Strategies for improving reproduction and milk yield of dairy cows through use of antioxidants and timed insemination


Material Information

Strategies for improving reproduction and milk yield of dairy cows through use of antioxidants and timed insemination
Physical Description:
xxiv, 166 leaves : ill. ; 29 cm.
Arechiga Flores, Carlos Fernando, 1967-
Publication Date:


Subjects / Keywords:
Dairy cattle -- Physiology   ( lcsh )
Milk yield   ( lcsh )
Dairy cattle -- Reproduction   ( lcsh )
Animal Science thesis, Ph.D   ( lcsh )
Dissertations, Academic -- Animal Science -- UF   ( lcsh )
bibliography   ( marcgt )
non-fiction   ( marcgt )


Thesis (Ph.D.)--University of Florida, 1997.
Includes bibliographical references (leaves 134-164).
General Note:
General Note:
Statement of Responsibility:
by Carlos Fernando Aréchiga Flores.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 028620579
oclc - 38855434
System ID:

Table of Contents
    Title Page
        Page i
        Page ii
        Page iii
        Page iv
        Page v
        Page vi
        Page vii
        Page viii
        Page ix
        Page x
        Page xi
    Table of Contents
        Page xii
        Page xiii
        Page xiv
        Page xv
    List of Tables
        Page xvi
        Page xvii
    List of Figures
        Page xviii
        Page xix
        Page xx
        Page xxi
        Page xxii
        Page xxiii
        Page xxiv
    Chapter 1. Foreword
        Page 1
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    Chapter 2. Literature review
        Page 5
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    Chapter 3. Effect of injection of B -carotene or vitamin E. and selenium on fertility of lactating dairy cows
        Page 61
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    Chapter 4. Effects of timed insemination and supplemental B-carotene on reproduction and milk yield of heat-stressed dairy cows
        Page 76
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    Chapter 5. Response of preimplantation murine embryos to heat shock as modified by developmental stage and glutathione status
        Page 107
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    Chapter 6. General discussion
        Page 125
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    Appendix A. Timed AI implementation at a commercial dairy
        Page 131
    Appendix B. Effect of media preincubated at elevated temperature on development of two-cell embryos and morulae
        Page 132
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    List of references
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    Biographical sketch
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Full Text







Copyright 1997


Carlos Fernando Ardchiga Flores

This professional achievement is not the result of personal effort alone, but

rather also reflects the effort, sacrifice and guidance of many people. This thesis is

dedicated especially

To my parents: Fernando Ar6chiga Flores and Zoila Flores Sandoval

for all their effort, dedication, sacrifice, and concern for family unity.

They nurtured a family proud of their origins and roots; a family that

demonstrated by their conduct and guidance the best means for education and a family

that taught that work with perseverance is the best tool to accomplish one's goals.

To my wife and daughter: Delia and Ailed Maciel

They are the shining light God brought to me to see the end of the tunnel ...

To my brothers: Evelyn, Victor Edgardo, and Jorge Alberto
I have faith in future life, in human improvement, in the utility of a virtue, and, in you.
(Jos6 Marti, Cuban poet).

To Grandma Domi: for her efforts on teaching her children and for

passing on her interest and curiosity for Science.

- To my adoptive families:

families Flores Flores, Valenzuela Flores, Garcds

Torres, RomAn Pr6spero, and Snchez Moreno.

- To my professors, friends and relatives for their trust and support.

- To Mexico ...

"Mexico, I give to you ofyour joy the key:

be always the same, loyal to your self image ".

(Ram6n L6pez Velarde, poeta Zacatecano).

--- especially to Zacatecas ... "Cruel skies and a reddish soil..."

where .... "Work can conquest everything".

---- and to my unforgettable "Caxcdn paradise" with its guava scent ... JALPA,

- To Florida ...

The sunchine state.

Carlos F. Ar6chiga
Fall, 1997
Gainesville, Florida

7en cuidad dce &4 cosau de ea T a;

0A~e4 Ceaa,
t46~Z4 C wc4aW
&NA foetdd4.

PCt omem,
quwe c4w,

eder, 4 co&", sd

Conae sodards a coocer.

(Poesfa del Mexico Prehispdnico).

Este logro en mi vida profesional no representa solamente un esfuerzo personal,
sino al contrario, refleja tambidn el esfuerzo, sacrificio, ejemplo y dedicaci6n de muchas
personas. Esta tesis es dedicada de manera muy especial

A mis padres: Fernando Ardchiga Flores y Zoila Flores Sandoval

Por todo su esfuerzo, dedicaci6n, sacrificio, y preocupaci6n por mantener una

familia unida. Unafamilia comprometida con sus origenes y orgullosa de sus raices.

Unafamilia que inculca la conducta y el ejemplo como la mejor manera de educar y una

familia que postula al trabajo como el principio indispensable para lograr sus


A mi esposa e hija: Delia y Ailed Maciel
Ellas son el resplandor que Dios me ha enviado para vislumbrar elfinal de iste tznel...

A mis hermanos: Evelyn, Victor Edgardo y Jorge Alberto
Tengofe en la vidafutura, en el mejoramiento humano
en la utilidad de la virtud y en Uds. ...

(Josd Martf, poeta cubano)

- A mi Mamd Domi:

por su preocupaci6n en la instrucci6n de sus hijos y
en transmitir ese interns y curiosidad por la Ciencia.

- A mis familias adoptivas: familias Flores Flores, Valenzuela Flores, Garcds

Torres, Romdn Pr6spero, y Sdnchez Moreno.

- A mis maestros, familiares y amigos y por su confianza y apoyo.

- A Mexico ...

"Patria, te doy de tu dicha la clave:

se siempre igual, fiel a tu espejo diario ".

(Ram6n L6pez Velarde, poeta Zacatecano).

