growth caused by LCFA. The results demonstrated that the addition of carotene increased bacteria l growth with the presence of LCFA. The active fo rm of vitamin A could be different for different functions (Hemken and Bremel, 1982) Al osilla et al. (2007) reported that feeding different commercial vitamin A sources to yearling beef cattle led to different retinol concentrations in liver indicating that some supplemental vitamin A sources had greater amounts of vitamin A reaching the du odenum for absorption and storage than others.

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29 Vitamin A plays an important role in resistance to infectious disease, especially mastitis (NRC, 2001) Johnston and Chew (1984) studied the peripartum c oncentrations of vitamin A and carotene in Holstein cows with or without mastitis. They reported that plasma concentrations of vitamin s A and carotene decreased rapidly before calving and reached the ir lowest point at calving (vitamin A) or on d 4 to d 6 postpartum ( carotene). In addition, c oncentration of vitamin A in plasma was lower in mastitic cows from d 0 to 7 and at wk 2 and 4 than nonmastitic cows, whereas carotene was greater in mastitic cows from prepartum to d 7. When feeding lactating Ho lstein cows carotene at a rate of 0 or 300 mg/d from 3 to 98 d postpartum concentration of carotene in serum declined in both control and carotene supplemented group s between 1 and 2 wk postpartum, but increased to 225 g/dl after 3 wk in supplemented group, whereas the concentration stayed the same in control group. Additionally, feeding carotene did not affect the length of first estrous cycle or peak concentration of progesterone in the first estrous cycle. However, the incidence of mastitis was less for the supplemented group (Wang et al., 1988) In a review paper Hemken and Bremel (1982) indicated that a deficiency of vitamin A was associated with a number of reproductive prob lems such as re tained placenta and abortions. Consumption of vitamin A to meet requirement could improve conception rate or reduce days open for cows. Pregnant Holstein cows were given 0, 300, or 600 mg/d of carotene or 120,000 IU/d of vitamin A from 4 wk before expec ted calving date to 4 wk postpartum (Michal et al., 1994) Blood lymphoc yte proliferation in response to 5 g/mL of concanavalin A ( Con A ) was greater at 1 wk before calving and at 2 wk postpartum in cows fed 600 mg/d of carotene compared to unsupplemented group. The phagocytic ability of

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30 Staphylococcus aureus by neutrophils was enhanced in cows fed 300 mg/d of carotene at 1 wk after calving compared with cows in other treatments. Tjoelker and co workers (1990) conducted a trial to evaluate the impact of supplementation of vitamin A carotene on neutrophil a nd lymphocyte function of dairy cows in the nonlactating period. Cows were assigned randomly to one of 3 treatments: 53,000 IU of vitamin A, 213,000 IU of vitamin A, or 53,000 IU vitamin A plus 400 mg of carotene per d from 6 wk before to 2 wk after dry off. Phagocytosis and bacterial killing ability of S. aureus by neutrophils were not different among treat ments but lymphocyte blastogenesis was stimulated by 10 g/mL of Con A on wk 2 for cows fed 53,000 IU of vitamin A but did not change in other treatm ent s throughout the experiment Whether supplemental vitamin A exerts these effect s through its role as an antioxidant is unknown c) Vitamin E Vitamin E is the term for a class of lipid soluble tocopherols ( ) and tocotrienols ( ), of which tocopherol has the highest biological activity (B rigelius Flohe and Traber, 1999; NRC, 2001) Vitamin E acts as a chain breaking antioxidant that limits the propagation of peroxidation by trapping free radicals ( Nockels et al., 1996 ; B rigeliu s Flohe and Traber, 1999; Goupy P., 2007; Gobert et al., 2009 ) It also exerts other functions involving cellular signaling, immunity and reproductive function. Vitamin E is absorbed in the small intestine and enters the circulation via the lymphatic syste m. It is absorbed with lipids, transported from the small intestine to the liver, packed in lipoproteins, and distributed via plasma to the rest of the body (Herdt and Smith, 1996) Although found in feedstuffs, the concentration and activity of vitamin

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31 E is easy to lose during processing and storage of feedstuffs. Therefore, supplementation of vitamin E in the diet is necessary The common form of supplemental vitamin E fed to dairy cattle is DL tocopheryl acetate. tocopherol was reduced during the last month prepartum and at 1 or 2 wk postpartum (LeBlanc et al., 2002; Rezamand et al., 2007) in dairy cows Goff et al. (2002) reported a similar pattern in that the concentration of vitamin E declined from 1 wk before calving to 3 DIM. A steady vitamin E state may be reached by supplementation of 3000 IU/d for 2 wk prepartum to pregnant heifers (Bouwstra et al., 2008) Oxidative damage during lipid peroxidation and its prevention by vitamin E can be analyzed by quant ification of 8 isoprostane (F 2 isoprostanes). 8 isoprostane concentration was reduced when overweight patients received 800 IU/d of vitamin E for 3 mon and 1200 IU/d for another 3 mon compared with a placebo group (Sutherland et al., 2007) indicating that supplementation of vitamin E may improve oxidative status in obese humans. Two studies (Rimm et al., 1993, Stampfer et al., 1993) in human species reported that high intakes of vitamin E reduced the risk of cardiovascular diseases. Supplementation or injection of vitamin E to animals/human s has reduc ed oxidative damage in the body. d) Selenium Many selenoproteins have Se in their structures to participate in the antioxidant defense system of cells (Cerri et al., 2009) Considerable evidence exists that Se functions by a similar mechanism as vitamin E in lipid peroxidation (Hamilton and Tappel, 1963) Selenium is an integral part of the enzyme GPx which functions to prevent oxidative damage to tissues or cells.

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32 Dietary s upplementation of 2 mg/d of Se in th e form of sodium selenite increased concentration of Se and GPx activity in blood of dairy cows during the periparturient period compared to cows n ot supplemented with Se (Grasso et al., 1990) However, feeding Se in the form of sodium selenite at 0.3 mg/kg o f dietary DM from 25 d before to 70 d after calving did not influence incidence of postpartum diseases (metritis, ketosis, and mastitis) and ovarian responses compared with cows fed Se yeast at the same rate (Cerri et al., 2009) due to the lack of effects of source of dietary Se on Se status A similar dietary supplementation of Se study was conducted by Silvestre ( 2006) in which they reported Se yeast reduced the risk of some postpartum uterine problems compared with that of a supplemental inorganic source of Se. The disparity of effects of supplementation of Se on health was likely due to differences in Se status due to location. Cows on the study of Silvestre (2006) were man aged in Florida using forages grown on Se deficient soils whereas cows used by Cerri et al. (2009) were managed in California on Se adequate soils. This was reflected by different blood concentrations of Se. Erskine et al. (1987) reported that somatic ce ll count ( SCC ) obtained from 9 dairy herds decreased as concentration of plasma Se increased in cows. The GPx activity was positi vely correlated with Se intake but negatively with SCC. Injection of 1 mg/kg of BW of Se at 21 d prior to estimated calving dat e for dairy cows reduced the incidence of mastitis by 12 % compared to cows not receiving Se injection (Smith et al., 1984) The effect of supplementation or injection of vitamin E and Se on mammary health has been review ed by Smith et al. (1997) who pointed out that vitamin E and Se deficiency were

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33 associa ted with greater incidence of mastitis and greater SCC. Therefore alleviating deficiencies of vitamin E and Se can enhance mammary health. 3) Synthetic Antioxidants Three commonly used synthetic antioxidants are ethoxyquin, butylated hydroxyanisole ( BHA ), and butylated hydroxytoluene ( BHT ). These synthetic antioxidants are used mainly by the feed industry to delay the peroxidation of feed lipids and to stabilize the formulation of vitamin A and vitamin D 3 in premixes and feeds In addition Kahl (1984) sum marized that synthetic antioxidants have been associated with a wide variety of molecular, cellular, and organ activity roughly divided into three categories: 1) modulation of growth, macromolecule synthesis and differentiation; 2) modulation of immune re sponse; and 3) interference with O 2 activation. Ethoxyquin is used widely as an antioxidizing agent in food formulation for animals including fish, livestock and pets. It functions as a scavenger of free radicals which are formed during lipid peroxidition (Yamashita, 2009) The FDA approved feeding rate of ethoxyquin for use in animal feeds is 150 ppm ( FDA, 2010 ) It has been used effectively to promote color retention and preserve fat soluble vitamins. However, its toxic effect in vitro on phagocytosis by leukocyte s isolated from swim bladder of tilapia was reported by Yamashita et al. (2009) when concentration of ethoxyquin in media was > 0.1 mg/L T hey also reported phagocytic activity by inflammatory leucocytes isolated from fish fed ethoxyquin at a rate of 150 mg/kg for 30 d was reduced compared with the activity in fish fed no ethoxyquin T wo additional synthetic antioxidants, BHA and BHT are used as food additives by the feed industry. Butylated hydroxyanisole is effective to preserve animal fats but not vegetable oils. It is approved for human and an imal use. Butylated hydroxytoluene

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34 has similar properties. Bj rkhem et al. (1991) reported that BHT had an antiatherogenic effect in cholesterol fed rabbits. However, organ proliferation and histopathological changes were induced by BHT and BHA in rat live r (Kahl, 1984) More studies of the effects and mechanism of action of synthetic antioxidants in animals need to be investigated in the future Overview of Immune Function The immune system can be divided simply into two systems: innate and adaptive systems In the innate system, foreig n bodies are destroyed and/or neutralized by an about that foreign body. In the adaptive system, immune responses are the result of a previous memory obtained by exposure t o a particular antigen. Therefore, adaptive immunity provides life long protection against reinfection by the same pathogens (Janeway, 2004) Both innate immunity and adaptive immunity responses depend upon the activities of WBC or leukocytes. Neutrophils, macrophages (the mature form of monocytes), and eosinophils are the primary cells to arrive at the infection site and phagocytize pathog ens or parasites without requiring memory (innate immunity). Phagocytosis by macrophages and neutrophils is triggered by the binding of ligand to the receptors, and subsequent destruction of pathogens takes place by complement or by the generation of toxic chemicals, such as superoxide radicals, hydrogen peroxide, and nitric oxide (Calder, 2007) Adaptive immune responses rely upon lymphocytes, i.e. B cells and T cells. Therefore, the components of the immune system communicate and work with each other to help hosts defend against infectious agents from the environment.

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35 Acute phase proteins as their names imply are synthesized by the liver and secreted into the blood to protect the host from local inflammation or stress. These proteins mimic the action of antibodies but are nonspecific Acid soluble protein is one acute phase protein which contains mainly acid glycoprotein It is an anti inflammatory agent that controls inappropriate or extended activation of the immune system (Jafari et al., 2006) Acid soluble protein has a dual immune modulatory effect in that it causes immune activation of macrophage s to secrete cytokine s or immune supp ression to control immune response Haptoglobin ( Hp ) is another acute phase protein which binds to hemoglobin and so inhibits bacterial proliferation by reducing the availability of iron ( Wassell, 2000 ; Huzzey, 2009 ) and functions as an antioxidant by virt ue of its ability to prevent hemoglobin driven oxidative damage of tissues (Melamed Frank et al., 2001) Many studies (Hirvonen et al., 1996; Wittum et al., 1996 ; Huzzey, 2009 ) have reported that an increase in plasma concentration of acute phase proteins, espec ially haptoglobin, is an indicator of severity or chronicity of sick ness in cattle. Effect of Feeding Antioxidants on the Immune S ystem Numerous studies have been conducted to evaluate the effects of supplementation of antioxidants on neutrophil functions, acute phase protein s and immune challenge A review of effects of vitamin E supplementation on health and fertility of dairy cattle (Allison and Laven, 2000) indicated that Holstein calves receiving up to 500 IU/d of dietary vitamin E increased the blastogenic responses of T cells and B cells (Reddy et al., 1987) Injection of 3000 IU of vitamin E at 10 to 5 d before calving increased the killing ability of bacteria by neut rophils at calving (Hogan et al., 1992) Weiss and Hogan (2005) reported that supplementing Se to provide 0.3 mg/kg of dietary DM from either sodium selenate or Se yeast with 500 IU of vitamin E did not

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36 affect the percentage of neutrophils that phagocyt ized E. coli in Holstein heifers or cows, but bacterial killing ability by neutrophils tended to be increased for cows fed selenite. Similarly, supplementation of vitamin E (400 to 600 mg/d) or Se (0.3 mg/kg of dietary DM ) alone to 21 multiparous Holstein cows increased the proportion of bacteria killed by neutrophils, but did not influence phagocytic ability (Hogan et al., 1990) Grasso et al. (1990) reported that cows supplemented with 2 mg/d of sodium selenite during the transition period had greater bacterial killing ability by neutrophils in milk and increased viability of neutrophils when challeng ed with S. aureus compared with cows not supplemented with Se. The percentage of neutrophils that phagocytized and killed Candida albicans was greater for cows that received sufficient Se (0.1 ppm of dietary DM) than cows given a deficient Se diet (Boyne and Arthur, 1979) However, the percentage of neutrophils that phagocytized C albicans did not differ between cows fed the two dietary treatments. Ascorbic acid (vitamin C) is considered to be the most abundant and important water soluble antioxidant. Functions of neutrophils (the proportion of neutrophils that phagocytized bacteria and number of intracellular bacteria per neutrophil) isolated from whole blood were not influenced by supplementation with either 0 or 30 g/d of vitamin C starting from 2 wk before calving through 7 DIM (W eiss and Hogan, 2007) Yamashita et al (2009) evaluated the effect of a synthetic antioxidant, ethoxyquin, on immunity of tilapia. The phagocytic activity of leucocytes in vitro was lower in leucocy te s exposed to 0.1 mg/L of ethoxyquin than leucocyte s not exposed to ethoxyquin. In summary, supplementation of natural antioxidants to dairy cows improved killing ability by neutrophil s measured as oxidative burst.

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37 B ull calves were assigned randomly to one of three supplementation rate s of vitamin E (285, 570, and 1140 IU/d, respectively) for 21 d. They were vaccinated with 4 mL of ovalbumin (2 m g/mL of PBS). A linear increase in IgG concentration with increased dietary supplementation of vitamin E was detected at 21 d after ovalbumin ingestion (Rivera et al., 2002) Similar to this finding, calves vaccinated with 125 IU of vitamin E at 7 wk of age had greater IgG values compared with those receiving no vitamin E (Reddy et al., 1987) The enhancement of serum IgG to Pasteurella haemolytica was detected in steers injected i.m. with 25 mg of Se and 340 IU of vitamin E (Droke and Loerch, 1989) Heifer calves were fed 2000 IU of v itamin E for 0, 7, 14, or 28 d ( Carter et al. 2002). No differences were detected in Hp concentrations in plasma among treatments on any sampling day However plasma concentrations of acid soluble protein were lower in calves fed 7, 14, or 28 d of vitami n E on d 7 of experimental period compared to concentration of calves not receiving vitamin E The effects of supplementation of vitamin E on responses of antibody and acute phase proteins were not consistant possibly due to differing initial concentration s of vitamin E that may have made the animals more or less susceptible to respond to additional vitamin E supplementation. Effect of Feeding Synthetic Antioxidants on P erf ormance Few experiments have been conducted to examine the effects of supplementatio n of synthetic antioxidants on the performance of dairy cattle. Feeding lactating Holstein cows a basal diet contain ing distillers grains (15% of dietary DM) supplemented without or with (0 or 0.02% of dietary DM) a blend of ethoxy q uin BHA and BHT (Agr ado Plus, Novus International, St. Louis, MO) did not affect DMI (26.6 0.5 kg/d) or milk yield (49.8 1.7 kg/d) (Preseault, 2008) Although milk fat depression occured in both control

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38 and Agrado Plus supplemented treatment groups, the exten t of milk fat depression was less for cows fed Agrado Plus (3.22 vs. 3.32%) In another abstracted study (He, 2008) milk yield of dairy cows was not influenced by adding Agrado Plus (0 or 0.025% of dietary DM) to six diets containing 1 of 5 vegetable oil s (control, palm, high oleic safflower, high linoleic safflower, linseed, or corn oil ) at 5% of dietary DM However Bowman et al. (2008) reported that dairy cows fed Agrado Plus at 250 mg/kg of dietary DM increased DMI (22.2 vs. 22 .7 kg/d) and tended to i ncrease milk yield (P < 0.10) (Bowman, 2008) Vzquez An et al. (2008) also detected increased DMI (20.2 vs. 20.9 kg/d, SEM = 0.22) and increased production of 3.5% FCM (27.3 vs. 28.3 kg/d, SEM = 0.36) when dairy cows (171 10 DIM) were fed Agrado Plus at 200 mg/ kg of dietary DM. A n in vitro study using continuous cultures (Vazquez Anon et al., 2008) was conducted to investigate the effect of presence or absence of Agrado Plus on nutrient digestibility, microbial N and fatty acid metabolism. This study was a 2 x 2 factorial design in which two types of oil (oxidized vs. u noxidized mixture of unsaturated oil) and supplementation or not of Agrado Plus at 20 0 mg/kg of dietary DM were combined. Feeding Agrado Plus increased NDF and ADF digest ion, and increased conversion of feed N to microbial N but tended to reduce the outflow of 18:3 in the effluent. Few experiments have been conducted to investigate the e ffect of supplementary synthetic antioxidants on performance of species other than dairy cattle. In poultry, feeding laying hens ethoxyquin at 250 mg/kg of food from 32 to 88 w k of age did not affect weight gain and egg production compared with hens fed control diet which contained 5 mg of vitamin E and 125 mg of ethoxyquin /kg of food (Bartov et al., 1991)