--- muy en especial a Zacatecas ... "Un cielo cruel y una tierra colorada..."

donde .... "El Trabajo lo Vence Todo ".

y a mi inolvidable terrufio caxctn con olor a guayaba .... JALPA,

- A Florida ...

El estado resplandeciente.

Carlos Fernando Ar6chiga Flores
Otofio de 1997
Gainesville, Florida


Once more, an opportunity is presented to formally demonstrate my gratitude to

people who have been mentors and supporters at the different stages of my life. Even

though we do not forget to express our gratitude verbally, publishing a dissertation is one

of the few opportunities to stamp those feelings on paper. Many thanks are expressed to

my parents, uncles and grandparents and, especially, to my uncles and veterinary

colleagues: Francisco, Gilberto, and Blanca Esthela Flores Sandoval. They are the people

that inspired me, awakened in me the curiosity for science and developed in me the

discipline to work and study with perseverance.

I wish to express my appreciation and most sincere gratitude to Dr. Peter J.

Hansen, chairman of my supervisory committee, for his invaluable assistance, constant

guidance, support and encouragement throughout these years. I have considered myself

very lucky to have him as an advisor. His scientific skills and special qualities have

helped me to develop gradually as a professional and as an individual. His scientific view

and life principles have created a comfortable environment for me to learn while giving

me the strength to withstand the difficult and trying moments of this learning process. His

lessons will be remembered throughout my life and my best effort will be put forward to

apply the principles he taught and to trust in myself. I can proudly say that Dr. Hansen


has not only been a good advisor but also a very good and supportive friend. I'm very

thankful for the kindness and support that he and his family have always showed towards

me and my family.

Appreciation and sincere gratitude are further extended to the members of my

supervisory committee, Drs. William W. Thatcher, Frank A. Simmen, Herbert H. Head,

and Peter J. Chenoweth, as well as other research collaborators Drs. Charles R. Staples,

Lee R. McDowell, Carlos A. Risco, M. Drost, Oscar 0. Ortiz, Sonia Vasquez-Flores, Joel

Hernandez, Antonio Porras, Alan D. Ealy, and Charles J. Wilcox, for their assistance,

support, dedication and friendship.

This feeling is expressed in the same way to all of the members of Dr. Hansen's-

Lab these past 6 years: as well as Wen-jun Liu, Fabiola Paula-Lopes, Alice S. De Moraes,

Jennifer P. Trout, Rocio M. Rivera, and Lannett Edwards for their valuable support,

assistance, friendship and help during field trials, special thanks go to Susan L. Gottshall

and Morgan and Maggie Peltier. Other fellow graduate students and friends deserve

thanks for their support, friendship, companionship and solidarity: Pedro Garc6s Yepez,

Rafael Rom.n, Andres Kowalski, Victor Monterroso, Luzbel de la Sota, Thais Diaz,

Alfredo Garcia, Diego Rochinotti, Juan Villanueva, Juan Velasquez, Elide Valencia,

Yaser Al-Katanini, Mario Binelli, Divakar Ambrose, Javier Rosales, Carlos Vargas, Juan

Valiente, Rey Acosta, Hugo Guillen, Javier y Bertha Sanchez, Leonel Espinoza, Juan

Pablo Mondrag6n, Celina Fernandez, Josd Luis Osomo, Dante Rodriguez and some other

friends at the University of Florida for their cooperation, patience, friendship and daily

support and advice.

The most sincere gratitude and respect is expressed to the members of

MEXICANS in GAINESVILLE who honored me as their founding president. I am

thankful for their trust, friendship, solidarity, encouragement, efforts and daily support.

My best wishes go to each of them. I hope to see them again soon because they have

become part of my family. There are no words to express all my gratitude to my family

and friends from Mexico, California and Florida. Their love, support, encouragement and

faith in me made this new achievement possible.

Sincerely acknowledged are the financial and moral support provided by the

CONACYT/Fulbright/Institute of International Education Program and the International

Center for Cell and Molecular Biology in Mexico, the Dairy Checkoff Program, USDA-

CBAG, Hoffmann-LaRoche and the University of Florida. Special thanks to Dr. Ted L.

Frye, Dr. Jonathan Wilson and Thelma Vicente at Hoffmann-LaRoche for providing [5-

carotene and performing feed analyses, Dr. Michael B. Coelho at BASF for donation of

LUCAROTIN, Dr. Charles R. Staples for help with design and implementation of the

feeding experiment, Dr. J.P. Everett at Purina Mills for preparation of the vitamin

premix, and Dr. Lee R. McDowell for assaying vitamin concentrations for many studies

reported here. Sincere thanks are extended to Dr. W.W. Thatcher for his moral support

and advice, help obtaining reagents, and for technical assistance with progesterone assays.

I also thank Upjohn Co. (Kalamazoo, Mi) for supplying Lutalyse, and Hoechst-Roussel

Agri-Vet (Somerville, NJ) for supplying Receptal. I have deep appreciation for the

cooperation and assistance provided by the owners and personnel of San Sebasti,.n and La

Palma Dairy in Mexico, and North Florida Holsteins, Genfarm III, ALC, Gainesville

Farms Inc., the Dairy Research Unit at the University of Florida, Levy Co. Dairy and

Larson's Dairy, Inc. in Florida. A special mention is made of the help and assistance of

the Larson and Skelton families, Tony Strickland, Ken Marion, Eddy Trejo and Don


I also extend my appreciation for their support and cooperation to the Facultad de

Medicina Veterinaria y Zootecnia of the Universidad Aut6noma de Zacatecas and the

Departamento de Reproducci6n, FMVZ-UNAM. Special gratitude goes to Donato F.

Cortrs, Miguel Castillo Pecina, Antonio Mejia Haro, Fco. Javier Escobar, Daniel

Rodriguez Tenorio, Sergio Mrndez de Lara, Ignacio Dtvila, Luis Zarco, Carlos Galina,

Javier Valencia, Rosa Piramo, Adriana Saharrea, Tere Sanchez (Colegio de

Postgraduados) and fellow graduate students. At the University of Florida, many thanks

are also extended for their help and moral support to Dr. John M. Sivinski at USDA-ARS

and Dr. Fedro Zazueta at IFAS-Information Techniques. My recognition and sincere

thanks are also extended to Maria Cruz at International Programs, and to Dr. Jack L. Fry,

former Assistant Dean of the College of Agriculture, for many years of support toward

international students.


ACKNOW LEDGMENTS ............................................... viii

LIST OF TABLES ..................................................... xvi

LIST OF FIGURES ................................................... xviii

ABBREVIATIONS .................................................... xix

A B STRA CT ......................................................... xxii


1 FO REW O RD ..................................................... 1

2 LITERATURE REVIEW ........................................... 5
Introduction ................. ............................... 5
The Bovine Estrous Cycle and Follicular Function ........................ 5
General Characteristics ....................................... 5
Follicular W aves ............................................ 7
Approaches to Regulate the Estrous Cycle through Manipulation of Follicular
Function ................................................... 9
Progesterone-Releasing Devices ................................ 9
GnRH and GnRH-Analogs ................................... 13
Synchronization of Ovulation ................................. 14
Fertility to Synchronized Ovulation ............................ 18
Effects of Heat Stress on Reproductive Function of Cattle ................. 22
Puberty ................................................... 22
Estrus Detection ............................................ 23
Length of Estrous Cycle ...................................... 24
Conception Rate ............................................ 26
Late Pregnancy ............................................. 29
Postpartum Anovulatory Period ................................ 30


Mechanisms by which Heat Stress Reduces Conception Rates in Cattle ...... 30
Detrimental Effects of Elevated Temperature on Sperm ............. 31
Detrimental Effects of Elevated Temperatures on Oocytes ........... 33
Detrimental Effects of Elevated Temperatures on Preimplantation
Em bryos .................................................. 35
Detrimental Effects of Heat Stress on Luteal Function .............. 37
Effects of Elevated Temperatures on Oviduct and Uterus ........... 39
Mechanisms to Protect Cells from Elevated Temperatures ................. 40
Protective Effects of Heat Shock Proteins ........................ 40
Control of Gene Expression ................................... 43
Ontogeny of Heat Shock Protein Synthesis on the Embryo ........... 45
Ontogeny of Thermotolerance in the Early Embryo ................ 46
Heat Shock and Free Radicals ................................. 48
Evidence That Free Radicals are Involved in Effects of Heat on
Em bryos ........................................... 51
Protective Effects of Antioxidants .............................. 52
G lutathione ................................................ 53
V itam in E ................................................. 56
1-carotene ................................................ 59

Introduction ..................................................... 61
M aterials and M ethods ............................................. 63
Plasma Concentrations of P-Carotene in Cows Injected with
P-Carotene .......................................... 63
Effect of P-Carotene on Fertility of Lactating Dairy Cows .......... 64
Effect of Injection of Vitamin E and Selenium on Reproductive
Function ............................................ 65
Statistical Analysis .......................................... 66
R esults ......................................................... 67
Plasma Concentrations of P-Carotene in Cows Injected with 1-Carotene
........ ........... .. .. .. .... .. .. ....... ..... .. ....6 7
Effect of P-Carotene on Fertility of Lactating Dairy Cows ........... 69
Effect of Injection of Vitamin E and Selenium on Reproductive
Function ............................................ 69
D iscussion ...................................................... 72

STRESSED DAIRY COWS ........................................ 76
Introduction ..................................................... 76
M aterial and M ethods ............................................. 78

Farms, Cows and Total Mixed Rations .......................... 78
P-Carotene Supplementation .................................. 81
Reproductive Management ................................... 81
Blood Collection and Analysis ................................ 82
Body Condition Scores ...................................... 83
Statistical Analyses ......................................... 83
R esults ......................................................... 85
Effects of TA I ............................................. 85
Effects of P-Carotene ........................................ 90
D iscussion ...................................................... 99
Conclusions .................................................... 106

GLUTATHIONE STATUS ........................................ 107
Introduction .................................................... 107
M aterials and M ethods ............................................ 110
M aterials ................................................ 110
Superovulation, Embryo Collection and Culture .................. 110
Effect of Developmental Stage on Resistance to Heat Shock ........ 111
Induced Thermotolerance in 2-cell and Morula-Stage Embryos ...... 111
Effect of BSO on Response of 2-cell Embryos and Morulae to Heat
Shock ........................................112
Statistical Analysis ......................................... 113
R esults ........................................................ 114
Effect of Heat Shock on 2-cell, 4-cell and Morula-Stage Embryos... 114
Induced Thermotolerance in 2-cell and Morula-Stage Embryos ...... 114
Effect of BSO on Responses of 2-cell Embryos and Morulae to Heat
Shock ............................................. 116
D iscussion ..................................................... 118

6 GENERAL DISCUSSION ......................................... 125



Introduction .................................................... 132
M aterials and M ethods ............................................ 132

Results ................................................... 133

LIST OF REFERENCES ................................................ 134

BIOGRAPHICAL SKETCH ............................................. 165


Table PAU

2-1. Effect of timed Al on pregnancy rates of lactating dairy cows and heifers ...... 17

2-2. Effects of elevated temperatures on duration of estrus in cows ................ 23

2-3. Effect of maternal heat stress at different stages of pregnancy on embryonic
survival of preimplantation embryos from different species ............... 36

2-4. Effects of strategic cooling around on pregnancy rates of cows in hot climates .. 38

3-1. Effect of administration of [-carotene on estrous synchronization, interval to
estrus, variation of interval to estrus and pregnancy rate ................... 69

3-2. Effect of postpartum administration of Vitamin E and selenium on reproductive
function of lactating dairy cows ...................................... 70