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39 Similar result s have been reported for chick s in that dietary ethoxyquin fed at 0, 125, 500, and 1000 ppm did not affect body weight and weight s of liver, spleen, kidney or heart. Average c arcass weights of broilers were greater when supplementing BHA at the rate of 12.5 mg/d/bird from 3 to 7 w k of age and BHT at the same rate for the last 5 d of the trial compared with those only fed u noxidized sunflower oil at 55 g/kg of dietary DM (Lin et al., 1989) Rainbow t rout were utiliz ed in a 2 x 2 x 2 factorial design with 2 feeding rates of oxidized fish oil (peroxide value of 5 and 120 meq/kg of oil), 2 feeding rates of tocopheryl acetate (0 and 33 mg/kg of food), and 2 feeding rates of ethoxyquin supplement ation (0 and 125 mg/kg of food). L ive weight gains and carcass composition were no t different among treatments (Hung et al., 1981) In s ummary feeding ethoxyquin did not affect performance of chicks and fish. Effect of Feeding Antioxidants on Oxidative Status and S tability Dunkley et al. (196 7) conducted a trial to evaluate the effects of supplementation of either tocopherol at 0.0025% of dietary DM or ethoxyquin at 0.0125% of dietary DM on oxidative stability of milk from dairy cows Oxidative stability of the milk fat was increased by the su pplementation of tocopherol but not by ethoxyquin. A second experiment was conducted to study the effect of feedin g increasing dietary concentrat ion s of ethoxyquin (0.015 and 0.15 % of dietary DM) on milk quality. T hey reported an increase in endogenous toc opherol concentration and oxidative stability of the milk when feeding either concentration of ethoxyquin but the improvement was greater when more ethoxyquin was fed The same research group also reported that supplement al ethoxyquin in the diet was transferred to milk and accompanied by appearance of an unidentified compound using a fluorimetric method (Dunkley et al., 1968)

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40 Oxidative status of mammals can b e monitored by several markers such as SOD GPx, and TBARS. The activity of erythrocyte SOD was decreased compared with the value before treatment after 3 injection s of Vitamin E (i.m., 500 IU/ injection ) and Se (i.m. 15 mg/ injection ) on alternate days up to the 5 th d ay as a therapy for subclinical ketosis. Comparing the activities of erythrocyte SOD after treatments, cows receiving additional vitamin E and Se had the lowest SOD activity followed by those receiving 5 injections of 25% dextrose daily at 540 mL plus 1 in jection of 4 mg/mL of dexamethasone at 2.5mL, whereas cows receiving no treatment had the highest SOD activity (Sa hoo et al., 2009) Vzquez An et al. (2008) reported that the activity of plasma SOD was decreased (22.02 vs. 19.34 U/g of protein) for cows with vs. without supplementation of 200 mg of Agrado Plus /kg of dietary DM when an u noxidiz ed blend of unsaturated oil was fed whereas SOD activity was increased (23.74 vs. 26.35 U/g of protein) by feeding Agrado Plus with oxidized oil compared to cows without Agrado Plus The varied responses of activity of SOD may due to differences in degrees of oxidative stress among animals used in the studies. Usual ly supplementation of Se is correlated positively with GPx activity in RBC or plasma. Hafeman et al. (1974) conducted a study of increasing concentrations of Se supplementation (0, 0.005, 0.1, 0.5, or 1.0 ppm) on erythrocyte GPx of the rat. A ctivity of GPx was increased markedly with increased concentration of Se in the diet. The difference among these g roups became greater as the diet s were fed over a longer period of time. A ctivity of plasma GPx was not different between cows fed an inorganic vs. organic source of Se ( 0.3 mg/kg of dietary DM) fro m 25 d before calving to 70 DIM (Cerri et al., 2009) The GPx activity in RBC of horses was increased with increasing the

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41 distance of endurance race up to 80 km for horses under tocopheryl acetate) supplementation and horses supplemented with 5000 IU/d of vitamin E plus 7 g/d of ascorbic acid (Williams et al., 2004) The supplementation of vitamin A (5000 IU/d), vitamin E (100 IU/d), and vitamin C (50 mg/d) resulted in decreased concentration of plasma TBARS in HIV infected patients compared to the patients who received a placebo (Jaruga et al., 2002) indicating the reduced production of lipid peroxides in liver. I n another human study involving 24 pa rticipants who were nonsmokers and not supplemented with vitamin (Jialal and Grundy, 1993) concentrations of plasma TBARS did not differ betw een volunteers who received 1.0 g/d of vitamin C, 800 IU/d of vitamin E, and 30 mg/d of vitamin A and those receiving placebo capsules Two amounts of vitamin E (0 or 1000 IU /d ) and of Se (0 or 3 m g/kg of dietary DM) were studied for 6 wk using multiparous dairy cows. C oncentration of TBARS in erythrocyte s was decreased by supplementation of vitamin E regardless of Se supplementation (Brzezinska Slebodzinska et al., 1994) Holstein steers were fed supplemental vitamin E at 4 concentrations (0, 250, 500, or 20 00 mg/d) for either 42 or 126 d. C oncentrations of TBARS in meat increased with increased length of display of retail cuts but accumulation of TBARS was less in beef from vitamin E supplemented steers than from control s (Liu et al., 1996) Descalzo and Sancho (2008) reported that meat from pasture fed steers had lower TBARS concentration than that from grain fed steers. They suggested that cattle grazed on good quality pasture incorporated enough vitamin E in their tissues to prevent lipid peroxidation in meat. A 2 x 2 factorial design was arraged with two types of corn oil (oxidized vs. unoxidized) and with o r without Agrado Plus to test the effect of synthetic

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42 antioxidants on shelf life of pork after pigs were or were not given antioxidants. After 21 d in display case, the concentrations of TBARS in loin chop were lowest from pigs fed fresh oil with Agrado w hereas TBARS were greatest in loin chop from pigs fed oxidized oil without Agrado (D. M. Fernndez Dueas, 2009) Supplementation of li pid soluble antioxidant effectively reduced the lipid peroxidation by scavenging peroxyl radicals generated through the chain reaction. Limited studies have been published on the effects of synthetic antioxidants on oxidative stability. Bartov et al. (1991 ) reported that initial oxidation of uterine tissue from laying hens after 30 d of frozen storage as measured by thiobarbituric acid ( TBA ) was reduced by supplement ing ethoxyquin at 250 mg/kg of food compared with those given 5 and 125 mg/kg of vitamin E and ethoxyquin, respectively Lin et al. (1989) reported that adding BHA at the rate of 12.5 mg/d/bird for 7 wk plus BHT at the same rate for the last 5 d before slaughter with unoxidized sunflower oil increased the oxidative stability of dark and while me at from boilers after 9 d of refrigerated storage in contrast with meat from boilers given only unoxidized sunflower oil In a study by Bailey et al. (1996) 4 feeding rates of ethoxyquin ( 0, 125, 500, and 1000 ppm ) were given to chicks from 30 d to 6 wk o f age Both 500 and 1000 ppm ethoxyquin rates resulted in lower TBARS concentration in liver and spleen tissues but not in kidney compared to 0 ppm indicating that ethoxyquin is effective to reduce tissue peroxid a tion. Murai and Andrews (1974) reported t hat c hannel c atfish fed oxidized menhaden oil had reduced lipid content of liver and lipid peroxidation of pelleted diets when supplement ed with 125 mg/kg of ethoxyquin

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43 Effect of Prepartum Heat Stress on Performance and Metabolites Commonly, temperature humidity index ( THI ) is used to indicate the degree of heat stress on cattle (Armstrong, 1994; West, 2003) A THI above 72 indicat es heat stress such that performance and physiological status will be altered Typically, m ajor responses to heat stress by dairy cattle include the following : reduced DMI and activity, increased sweating, water intake, respiratory rate body temperature and maintenance requirement and change s in metabolic and hormonal status ( Fuquay, 1981; Armstrong, 1994; West, 2003) Ultima tely, heat stress results in loss of milk production (Collier et al., 1982, Rhoads et al., 2009) Avendao Reyes et al. (2006) reported that non cool cows which were only under shades tended to have a greater respiratory rate (95.7 vs. 89.5 breaths/min) than cooled cows which were cooled by soaking the entire body with a hose that delivered approximately 25 L of 27 C water/cow/cooling event for every 2 min each day from 1130 to 1430 h for a 60 d nonlactating period but rectal temperatures and BW were not different between treatment groups After calving, cows were moved to a common pen with only shade. Milk production was numerically greater for cows exposed to the cooled environment relative to cows exposed to hea t stress (22.3 vs. 20.2 kg/d, but proportion of milk fat and milk fat yield were not affect ed by treatment. Amaral et al. (2009) reported that rectal temp eratures in the afternoon were greater for cows offered shade alone from dry off (60 d) until calving than cows offered fans and sprinklers. Milk yield, milk fat content, and milk fat yield were greater for cows under prepartum evaporative cooling even though all cows were cooled after calving The DMI (as % of BW) of cooled vs. noncooled cows were not dif ferent during the nonlactating period, but was greater for cows under prepartum heat stress compared to those given evaporative

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44 cooling from calving to 14 DIM Pregnant dairy c ows were assigned randomly to either a shad ed or a non shad ed group during the l ast 60 d of pregnancy (Collier et al., 1982) A fter calving all cows were cooled with fans and sprinklers The shaded cows yielded 13.6% more milk for a 305 d lactation. Wolfenson et al. (1988) reported that prepartum cows under shade alone had greater rectal temperature in the afternoon period than cows under shade with fans and sprinklers (39.2 vs. 38.7 C) but BC S did not differ between treatments. Milk yield corrected for fat was increased by prepartum cooling compared with noncooled cows during 150 d of lactation. A 2 x 2 factorial trial was conducted using 112 growing replacement heifers in which animals were a ssigned to one of 4 treatments: no shad e or misting, only misting, only shad e or shad e and misting. Misted heifers had lower rectal temperatures and respiratory rate s than unmisted heifers. After 131 d on tr eatment, BW was greater (520 vs. 547 kg) for sha ded than for the unshaded heifers (Mitlohn er et al., 2001) Amaral et al. (2009) reported that prepartum cows under shade alone had lower NEFA in plasma at parturition and for the following 28 DIM compared with cows exposed to shade plus fans and sprinklers T his result was due to lower DMI and greater BCS for the cooled cows. They also detected lower BHBA in plasma from 14 to 28 DIM for prepartum cows only under shade than cows under shade with fans and sprinklers. Pregnant multiparous Holstein cows were assigned random ly to 2 study pens where cows were provided with either sprinklers over the feed bunk or sprinklers, fans, and shade over the feed bunk around 30 d before calving during summer. After parturition, cows were housed under identical condition s Body condition scores and serum NEFA were not different between treatment group s (Urdaz et al., 2006)

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45 The somatic cell count ( SCC ) response of cows responding to heat stress is consistent. Wegner et al. (1976) reported that cows under mild to severe heat stress from June to November had increased SCC during t he hot summer season (August to October) than the rest of the study period. Mohammed and Johnson (1985) also reported that the number of somatic cells increased 56% when cows were exposed to heat stress (28.9 C and 55% RH) Effect of Prepartum Heat Stress on the Immune System Nardone et al. (1997) conducted a trial in which primiparous Holsteins were housed either in a cool (THI = 65) or hot environment (THI = 82 from 0900 to 2000 h, THI = 76 from 2100 to 0800 h) from 3 wk befor e calving to 36 h after calvi ng. Concentration of IgG in colostrum was lower for cows housed in the hot vs. cool prepartum environment. S ecretion of IgM by PBMC isolated from cows calving in summer (THI = 79 ) was greater than those from cows calving in spring (Lacetera et al., 2005) Several studies have been carried out to evaluate the effect of heat stress on immune cell function in the bovine. However, the results of these studies are inconsistent regarding lymphocyte function in cows expose d to a hot environment. Soper et al. (1978) tested PBMC immunostimulation on lactating dairy cows every 2 wk for 1 yr. The greatest PBMC response to mitogens was shown for 4 to 6 yr old cows in August when heat stress occured whereas the least response was shown for 7 to 9 yr old cows in Feburary Contrary to this result, a fter in vivo heat stress of lactating Holstein cows, the responses of polymorphonuclear leukocytes in vivo was not influenced by heat stress, but the decrease in proliferation of lymphocyte s isolated from cows exposed to heat stress was less at 42 C (Elvinger et al., 1991) The same lab

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46 (Kamwanja et al., 1994) also reported that proliferation of phytohemagglutinin stimulated lymphocyte s isolated from 3 Holstein cows was decreased when cells were exposed to 42 C compared with 38.5 C. Lymphocytes were isolated from Holstein cows and stimulated by Con A. C ows provided only shade prepartum had less proliferation compared with those provided shade plus fans and sprinklers ( Amaral et al. 2009 ) Yet Lacetera et a l. (2002) reported that the response of Con A treated PBMC isolated from calves exposed to a constant ly hot environment (35 C) was not different from those of calves exposed to thermoneutral condition s These variations may be due to the duration of the exposure, intensity of heat stress, and immune function variables measured (Kelley et al., 1982) The Effect of Cooling System s for Dairy Cows in Hot Environment s The characteristic climate in the southeastern United States is the high ambient temperature and relative humidity. H ot and humid conditions are associated with numerous physiologic al changes that occur in the digestive system, acid base chemistry, and blood hormones (West, 2003). In order to improve cow performance in such conditions, alter ations of the have been developed. Shad ing. First ly, shade structures should be used to reduce the direct and indirect solar radiation reaching the animals during the day (Ryan and Boland, 1992; West, 2003). They have little effect on changing ambient temperature and humidity (Buffington et a l., 1981). Shading during the prepartum period. Reducing the negative effect of heat stress by shading for nonlactating and pregnan t cows has shown benefits. Two studies evaluated the effect of heat stress relief by shading during the last 80 d of pregnancy on

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47 prepartum and postpartum responses of Holstein cows (Collier et al., 1982; Lewis et al., 1984) U pon calving, all cows were ma naged uniformly. Cows with shade during the last 80 d of gestation had numerically greater milk yields in the subsequent lactation, lower rectal temperatures and respiratory rates, greater thryoxine and lower NEFA concentration in plasma and gave birth to heavier calves (the difference about 3 kg) compared with cows offered no shade (Collier et al., 1982). Prepartum shading reduced postpartum plasma concentrations of prostaglandin F but increased rectal temperatures compared with non shaded cows (Lewis et al., 1984 ). Shading during the postpartum period. Providing shade to cows in midlactation for 11 wk during summer in Florida resulted in lower rectal temperatures (38.9 vs. 39.4 C), reduced respiratory rate s (54 vs. 82 breaths/min), and about 10% m ore milk yield ( R oman Ponce et al., 1977). Similarly, mid lactating cows with shade for 102d had lower rectal temperatures (38.7 vs. 39.6 C) and respiratory rate s (79 vs. 115 breaths/min), increased ruminal contractions (2.3 vs. 1.6 times/min), and greater milk yield corrected for stage of lactation (15.1 vs. 12.7 kg/d) compared with cows with no shade indicating that environmental modification altered physiological responses of the cows (Collier et al., 1981). Mechanical Cooling. Air movement (fans), we tting the cow (sprinklers and sprayers), evaporation to cool the air (misting), and their combinations are effective to enhance heat dissipation. Mist precools the air by evaporation before it reaches the hair coat and respiratory system of the cows where as sprinklers and sprayers dampen the hair and skin of the cows, increase the rate of evaporation and subsequent heat removed from the skin T he main disadvantage of sprinklers is that they create an environment saturated with water

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48 which markedly reduces t he capability of animals to dissipate heat by evaporation (Flamenbaum et al., 1986). Cooling during the prepartum period. Cooling with sprinklers and fans during the last 60 d of gestation reduced rectal temperatures (38.7 vs. 39.0 C), increased 150 d lac tation mean milk yield by 3.5 kg/d, and increased cal f birth weights by 3.3 kg compared to cows managed under shade only. Prepartum cooling may alleviate any detrimental effect s of heat stress on the development of mammary parenchyma or its lactogenic capacity. The effect of adding shade and fans to a sprinkler system for p e r i parturient cows was evaluated in California (Urdaz et al., 2006). Shade with sprinklers and fans during t he last 3 wk of gestation did not affect either the incidence of postparturient disorder s /disease s or serum concentration of NEFA in the prepartum period compared with cows ma naged with sprinklers only but milk yield was increased by 1.4 kg/d. Cooling dur ing the postpartum period. Two evaporative cooling system s (Korral Kool vs. fan s and sprinklers ) were evaluated for lactating dairy cows for a 142 d period from the end of May through the middle of October (Ryan and Boland, 1992). The mean rectal temperat ure (39.0 vs. 38.8 C) and milk production (27.7 vs. 26.8 L/d) of cows managed with Korral Kool compared with cows managed with fan s and sprinklers Correa Calderon et al. (2004) also reported that using a Korral Kool cooling system for lactating Holstein c ows for 18 wk reduced rectal temperature by 0.9 C compared with cows under shade only. Flamenbaum et al. (1986) reported that mean rectal temperature of Israeli Holstein lactating cows was reduced by 0.6 C by cooling (sprinkler s and forced ventilation) for 5 cooling periods of 30 min each during the day

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49 compared with cows under no cooling system. Shading and cooling cows during late gestation and postpartum will improve subsequent lactation performance and may result in heavier calves at birth.

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50 CHA PTER 3 EFFECT OF FEEDING ANTIOXIDANTS AND PRE PARTUM EVAPORATIVE COOLING ON PERFORMAN CE OF TRANSITION HOL STEIN COWS DURING SUMMER IN FLORIDA Introduction Dairy cattle undergo tremendous physiological and nutritional changes during the periparturient period. As a result they might experience a variety of metabolic disorder s (ketosis, hepatic lipidosis, displaced abomasum, and hypocalcemia) and infectious di seases (retained fetal membranes, mastitis, endometritis/metritis) ( Goff and Horst, 1997; Bernabucci et al., 2005; Sordillo et al., 2007; Sordillo and Aitken, 2009). Many disorders and diseases during this special period are associated with suppressed host defense mechanisms (Sordillo, 2005; Sordillo et al., 2009) and oxidative stress. Oxidative stress results from excess production of reactive oxygen species ( ROS ) and insufficient antioxidant production to remove the se ROS (Miller et al., 1993; Bernabucci et al., 2002, 2005; Castillo et al., 2005; Sordillo et al., 2007). Recently, clinical medicine has given increased attention to the detection and protection of ROS (Castillo et al., 2003). Oxidative status can be monitored by several biomarkers, such as s uperoxide dismutase ( SOD ) which is the enzyme that catalyzes the dismutation of superoxide radicals to hydrogen peroxide and oxygen, glutathione peroxidase ( GPx ) which is a Se dependent enzyme that decompose s hydrogen peroxide, thiobarbituric acid reactive substances ( TBARS ) which present a composite number of lipid oxidative end products including malondialdehyde and indicates the level of lipid peroxidation ( Trevisan et al., 2001; Bernabucci et al., 2005 ), and total antioxidant status ( TAS ) which provides more overall evaluation of oxidative status.