4-1. Ingredient composition of experimental diets ............................. 80

4-2. Effect of a timed insemination protocol on reproductive function of lactating
H olsteins ...................................................... 86

4-3. Effect of feeding supplemental P-carotene on reproductive function of lactating
H olsteins ....................................................... 93

4-4. Effect of feeding supplemental P-carotene for at least 90 d on reproductive
function of lactating Holsteins ...................................... 95

4-5. Interactions between feeding of supplemental P-carotene and a timed
insemination program on reproductive function of lactating Holsteins ....... 97

4-6. Effect of feeding supplemental P-carotene on milk yields of lactating Holsteins 98

4-7. Effect of supplemental P-carotene feeding period on milk yields of lactating
H olsteins .................................................. 100

5-1. Induced thermotolerance in 2-cell embryos and morulae ................... 117

5-2. Effect of BSO and heat shock of 41 C/I h on development of murine 2-cell
em bryos and m orulae ............................................. 119

5-3. Effect of BSO and heat shock and stages of development on percent
development to blastocyst of murine embryos evaluated at 72 and 96 h after
detection of vaginal plugs in dams ................................... 122

B-1. Percent development of murine embryos to the blastocyst stage in heat-shock
pretreated M16 and KSOM media at 72 and 96 h after detection of vaginal
plugs .......................................................... 133



1-1. Seasonal variation in pregnancy rate on a commercial dairy in South Florida in
which cows were exposed to environmental modifications through the use of
shade, fans, and sprinklers .......................................... 4

3-1. Plasma concentrations of n-carotene, retinyl palmitate and retinol from heat-
stressed dairy cows injected, i.m., with 800 mg n-carotene or saline at days 0,
3 an d 6 ........................................................ 68

3-2. Frequency distribution of services per conception for control cows and cows
treated with vitamin E and selenium .................................. 71

4-1. Frequency distribution of interval from calving to first service (days) of
lactating Holstein cows bred at observed estrus or subjected to timed-artificial
insemination for first breeding ....................................... 88

4-2. Cumulative pregnancy rate of lactating Holstein cows bred at observed estrus or
subjected to timed-artificial insemination for first breeding pregnancy rate .... 89

4-3. Changes in plasma concentrations of vitamins as affected by supplemental
feeding of n-carotene ............................................. 91

5-1. Development of murine embryos exposed to 37 C continuously or a heat shock
of 41 C for 1 h or 41 C for 2 h at the indicated stage of development ........ 115



ACTH Adrenocorticotropic hormone
ADF Acid detergent fiber
Al Artificial insemination
ATP Adenosine triphosphate
ATPases Adenosine triphosphatases
BOE Breeding at observed estrus
BSA Bovine serum albumin
BSO D,L-buthionine-[S,R]-sulfoximine
BST Bovine somatotropin
cDNA DNA complementary to RNA
CL Corpus luteum
CP Crude protein
CR Conception rate
DAPI 4,6-Diamidino-2-phenylindole
DM Dry matter
DMEM Dulbecco's Modified Eagle's Medium
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
D-PBS Dulbecco's Phosphate Buffered Saline
EB Estradiol benzoate
EDR Estrus detection rate
EDTA Ethylenediaminetetraacetic acid
eIF-2a Eukaryotic initiation factor-2a
FCS Fetal calf serum
FSH Follicle-stimulating hormone
GnRH Gonadotropin releasing hormone
GnRH-a Gonadotropin releasing hormone-analog
GSH Reduced glutathione
GSHPx Glutathione peroxidase
GSH-tx Glutathione-S-transferase
GSSG Oxidized glutathione
hCG human chorionic gonadotropin
HSC Heat shock constitutive (Cognate)
HSE Heat shock element
HSP Heat shock protein


Heat-treated fetal calf serum
Insemination at detected estrus
In vitro fertilization
Luteinizing hormone
Least square means
Least square means
Messenger ribonucleic acid
Molecular weight
Milk yield
Cumulative milk yield at last test
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate reducing equivalents
Neutral detergent fiber
Net energy for lactation
Superoxide anion radical
Hydroxyl radical
Predicted milk yield at 305 d of lactation
Prostaglandin F-2 alpha
13,14-dihydro- 15-keto PGF2,
Pregnant mare serum gonadotropin
Pregnancy rate
Polyunsaturated fatty acids
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Standard error of the mean
Superoxide dismutase
Manganese superoxide dismutase
Copper/Zinc superoxide dismutase
Disulfide bond
Timed artificial insemination
Timed insemination
Total mixed ration


IU International unit
kg Kilogram
g Gram
Mcal Megacalories
mg Milligram
119 Microgram
1 Liter
ml Milliliter
P1l Microliter
M Molar
mM Millimolar
giM Micromolar
nM Nanomolar
mo Month
wk Week
d Day
h Hour
min Minutes
kDa Kilodaltons

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



Carlos Fernando Ar6chiga Flores

December, 1997

Chairperson: Peter J. Hansen
Major Department: Animal Science

Herd pregnancy rate, that is the product of estrus detection rate and pregnancy rate

per insemination, is reduced during heat stress. Environmental modification through the

use of shade, fans or evaporative cooling has not completely eliminated the negative

consequences of heat stress. Therefore, other strategies for increasing pregnancy rates in

heat-stressed cows offer the possibility for further improvements in reproductive function.

The goal of this dissertation was to develop strategies to increase herd pregnancy rates of

dairy cattle by influencing both estrous detection rate and pregnancy rate per


First, it was tested whether implementation of a timed artificial insemination

program would increase pregnancy rates at a fixed time postpartum by reducing losses


associated with a low estrous detection rate during heat stress. Three experiments tested

the efficacy of timed insemination during the summer/fall (exp. 1 and 2) and

winter/spring (exp. 3). Cows were inseminated at each estrus after d 70 (exp. 1) or 50

(exp. 2 and 3) or included in the timed insemination program [d 0 (i.e., 40 or 60 d

postpartum), 8 ptg Buserelin (a GnRH agonist) i.m.; d 7, 25 mg PGF2. i.m.; d 9, 8 p.g

Buserelin i.m., d 10, Al] for first breeding followed by insemination at detected estrus for

subsequent breedings. Pregnancy rates at first insemination were similar between groups.