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51 The relationship between heat stress and oxidative status in dairy cattle has been examined only minimally. Activities of SOD, GPx and concentration of TBARS in erythrocytes as indicators of increased oxidative challenge were increased during heat stress (Bernabucci et al., 2002; Saker et al., 2004), whereas a decrease in GPx activity by peripheral blood mononuclear cells ( PBMC ) was detected in spite of heat stress (Burke et al., 2007). Supplementation with 200 mg/kg of dietary synthetic antioxidant (Agrado Plus, Novus International, St. Charles MO) increased plasma SOD activity when cows were fed oxidized rather than fresh oil and increased plasma GPx activity across both types of oil fed (Vzquez An et al., 2008). The effect of supplementation of synthetic antioxidants on oxidative status and performance of dairy cattle during the transition period is limited. The aim of this study was to evaluate whether the supplementation with a blend of synthetic antiox idants (Agrado Plus ) would ameliorate the expected negative effect of heat stress on performance and oxidative status of Holstein cows. Material and Methods Animals, Treatments, and Management The e xperiment was conducte d at the University of Florida Dairy Research U nit (Hague, FL) during the months of July through December 2008. All experimental animals were managed according to the guidelines approved by the University of Florida Animal Research Committee. Periparturient Holstein primiparous (n = 22) and multiparous (n = 13) cows were blocked by parity and were assigned to treatments at 29 7 days prior to their calving date. Four treatments were arranged in a 2 x 2 factorial design including 2 dietary concentrations of antioxidants (0 vs. 250 mg/kg of dietary DM,

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52 AO ) and 2 environmental housing conditions for pregnant cows. The AO is a liquid mixture of ethoxyquin and tertiary butyl hydroquinone which was added to corn oil by the manufacture r and shipped to the research site. The corn oil was mixed w ith ground corn just prior to preparing the concentrate portion of the diet in 909 kg batches. The corn oil was devoid of commercial antioxidants except AO Pregnant cows were housed in an open sided free stall barn with sand bedding equipped with or witho ut fans (J & D Manufacturing Eau Claire, WI) and sprinklers (Rainbir d Manufacturing, Glendale, CA) in cooled vs. noncooled treatments, respectively Sprinklers were intermittently operated every 6 min for 1.5 min. Lights were operational from 0600 h to 200 0 h. Calan gates ( American Calan Inc., Northwood, NH ) were used to measure DMI of individual cows. Pregnant cows were fed twice daily ad libitum amounts of a bermuda grass silage corn silage based TMR (Table 3 1) Refusals of TMR were measured daily. Recta l body temperatures were recorded daily between 1430 and 1530 h using a GLA M700 digital thermometer (GLA Agriculture Electronics, San Luis Obispo, CA) After calving, all animals were moved to a sand bedded, open sided, free stall barn equipped with fans and sprinklers and Calan gates. Cows were milked twice daily at 0700 and 2000 h and fed in ad libitum amounts a corn silage alfalfa hay based TMR twice daily at 0800 and 1300 h for 7 weeks. Dry matter intake was recorded daily. Prepartum and postpartum c ow s were weighed on the same day each week before the morning feeding. Rectal body temperature was measured on 4, 7, and 12 DIM using a GLA M700 digital thermometer Sample C ollection and Analysis Representative samples of corn silage, bermuda grass silage, alfalfa hay and grain mixes were collected weekly, composited monthly and ground through a 1 mm Wiley

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53 mill screen (A. H. Thomas, Philadelphia, PA). Silage and hay samples were ground before compositing whereas grain mix samples were composited before grind ing. Composited feed samples were analyzed for CP using a macro elemental analyzer vario MAX CN (Elementar Analysensystene GmbH, Hanau, Germany), NDF (Mertens, 2002), ADF (AOAC, 1995), ether extract (AOAC, 2003) and minerals (Dairy One, Ithaca, NY). The chemical composition of diets are shown in Table 3 2. Milk samples were collected from two consecutive milkings weekly using bronopol B 14 as a preservative, and analyzed for true protein, fat, and SCC by Southeast Milk laboratory (Belleview, FL) using a Bently 2000 NIR analyzer. Final concentrations of fat and protein were calculated after adjusting for milk production during those 2 milk collections Blood samples were collected at 0900 h on Monday, Wednesday and Friday weekly from the coccygeal vesse ls into sodium heparinized tubes (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) from calving until 49 DIM. Blood samples were placed on ice immediately after collection until centrifuged at 1200 g at 4C for 15 min (Allegra X 15R Centrifuge, Beckman Coulter). Plasma was separated after centrifugation and stored at 20C for subsequent metabolite and hormone analyses. Plasma concentrations of NEFA (NEFA C kit; Wako Diagnostics, Inc., Richmond, VA; as modified by Johnson, 1993) and BHBA (Wako Autokit 3 HB; Wako Diagnostics, Inc., Richmond, VA ) were determined weekly for 7 wk. A Technicon Autoanalyzer (Technicon Instruments Corp., Chauncey, NY) was used to determine weekly concentrations of plasma BUN (a modification of Coulombe and Favreau, 1963 and Marsh et al., 1965) and plasma glucose (a modification of Gochman and Schmitz,

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54 1972). Concentrations of progesterone were determined on all plasma sample s collected using Coat A Count Kit (DPC Diagnostic Products Inc., Los Angeles, CA) solid phase 125I RI A. The sensitivity of the assay was 0. 02 ng/mL and the intra assay CV was 3.8% Processing of Red Blood C ell (RBC) Approximately 7 mL of blood was collected on 15, 1, 8, 15, and 29 DIM from the coccygeal vessels into evacuated tubes containing 17.55 mg o f K 2 EDTA (Vacutainer Becton Dickinson, Franklin Lakes, NJ, USA). The blood samples were placed on ice immediately following collection and trans ported to the lab oratory within 3 h To measure haematocrit, blood was drawn into a haematocrit capillary tub e ( Fisher Scientific, Cat. No. 22 362 566 ) and centrifuged for 2.5 min. Plasma and RBC were separated using a refrigerated centrifuge operating at 1200 g for 10 mins at 4 C (Allegra X 15R Centrifuge, Beckman Coulter). Plasma was stored at 80C until a nalyz ed for TBARS The RBCs were processed as follows. T he buffy coat (leukocytes) was discarded via pipette The RBC (2 mL) were transferred using a positive displacement pipette to a 1 3 x 100 mm polypropylene tube. C old (4C) physiological saline (4 mL) was added to the RBC cap ped, and gently mix ed The mixture was centrifuged at 1200 g for 10 min and saline pippetted off. This washing process was repeated 2 more times fo r a total of 3 saline washes. After the third saline wash, the washed RBC were ly sed by adding 2 mL of cold (4C) UltraPure water ( Cayman Chemical, Catalog No. 400000 Ann Arbor, MI) at which time a dark maroon color was achieved upon vortexing Samples (400 uL) were stored in triplicate vials at 80C for analy sis for superoxide dismu tase ( SOD ) and glutathione peroxidase ( GPx ) respectively

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55 Thiobarbituric Acid Reactive Substances (TBARS) Assay Plasma concentrations of TBARS were determined by the metho d modified by Armstrong et al. ( 1998 ) Briefly, fresh thiobarbituric acid (TBA) bu ffer was made ( 200 mL ) by a dding 40 mL of glacial acetic acid and 1.060 g of TBA to 160 mL distilled water. S odium dodecyl sulfate (SDS) reagent ( 8.1% ) was prepared by mixing 8.1 g of SDS with 100 mL of distilled water. Malondialdehyde (MDA) stock solution (100 nmol/mL) was made by mixing 82 uL of MDA (S igma Aldrich Inc. cat no. 108383 St. Louis, MO ) and 1 mL of concentrated HCl with 100 mL of distilled water. One portion of stock MDA and 9 portions of distilled water were combined to make the MDA working solution (10 nmol/mL) W orking MDA standards of 0, 1, 2, 3 and 4 nmol/mL were prepared by adding 0, 10, 20, 30, and 40 uL of MDA working solution respectively to 100, 90, 80, 70, and 60 uL of distilled water. In each labeled test tube, 100 uL of diluted sample (1:100) or working MDA standards, 100 uL of SDS reagent and 2.5 mL of TBA buffer were added by force in order to mix them well. Tubes were covered with a marble and incubated at 95 C for 1 h. Aft er incubation, tubes were placed in an ice water bath for 10 min. Lastly, fluorescent readings were obtained using a florescence spectrophotometer (RF 1501, Shimadzu International, Columbia ). Superoxide Dismutase (SOD) Assay The SOD activities of RBC were measured using the Superoxide Dismutase Assay Kit (Cayman Chemical, Catalog No. 706002 Ann Arbor, MI ). This assay utilizes a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. All 3 types of SOD (Cu/Zn SO D Mn SOD and FeSOD) are measured in this 96 well microplate assay. Briefly, 2.5 mL of concentrated assay buffer was diluted with 22.5 mL of HPLC grade water. C oncentrated sample buffer (2.5 mL) was diluted

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56 with 22.5 mL of HPLC grade water. Diluted assay buffer and sample buffer was kept at room temperature while performing the assay. Erythrocyte lysate samples (10 L) from 15 d before expected calving date 1, 8, 15 and 29 DIM were thawed and diluted 5000 times with di luted sample buffer. The SOD Stock standard was prepared by diluting 20 L of concen trated SOD standard with 1.98 mL of diluted sample buffer. Working s tandard s of 0, 0.025, 0.0 5, 0.1, 0.15, 0.2, and 0.25 U/mL were made by adding 0, 20, 40, 80, 120, 160, a nd 200 L respectively of SOD standard stock to 1000, 980 960, 920, 880, 840, and 800 L of diluted sample buffer. Diluted samples and working standards were kept on ice. Diluted radical detecto r was obtained by diluting 50 L of concentrated radical dete ctor with 19.95 mL of diluted assay buffer. The wells on a 96 well plate were categorized as SOD standard wells and sample wells. Each standard/sample was measured in duplicate. For the SOD standard wells, 200 uL of the diluted radical detector and 10 uL o f working SOD standard (7 standards) were added to each correspond ing well. For sample wells, 200 L of the di luted radical detector and 10 L of the diluted sample were added to each corresponding well. After this step, xanthine oxidase was prepared by ad ding 50 L o f the supplied enzyme to 1.95 mL of sample buffer. A s quickly as possible 20 L of xanthine oxidase was added to each well to initiate the reaction. The p late was covered incubated on a shaker for 1 h and read at 450 nm using a plate reader (SpectraMax 340PC 384 Molecular Devices, CA). Superoxide dismutase activity was calculated based on the standard curve. Enzyme activity was expressed as units per milliliter of packed cell volume (PCV) Plates contained samples from each tr eat m ent and al l the samples from the same cow were

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57 analyzed in the same plate. This method was used for all variables measured using the microplate reader. Glutathione peroxidase (GPx) Assay The GPx activities of erythrocyte s were measured using the assa y kit supplied by Cayman Chemical Company (catalog no. 703102 Ann Arbor, MI ). This assay measures GPx activity indirectly by a coupled reaction with glutathione reductase (GR). The proce dure of this assay is as below. The a ssay buffer and sample buffer were diluted 10 fold and kept at room temperature. Erythrocyte lysate samples from 15 d before expected calving date 1, 8, 15 and 29 DIM were thawed and diluted 500 times with diluted sam ple buffer. Ten l of supplied glutathione peroxidase was d iluted with 490 uL of diluted sample buffer. Cayman sells a 96 well kit and a 480 well kit. The 96 well kit contains 3 vials of co substrate mixture. E ach vial was r econstitute d by adding 2 mL of HPLC grade water. The 480 well kit contains 5 vials of co substrate m ixture. E ach vial was r econstitute d by adding 6 mL of HPLC grade water. The diluted samples, enzyme, and co substrate mixture were kept on ice at all time s The wells in a 96 well plat e were categorized as background wells, positive control wells, and sample we lls. For background wells, 50 L of co substrate mixture and 120 L of assay buffer were added to each well. F or positive control wells, 20 L of diluted enzyme, 50 L of co subst rate mixture and 100 u L of assay buffer were added to ea ch well. For sample wells, 20 L of diluted RBC sample, 50 L of co substrate mixture and 100 uL of assay

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58 buffer were added to each well. The reaction was initiated by adding 20 L of cumene hydroper oxide to all wells as quickly as possib le. The p late was covered and shaken for a few seconds. Lastly, plate was read at 340 nm once every minute for at least 6 min GPx activity was calculated using the formula below: GPx activity = sample dilution = nmol/min/ml was calculated by subtracting the change in absorbance per minute for the background from the change in absorbance per minute for the sample. Enzyme activity was expressed as nmol/min/mL, which is the a mount of enzyme used to oxidize 1.0 nmol of NADPH to NADP + per minute at 25 C. Enzyme activity was expressed as nmol per min per mL of PCV Acute Phase Protein Assay s Plasma samples collected on Monday Wednesday Friday were used to determine the concentrations of acid soluble protein ( ASP ) and haptoglobin ( Hp ). A cid soluble protein was extracted from 50 L of plasma with 1 mL of 0.6 M perchloric acid after 20 min of incubation at room temperature in duplicate. Tubes were centrifuged at 1200 g fo r 30 min at room temperature. Supernatant was analyzed with the bicinchoninic acid kit (Sigma Aldrich, Saint Louis, MO; Cat No. 096k9802). Concentrations of unknowns were obtained from the standard curve. The inter assay variation was 9.2%. If CV% between replicate s was greater t han 10%, samples were re analyzed. Concentrations of p lasma Hp were determined by measuring the differences of hydrogen peroxide activity with haptoglobin hemoglobin (Hb) complex (modified by Tarukoski 1966). Briefly, O dianisidine solution (4 L) was prepared by adding 2.4 g of O dianisidine, 2.0 g of Na 2 EDTA, and 55.2 g of NaH 2 PO 4 to 4 L of distilled water and

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59 adjusted to pH of 4.1. H emoglobin stock solution (25 uL) was added to each tube to form a Hb Hp complex with Hp from 5 uL o f plasma sample. O dianisidine solution (7.5 mL) was added to each tube. Tubes were incubated in a water bath for 45 min at 37C. Then 100 uL of hydrogen peroxide were added to react with Hb Hp complex to liberate oxygen which oxidized O dianisidine to a yellow color compound. Samples from the tubes were transferred to the 96 well plate and read at 450 nm using the microplate reader Neutrophil F unction WBC and Lymphocyte Blood (6 mL) was collected from coccygeal vessels at 21, 0, 7, and 14 DIM in vacutainer (Vacutainer, Becton Dickinson, Franklin Lakes, NJ) tubes containing acid citrate dextrose. Tubes were not put on ice but were gently mixed by hand appro ximately every 15 min. Counts of WBC, lymphocytes, and neutrophils were done using a Bayer Advia 120 cell counter ( Fisher Diagnostic, Middletown, VA ) within 3 h of collection. Pha gocytosis and oxidative burst by neutrophil s were assessed within 3 h after blood collection. Neutrophil WBC and lymphocyte concentrations were estimated by a demacytometer. W hole blood (100 uL) was pipe ted into each of 3 tubes. Then 10 Aldrich, Saint Louis, MO) was added to all tubes Tubes were vortexed and incubated in an oven at 37C for 10 min with constant rotation using the Clay Adams nutator (BD Bioscience San Jose, CA). Ten myristate, 13 a cetate (PMA) (Sigma Aldrich St. Louis, MO ) were a dded to the second tube only. An Escherichia coli bacterial suspension (10 9 cells/ L ) labeled with propidium iodide (Sigma Aldrich) was added t o the third tube to establish bacteria to neutrophil ratio of 40:1 ( Escherichia coli strain were isolated from a dairy cow with mastitis and grown in vitro for labeling) Tubes were

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60 vortexed and incubated in oven at 37 o C for 30 min with constant rotation using the Clay Adams nutator (BD Bioscience San Jose, CA). Then all tubes were removed and placed immediately on ice to stop phagocytosis and oxidative burst activity. Tubes were processed in a Q Prep Epics immunology workstation on the 35 cyc le. Cold distilled ) were added to each tube. Then tubes were vortexed, kept on ice and 10,000 neutrophil cells were read using the Facsort flow cytom eter (BD B iosciences, San Jose, CA). The percentage of total neutrophils able to phagocytize E. coli undergo oxidative burst, and efficiency of bacteria killing ( mean fluorescence intensi ty MFI ) were measured using the flow cytometer. Ovalbumin C hallenge All cows were injected i.m. with 1 mg of ovalbumin (Sigma Aldrich, Saint Louis, MO) diluted in 1 mL of sterile Quil A adjuvant (0.5 mg o f Quil A/ mL of PBS) (Accurate C hemical & Scientif ic Corp. Westbury, NY) at 4 and 2 wk relative to expected calving date and at calving Blood samples (8 mL) for measurement of anti ovabumin IgG were collected at 4, 2, 0, 1, 2, 3, 4, and 7 wk relative to calving. Samples were taken in vacutainer tubes containing no anticoagulant before the ovalbumin injection. Serum concentration of anti ovalbumin IgG was measured by an Enzyme Linked ImmunoSorbent Assay (ELISA) as described by Mallard et al. (1997). Briefly, flat bottom 96 well polystyrene plates (Immu lon 2, Dynex Tech., Chantilly, VA) were coated with a solution of ovalbumin dissolved in carbonate bicarbonate coating buffer (1.4 mg OVA/ mL of carbonate bicarbonate buffer). Plates were incubated at 4C for 48 h, then washed with PBS and 0.05% Tween 20 s olution (pH = 7.4). Plates were blocked with a PBS 3% Tween 20 and bovine serum albumin (Sigma Chemical, St. Louis, MO) solution and incubated at room temperature for 1 h. Plates were washed and serum

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61 samples and control sera diluted at l/50 and l/200 were added in duplicate using a quadrant system (Wright, 1987). Positive and negative control sera to anti ovalbumin IgG were obtained from a pool of sera of known high (21 d after the third injection of ovalbumin) and low (no ovalbumin injection) concentratio ns, respectively. All samples from the same cows were analyzed in the same plate and plates contained balanced number of animals from each diet group. Plates were incubated at 24 C for 2 h and washed with the previously described buffer solution. Subsequen tly, alkaline phosphatase conjugate rabbit antibovine IgG whole molecule (Sigma Chemical, St. Louis, MO) was dissolved in Tris/HCl Buffer, added to the plates and incubated for 1 h at room temperature. After incubation, plates were washed 4 t imes and subst rate solution [P nitrophenyl phosphate d isodium (Sigma Chemical, St. Louis, MO)] was added and the plate was incubated at room temperature for 30 min. Plates were read on an automatic ELISA plate reader (MRX Revelation; Dynex Technologies Inc., Chantilly, VA) and the optical density was recorded at 405 nm and the reference at 650 nm. Vaginoscopy Cows were evaluated for cervical discharge on 7, 16, and 25 DIM using the metricheck (Metricheck, Simcro, New Zealand) tool. The vulva was cleaned using a povidon e iodine scrub (0.75% titratable iodine and 1% povidone solution, Agripharm, Mem phis, TN, USA) and dried with clean gauz e. The metricheck was inserted in the vagina close to the cervix. The floor of the vagina was scraped, the discharge collected in a 50 m l conical tube (Fisher Diagnostics, Middletown, VA), and scored according to Sheldon et al. (2006). Scoring system was as follows: 0 = translucent or clear, 1 = flecks of white or off white pus, 2 = discharge containing < 50% white or off white

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62 mucopurul ent material, 3 = discharge containing > 50% white or yellow mucopurulent material, 4 = discharge containing > 50% sanguineous mucopurulent material. Uterine C ytology At 40 2 days postpartum, one of the uterine horns was flushed with saline A fter thorough sanitation of the vulva and entrance of the vagina using chlor hexidine d iacetate (Nolvasan, Fort Dodge, Overland Park, KS) an 18 French, 30 mL 56 cm, 2 way Foley catheter was placed randomly in 1 uterine horn at approximately 2 cm past the bifur cation of the uterus. The cuff of the catheter was i nflated with 7 to 10 mL of air according to the diameter of the uterine horn, and 20 mL of sterile isotonic saline solution was infused into the uterine horn and then recovered using a 35 mL sterile syrin ge. The aspirated fluid was transferred to a 50 mL sterile conical tube, placed in ice and transported to the lab oratory within 4 h of collection. In the laboratory the aspirated fluid was centrifuged at 750 x g for 10 min and the supernatant discarded. T he pellet was resuspended with 2 mL of saline, and an aliquot of 20 uL was pipette d onto glass slides and smeared in triplicate Smears were air dried and stained using a Diff Quick (Fisher Diagnostics, Middletown, VA) stain. Slides were examined under a m icroscope and the number of total leukocytes, epithelial endometrial cells, and neutrophils were counted to complete a 100 cell count per slide, and percentage neutrophiles was calculated. Subclinical endometritis was diagnosed when the proportion of neutr ophil s exceeded 5% after 30 DIM (Gilbert et al., 2005). Statistical A nalyses Treatments were arranged in a 2 x 2 factorial completely randomized design Repeated measurements were made on nearly all variables and were analyzed using the PROC MIXED proced ure of SAS ( Release 9.2) according to the following model:

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63 Y ijkl i j ij k ik jk ijk + C l(ijk) + W m im + jm ijm km ikm jkm ijkm ijklm where Y ijk l is the observation, is overall mean i is the fixed effect of diet ( i = 1, 2 ), j is the fixed effect of environment ( j = 1, 2 ), ij is the interaction of diet and environment k is the fixed effect of parity ( k = 1, 2 ), ik is the interaction of diet and parity jk is the interaction of environment and parity, ijk is the interaction of diet environment and parity, C l(ijk) is the random effect of cow within diet and environment l is the fixed effect of week (l = 1, 2, 3, 4, 5, 6, 7), ( W) i l is the interaction of diet and week, ( W) j l is the interaction of environment and week, W ) ij l is the interaction of diet, environment, and week, ( W) k l is the interaction of parity and week, W ) ik l is the interaction of diet, parity, and week, W ) jk l is the interaction of environment, parity, and week, W ) ijk l is the interaction of diet, environment, parity, and week, and ijklm is the r esidual error. Different temporal responses to treatments were further examined using the SLICE option of the MIXED procedure. Mean treatment, parity, and time (week or day relative to calving) effects are presented a s least square means. A covariate representing the initial measurement for IgG against ovalbumin and BCS of i ndividual cow s was includ ed in the model Data were tested to determine the structure of best fit, namely AR(1), ARH(1), CS, or CSH, as indicated by a lower Schwartz Baesian information criterion value (Littell et al., 1996) If repeated measures were taken on unequally spaced in tervals, the sp(pow) covariance structure was used. Measures of blood cell numbers, neutrophil function, acute phase proteins, oxidative markers, and IgG against ovalbumin were tested for normality before and after

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64 transformation (log or square root transf ormation ) by using the PROC UNIVARIATE procedure of SAS (SAS Institute, 2007). Probability values > 0.05 using the Shapiro Wilk test for variables were considered normal. After data were transformed, the PROC MIXED procedure with the same model described above was used. Progesterone data (DIM of first ovulation, number of ovulations, peak concentration of progesterone in the first ovulation, the length of the first cycle, and accumulated progesterone over 49 DIM) were analyzed using Proc MIXED of SAS wit h t he accumulated progesterone analysis requir ing the repeated measures function. Metricheck scores were analyzed using logistic regression with the Odds Ratio option. If cervical discharge of cows was scored as 0 or 1 cows were classified as healthy whereas scores of 2, 3, or 4 resulted in a diagnosis of metritis /endometritis These reclassified scores were analyzed as binomial data. Uterine cytology data were used to classify cows as either clean or with subclinical endometritis. This binary data also was analyzed using logistic regression techniques. Differences discussed in the text were significant at P be significant at 0.05 < P 5 Results and Discussion Body T emperature, BW, BCS, and DMI In the p repartum period, cows stayed in the assigned freestall barn area for 29 7 d before calving. However the data for body temperature, BW, and DMI are reported only for the last 21 d prepartum because most cows contributed data during this time period. Mean rectal temperature over the last 21 d prior to calving was less for c ool ed vs noncooled cows ( 39. 2 vs. 39.6C P < 0.001 Table 3 3 ) as exp ected. This difference was consistent across prepartum days as tests of all interactions of week with treatments were not significant. Rectal temperatures above 39.2C may represent

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65 reduced ability of lactating dairy cows to adapt to thermal stress resulting in reduced performance (Staples and T hatcher, 2003). Therefore providing fans and sprinklers to sh aded freestalls helped reduce heat stress and established the planned differences of prepartum environments for cows assigned to the study. Prepartum intake of DM did not differ among the treatment groups, averaging 10 kg/cow per day (Table 3 3). However effect of treatment on DM intake when expressed as a percentage of BW differed according to parity. Feed intake (% of BW) by primiparous cows was unaffected by treatments but DMI by multiparous cows fed the control diet was decreased by cooling whereas in take was increased by cooling when cows were AO (diet by cooling by parity interaction, P = 0.03, Figure 3 1). Typically cows under significant heat stress reduce DMI. Multiparous cows fed the AO diet demonstrated this response. The DMI of multiparous cows fed the control diet and evaporatively cooled responded unexpectedly as mean DMI was only 1.09% of BW. Multiparous cows on this treatment were much heavier than those in the other groups, averaging 843 kg, about 147 kg heavier than multiparous cows on the other treatments (Table 3 3). Mean prepartum BW of primiparous cows did not differ across treatments resulting in a diet by environment by parity interaction ( P = 0.001, Figure 3 2). This disparity in BW across treatments likely occurred due to the removal of some lighter BW cows from this treatment (control diet cooled environment) bec ause of poor health postpartum. Overconditioned cows may consume less DM postpartum (Jones and Garnsworthy, 1989) and both multiparous cows on the control diet cool treatment in this study were poor eaters and had a mean prepartum BCS of 3.75 whereas othe r 3). Cows maintained under evaporative cooling

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66 had greater body condition throughout the prepartum period (3.48 vs. 3.18, Table 3 3, P = 0.05) This difference was a result of removing cows assigned to this treatment from the experiment due to health reasons. Evaporative cooling of prepartum cows did not affect BW change although BW gain between 3 and 1 wk before calving was numerically greater for cooled vs noncooled cows ( 1.17 vs. 0.25 kg /d P = 0.24, data not shown ). As expected, mean DMI during the first 7 wk postpartum was greater ( P < 0.001) for multiparous compared with primiparous cows (16.8 vs. 13.8 kg/d, Table 3 4). However no difference in DMI was detected between parities when DMI was expressed as a perce ntage of BW (2.67 vs. 2.69%) thus indicating that more DM can be consumed by larger animals (BW of 643 vs. 516 kg for multiparous and primiparous cows, respectively). Effect of parity by diet by environment interaction on DMI was the same whether expresse d as amount ( P < 0.01) or as a proportion of BW ( P = 0.001, Table 3 4). Postpartum DMI was greater when primiparous cows were evaporatively cooled prepartum and fed the control diet compared to the uncooled cows fed the control diet (2.98 vs. 2.62% of BW) but prepartum cooling had no effect on postpartum DMI of primiparous cows fed AO (2.49 vs. 2.68% of BW). However prepartum evaporative cooling of multiparous cows fed the control diet resulted in less DMI compared to those not cooled (1.99 vs. 3.24% of B W) whereas prepartum cooling had no effect on postpartum DMI of multiparous cows fed AO (2.83 vs. 2.64% of BW, Figure 3 3, diet by environment by parity interaction, P = 0.001). The cooled multiparous cows fed the control diet were the heaviest (Figure 3 4) and the heaviest conditioned at calving which they maintained throughout the study (Figure 3 5). Cows on this treatment had a BCS

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67 of 3.75 at calving whereas the BCS of the other treatment groups ranged from 3.06 to 3.37 at calving (data not shown). Th ese cows were the poorest eaters (DMI at 1.99% of BW), continuing their prepartum pattern of poor DMI (Table 3 3). Therefore the interaction effect of treatment with parity on postpartum DMI was likely largely due to greater body condition for the one gro up of animals at calving. Amaral et al. (2009) reported lower DMI by multiparous cows during the first 14 DIM when they were evaporatively cooled prepartum compared to cows provided shade only. Similarly, cows fed the control diet in the current study co nsumed less DM postpartum when cooled prepartum compared to those not cooled (2.57 vs. 2.79% of BW). However postpartum DMI of cows fed AO was not affected by prepartum cooling (2.66 vs. 2.66% of BW, diet by environment interaction, P = 0.05, Table 3 4). Mean DMI postpartum was not affected by diet although DMI was lower during the first week postpartum for cows fed AO compared to control cows (1.36 vs. 1.72% of BW, diet by week interaction, P = 0.10, Figure 3 6). This difference in DMI at wk 1 postpartu m may have been due to a greater incidence of serious health disorders (mastitis, metritis, retained fetal membranes, milk fever, or displaced abomasum) for cows fed AO (n = 11/20) vs. the control diet (n = 3/15) (Table 3 9). In contrast to the current st udy, Vzquez An et al. ( 2008 ) reported that feeding AO to lactating dairy cows increas ed DMI Neither mean BCS (3.21) nor chan ge in BCS over 7 wk postpartum were influenced by treatments (Table 3 4). This result is consistent with a study in which the f eeding of AO did not influence BCS of lactating dairy cows (Vzquez An et al., 2008).

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68 Milk Production and M ilk C omposition As expected m ultiparous cows produced more milk (Table 3 4) than pr i miparous cows (32.8 vs. 24.4 kg/d, P = 0.001) likely due to greater DMI by multiparous cows Mean and pattern of m ilk yield measured over the first 7 wk were not different among treatments (Table 3 4 and Figure 3 7) This is consistent with 3 studies in which milk yield was not influenced by d ietar y supplementation with AO ( Bowman et al., 2008; He et al., 2008; Preseault et al., 2008). However, lactating cows did respond to supplemental AO by increasing milk yield adjusted for fat (3.5% FCM) (Vzquez An et al., 2008). Smith et al. (2002) is th e only group to report improvements in uncorrected milk yield (38.5 vs. 32.3 kg/d, P < 0.05) despite a 2.6 kg/d decrease in DMI when adding synthetic antioxidants (50 ppm) in the form of ethoxyquin to the diet for 2 wk. Possibly differences in concentrati on of natural dietary antioxidants such as vitamins A and E and Se in the control diets may account for differences in milk yield responses to AO across these studies. In the current study, mean prepartum and postpartum intakes were 174,853 and 83,781 IU/ d for vitamin A, 848 and 446 IU/d for vitamin E, and 4.6 and 4.7 mg/d for Se, respectively. Mean concentration (3.25 vs. 3.61%, P = 0.03) and yield (0.87 vs. 1.04 kg/d, P = 0.04) of m ilk fat were less for cows fed AO compared to control cows (Table 3 4). Smith et al. (2002) reported numerical decreases in milk fat concentration when ethoxyquin was fed at 0, 50, 100, or 150 ppm (3.6, 3.2, 3.5, and 3.4%, respectively) although milk fat production was unchanged. Milk fat depression is usually associated with changes in biohydrogenation of long chain polyunsaturated fatty acids to conjugated linoleic fatty acids by ruminal bacteria in cows fed high concentrate/low forage diets and/ or diets rich in plant oil supplementation (Bauman and Griinari, 2001; Zebeli an d Aetaj, 2009)

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69 Vzquez An et al. (2007) reported a trend for reduced escape of linolenic acid from continuous cultures fed AO indicating that AO may stimulate some of the steps in the biohydrogenation process, although the outflow of cis 9 trans 11 CLA was not changed by adding AO. Likewise concentration of trans 10 C18:1 and the 3 CLA isomers ( cis 9, trans 11 CLA, trans 10, cis 12 CLA, and trans 9, trans 11 CLA) in milk fat were not changed by feeding AO (Vzquez An et al.,2008). Indeed milk f at yield was increased (0.95 vs. 1.00 kg/d) in dairy cows fed AO in that study. Their reported improvements in milk fat yield might be associated with positive effects of AO on efficiency of ruminal microbes (Vzquez An et al., 2007). The AO appears to act as a microbial modifier in the rumen because feeding AO improved fiber digestibility and conversion of dietary N to microbial N in continuous culture systems (Vazquez Anon et al., 200 7 ), Cows in the current study were fed diets of low NDF and ADF conce ntrations ( Table 3 2; NRC, 2001) and supplemented with polyunsaturated fat in the form of corn oil A more acidic ruminal environment in the presence of additional polyunsaturated fatty acids may be a situation in which a microbial modifier such as AO can shift fatty acid metabolism toward CLA and result in reduce milk fat production. Although the effect of prepartum cooling did not affect mean concentration or yield of milk fat, the pattern of milk fat concentration and yield over weeks postpartum differed; cows not evaporative ly cool ed prepartum experienced decreased milk fat % at wk 3 and 4 postpartum compared to cows evaporatively cooled prepartum (environment by week interaction, P = 0.01, Figure 3 8). Production of milk fat over time responded in a similar fashion (environment by week interaction, P = 0.02, Figure 3 9). Production of 3.5% FCM was reduced at wk 3 and 4 postpartum due to lack of prepartum cooling

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70 (environment by week interaction, P = 0.07, Figure 3 10). Dairy cows under similar management conditions (Amaral et al., 2009) also tended to produce milk of less fat concentration due to lack of prepartum cooling (3.5 vs. 3.9% milk fat) and yielded less milk fat (0.9 vs. 1.3 kg/d) Avenda o Reyes et al. (2006) reported that cows coole d with water sp r ay and fans during pregnancy had an increase in milk fat yield in the subsequent lactation compared with cows managed under heat stress. It is unknown why lack of evaporative cooling prepartum would depress milk fat postpartum. Concentration of milk fat often is reduced when lactating cows undergo moderate serious heat stress, thought to be due to respiratory alkalosis Collier et al. (1982 ) indicated that the effects of heat stress during the last trimester of pregnancy reduced placental and maternal hormone concentration which in turn reduced mammary gland growth and function that may have led to the reduction in milk fat concentration Mean concentration of milk true protein tended to be greater for cows fed AO (3.07 vs. 2.94%, P = 0.06, Table 3 4). Much of this increase was due to a difference detected at wk 1 postpartum (diet by week interaction, P = 0.04, Figure 3 11) therefore AO d id not improve milk protein long term When feeding AO to lactating cows, no dietary effec t on milk protein content and yield were reported (Vzquez An et al., 2008). Prepartum cooling did not affect concentration or yield of milk protein. Milk protein concentration dropped acutely when cows were housed under heat stress for 5 d compared wit h the same cows housed under thermoneutral conditions for 5 d (Ominski et al., 2002). Somatic cell count was greater in milk produced by cows without prepartum evaporative cooling at wk 5 and 6 after calving ( environment by week interaction, P =

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71 0.03 Fi gure 3 12 ). This was largely driven by a cow diagnosed with mastitis during this time postpartum although 5 additional animals had elevated SCC during this same time as well. The SCC was numerically greater at 5 of the 7 wk postpartum for cows not cooled prepartum. Partially relieving heat stress during the immediate prepartum period may reduce the susceptibility of cows to mammary gland infections postpartum. Wegner et al. (1976) reported that 64 cows housed in mild to severe heat stress starting in Ju ne, through the hot summer months of July and August, and into the cooler month of November had increased SCC from August to October. The number of somatic cells in milk from cows exposed to heat stress for 5 d was elevated compared with th at from cows exposed to thermo neutral condition s for the same period ( Mohammed and Johnson, 1985 ). Plasma Metabolites The mean concentration of plasma NEFA tended to be greater for cows fed the control diet compared to that of cows fed AO (325 vs. 246 Eq/L P = 0.0 7 ). However this dietary difference in plasma NEFA concentration was for multiparous cows only. Mean plasma concentrations of NEFA tended to be greater for multiparous cows fed the control diet (469 vs. 305 Eq/L) but the mean plasma concentrations of NEFA for primiparous cows were not affected by diet (182 vs. 187 Eq/L, diet by parity interaction, P = 0.06). These greater concentrations of plasma NEFA for multiparous cows in control cool treatment group occurred only during the first 3 wk post partum (diet by environment by parity by week interaction, P = 0.03 ; Figure 3 13 ). Therefore feeding AO to multiparous cows reduced plasma concentrations of NEFA during the early postpartum period. Elevated concentrations of plasma NEFA during the first 3 wk