At 90 d postpartum, pregnancy rates were greater for the timed insemination group in

Exp. 1 (16.6 vs 9.8%) and 2 (34.3 vs 14.3%) but not for exp. 3 (24.1 vs 28.7%).

Pregnancy rate at 120 d PP was greater only for exp. 2 (62.9 vs 37.1%).

Other experiments evaluated whether pregnancy rates could be improved by

reducing free radical levels in embryonic and maternal cells. Two series of experiments

evaluated the effectiveness of administration of P-carotene during heat stress. Cows

receiving supplemental 3-carotene through feeding (400 mg/cow/d) for at least 90 d

during warm months had higher pregnancy rates at 120 days postpartum than control

cows (35.4% vs 21.1%). Feeding supplemental P-carotene was not beneficial in an

experiment performed during the cool time of the year, or in an experiment during heat

stress in which supplemental feeding was for less than 120 days. In all three experiments,

cows fed supplemental P-carotene had higher milk yields than controls. Injection of P-

carotene at -6, -3 and 0 days before insemination did not improve pregnancy rates in

heat-stressed cows. Thus, long-term, but not short-term, administration of P-carotene may

improve reproductive function in summer.


Efficacy of another antioxidant treatment, injection of vitamin E (500 mg) and

selenium (50 mg) at 30 d postpartum, in improving reproduction was evaluated in non

heat-stressed cows. Vitamin E/selenium did not affect the interval from calving to first

breeding or the proportion of cows pregnant at first service, but increased the pregnancy

rate at second service (69.8 vs 52.1%) and reduced services per conception (1.7 vs 2.0)

and interval from calving to conception (84.6 vs 98.1%). Thus injection of vitamin E and

selenium increased fertility in cattle not becoming pregnant at first insemination.

The mouse was used as a model in several experiments to determine differences

in thermotolerance associated with embryo developmental stage and to test whether

intracellular amounts of the antioxidant glutathione were related to embryonic resistance

to heat shock. Embryos were heat shocked (41 C for 1 or 2 h) at the early 2-cell (24-27 h

after detection of vaginal plugs), late 2-cell and 4-cell (28-32 h), and morula stages. Late

2-cell embryos were the most affected by heat shock. Since these embryos are retarded in

development, these findings raise the possibility that maternal heat stress is most likely to

compromise development of retarded embryos. Inhibition of synthesis of the intracellular

antioxidant glutathione affected the proportion of embryos developing to the hatched

blastocyst stage but did not make embryos more sensitive to heat shock, suggesting that

synthesis of glutathione is not important for resistance to heat shock under the conditions


Overall, results obtained suggest that implementation of timed artificial

insemination and appropriate antioxidant treatments may improve the herd pregnancy rate

in heat-stressed dairy cows.



Exposure to hot and humid environments has long been associated with disruption

of reproductive function in dairy cows. Heat stress can disrupt reproduction by altering

occurrence and timing of ovulation, intensity of estrous activity, viability of gametes,

embryo survival, and fetal development. As a consequence, herd pregnancy rates, that is,

the product of estrus detection rate and pregnancy rate per insemination, are reduced

drastically during heat stress. The magnitude of heat stress effects is large. For example,

in a large dairy herd in Florida, the estimated frequency of missed estruses was

calculated as 80% during the hot and humid months of June to September versus 66% for

the months of October to May (Thatcher and Collier, 1982; 1986; Thatcher et al., 1986).

Similarly, conception rates in Florida were reduced from 40 to 50% during the cool

months of the year to less than 10 to 20% during the hot months of the year (Badinga et

al., 1985).

The major strategy for reducing effects of heat stress on reproduction has been to

alter the environment of the cow through the use of shade, fans or evaporative cooling

(Bucklin et al., 1991). Although somewhat effective, this approach has not completely

eliminated the negative consequences of heat stress. Recent data on conception rates of

dairy cows obtained from a commercial dairy farm in South Florida illustrate the problem

(Fig. 1-1). Despite the fact that housing facilities were equipped with fans, sprinklers,

and were constructed to maximize ventilation, pregnancy rates in the summer were as low

as 10-15 %. Therefore, cooling cows is not a completely effective method for improving

reproductive function during periods of heat stress, and development of other strategies

for increasing pregnancy rates in heat-stressed cows offers the possibility for further

improvements in reproductive function.

Cattle and other homeotherms establish physiological priorities to maintain

critical functions such as body temperature. As a result, other physiological functions are

compromised. For example, one homeokinetic mechanism to prevent hyperthermia in

animals exposed to environmental stress is to reduce feed consumption (Knapp and

Grummer, 1991). As a consequence, there is also a reduction in rumen motility (Attebery

and Johnson, 1969) and decreased growth rate (Collier et al., 1982b). Additionally, failure

or insufficient ability to regulate body temperature also can have negative consequences,

especially for reproduction (Fallon, 1962) because elevated temperature causes disruption

of spermatozoa (Howarth, 1969; Howarth et al., 1964; 1965; Ulberg and Burfening, 1967;

Monterroso et al., 1995), oocytes (Woody and Ulberg, 1964; Alliston et al., 1965;

Baumgartner and Chrisman, 1987; Edwards and Hansen, 1996; 1997) and

preimplantation embryos (Alliston et al., 1965; Edwards et al., 1997). As more is learned

about the biochemical mechanisms through which heat stress compromises embryonic

development, new methods for prevention of embryonic mortality may result. For

example, free radicals in embryonic cells may be elevated in response to high

temperatures; if so, provision of antioxidants might improve embryonic survival.