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72 postpartum were likely due to greater mobilization of adipose tissue in support of milk and maintenance requirements. Loss of BW and feed efficiency as we ll as energy balance (figure 3 14 diet by environment by parity by week interaction, P = 0.03) during the first 3 wk postpartum for these 4 treatment groups followed rather closely the same ranking as the plasma concentrations of NEFA; namely 2.05, 1.95, 1.69, 0.86 kg /d for BW loss; 2.55, 2.12, 1.90 1.73 kg of 3.5% FCM/kg of DMI, and 802, 492, 259, and 270 Eq/L of NEFA for multiparous control diet, multiparous AO diet, primiparous control diet, and primiparous AO diet groups, respectively. The weekly postpartum pattern of concentration of plasma BHBA was similar to that of plasma NEFA; that is, pla sma concentrations of BHBA were greater in multiparous cows fed the control diet during the first 3 wk postpartum (diet by parity by week interaction, P < 0.01, Figure 3 15 ). Greater loss of BW during this time for this group of cows likely accounted for t he elevated BHBA concentrations. Thus feeding AO to multiparous cows reduced plasma concentrations of BHBA but not those of primiparo us cows. Urine of all cows were checked for ketosis using a ketostik at 4, 7, and 12 DIM. Ketosis was diagnosed for 22 o f the 35 cows at least once during the 3 d. A greater proportion of multiparous cows were diagnosed with keto nemia than primiparous cows (10/13 vs. 12/22) and mean concentration of BHBA was greater for multiparous cows (8.9 vs. 6.1 mg/ d L, P < 0.01, Table 3 5). Incidence of ketosis was not different between the 4 treatment groups; namely 4/7 for control cool, 5/8 for control noncool, 6/10 for AO cool, and 7/10 for AO noncool. Mean plasma concentration of glucose was greater for cows fed AO com pared with that of control cows (65.7 vs. 62.7 mg/ 100 mL, P = 0.03, Table 3 5 ). This

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73 increased glucose concentration may be due to greater glucose synthesis or less glucose utilization. Glucose production from propionate was likely similar between the 2 dietary groups because DMI did not differ. However the inclusion of AO in the diet may have caused a shift towardsbacterial species that produce propionic acid, such that glucose synthesis was increased. The reduced production of milk fat by cows fed AO m ay suggest a bacterial shift in the ru men against fiber digesters. On the other hand, glucose utilization may have been less for cows fed AO. There was a weak tendency detected ( P = 0.11) for cows fed AO to produce 3.3 kg/d less 3.5% FCM compared with con trols (26.2 vs. 29 .5 kg/d, Table 3 4). If glucose production was similar between the 2 groups but glucose utilization was less due to lowered milk production, concentrations of plasma glucose could be elevated. The cost of energy to maintain the immune s ystem should be taken into account in energy expense equations (Lochmiller and cells, glucose utilization would be less and concentrations of plasma glucose could rise. Ne ither mean nor weekly pattern of concentration of plasma BUN differed among treatment groups (10.9 mg/ d L, Table 3 5). The overall mean concentrations of BUN did change with week postpartum ( P < 0.001), increasing from 9.9 mg/ d L at wk 1 to 12.2 mg/100 mL a t wk 7 postpartum. This probably reflected increasing intake of CP with increasing intake of DM. Blood urea nitrogen is a major end product of N metabolism by ruminal microorganisms and can be an indicat or of efficien cy of utilization of dietary N (Nousia inen et al., 2004). Postpartum Body Temperature and Oxidative Markers i n Blood Mean rectal temperatures of postpartum cows (taken at 4, 7, and 12 DIM) were greater for primiparous than multiparous cows (39.0 vs. 38.7C, P = 0.01, Table 3 6).

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74 This elevat ed temperature may reflect greater stress typically experienced by primiparous cows from the new experiences of calving and lactating for the first time. C ow s without evaporative cooling prepartum had reduced mean body temperature postpartum compared with cows provided evaporative cooling ( 38.6 vs. 3 9.0 C, P < 0.01). al. (1969) reported that cows exposed to heat stress for 2 wk experienced improved surface evaporation compared to cows exposed for only 1 wk. After calving, all cows were under the same environmental conditions with fans and sprinklers. Assuming that heat production by the 2 groups of cows were similar postpartum, those that lacked cooling prepartum had greater evaporative heat loss than cooled cows which resulted in lower body temperature postpartum. Plasma TBARS represented a composite number of lipid peroxidative end products (Bernabucci et al., 2005) but it should perhaps be considered as an index of oxidati ve stress (Armstrong and Browne, 1994) Plasma concentration of TBARS was greatest at 15 d prepartum and at 1 DIM and then decreased through 29 DIM (effect of week, P < 0.001, Figure 3 16 ). This pattern was followed by both parities and all treatment groups (tests of interactions of week with other independent variables were not significant). Data suggest that these postpartum cows were under less oxidative stress than prepartum cows. B ernabucci et al. (2005) collected 5 prepartum blood samples (30 to 4 d prepartum) and 5 postpartum blood samples (4 to 30 DIM) from 24 Holstein cows for analysis of oxidative markers. Plasma concentrations of TBARS were not different between prepartum and postpartum cows with the exception that cows exceeding a BCS of 3.0 had greater plasma concentrations of TBARS post vs.

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75 prepartum (2.1 vs.1.5 nmol/mL). Cows calving in the summer in Italy had numerically greater plasma concentrations of TBARS in the prep artum (21 and 3 d prepartum) compared to the postpartum period (1, 3, and 35 DIM, Bernabucci et al., 2002). Cows in the current study may have been consuming more antioxidants than those in Italy and thus responded differently. Prepartum cooling did not reduce oxidative stress as measured with TBARS (2.05 vs. 1.79 nmol/mL for cooled and hot respectively, Table 3 6). Likewise, Bernabucci et al. (2002) reported that moderate heat stress (summer vs. spring calving cows) had no effect on concentration of pl asma TBARS in cows during the transition period. Feeding AO tended to reduce mean plasma concentration of TBARS when cows were evaporatively cooled prepartum (2.33 vs. 1.78 nmol/mL) but diet had no effect when prepartum cows were offered shade alone (1.74 vs. 1.83 nmol/mL, diet by environment interaction, P = 0.07, Figure 3 17 ). Bernabucci et al. (2005) suggested that plasma concentrations of TBARS were positively correlated with NEFA and BHBA values. In the current study, when the NEFA values for the fir st 4 wk postpartum were examined (same time period of measurement for TBARS), the mean concentration for the cooled cows fed the control diet were only numerically greater compared with that of the other 3 treatments (503 vs. 321, 390, and 325 nmol/mL for control cool, AO cool, control hot, and AO hot, respectively, P = 0.26 for diet by environment interaction). The same treatment group (control cool) had the greatest 4 wk mean concentration of plasma BHBA (8.4 vs. 6.8, 7.2, and 5.7 mg/100 mL) but the test of diet by environment interaction was P = 0.95). Therefore newly calved cows that are mobilizing more

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76 adipose tissue may be under greater oxidative stress but plasma concentrations of NEFA and BHBA are imperfect indicators of that stress. Activity of GPx per mL of erythrocyte decreased from 15 d prepartum to 8 DIM by 7% and then plateaued (effect of week, P = 0.02; Figure 3 18 ). Bernabucci et al. (2005) also reported GSH Px activity of erythrocytes to drop between 4 d and +11 d of calving. This peri parturient pattern followed that of TBARS. The effect of prepartum cooling on m ean activity of erythrocyte GPx uncorrected and corrected for PCV was influenced by the feeding of AO. Heat stress of multiparous cows fed the control diet elevated GPx correct ed for PCV (8,854 vs.12,247 nmol/min/mL). Including AO in the diet of multiparous cows reversed this effect of heat stress by reducing GPx activity (10,720 vs. 8,697 nmol/min/mL, Table 3 6) whereas the activity of erythrocyte GPx corrected for PCV of prim iparous cows was not affected by diet or environment (diet by environment by parity interaction, P = 0.01, Figure 3 19 ). The same 3 way interaction for GPx activity per mL of erythrocyte w as detected. Lack of evaporative cooling of multiparous cows fed t he control diet elevated GPx (34,960 vs.40,505 nmol/min/mL of RBC). Including AO in the diet of multiparous cows reversed this effect of heat stress by reducing GPx activity (35,716 vs. 30,203 nmol/min/mL of RBC) whereas the activity of erythrocyte GPx pe r mL of erthrocyte of primiparous cows was not affected by diet or environment (diet by environment by parity interaction, P = 0.05, Figure 3 20 ). The mechanism by which AO alleviated the negative effect of heat stress by reducing the GPx activity for mul tiparous cows is unclear. It is possible that AO scaveng ed ROS molecules and reduc ed the load of peroxides generated from increased panting under lack of evaporative cooling as a result, GPx activity was

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77 suppressed. Vazquez Anon et al. (2008) reported th at feeding AO at 200 mg/kg increased the activity of plasma GPx (0.39 vs. 0.55 U/mg of protein for the absence and presence of AO, respectively) for mid to late lactation dairy cows regardless of the type of soybean oil (oxidized vs. unoxidized) fed. Hafeman et al. (1974) reported that activity of erythrocyte GPx was increased when increasing Se supplementation, indicating that there is a c orrelation between the concentration of Se in diet and the activity of GPx In current study, the concentration of Se that multiparous cows consumed postpartum followed the same pattern as the activity of erythrocyte GPx corrected for PCV (4.3, 5.8, 5.4, and 5.1 mg /d for control cool, control hot, AO cool, and AO hot, respectively). Concentration of erythrocy te SOD corrected for PCV increased 22%, from 2,412 U/mL on d 15 prepartum to 2937 U/mL on 8 DIM (effect of week, P = 0.02, Figure 3 21 ). Bernabucci et al. (2005) reported this same dependent variable to increase about the same percentage as the current st udy from 17 d to 4 d prepartum but then decreased thereafter until 30 DIM. The periparturient pattern of SOD followed the opposite pattern of TBARS and GPx. Mean concentration of SOD activity corrected for packed cell volume w as 2955 and 2495 U/mL for m ultiparous and primiparous cows, respectively ( P = 0.05). High producing dairy cows were more likely to suffer from oxidative stress (Castillo et al., 2005; Lohrke et al., 2005) which can be monitored by elevated activities of antioxidant enzymes. Parity affected SOD activity in the opposite way compared to that of plasma TBARS.

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78 Feeding AO resulted in an increase in SOD per mL of erythrocytes at 8 DIM, reversing the trend for cows not fed AO (diet by week interaction, P = 0.03, Figure 3 22 ). Contra r y to current study, S ahoo et al. (2009) reported that t he activity of erythrocyte SOD was decreased compared with the value before treatment after 3 injections of Vitamin E (i.m., 500 IU/injection) and Se (i.m., 15 mg/injection) on alternate days up to the 5 th day as a therapy for subclinical ketosis. Vzquez An et al. (2008) reported that the activity of plasma SOD was decreased (22.02 vs. 19.34 U/g of protein) for cows with vs. without supplementation of 200 mg of Agrado Plus per kg of dietary DM when an uno xidized blend of unsaturated oil was fed, whereas SOD activity was increased (23.74 vs. 26.35 U/g of protein) by feeding Agrado Plus with oxidized oil compared to cows without Agrado Plus. The different responses of SOD and GPx to feeding of AO may be because AO might do a better job of scavenging H 2 O 2 than O 2 radicals along the sequence of reactions of reduction of oxygen in the electron transport chain. White Blood Cells The predominant cells comprising white blood cells (WBC) are lymphocytes and ne utrophils, making up from 86 to 91% of the WBC in the current study (Table 3 7). The lymphocytes are mononuclear cells that are part of the adaptive immune system in that they produce antibodies against antigens to help the host organism resist infection by foreign pathogens. The neutrophils are polynuclear cells that are part of the innate immune system that are the first cells to arrive at sites of infection. The concentration of WBC decreased from 10,887 per L of blood at 15 d prepartum to 7711 per L of blood at 7 DIM (effec t of DIM, P < 0.001, Figure 3 23 ). The type of WBC responsible for this decrease was the neutrophil. Neutroophils migrate to the mammary gland and the

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79 uterus in response to the presence of pathogenic microorganisms after calving The concentration of blood neutrophils was greatest at 15 d prior to calving (4617/L) and decreased to 2272/L by 7 DIM (effect of DIM, P < 0.001, Figure 3 22). Concentration of lymphocytes did not decrease over this same time period (Figure 3 22). Pr epartum cooling of cows tended to reduce mean concentration of WBC for the periparturient cow (7900 vs. 10,176 per L of whole blood, P = 0.09, Table 3 7). The reduction was detected only at 7 and 14 DIM (environment by DIM interaction, P = 0.05, Figure 3 24 ) in which the concentration dropped 32% from 9577 to 6524 per L of whole blood as cows transitioned from the prepartum to postpartum period. The WBC concentration of noncooled cows remained the same throughout the periparturient period (mean of 10,22 0 per L of whole blood. The blood cell type that was responsible for this postpartum drop in WBC of cooled cows was the neutrophil. Concentration of blood neutrophils decreased from 0 to 7 DIM by 57% when cows were cooled prepartum but only by 24% when cows were not evaporatively cooled (environment by DIM interaction, P = 0.11, Figur e 3 25 ). On the other hand, the concentration of lymphocytes did not change (mean of 3455 per L of whole blood) during the transition period for co ws cooled prepartum (Fig ure 3 26 ). Therefore the innate immune system of bovine appeared to be more sensitive than the adaptive system to parturition. Lack of prepartum cooling appeared to minimize the typical decrease in immuno suppression reported for cows in the first 2 wk p ostpartum. Possibly the prepartum exposure of cows to hotter environmental conditions prepared their immune system for the stress of parturition. The cows with the elevated WBC count postpartum had a lower mean rectal temperature at 4, 7, and 12 DIM (Tab le 3 6) suggesting that elevated body temperature

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80 postpartum may reflect a degree of suppressed immune response. Cows not offered evaporative cooling prepartum had a 53% greater mean concentration of lymphocytes throughout the study (5411 vs. 3455 per L of whole blood, P = 0.03, Table 3 7). The concentration of blood lymphocytes tended to decrease on the day of calving and then rebound for noncooled cows whereas that of the cooled cows did not change over time (environment by DIM interaction, P = 0.09, F igure 3 25). Again, hotter environmental conditions resulted in elevated blood concentration of immune cells. Function of Blood Neutrophils Effect of calving. Measures of neutrophil numbers and activities were lowest at 7 or 14 DIM compared with 15 d or day of calving indicating a partially suppressed immune system within a week or 2 after calving (effect of day P < 0.001, Figure 3 22). The proportion of neutrophils carrying out phagocytosis against labeled E. coli was less at 7 compared to 0 DIM (72.6 vs. 83.3%, effect of day P < 0.001) and the MFI for phagocytosis also decreased from 60.4 to 33.5 on these same days (effect of day, P < 0.001, Figure 3 27 ). Proportion of neutrophils undergoing oxidative burst was less at 7 compared to 0 DIM (84 vs. 78%, effect of day, P < 0.01, Figure 3 28 ) and the MFI for oxidative burst also decreased progressively over time from 1687 at 15 DIM to 868 at 14 DIM (effect of day, P < 0.01, Figure 3 27). This decrease after calving has been reported by other investigators as indicators of suppressed immune response immediately postpartum. Effect of parity. In general, the immune system of multiparous cows appeared to be more suppressed than that of primiparous cows. The concentration of neutrophils (2894 vs. 3545/L of blood, P = 0.11), the MFI of phagocytosis (36 vs. 62, P < 0.001), the percentage of neutr ophils conducting oxidative burst (76 vs. 82, P = 0.03), and the

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81 MFI of neutrophils conducting oxidative burst (1041 vs. 1431, P = 0.03) were lower for multiparous cows (Table 3 7). This effect of parity for these dependant variables was consistent across all days of measure with the exception of percentage of neutrophils conducting oxidative burst. Multiparous cows had a reduced percentage only at 7 DIM compared with primiparous cows (71 vs. 86%, parity by week interaction, P = 0.05, Table 3 7). Effect of treatments. The concentration of blood neutrophils (number per L) throughout the study was unaffected by diet or prepartum evaporative cooling. Prepartum cooling increased phagocytosis of E. coli by blood neutrophils of multiparous cows (77.1 vs. 71 .6%) but the reverse occurred for primiparous cows (77.1 vs. 71.6%, cooling by parity interaction, P = 0.03, Figure 3 29 ) This decrease in % neutrophils undergoing phagocytosis due to the cooling of primiparous cows was matched with a decrease in MFI for phagocytosis (69 vs. 55) but MFI for phagocytosis by neutrophils of multiparous cows was not affected by prepartum cooling (37 vs. 34, cooling by parity interaction, P = 0.05, Figure 3 30 ). Feeding AO did not influence the proportion of neutrophils that phagocytized E. coli However the MFI for phagocytosis was affected by diet. The MFI for phagocytosis was reduced by feeding AO to primiparous cows (69 vs. 55) but was unchanged by feeding AO to multiparous cows (33 vs. 39, diet by parity interaction, P = 0.02, Figure 3 31 ). This reduction by AO on MFI of phagocytosis as cows transitioned from prepartum to 7 and 14 DIM was greater for primiparous than for multiparous cows (diet by parity by time interaction, P = 0.04, Figure 3 32 ).

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82 The consumption of oxygen during the generation of ROS is a critical process termed oxidative burst to kill bacteria after phagocytosis. Killing ability by neutrophils (oxidative burst) tended to be compromised by feeding AO when prepartum cows were cooled (83 vs. 77%) vs. noncooled (77 vs. 80%, diet by environment in teraction, P = 0.10, Figure 3 33 ). This effect tended to occur at 7 and 14 DIM (diet by environment by day interaction, P = 0.08, Figure 3 34 ). Including AO in the diet of hotter cows prepartum resulted in a similar pattern of oxidative burst by neutrophils as cows fed the control diet and kept with evaporative cooling. Feeding ethoxyquin at 150 ppm inhibited phagocytosis of leucocytes of tilapia (Yamashita et al. 2009) indicating th at to some extent, the synthetic antioxidants can suppress partially neutrophil activity Several studies have examined the impact of feeding vitamin antioxidants on neutrophil function When cows were inject ed with 3000 IU of vitamin E at 10 and 5 d bef ore calving, intracellular kill of bacteria by neutrophils was increased at calving (Hogan et al., 1990). The same research group (1992) also reported that supplementation of vitamin E (400 to 600 mg/d) or Se (0.3 mg/kg of dietary DM) increased the propor tion of bacteria killed by neutrophils. Weiss and Hogan (2005) also reported that bacterial killing ability by neutrophils tended to be increased for cows fed selenite at 0.3 mg/kg of dietary DM with 500 IU/d of vitamin E compared with cows fed an organic source of Se. Boyne and Arthur (1979) concluded that the percentage of neutrophils that phagocytized and killed bacteria was greater for cows that received sufficient Se (0.1 ppm of dietary DM) than cows given a Se deficient diet. Grasso et al. (1990) re ported that cows supplemented with 2 mg/d of sodium selenite during the transition period had greater bacterial killing ability by neutrophils in milk compared with

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83 cows not supplemented with Se. However function of neutrophils (the proportion of neutrophi ls that phagocytized bacteria and number of intracellular bacteria per neutrophil) isolated from whole blood were not influenced by supplementation with either 0 or 30 g/d of vitamin C starting from 2 wk before calving through 7 DIM (Weiss and Hogan, 2007) No published study has reported on the effect of feeding synthetic antioxidants on neutrophil function. Ovalbumin Challenge and A cute phase P rotein s Cows were given 3 separate injections of ovalbumin at approximately 4 and 2 wk before calving and on the day of calving. Plasma concentrations of IgG for ovalbumin increased after each in jection as expected (F igure 3 35 ). The IgG concentration at wk 4 before calving was used as a covariate in the statistical analysis because analysis of wk 4 alone in a reduced model resulted in a P = 0.12 for the test of diet by environment interaction. The mean circulating c oncentrations of IgG against injected ovalbumin was less for multiparous cows fed AO vs. the control diet (0.80 vs. 0.63 optical density) whereas the response to diet was unchanged for primiparous cows (0.68 vs. 0.73 OD, diet by parity interaction, P = 0.0 5). This difference between multiparous cows occurred primarily from calving to 7 wk postpartum (Figure 3 36 ). From my knowledge, this is the first study to report the effect of feeding synthetic antioxidants on IgG responses to ovalbumin injections of b ovine. Under the current conditions of this trial, supplementing synthetic antioxidants appeared to suppress adaptive immunity responses of multiparous cows. A linear increase in IgG concentration with increased dietary supplementation of vitamin E (285, 570, and 1140 IU/d, respectively) for bull calves was detected at 21 d after ovalbumin ingec tion (Rivera et al., 2002) Similar to this finding,

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84 calves injected with 125 IU of vitamin E at 7 wk of age had greater IgG values compared with those receiving no vitamin E (Reddy et al., 1987) Primiparous cows had greater mean plasma concentration of ASP than multiparous cows (49.5 vs. 39.8 g/mL, P = 0.02, Table 3 7) with the greater values occurring during the first 2 wk after calving (parity by week interaction, P < 0.001, Figure 3 37 ). This result is in agreement with our previous studies (Amaral et al. 2008). Primiparous cows had a greater mean rectal temperature than multiparous cows on 4, 7, and 12 DIM (Table 3 6) suggesting that primiparous cows were experiencing more stress and responding with greater circulation of acid soluble protein. When plasma ASP values were plotted for cows diagnosed as hea lthy (n = 1 8 ) vs. unhealthy (mastitis metritis or retained fetal membranes in the first 14 DIM n = 17 ), unhealthy cows ha d greater mean concentrations of plasma ASP ( 51.2 vs. 4 0.1 g/mL, P < 0.0 1 ) and had greater peak concentrations of plasma ASP betwee n 6 to 22 DIM ( 78.0 vs. 5 0.9 g/mL, healthy vs. unhealthy by time interaction, P = 0.0 01 Figure 3 38 ). Lastly, cows supplemented with AO had greater plasma concentrations of ASP from 6 to 15 DIM compared with the control group (diet by DIM interaction, P = 0.03, Figure 3 39 ). This dietary effect on plasma ASP during the first 2 wk postpartum may have resulted from a greater incidence of endometritis (10/20 vs. 3/15) and retained fetal membranes (2/10 vs. 0/15) for cows fed AO vs. control, respectively (T able 3 9). Similarly to ASP, plasma concentrations of Hp in early in lactation tended to be greater for primiparous compared to multiparous cows (parity by day interaction, P = 0.09, Figure 3 40 ). Also in agreement with ASP responses to disease was the ef fect of mastitis and metritis on plasma Hp. Plasma concentrations of Hp were greater between

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85 6 and 15 DIM in unhealthy vs. healthy cows (healthy vs. unhealthy by time interaction, P < 0.0 1 Figure 3 41 ). Plasma concentrations of Hp may not be as sensitiv e an acute phase protein to reflect disease as plasma concentrations of ASP ( 6 vs. 4 significant time points). Diet did not affect mean or weekly pattern of plasma Hp. Although prepartum cooling did influence plasma concentrations of Hp over DIM, the in fluence was mixed (environment by DIM interaction, P = 0.03, Figure 3 42 ); namely that cooled cows had greater plasma Hp at 6 DIM but less at 13 DIM. Progesterone The reproductive system of multiparous cows appeared to respond better than primiparous cows in the first 7 wk postpartum. Multiparous cows had a longer first estrous cycle (19.3 vs. 15.1 d, P = 0.06) and a greater peak concentration of plasma progesterone in the first cycle (8.2 vs. 5.8 ng/mL, P = 0.04) although the number of ovulations were fewer (1.3 vs. 1.9, P = 0.04, Table 3 8). Thirty two of the 35 cows ovulated within the first 49 DIM. When considering only the 32 cows, treatment did not influence the day of first ovulation which averaged 19.7 2.6 DIM (Table 3 8). However when the 3 cows that did not ovulate were assigned 49 DIM as their day of first ovulation, the day of first ovulation was affected by treatment. Providing evaporative cooling to cows fed the control diet tended to inc rease the number of days to first ovulation compared to noncooled cows (26.0 vs. 19.8 DIM) whereas cooling of cows fed AO tended to decrease time to first ovulation (20.5 vs. 28.9 DIM, diet by environment interaction, P = 0.08). This pattern was repeated for length of first estrous cycle in that providing evaporative cooling to cows fed the control diet resulted in a reduced length of cycle compared to noncooled cows (14.6 vs. 19.1 DIM) whereas cooling of cows fed AO increased length of first cycle (20.1 v s. 15.0 DIM, diet by environment interaction, P =

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86 0.04). Likewise the plasma progesterone values accumulated over all 21 d of sampling times tended to be greater for these same 2 groups of cows ( P = 0.10). Therefore noncooled cows fed the control diet and cooled cows fed AO had an earlier ovulation, a longer first estrous cycle, and produced more progesterone over 7 wk than the other 2 treatment groups. This effect can not be explained adequately by treatment differences in plasma concentrations of NEFA o r in feed efficiency, nor by lower incidence of health disorders (Table 3 10). Multiparous cows fed the control vs. the AO diet had greater peak concentrations of plasma progesterone in the first estrous cycle (10.6 vs. 5.8 ng/mL) whereas that of primipar ous cows did not differ between diets (6.0 vs. 5.7 ng/mL, parity by diet interaction, P = 0.05, Figure 3 43 ). V a ginoscopy and Uterine cytology Cervical discharge was scored based upon extent of presence of pus on 7, 16, and 25 DIM Cows having clear disch arge or only specks of pus were classified as healthy whereas significant amounts of pus resulted in diagnosis of metritis (7 and 16 DIM) or endometritis (25 DIM) (Sheldon et al., 2006), creating a binomial set of data. Compared with multiparous cows, pri miparous cows had a greater incidence of uterine disease at 7 DIM (96 vs. 62%), at 16 DIM (91 vs. 42%), and at 25 DIM (59 vs. 23%). Primiparous cows were 18.2 ( P = 0.02), 12.8 ( P = 0.01), and 11.5 ( P = 0.01) times more likely to have a uterine infection on 7, 16, and 25 DIM than multiparous cows based on odds ratio analysis. At 25 DIM, cows fed AO had a greater incidence of endometritis than cows not fed AO (60 vs. 27%, odds ratio of 9.6, P = 0.02). At 40 2 DIM, one uterine horn was flushed and the pro portion of neutrophils calculated after counting 100 stained cells identified using a microscope. Neutrophils comprised a greater proportion of cells when cows were fed AO (21.9 vs. 6.8%, P =

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87 0.02). Cows were diagnosed as having subclinical endometritis when the proportion of neutrophils exceeded 5% ( Gilbert et al., 2005 ) thus creating a binominal data set. The incidence of subclinical endometritis was greater in cows fed AO (80 vs. 33%, odds ratio of 3.4, P < 0.01). This negative effect of AO on increa sing uterine subclincial 25 DIM as reported in the previous paragraph. Primiparous cows tended to have more cases of endometritis than multiparous cows (68 vs. 46%, odds ratio = 9.3, P = 0.06), a pattern that agrees with the greater incidence of metritis for primiparous vs. multiparous cows. Lastly, cows lacking prepartum evaporative cooling tended to have a greater incidence of subclinical endometritis (72 vs. 47%, P = 0.08). Summary Parity. Primiparous cows appeared to be under greater stress postpartum than multiparous cows as evidenced by greater or tendency for greater mean body temperature at 4, 7, and 12 DIM (39.0 vs. 38.7C), greater metricheck score which is greater than 1 at 7 DIM (95 vs. 62%), 16 DIM (91 vs. 42%), and 25 DIM (59 vs. 23%), greater incidence of subclinical endometritis around 40 DIM (68 vs. 46%), and increasing concentrations of BHBA with increasing DIM. As a consequence of a greater incidenc e of uterine diseases, the immune responses were stimulated; namely, an increase in the concentration of blood neutrophils (3545 vs. 2894 per L), in MFI for phagocytosis (62 vs. 36) and for oxidative burst (38 vs.32) by neutrophils, in % of neutrophils un dergoing oxidative burst (82 vs. 76%), and in plasma concentration of acute phase proteins (acid soluble protein and haptoglobin) in the first 2 wk postpartum. Also as a result of increased metritis and endometritis, ovarian activity was affected negativ ely; namely, a shorted length of the first estrous cycle (15.1 vs. 19.3 d) and a

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88 lower peak concentration of progesterone in the first cycle (5.8 vs. 8.2 ng/mL). Lastly, oxidative markers in the blood (TBARS, SOD, and GPx) reacted differently. Mean plasma concentrations of TBARS were greater (2.12 vs. 1.71 nmol/mL) whereas mean RBC concentrations of SOD corrected for packed cell volume were lower (2495 vs. 2955 U/mL) for primiparous vs. multiparous cows. Activity of GPX per mL of RBC were lower at 15 d be fore parturition (34,601 vs. 40,096 nmol/min/mL) but greater at 29 DIM (36,629 vs. 32,440 nmol/min/mL) for primiparous vs. multiparous cows. Cooling. Prepartum evaporative cooling during the 3 wk prior to calving had or tended to have several positive bene fits. Prepartum cooling resulted in a lower mean rectal temperature during the prepartum period (39.2 vs. 39.6C) but a greater mean rectal temperature at 4, 7, and 12 DIM (39.0 vs. 38.7C). This slightly greater mean rectal temperature was not increased enough to negatively affect production or health. Indeed prepartum cooling resulted in greater mean concentration of milk fat during 7 wk (3.54 vs. 3.32%) and mean production of 3.5% FCM during the first 4 wk postpartum (26.5 vs. 23.0 kg/d). Additionall y, incidences of mastitis (1/17 vs. 6/18), displaced abomasum (0/17 vs. 3/18), and subclinical endometritis (47 vs. 72%) were reduced due to prepartum cooling. Associated with the reduction in health disorders, the number and activity of select immunity c ells were affected as evidenced by lower concentration of circulating WBC postpartum (2049 vs. 2804 per L of blood) and of circulating lymphocytes (3455 vs. 5411 per L of blood) and increased proportion of neutrophils undergoing oxidative burst of E. col i of cows fed the control diet (83 vs. 77%). Assuming that an elevated concentration of oxidative markers in blood is indicative of greater oxidative stress, prepartum cooling of multiparous cows resulted in less

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89 oxidative stress as evidenced by lower act ivity of GPx in RBC (8,854 vs. 12,247 nmol/min/mL) and of SOD in RBC (2503 vs. 3111 U/mL). The only negative effects of prepartum cooling were a greater plasma concentration of TBARS for control fed cows (2.33 vs. 1.74 nmol/mL), a decreased length of the first estrous cycle of control fed cows (14.6 vs. 19.1 d), and a reduction in phagocytosis by neutrophils (74 vs. 81% and 55 vs. 67 MFI) in primiparous cows. Synthetic antioxidants. Feeding AO did not influence DM intake or uncorrected milk production. Ho wever supplemental AO had mixed effects on milk composition. Concentration of milk true protein was increased (3.07 vs. 2.94%) but concentration of milk fat was decreased (3.25 vs. 3.61%) resulting in less production of milk fat (0.88 vs. 1.04 kg/d) and o f 3.5% FCM (26.2 vs. 29.5 kg/d). Therefore AO seems to influence the microbial population in the rumen. In addition, cows fed AO had a greater incidence of endo metritis (60 vs. 27%) at 25 DIM and of subclinical endometritis at 40 DIM (80 vs. 33%). These reproductive disorders may have contributed to a lower peak concentration of plasma progesterone in the first estrous cycle of multiparous cows (5.8 vs. 10.6 ng/mL ). A g reater peak concentration of plasma ASP postpartum in cows fed AO vs. control diet m ay reflect greater stress The effect of AO on oxidative markers or immune cells was not consistent. Under 4 different scenarios of more stressful (primiparous or prepartum noncooling) or less stressful (multiparous or prepartum cooling) scenarios cows f ed AO responded differently. Results are the following:

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90 Nonstressful situation improved F eeding AO to cows managed under prepartum evaporative cooling conditions resulted in 1. a longer first estrous cycle (20.2 vs. 14.6 d) 2. A d ecrease in plasma concentration of TBARS (1.78 vs. 2.33 nmol/mL). Nonstressful situation aggravated F eeding AO to cows managed under prepartum evaporative cooling conditions resulted in 1. a decrease p roportion of neutrophils undergoing oxidative burst (77 vs. 83%). Stressful situation improved F eeding AO to multiparous cows managed without prepartum evaporative cooling resulted ina reduced activity of GPX in RBC corrected for packed cell volume ( 8,697 vs. 10,720 nmol/min/mL). Stressful situa tion aggravated : Feeding AO to primiparous cows 1. reduced MFI of phagocytosis by neutrophils (36 vs. 57%) 2. Feeding AO at 250 mg per kg of dietary DM to periparturient Holstein cows fed diets of minimal fiber density resulted in reduced production of fat corrected milk, increased incidence of uterine infections, reduced neutrophil activity, and mixed effects on oxidative markers in bloo d.

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91 Table 3 1. Ingredient composition of diets fed to nonlactating and lactating Holstein cows. Ingredient, % o f DM Nonlactating Lactating Corn silage 30.0 39.5 Bermuda grass silage 35.0 Alfalfa hay 12.5 Ground corn 9.5 18.9 Soybean meal 9.1 7.8 Soy Plus 1 1.2 7.3 Citrus pulp 6.7 4.0 Corn gluten feed 4.4 Corn oil 2.0 2.0 Mineral/vitamin mix 6.5 2 3.7 3 1 West Central Soy, Ralston, IA. 2 Contained 18.3% CP, 20.5% Ca, 0.3% P, 3.1% Mg, 9.6% Cl, 0.3% K, 1.6% Na, 2.4% S, 11 ppm Co, 176 ppm Cu, 8 ppm I, 158 ppm Fe, 142 ppm Mn, 7 ppm Se, 268,00 IU vitamin A, 40 IU vitamin D, and 1300 IU of vitamin E (DM basis). 3 Contained 24% CP, 9% Ca, 1% P, 4% K, 3% Mg, 10% Na, 1.1% S, 1200 ppm Mn, 158 ppm Fe, 500 ppm Cu, 1500 ppm Zn, 8.25 ppm Se, 2% Cl, 20 ppm I, 147,756 IU/kg of vitamin A, 43,750 IU/kg of vitamin D, and 787 IU/kg of vitamin E (DM basis).