As mentioned before, overall herd pregnancy rate is a product of estrous detection

rate and pregnancy rate per insemination. The goal of this dissertation was to develop

methods to increase herd pregnancy rates of dairy cattle by influencing both estrous

detection rate and pregnancy rates per insemination. First, it was tested whether

implementation of a timed artificial insemination program (Pursley et al., 1995; 1997a,

1997b; Burke et al., 1996; Schmitt et al., 1996c; Stevenson et al., 1996) to eliminate the

detection of estrus would increase pregnancy rates at fixed breeding time postpartum by

reducing losses associated with low estrous detection rate. This innovative protocol holds

promise one to revolutionize reproductive management in all environments by

eliminating the need for estrous detection. It may be especially valuable in environments

where environmental stress obscures estrous behavior and exacerbates the frequency of

missed estrus. The remaining experiments with cattle evaluated whether pregnancy rates

could be improved by reducing free radical levels in embryonic and maternal cells. The

main goal was to evaluate the effectiveness of administration of P-carotene during heat

stress. Additionally, another antioxidant treatment, vitamin E and selenium, was also

evaluated in non heat-stressed cows. The mouse was used as a model in several

experiments. One goal was to determine whether intracellular amounts of one

antioxidant, glutathione, were related to embryonic resistance to heat shock. An

additional goal was to determine differences in thermotolerance associated with

developmental stage. This latter experiment was viewed as the first step in identifying

the developmental processes by which the embryo acquires biochemical protection from

elevated temperatures.

35 -

30 -


(U 15
10 -


1994 1995 1996

Figure 1-1. Seasonal variation in pregnancy rate (percentage of inseminations in which
pregnancy was established) on a commercial dairy in South Florida in which cows were
exposed to environmental modifications through the use of shade, fans, and sprinklers.
(reproduced from Hansen et al., 1997).



The following sections are presented to give an overview of the bovine estrous

cycle, especially in relation to ovulation control, and to review current knowledge of the

magnitude and range of deleterious effects of heat stress on various aspects of

reproduction. The focus will be on cattle. An additional goal is to describe the

physiological and cellular basis for the effects of heat stress on embryonic survival and to

discuss tentative strategies for reducing the detrimental effects of heat stress on the

reproductive function of cows.

The Bovine Estrous Cycle and Follicular Function

General Characteristics

Estrus is known as the period of sexual receptivity during which mating and

ovulation occur. The estrous cycle begins at puberty and is considered as the interval

between two estrous periods when the female is not pregnant. The average length of the


estrous cycle is considered to be about 20 days in dairy heifers and 21 days in dairy cows

(Asdell, 1946). It is commonly accepted that the cow estrous cycle ranges within 21 + 4d

(Salisbury and Vandemark, 1961). In fact, about 60 % of the estrous cycles fell into the

range of 17-24 d in one study (Chapman and Casida, 1937). Approximately 16% of the

cows showed cycles shorter or longer than 18-24 days in another study (Asdell et al.,

1949), and 30 % of cows had estrous cycles shorter or greater than 17-25 d in a third

study (Ellemberger and Lohmann, 1946).

Hammond (1927) reported an average duration of estrus of 19.3 h in cows and

16.1 h in heifers, within a wide range of 6-30 h. Recently, it was reported by Walker et

al. (1996) that estrus of lactating cows averaged only 9.5 h (with an average of 10.1

mounts during estrus). Duration of estrus was similar for estrus induced with PGF2. or

occurring spontaneously. The apparent reduction in duration of estrus compared with

early studies might be associated with the increased lactational output of modem dairy


In a recent study (Walker et al., 1996), the mean ovulation time relative to the

first mount of estrus in dairy cattle was determined to be 27.6 + 5.4 h with no differences

between spontaneous or induced estruses. This value agrees closely with text book

values of ovulation occurring within 24-30 h after the onset of estrus, that is, 10-11 h after

end of estrus (Levasseur and Thibault, 1980).

Follicular Waves

The estrous cycle of cattle is characterized by the occurrence of two or three

follicular waves as determined by transrectal ultrasonographic imaging (Pierson and

Ginther, 1984; Ginther et al. 1989a; 1989c). Each of these waves is associated with the

processes of follicular recruitment, selection and dominance (Thatcher et al., 1996).

Recruitment is a process in which a pool of antral follicles start to grow under the

influence of FSH and LH to allow development for a potential ovulation. Selection is a

process in which a unique follicle avoids atresia and continues growth. Dominance is the

process by which the selected follicle inhibits recruitment of a new pool of follicles

(Thatcher et al., 1996).

The first follicular wave begins around the day of ovulation when a wave of

follicles of 4-5 mm in diameter emerges from the pool of small follicles to initiate

growth. This process of recruitment is followed by selection whereby a single follicle

from the cohort of recruited follicles undergoes enhanced growth. The selected follicle (2

mm greater than largest subordinated follicle) exerts dominance over the other

subordinate follicles, so that these follicles undergo atresia. The selected or dominant

follicle grows to a size of 10-15 mm in diameter between d 8-10 of the estrous cycle;

afterward the follicle begins to regress (Ginther et al., 1989a). Subordinate follicles can

become as large as 7-9 mm of diameter but undergo regression because the selected

follicle exerts its dominance through inhibition of recruitment of additional follicles for

the following wave (Adams et al., 1992a; 1992b; Savio et al., 1993a).

Subsequently, there is regression of the first dominant follicle and initiation of a

second follicular wave and growth of the second dominant follicle. The emergence of

these follicular waves during the recruitment phase has been associated with surges in

FSH concentrations (Turzillo and Fortune, 1990; Adams et al., 1992b; Sunderland et al.,

1994; Gong et al., 1995; Bodensteiner et al., 1996). Maturation of the second dominant

follicle generally is associated with regression of the CL and luteolysis on approximately

d 18. This follicle for a two-wave cycle, then grows to ovulatory size until the day of

estrus (d 0) and ovulation on day 1 of the following estrous cycle (Matton et al., 1981;

Walker et al., 1996).

In some cases, the second dominant follicle can undergo atresia, and as a result,

the emergence of a new dominant follicle or a third follicular wave occurs around day 16

of the estrous cycle (Ginther et al., 1989c). Savio et al. (1988) characterized follicular

development in heifers. The first dominant follicle was observed by d 4, with maximum

growth by d 6, a period of stability between d 6-10 and a subsequent decrease in size

until its disappearance around d 15. The second dominant follicle appeared on d 12 with

maximum growth by d 16 (or 19 if there were 2 follicular waves only) and disappearance

by d 19. The third dominant follicle was observed by d 16 (range 10-19) and reached its

maximum size by d 21.

Most cows have estrous cycles with two or three follicular waves (Pierson and

Ginther, 1988; Ginther et al., 1989a,b,c; Knopfet al., 1989; Savio et al., 1988; Sirois and

Fortune, 1988), estrous cycles with four waves of follicle growth have also been reported

(Savio et al., 1988; Sirois and Fortune, 1988). Interestingly, in heifers there is a tendency


for estrous cycles with three waves to predominate (Savio et al., 1988; Sirois and Fortune,

1988; Knopfet al., 1989). For example, Savio et al. (1988) observed that approximately

81% of the heifers had three follicular waves, 15% had 2 follicular waves and 4% had one

single follicular wave. The difference in timing between a 2 vs 3 wave cycles is that for

the latter there is a shorter second follicular wave and a slight prolonged estrous cycle

(Ginther et al., 1989c; Taylor and Rajamahendran, 1991; Fortune, 1994; Savio et al.,


Heat stress alters secretion patterns of LH and FSH during the estrous cycle. LH

concentrations have decreased in cycling cows exposed to heat stress (Madan and

Johnson, 1973) compared to non heat-stressed cows, or remain unaltered (Vaught et al.,

1977; Gwazdauskas et al., 1981). A reduced amplitude of LH pulses by day 5 of the

estrous cycle of dairy cows has been reported, but no difference was seen on day 12 of

the cycle (Wise et al., 1988a; 1988b).

Approaches to Regulate the Estrous Cycle through Manipulation of Follicular Function

Progesterone-Releasing Devices

Implantation of progesterone-releasing devices prolongs lifespan of the dominant

follicle with absence of a CL (Sirois and Fortune, 1990; Savio et al., 1993b). Withdrawal

of progesterone is accompanied by estrus and spontaneous ovulation about 24 to 36 h

upon removal of the progesterone-releasing devices (Tjondronegoro et al., 1987; Sirois

and Fortune, 1990; Savio et al., 1993b; de la Sota et al., 1996; 1997). Thus, some degree

of ovulation or estrus control can be achieved with this approach. The turnover of

ovarian follicles during the estrous cycle is exerted through a mechanism regulated by a

high progesterone environment that permits a low LH pulse frequency and turnover of the

follicle (Savio et al., 1993b). For a low progesterone environment (device but not CL),

the first-wave dominant follicle continues to grow and prolongs its dominance. Perhaps,

this effect occurs through increased secretion of LH that maintains thecal production of

androstenedione for the synthesis of estradiol by the granulosa cells (Thatcher et al.,

1996). Also, progesterone delays ovulation and allows luteolysis to occur without a new

ovulation; when the progesterone is withdrawn, there is no CL and ovulation can occur.

The importance of a low progesterone environment was demonstrated by one study

(Savio et al., 1993b) in which on d 8 of the cycle cows received implants that either were

a 1) new CIDR implant with higher levels of progesterone (1.9 g progesterone) or 2) a

used CIDR with lower levels of progesterone (1.2 g progesterone). Controls received a

new CIDR on d 8 and a PGF2. injection on d 17. All CIDRs were removed on d 17

(Savio et al., 1993b) and 1 of 6, 6 of 6 and 0 of 6 cows maintained the first dominant

follicle and ovulated spontaneously upon removal of the CIDR for the new, used and

control CIDR, respectively. Therefore, low levels of progesterone resulted in persistence

of the dominant follicle and the withdrawal of progesterone by removing the implant is

responsible for ovulation of the persistent follicle (Sirois and Fortune, 1990; Savio et al.,

1990a,b; 1993b; de la Sota et al., 1997). In the study by Savio et al. (1993b), fertility was

impaired temporarily. Pregnancy rates at first Al were lower (37%) for a persistent


dominant follicles compared to a new dominant follicle (65%). Pregnancy rates at second

service (53% vs 50%) were normal.

Several groups have evaluated the low progestin environment scenario as a way to

synchronize ovulation. In one study (Tjondronegoro et al., 1987), lactating Holstein cows

(n1 29, 7-8 wk PP) were exposed to three treatments: 1) a progesterone-releasing

intravaginal device (PRID) for 12 days with a capsule of estradiol benzoate (EB)

attached; 2) a CIDR for 9 days; 3) a CIDR for 12 d. The percentage of cows in estrus

following removal of the progesterone source was 81%, 58%, and 64%, respectively.

Estrus was observed in an average of 24, 32 and 36 h. Thus, the combined use of PRID +

EB increased the percentage of cows showing estrus compared to the CIDRs.