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92 Table 3 2. Chemical composition of diets fed to nonlactating and lactating Holstein cows. Chemical % DM Nonlactating cows Lactating cows Control Antioxidant 1 Control Antioxidant CP 15.1 15.3 17.9 17.7 NDF 37.6 37.7 26.0 25.8 ADF 23.5 23.6 15.8 16.1 Ether extract 3.9 4.0 4.5 4.7 NEL, Mcal/kg 1.31 1.34 1. 75 1. 75 Ca 1.86 1.85 0.83 0.95 P 0.32 0.33 0.40 0.39 Mg 0.43 0.44 0.33 0.35 K 1.25 1.25 1.46 1.45 Na 0.15 0.15 0.31 0.31 S 0.34 0.33 0.23 0.24 Cl 0.99 0.93 0.38 0.45 Cu 29 28 25 30 Fe 256 252 18 18 Mn 107 100 78 87 Zn 46 48 69 59 1 Agrado Plus (Novus Internat ional Inc., St. Charles, MO)

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93 Table 3 3. Effect of feeding synthetic antioxidants (AO) and p repartum c ooling on body temperature, body weight, BCS, and DMI of nonlactating pregnant Holstein cows during summer in Florida. Treatment Measure Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Number of a nimals 5 2 6 2 5 5 6 4 Body temperature, C 39.2 0.1 39.4 0.2 39.5 0.1 39.8 0.2 39.2 0.1 39.2 0.1 39.6 0.1 39.6 0.1 DMI, kg/d 10.6 0.6 9.2 0.9 10.4 0.5 9.4 0.9 10.3 0.7 11.7 0.6 9.7 0.5 9.0 0.6 BW, kg 568 18 843 28 585 16 680 28 596 18 691 18 587 16 720 20 DMI, % of BW 1.86 0.10 1.09 0.16 1.77 0.09 1.41 0.16 1.69 0.12 1.70 0.10 1.62 0.09 1.25 0.11 BCS 2 3.37 0.17 3.75 0.27 3.21 0.16 3.06 0.27 3.37 0.16 3.41 0.19 3.35 0.16 3.09 0.19 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO). 2 Least squares mean of BCS at the time of enrollment in the study and at calving. 3 N / A = Not available

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94 Table 3 3. Continued P values Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P Week (W) Diet by W Cooling by W Diet by cooling by W P by W Diet by P by W Cooling by P by W Diet by cooling by P by W Number of a nimals N / A 3 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A Body temperature, C 0.38 <0.001 0.77 0.19 0.15 0.85 0.76 0.53 0.48 0.35 0.39 0.81 0.98 0.44 0.34 DMI, kg/d 0.56 0.09 0.10 0.33 0.12 0.39 0.18 <0.001 0.23 0.49 0.22 0.85 0.73 0.96 0.47 BW, kg 0.20 0.04 0.01 <0.001 0.02 0.02 0.001 0.21 0.14 0.40 0.43 0.47 0.36 0.95 0.97 DMI, % of BW 0.73 0.42 0.03 <0.001 0.03 0.91 0.03 <0.001 0.36 0.64 0.18 0.56 0.85 0.97 0.75 BCS 0.77 0.05 0.37 1.00 0.43 0.16 0.69 0.03 0.39 1.00 0.34 0.03 0.77 0.56 0.70

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95 Figure 3 1. Least squares means for mean dry matter intake of prepartum primiparous (n = 22) and multiparous (n = 1 3 ) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of d iet by environment by parity interaction ( P = 0.0 3 ). 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 Primiparous Multiparous DM intake (% of BW) Parity Control Cool Control Hot AO Cool AO Hot

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96 Figure 3 2. Least squares means for mean body weight o f prepartum primiparous (n = 22) and multiparous (n = 1 3 ) Hols tein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of d iet by environment by parity interaction ( P = 0.0 01 ). 0 100 200 300 400 500 600 700 800 Primiparous Multiparous BW (kg) Parity Control Cool Control-Hot AO Cool AO Hot

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97 Table 3 4. Effect of feeding synthetic antioxidants (AO) and prepart um cooling on performance of lactating pregnant Holstein cows during summer in Florida. Measure Treatment Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Number of a nimals 5 2 6 2 5 5 6 4 BW, kg 504 16 724 26 518 15 590 26 516 16 625 16 526 15 631 18 BCS 3.23 0.13 3.46 0.20 3.12 0.12 3.08 0.20 3.19 0.12 3.33 0.14 3.19 0.12 3.08 0.14 DMI, kg/d 15.0 0.91 14.1 1.4 13.5 0.8 18.9 1.4 12.7 0.9 17.7 0.8 14.1 0.8 16.6 1.0 DMI, % of BW 2.98 0.14 1.99 0.21 2.62 0.12 3.24 0.21 2.49 0.14 2.83 0.14 2.68 0.12 2.64 0.15 Milk, kg/d 27.7 2.8 31.1 2.5 24.5 2.8 34.4 4.4 23.6 2.5 36.0 4.4 22.0 2.8 29.8 3.1 Milk fat, % 3.35 0.19 4.21 0.31 3.21 0.17 3.67 0.30 3.22 0.19 3.38 0.19 3.12 0.17 3.29 0.21 Milk protein, % 2.97 0.08 2.76 0.14 3.02 0.07 3.00 0.14 2.98 0.08 3.10 0.08 3.09 0.07 3.09 0.09 Milk fat, kg/d 0.9 0.1 1.3 0.1 0.7 0.1 1.2 0.1 0.7 0.1 1.2 0.1 0.7 0.1 1.0 0.1 Milk protein, kg/d 0.8 0.1 0.9 0.1 0.7 0.1 0.9 0.1 0.7 0.1 1.1 0.1 0.7 0.1 0.9 0.1 Milk SCC, x1000 174 21 182 28 89 20 300 28 105 21 47 21 190 20 177 22 3.5% FCM, kg/d 26.8 2.4 33.8 3.8 22.5 2.2 34.8 3.8 22.0 2.4 34.3 2.4 20.1 2.2 28.4 2.7 Feed efficiency 1.81 0.16 2.48 0.25 1.72 0.15 2.00 0.25 1.79 0.16 1.97 0.16 1.45 0.14 1.79 0.18 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO). 2 N/A = Not Available.

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98 Table 3 4. Contiu ned Measure P values Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P Week (W) Diet by W Cooling by W Diet by cooling by W P by W Diet by P by W Cooling by P b y W Diet by cooling by P by W Number of a nimals N/A 2 N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A BW, kg 0.48 0.06 0.02 < 0.001 0.16 < 0.01 0.01 < 0.001 0.12 0.56 0.29 0.52 0.80 0.16 0.41 BCS 0.83 0.10 0.59 0.62 0.72 0.23 0.97 0.001 0.59 0.25 0.18 0.58 0.32 0.82 0.53 DMI, kg/d 0.88 0.25 0.34 < 0.001 0.33 0.22 < 0.01 < 0.001 0.04 0.14 0.10 0.02 0.38 0.65 0.07 DMI, % of BW 0.69 0.06 0.05 0.87 0.14 0.01 0.001 < 0.001 0.10 0.21 0.26 0.28 0.60 0.72 0.30 Milk, kg/d 0.50 0.41 0.41 0.001 0.46 0.84 0.24 < 0.001 0.39 0.36 0.20 < 0.001 0.13 0.20 0.32 Milk fat, % 0.03 0.18 0.43 0.01 0.13 0.55 0.51 < 0.001 0.10 0.01 0.95 0.02 0.36 0.47 0.94 Milk protein, % 0.06 0.17 0.49 0.70 0.19 0.78 0.27 < 0.001 0.04 0.15 0.41 0.94 0.21 0.33 0.53 Milk fat, kg/d 0.04 0.11 0.86 < 0.001 0.72 0.96 0.40 < 0.001 0.17 0.02 0.83 < 0.01 0.48 0.33 0.55 Milk protein, kg/d 0.69 0.38 0.36 < 0.001 0.27 0.97 0.13 < 0.001 0.84 0.79 0.54 0.10 0.27 0.27 0.64 Milk SCC, x1000 0.35 0.31 0.23 0.82 0.22 0.27 0.80 < 0.001 0.15 0.03 0.68 0.27 0.26 0.68 0.40 3.5% FCM, kg/d 0.11 0.18 0.58 < 0.001 0.86 0.88 0.26 < 0.001 0.19 0.07 0.65 < 0.001 0.61 0.22 0.39 Feed efficiency 0.06 0.05 0.90 0.01 0.42 0.66 0.30 < 0.001 0.37 0.08 0.05 0.15 0.68 0.52 0.03

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99 Figure 3 3. Least squares means for mean dry matter intake of postpartum primiparous (n = 22) and multiparous (n = 1 3 ) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of d iet by environment by parity interaction ( P = 0.0 01 ). 0 0.5 1 1.5 2 2.5 3 3.5 Primiparous Multiparous DM intake (% of BW) Parity Control-Cool Control-Hot AO-Cool AO-Hot

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100 Figure 3 4. Least squares means for mean body weight (BW) of postpartum primiparous (n = 22) and multiparous (n = 1 3 ) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or wit h dietary antioxidant s (AO) Effect of d iet by environment by parity interaction ( P = 0.0 1 ). 0 100 200 300 400 500 600 700 800 Primiparous Multiparous BW (kg) Parity Control Cool Control Hot AO Cool AO Hot

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101 Figure 3 5. Least squares means for weekly body weight (BW) of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of parity by diet by environment by week ( P = 0.41 ). 460 480 500 520 540 560 580 0 1 2 3 4 5 6 7 BW (Kg) Week postpartum Control Cool Control Hot AO Cool AO Hot A A 520 570 620 670 720 770 820 0 1 2 3 4 5 6 7 BW (Kg) Week postpartum Control Cool Control Hot AO Cool AO Hot B

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102 Figure 3 6. Least squares means for weekly dry matter intake of postpartum Holstein cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of diet by week interaction ( P = 0.10). Week with asterisk i ndicates that diets differed ( P < 0.05). 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1 2 3 4 5 6 7 DM intake (% of BW) Week postpartum Control Cool Control Hot AO Cool AO Hot

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103 Figure 3 7. Least squares means for weekly milk production of Holstein cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls and fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of diet by environment by week ( P = 0.20 ). Week with dagger indicates that means differed ( P < 0.10). 5 10 15 20 25 30 35 40 45 0 1 2 3 4 5 6 7 Milk (kg/d) Week postpartum Control Cool Control Hot AO Cool AO Hot

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104 Figure 3 8. Least squares means for weekly concentration of milk fat of Holstein cows (n = 35) housed in c ooled (Cool) or noncooled (Hot) freestalls. Effect of environment by week interaction ( P = 0.0 1 ). Week with asterisk indicates that means differed using slice command ( P < 0.05). 2 2.5 3 3.5 4 4.5 5 1 2 3 4 5 6 7 Milk fat (%) Week postpartum Cool Hot

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105 Figure 3 9. Least squares means for weekly milk fat production of Holste in cows (n = 35) housed in cooled (Cool) or noncooled (Hot) freestalls. Effect of environment by week interaction ( P = 0.0 2 ). Week with asterisk indicates that means differed using slice command ( P < 0.05). 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1 2 3 4 5 6 7 Milk fat yield (kg/d) Week postpartum Cool Hot

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106 Figure 3 10. Least squares means for production of 3.5% fat corrected milk by postpartum Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of cooling by week interaction ( P = 0.0 7 ). Week with dagger indicates that means d iffered using slice command ( P < 0.10). 10 15 20 25 30 35 40 1 2 3 4 5 6 7 3.5% FCM (kg/d) Week postpartum Cool Hot

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107 Figure 3 11. Least squares means for weekly concentration of milk protein of Holstein cows (n = 35) fed diets supplemented with out (Control) or with dietary antioxidant s (AO) Effect of diet by week interaction ( P = 0.0 4 ). Week with asterisk indicates that means differed using slice command ( P < 0.05). 2.25 2.75 3.25 3.75 4.25 4.75 1 2 3 4 5 6 7 Milk true protein (%) Week postpartum Control AO

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108 Figure 3 12. Least squares means for somatic cell counts of postpartum Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of cooling by week interaction ( P = 0.0 3 ). Week with asterisk indicates that means differed using slice command ( P < 0.05), with dagger differed ( P < 0.10). 0 100 200 300 400 500 600 700 800 900 1000 1 2 3 4 5 6 7 Somatic Cell counts (x1000) Week postpartum Cool Hot

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109 Table 3 5 Effect of feeding s ynthetic antioxidants (AO) and p repartum c ooling on postpartum plasma concentration of metabolites of lactating Holstein cows during summer in Florida. Measure Treatment Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous NEFA, eq/L 195 47 557 74 168 44 381 74 222 48 288 47 152 43 321 52 BHBA, mg/dL 7.22 0.97 10.46 1.54 5.17 0.94 9.28 1.54 6.53 1.00 8.62 0.97 5.55 0.91 7.40 1.09 Glucose, mg/dL 64.2 1.6 58.2 2.6 68.5 1.5 59.9 2.6 69.9 1.6 60.6 1.6 70.8 1.5 61.5 1.8 BUN, mg/dL 11.8 0.8 11.1 1.2 10.4 0.7 9.5 1.2 11.5 0.8 10.9 0.8 10.7 0.7 11.0 0.9 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO).

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110 Table 3 5. Continued P values Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P Week (W) Diet by W Cooling by W Diet by cooling by W P by W Diet by P by W Cooling by P by W Diet by cooling by P by W NEFA, eq/L 0.07 0.16 0.31 < 0.001 0.06 0.78 0.14 <0.001 <0.01 <0.01 <0.01 <0.001 0.03 < 0.01 0.02 BHBA, mg/dL 0.22 0.11 0.75 < 0.01 0.30 0.85 0.73 <0.01 0.03 0.87 0.15 0.01 <0.01 0.61 0.22 Glucose, mg/dL 0.03 0.16 0.45 < 0.001 0.45 0.63 0.63 0.08 0.63 0.49 0.67 0.12 0.37 0.70 0.18 BUN, mg/dL 0.61 0.15 0.37 0.46 0.59 0.79 0.70 <0.001 0.38 0.52 0.08 0.59 0.94 0.98 0.19

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111 Figure 3 13. Least squares means for weekly plasma concentrations of NEFA of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets supplemented without (Control, C) or with synthetic antioxidants (AO) and housed in shade d freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of parity by diet by environment by week interaction ( P = 0.02). Week with one asterisk indicates that means for the 8 treatments differed for that week using slice command ( P < 0.05); with two asterisks differed at P < 0.001. 0 100 200 300 400 500 600 700 800 0 1 2 3 4 5 6 7 NEFA ( EQ/L) Week postpartum C Cool C Hot AO Cool AO Hot A 0 200 400 600 800 1000 1200 1400 0 1 2 3 4 5 6 7 NEFA ( EQ/L) Week postpartum C Cool C Hot AO Cool AO Hot B *

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112 Figure 3 14 Least squares means for weekly energy balance of postpartum primiparous (A, n = 22) and multiparous (B, n = 13) Holstein cows fed diets supplemented without (Control ) or with syntheti c antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of parity by diet by environme nt by week interaction ( P = 0.03 ). Week with one asterisk indicates that 8 treatment means differed for that week using slice command ( P < 0.05); with tw o asterisks differed at P < 0.0 1 ; dagger differed at P < 0.10 -10 -8 -6 -4 -2 0 2 4 6 8 10 0 1 2 3 4 5 6 7 Energy balance (Mcal) Week postpartum Control Cool Control Hot AO Cool AO Hot A -17 -14 -11 -8 -5 -2 1 4 7 10 0 1 2 3 4 5 6 7 Energy balance (Mcal) Week postpartum Control Cool Control Hot AO Cool AO Hot B

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113 Figure 3 15 Least squares means for weekly plasma concent ration of beta hydroxyl butyric acid of primiparous ( primi, n = 22) and multiparou s ( multi, n = 13 ) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). Effect of d iet by parity by week interaction ( P < 0.01) Week with two asterisks indicates that means differed for that week using slice command ( P < 0.01). 0 2 4 6 8 10 12 14 16 0 1 2 3 4 5 6 7 BHBA (mg/dl) Week postpartum Primi Control Primi AO Multi Control Multi AO ** ** **

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114 Table 3 6. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on postpartum body temperature and plasma concentration of oxidative markers of lactating Holstein cows during summer in Florida. Measure Treatment Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Body temp, C 39.0 0.1 39.0 0.2 38.7 0.1 38.4 0.2 39.0 0.1 38.9 0.1 39.0 0.1 38.4 0.2 TBARS 2 nmol/mL 2.32 0.19 2.34 0.29 2.08 0.17 1.39 0.31 1.95 0.19 1.61 0.19 2.15 0.17 1.52 0.21 GPx 3 nmol/min/mL 10,302 720 8854 1133 9384 659 12,247 1157 9305 723 10,720 726 9733 659 8697 801 GPx 4 nmol/min/mL 35,985 2181 34,960 3444 35,802 1993 40,505 3464 33,456 2184 35,716 2187 37,214 1993 30,203 2435 SOD 5 U/mL 2635 272 2503 427 2486 248 3111 439 2405 271 2759 275 2454 248 3448 302 SOD 6 U/mL 9247 1078 9709 1701 9635 985 10,634 1713 8806 1079 9120 1081 9484 985 11,839 1203 PCV 7 % of blood 28.6 1.1 25.5 1.8 26.0 1.0 30.3 1.8 27.4 1.1 29.8 1.1 26.2 1.0 29.1 1.2 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO). 2 TBARS = thiobarbituric acid reactive substances. 3 GPx = glutathione peroxidase expressed as pe r ml of blood corrected for PCV 4 GPx = glutathione peroxidase expressed per ml of red blood cell. 5 SOD = superoxide dismutase expressed as per ml of blood corrected for PCV. 6 SOD = superoxide dismutase expressed per ml of red blood cell. 7 Packed cell volume.

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115 Table 3 6. Continuned P values Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P Week (W) Diet by W Cooling by W Diet by cooling by W P by W Diet by P by W Cooling by P by W Diet by cooling by P by W Body temp, C 0.70 < 0.01 0.51 0.01 0.26 0.12 0.52 0.56 0.79 0.87 0.55 0.88 0.68 0.95 0.15 TBARS 0.19 0.13 0.07 0.02 0.68 0.15 0.52 <0.001 0.51 0.54 0.70 0.71 0.77 0.25 0.62 GPx 0.34 0.72 0.10 0.46 0.67 0.44 0.01 0.45 0.83 0.46 0.91 0.24 0.24 0.83 0.76 GPx 0.15 0.62 0.33 0.88 0.25 0.63 0.05 0.02 0.72 0.37 0.69 <0.001 0.35 0.89 0.98 SOD 0.72 0.20 0.76 0.05 0.35 0.13 0.90 0.02 0.39 0.73 0.71 0.83 0.10 0.73 0.92 SOD 0.99 0.20 0.56 0.26 0.74 0.48 0.68 0.82 0.03 0.91 0.22 0.17 0.09 0.69 0.25 PCV 0.57 0.94 0.28 0.10 0.27 0.04 0.07 <0.01 0.91 0.43 0.82 0.78 0.22 0.42 0.67

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116 Figure 3 16 Least squares means for plasma thiobarbituric acid reactive substances (TBARS) on 15, 1, 8, 15, 29 d relative to calving. Effect of time ( P < 0.0001). 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 -15 -10 -5 0 5 10 15 20 25 30 TBARS (nmol/mL) Days relative to calving

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117 Figure 3 17 Least squares means for mean plasma concentration of thiobarbituric acid reactive substances (TBARS) of Holstein cows (n = 35) fed diets supplemented without (Control) or with dietary antioxidant (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Diet by envi ronment interaction ( P = 0.07). 0 0.5 1 1.5 2 2.5 3 Control Cool Control Hot AO Cool AO Hot TBARS (nmol/ml) Treatment

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118 Figure 3 18 Least squares means for weekly activity of glutathione peroxidase (GPx) per mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented without or with synthetic antioxidants and housed in shaded freestalls equipped with fans and sprinklers or just shade on 15, 1, 8, 15, and 29 d relative to calving. Effect of time ( P = 0.02). 33000 34000 35000 36000 37000 38000 39000 -15 -10 -5 0 5 10 15 20 25 30 GPx (nmol/min/mL) Days relative to calving

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119 Figure 3 19 Least squares means for mean activity of glutathione peroxidase corrected for pack cell volume of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of diet by environment by parity interaction ( P = 0.01). 0 3000 6000 9000 12000 Primiparous Multiparous GPx (nmol/min/ml) Parity Control Cool Control Hot AO Cool AO Hot

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120 Figure 3 20 Least squares means for mean activity of glutathione peroxidase per mL of erythrocyte of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented w ithout (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of diet by environment by parity interaction ( P = 0.05). 25000 27000 29000 31000 33000 35000 37000 39000 41000 43000 45000 Primiparous Multiparous GPx (nmol/min/mL) Parity Control Cool Control Hot AO Cool AO Hot

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121 Figure 3 21 Least squares means for activity of superoxide dismutase (SOD) corrected for pack cell volume of Holstein cows (n = 35) fed diets supplemented without or with synthetic antioxidants and housed in shaded freestalls equipped with fans and sprinklers or just shade on 15, 1, 8, 15, and 29 d relative to calving. Effect of time ( P = 0.02). 2000 2200 2400 2600 2800 3000 3200 -15 -10 -5 0 5 10 15 20 25 30 SOD (U/mL) Days relative to calving

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122 Figure 3 22 Least squares means for activity of superoxide dismutase (SOD) per mL of erythrocyte of Holstein cows (n = 35) fed diets supplemented without (Control) or with dietary antioxidants (AO) on 15, 1, 8, 15, and 29 d relative to calving. Effect of diet by time (P = 0.03). 8000 8500 9000 9500 10000 10500 11000 11500 12000 -15 -10 -5 0 5 10 15 20 25 30 SOD (U/mL) DIM Control AO

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123 Table 3 7. Effect of feeding synthetic antioxidants (AO) and prepartum cooling on concentration of white blood c ells (WBC), lymphocytes, and neutrophils, function of blood neutrophils, and plasma concentration of acid soluble protein (ASP) and haptoglobin (Hp) of periparturient Holstein cows during summer in Florida. Treatment Measure Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous WBC, per L 9,273 1665 5,998 1762 10,698 1766 12,917 3795 8,488 1547 8,249 1481 8,969 1470 8,652 1746 Lymphocytes, per L 4272 1081 2208 944 4911 1131 8145 3484 3912 996 3862 978 4700 1080 4559 1304 Neutrophils, per L 3,728 563 2,983 730 4,571 661 3,010 737 3,031 489 2,819 426 3,056 429 2,772 470 Phagocytosis, % 77.0 3.3 80.4 5.2 80.3 3.2 70.4 5.0 71.1 3.0 73.8 3.6 81.4 3.3 72.8 3.6 Phagocytosis MFI 2 63.3 5.9 34.5 5.1 75.3 6.8 31.2 4.7 48.5 4.4 40.4 4.2 62.7 6.0 36.7 4.0 Oxidative burst, % 84.1 3.0 81.2 4.6 84.7 2.8 70.5 4.3 77.1 2.7 76.1 3.2 81.8 2.9 77.4 3.3 Oxidative burst, MFI 1374 211 1098 321 1645 219 1094 297 1146 183 950 196 1589 222 1025 209 Acute phase proteins ASP, g/mL 45.1 4.6 39.9 7.3 48.6 4.2 35.3 7.3 53.5 4.6 41.5 4.6 50.7 4.2 42.6 5.1 Hp, arbitrary units 0.015 0.002 0.015 0.002 0.013 0.002 0.012 0.002 0.012 0.003 0.011 0.003 0.011 0.002 0.013 0.002 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO). 2 MFI = Mean florescence intensity, to test the phagocytic ability of neutrophils.

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124 Table 3 7. Continuned P values Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P Week (W) Diet by W Cooling by W Diet by cooling by W P by W Diet by P by W Cooling by P by W Diet by cooling by P by W WBC, per L 0.56 0.09 0.18 0.60 0.76 0.30 0.29 <0.001 0.67 0.05 0.41 0.67 .042 0.97 0.83 Lymphocytes per L 0.85 0.03 0.18 0.81 0.89 0.16 0.15 0.07 0.85 0.09 0.73 0.14 0.96 0.94 0.57 Neutrophils, per uL 0.14 0.68 0.66 0.11 0.35 0.66 0.74 <0.001 0.47 0.11 0.24 0.50 0.54 0.98 0.97 Phagocytosis, % 0.42 0.83 0.15 0.27 0.93 0.03 0.86 0.001 0.41 0.97 0.34 0.29 0.60 0.65 0.15 Phagocytosis, MFI 0.68 0.16 0.79 <0.001 0.02 0.05 0.79 <0.001 0.93 0.93 0.61 0.40 0.04 0.11 0.17 Oxidative burst, % 0.46 0.64 0.10 0.03 0.24 0.13 0.40 <0.01 0.36 0.73 0.08 0.05 0.86 0.96 0.31 Oxidative burst, MFI 0.46 0.29 0.70 0.03 0.96 0.40 0.90 <0.01 0.81 0.47 0.77 0.22 0.23 0.96 0.99 Acute phase proteins ASP, g/mL 0.22 0.85 0.97 0.02 0.92 0.78 0.43 <0.001 0.03 0.72 0.47 <0.001 0.74 0.89 0.86 Hp, arbitrary units 0.65 0.99 0.73 0.20 0.36 0.68 0.65 <0.001 0.24 0.03 0.72 0.09 0.97 0.60 0.35

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125 Figure 3 23 Least squares means for number of white blood cells (WBC), neutrophils, 15, 0, 7, 14 d relative to calving. Effect of time for WBC ( P < 0.001), for neutrophil ( P < 0.001), and for lymphocyte ( P = 0.07). 17 2017 4017 6017 8017 10017 12017 14017 -15 -10 -5 0 5 10 15 Hematological concentration Days relative to calving WBC (per L) Neutrophil (per L) lymphocyte (per L)

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126 Figure 3 24 whole blood of Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by week interaction ( P = 0.05). Days relative to calving with asterisk indicates that means differed ( P < 0.05) using slice command. 17 2017 4017 6017 8017 10017 12017 14017 -15 -10 -5 0 5 10 15 WBC (per L of blood ) Days relative to calving Cool Hot

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127 Figure 3 25 Least squares means for number of Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by week interaction ( P = 0.11). 17 1017 2017 3017 4017 5017 6017 -15 -10 -5 0 5 10 15 Neutrophil (per L of blood ) Days relative to calving Cool Hot

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128 Figure 3 26 Least squares means for number o Holstein cows (n = 35) housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by week interaction ( P = 0.09). Days relative to calving with asterisk indicates t hat means differed ( P < 0.05) using slice command. 17 1017 2017 3017 4017 5017 6017 7017 -16 -11 -6 -1 4 9 14 Lymphocyte (per L of blood ) Days relative to calving Cool Hot

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129 Figure 3 2 7 Least squares means for percentage of neutrophils with phagocytic activity (solid line) and neutrophil mean florescence intensity (MFI, indication of number of bacteria phagocytised p er neutrophil, dash line) of Holstein cows (n = 35) on 15, 0, 7, 14 d relative to calving. Effect of time ( P = 0.001) for percentage of neutrophils with phagocytic activity, effect of time ( P < 0.001) for neutrophil MFI. 0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80 90 -15 -10 -5 0 5 10 15 MFI of phagocytosis Phagocytosis (%) Days relative to calving Phagocytosis % Phagocytosis MFI

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130 Figure 3 28 Least squares means for percentage of neutrophils with oxidative burst activity (solid line) and neutrophil mean florescence intensity (MFI, indication of intensity of reactive oxygen species produced per neutrophil, dash line) of Holstein cows (n = 35) on 15, 0, 7, 14 d relative to calving. Effect of time ( P < 0.01). 0 200 400 600 800 1000 1200 1400 1600 1800 2000 0 10 20 30 40 50 60 70 80 90 100 -15 -10 -5 0 5 10 15 MFI of Oxidative burst Oxidative burst (%) Days relative to calving oxidative burst % oxidative burst MFI

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131 Figur e 3 29 Least squares means for mean percentage of neutrophils with phagocytic activity of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows hou sed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by parity interaction ( P = 0.03). 0 10 20 30 40 50 60 70 80 90 Primi Cool Primi Hot Multi Cool Multi Hot Phagocytosis (%) Treatment

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132 Figure 3 30 Least squares means for neutrophil mean fluorescence intensity (MFI, indication of number of b acteria phagocytised per neutrophil) of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of environment by parity interaction ( P = 0.0 5). 0 10 20 30 40 50 60 70 80 Primi Cool Primi Hot Multi Cool Multi Hot MFI of phagocytosis Treatment

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133 Figure 3 31 Least squares means for neutrophil mean fluorescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil) of primiparous ( primi, n = 22) and multiparous ( multi, n = 1 3 ) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). Effect of diet by parity interaction ( P = 0.02 ). 0 10 20 30 40 50 60 70 80 Primi Control Primi AO Multi Control Multi AO MFI of phagoctosis Treatment

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134 Figure 3 32 Least squares means for neutrophil mean fluorescence intensity (MFI, indication of number of bacteria phagocytised per neutrophil) of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) on 15, 0, 7, 14 d relative to calving. Effect of diet by parity interaction ( P = 0.0 4). Days relative to calving with two asterisks indicates that means differed for that day using slice command ( P < 0.01), with three asterisks differed ( P < 0.001), with dagger differed ( P < 0.10). 0 20 40 60 80 100 120 -15 -10 -5 0 5 10 15 MFI of phagocytosis Days relative to calving Primi Control Primi AO Multi Control Multi AO *** ** ***

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135 Figure 3 33 Least squares means for mean percentage of neutrophil with oxidative burst activity of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot). Effect of diet by environment interaction ( P = 0.10). 68 70 72 74 76 78 80 82 84 86 Control cool Control hot AO cool AO hot Oxidative burst (%) Treatment

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136 Figure 3 34 Least squares means for percentage of neutrophil with oxidative burst activity of Holstein cows (n = 35) fed diets supplemented without (Control) or with synthetic antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot) on 15, 0, 7, 14 d relative to calving. Effect of diet by environment by time interaction ( P = 0.08). Days relative to calving with dagger indicates that means differed for that day using slice command ( P < 0.10). 65 70 75 80 85 90 95 -15 -10 -5 0 5 10 15 Oxidative burst (%) Days relative to calving Control Cool Control Hot AO Cool AO Hot

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137 Figure 3 35 Least squares means for IgG response against ovalbumin of Holstein cows (n = 35) fed diets supplemented without (Control) or with syntheti c antioxidants (AO) and housed in shaded freestalls equipped with fans and sprinklers (Cool) or just shade (Hot) on 4, 2, 0, 1, 2, 3, 4, and 7 wk relative to calving. Arrows on the top of week indicate the week of ovalbumin injection. Effect of diet by e nvironment by week interaction ( P = 0.69). 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -4 -3 -2 -1 0 1 2 3 4 5 6 7 IgG (optical density) Week relative to calving Control Cool Control Hot AO Cool AO Hot

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138 Figure 3 36 Least squares means for IgG response against ovalbumin of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). Effect of diet by parity interaction ( P = 0.05). Week with two asterisks indicates that means differed for that week ( P < 0.01) using slice command. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 -4 -3 -2 -1 0 1 2 3 4 5 6 7 IgG (optical density) Week relative to calving Prim-Control Prim-AO Multi-Control Multi-AO **

PAGE 139

139 Figure 3 37 Least squares means for concentrations of acid soluble protein (ASP) of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fa ns and sprinklers or just shade. Parity by DIM interaction ( P < 0.001). Days in milk with asterisks indicates that means differed for that day using slice command ( P < 0.05). 0 10 20 30 40 50 60 70 80 90 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 ASP (ug/mL) DIM Primiparous Multiparous

PAGE 140

140 Figure 3 38 Least squares means for concentrations of acid soluble protein of Holstein cows (n = 35) diagnosed as healthy (n = 14) or unhealthy (metritis, mastitis, or retainted fetal membranes, n = 21). Treatment by DIM interaction ( P = 0.04). Days in milk with one asterisk indicates that means differed for that day using slice command ( P < 0.05), with two asterisks differed ( P < 0.001). 0 10 20 30 40 50 60 70 80 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 ASP (ug/mL) DIM Healthy Unhealthy

PAGE 141

141 Figure 3 39 Least squares means for concentrations of acid soluble protein (ASP) of Holstein cows (n = 35) fed diets supplemented without (Control) or with synthetic antioxidants (AO ). Diet by days interaction ( P = 0.03). Days in milk with dagger indicates that means differed for that day using slice command ( P < 0.10), with one asterisk differed ( P < 0.05), with two asterisks differed (P < 0.01). 0 10 20 30 40 50 60 70 80 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 ASP (ug/mL) DIM control AO **

PAGE 142

142 Figure 3 40 Least squares means for concentrations of haptoglobin of primiparous (n = 22) and multiparous (n = 13) Holstein cows fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fans and sprinklers or just shade. Parity by DIM interaction (P = 0.09). Days in milk with asterisk indicates that means differed for that day using slice command (P < 0.05), with dagger differed (P < 0.10). 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Hp (optical Density) DIM Primiparous Multiparous

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143 Figure 3 41 Least squares means for concentrations of ha ptoglobin of Holstein cows (n = 35) diagnosed as healthy (n = 14) or unhealthy (metritis, mastitis, or retainted fetal membranes, n = 21). Treatment by DIM interaction ( P = 0.03). Days in milk with one asterisk indicates that means differed for that day us ing slice command ( P < 0.05), with three asterisks differed ( P < 0.001), with dagger differed ( P < 0.10). 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Hp (optical Density) DIM Healthy Unhealthy ***

PAGE 144

144 Figure 3 42 Least squares means for concentrations of haptoglobin of Holstein cows (n = 35) fed diets supplemented with or without dietary antioxidant (Agrado Plus) and housed in shaded freestalls equipped with fans and sprinklers (cool) or just shade (hot). Environment by DIM interaction ( P = 0.03). Days in milk with dagger indicates that means differed for that day using slice comma nd ( P < 0.10). 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Hp (optical Density) DIM Cool Hot

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145 Table 3 8 Profile of plasma progesterone of postpartum dairy cows fed with or without synthetic antioxidants (AO) and cooled or noncooled during the prepartum period during the summer season in Florida. Treatment Measure Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous First ovulation, DIM 2 21.6 3.8 12.0 8.6 18.7 3.5 21.0 6.1 24.0 3.8 17.0 3.8 23.0 3.5 20.5 6.1 Length of first cycle, d 12.2 2.2 17.0 5.0 15.2 2.0 23.0 3.5 19.5 2.5 20.8 2.2 13.5 2.0 16.5 3.5 Peak progesterone in first cycle, ng/mL 5.0 1.1 10.5 2.5 6.9 1.0 10.6 1.8 5.6 1.3 5.5 1.1 5.7 1.0 6.0 1.8 Number of ovulations 2.0 0.3 1.5 0.5 2.0 0.3 1.5 0.5 1.6 0.3 1.6 0.3 2.0 0.3 0.7 0.4 Accumulated progesterone from 1 to 49 DIM, ng/ml 26.1 8.0 32.0 12.6 35.1 7.3 51.4 12.6 26.8 8.0 41.3 8.0 33.0 7.3 19.0 8.9 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO). 2 Based on 32 of 35 cows ovulating in the first 49 DIM.

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146 Table 3 8. Continuned P values Measure Diet, Control vs. AO Cool vs. noncool Diet by cooling Parity (P) Diet by P Cooling by P Diet by cooling by P First ovulation 0.45 0.57 0.81 0.26 0.88 0.27 0.62 Length of first cycle, d 0.74 0.88 0.04 0.06 0.35 0.59 0.88 Peak progesterone in first cycle, ng/mL 0.03 0.54 0.75 0.04 0.05 0.74 0.60 Number of ovulations 0.33 0.68 0.68 0.04 0.81 0.25 0.25 Accumulated progesterone from 1 to 49 DIM, ng/ml 0.36 0.64 0.10 0.40 0.42 0.50 0.15

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147 Figure 3 43 Least squares means for mean peak concentration of progesterone (P4) of the first cycle of primiparous (primi, n = 22) and multiparous (multi, n = 13) Holstein cows fed diets supplemented without (Control) or with synthetic antioxidants (AO). Effect of diet by parity interaction ( P = 0.05). 0 2 4 6 8 10 12 14 Primi Control Primi AO Multi Control Multi AO Peak P4 (ng/mL) Treatment

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148 T able 3 9 Incidence of postpartum health disorders of dairy cows fed with or without synthetic antioxidants (AO) and cooled or noncooled during the prepartum period during the summer season in Florida. Measure Control diet AO diet 1 Cooled Non cooled Cooled Non cooled Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Primiparous Multiparous Number of animals 5 2 6 2 5 5 6 4 Endom etritis 1 2 3 2 3 2 Retained fetal membranes 1 1 Mastitis 2 1 1 2 1 Ketosis 2 2 4 1 3 3 3 4 Displaced abomasum 1 1 1 Milk fever 1 1 Agrado Plus (Novus Internat. Inc., St. Charles, MO).

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162 BIOGRAPHICAL SKETCH Dan Wang was born in Beijing, China on 1983. She is the daughter of Ruilin Wang and Jinhua Fe i. Dan Wang started her Bachelor of Science degree at Capital Normal University in 2002 and graduated in 2006. Later sh e pursued her Master of Science major ing in microbiology at the same university. But one year later, she mar ried with Zheng (Alex) Fu and moved to Gainesville with her husband in 2007. Dan Wang did a volunteering work in the Department of Animal Sciences in University of Florida from March to June in 2008. Then she started her Master of Science at University of Florida, Department of Animal Science, under the guidance of Dr. Charles R. Staples. Her research focused on the oxidative status of dairy cows during the transition period. After graduation, she is going to stay on her Ph.D. under Dr. Charles R. Staples. Dan Wang has a lovely daughter named Eunice and she really enjoys the life with this baby girl.