Xu et al. (1996) evaluated the effectiveness of PRID +EB treatment for

synchronizing estrus in dairy cattle. The synchronized group received an intravaginal

progesterone-releasing device containing progesterone (1.9 g) and a capsule of estradiol

benzoate (10 mg) at 10 d before the start of the planned breeding season (d 0). The device

was removed 8 d later (i.e., d -2) and PGF2a was administered 2 d before removal of the

device (i.e., d -4). Cows in the control group remain untreated. A total of 2681 cows

were used. A total of 89% of the cows in the synchronized group were inseminated

during the first 5 d compared to 29.7% of the controls. Cows returning to estrus were

reimplanted with the device during d 16-21 of the breeding season. Conception rates at

first (53 vs 64%) and second (52 vs 63%) insemination were lower in the synchronized

group than in the controls. At the end of the breeding season, there was a higher

percentage of non-pregnant cows in the synchronized group (7 vs 5%) and services per

conception were also higher for this group (2 vs 1.6). The interval from calving to

conception was 9.9 d in the synchronized cows and 21.2 d in the controls. Thus, the

synchronization regime did not increase reproductive efficiency to a large degree, at least

in part because of the reduction in fertility in the synchronized group (Xu et al., 1996).

Other studies also indicate lower pregnancy rates in response to ovulation of the

persistent follicle as compared to ovulation of a new follicle (Savio et al., 1993b;

Wehrman et al., 1993). Poor fertility observed with the use of progestins could result

because bovine oocytes from prolonged dominant follicles undergo premature maturation

in vivo (Revah and Buttler, 1996). Also, the pattern of proteins secreted by the oviduct is

altered for cows having a persistent follicle as compared to the pattern of proteins

secreted from oviducts with a fresh dominant follicle (Binelli et al., 1996).

Administration of melengestrol acetate (MGA) also has been used to produce a

persistent dominant follicle (Yelich et al., 1997). In this study, non-lactating beef cows

received 5 mg/cow/d of MGA from d 0-14. On d 6 and 8, cows received a PGF2

injection to regress the CL. On d 11 of treatment, half of the MGA cows received an

estradiol valerate (EV) injection. A persistent dominant follicle was observed by d 10 of

treatment in 90% of the cows receiving MGA. Cows receiving MGA alone ovulated the

persistent dominant follicle whereas cows receiving the EV injection regressed the

persistent dominant follicle and recruited a new follicle that ovulated. Estrous responses

were 90%, 85% and 64% by 7 d after MGA: 1) control; 2) MGA from d 0 to 14; and 3)

MGA-EV groups. There were no differences in the synchronized conception rate (i.e.,

number pregnant within 7 d after treatment divided by number inseminated).


Synchronized pregnancy rate (i.e., number pregnant within 7 d after treatment divided by

total in group) was higher for controls (61%) than for MGA-EV(36%); the MGA group

was intermediate (49%) and not significantly different from other groups.

Anderson and Day (1994) used another approach to synchronize estrus with

MGA. MGA was fed for 11 d (0- 11), and cows received a PGF2,a injection on d 2 to

regress the CL. On d 9, progesterone (200 mg) was administered to induce turnover of

the persistent follicle. Systemic concentrations of estradiol were reduced and the newly

recruited follicle was ovulated. When heifers received MGA supplementation for 14 d

(i.e., 0-14) and either progesterone or a vehicle on d 12, 95% of cows were detected in

estrus in both groups by 10 d after MGA withdrawal. In addition, synchronized

conception rates (50% vs 17%) and pregnancy rates (94% vs 57%) were greater in the

group receiving progesterone compared to cows receiving the vehicle. This increase in

fertility was only seen for those cows not showing a CL on d 12 when the respective

injections were given. For cows showing a CL on d 12, there were no differences in

synchronized pregnancy rate or pregnancy rate within treatments.

GnRH and GnRH-Analogs

Injection of GnRH or one of its analogs at any stage of the estrous cycle increases

concentrations of LH and FSH in the peripheral circulation within 2-4 h (Chenault et al.,

1990; Rettmer et al., 1992; Stevenson et al., 1993). Such an injection also synchronizes

follicle development (Thatcher et al., 1989; Macmillan and Thatcher, 1991) and interrupts

the occurrence of spontaneous estrus for 6-7 d after treatment (Thatcher et al., 1989;

Twagiramungu et al., 1992b,c; 1995b). Apparently both gonadotropins eliminate the

dominant follicle by inducing its ovulation or luteinization and subsequent formation of a

new CL (Twagiramungu et al., 1992a; 1995a,c). Additionally, the initiation of a new

follicular wave and recruitment of a new dominant follicle occurs within 3-4 d after

GnRH administration (Twagiramungu et al., 1995b). The emergence of a new follicular

wave reduces further growth of the emerging follicles from the previous wave so that they

suffer atresia (Twagiramungu et al., 1995a; 1995b) and the resulting new dominant

follicle will become the ovulatory follicle.

Injection of a GnRH agonist followed 6 or 7 d later by a PGF2,-injection has been

used to synchronize estrus (Thatcher et al., 1989; Twagiramungu et al., 1992a). The

percent of cows showing estrus was similar (88% vs 85%) for the cows receiving

treatment compared to the controls (Twagiramungu et al., 1992a). Pregnancy rate at first

service (85% vs 92%, GnRH vs control) and herd pregnancy rate (74% vs 71%) were

similar to the controls receiving PGF2a (Twagiramungu et al., 1992a).

Wolfenson et al. (1994) synchronized estrus in lactating dairy cows by

administration of a GnRH-agonist prior to injection of PGF2 Cows were injected on d

12 of the estrous cycle (estrus= d 0) with the GnRH-agonist Buserelin (buserelin acetate)

followed by a PGF2.-injection 7 d after (i.e., d 19). Cows in the control group received a

PGF2,-injection on d 12. In the group receiving GnRH agonist 7 of 8 cows ovulated a

dominant follicle by d 15. On the day of estrus, GnRH-treated cows had larger ovulatory

follicles (16 vs 13 mm) and a greater size difference between the ovulatory and the

second largest follicle (11 vs 6 mm) compared to the control group.

Synchronization of Ovulation

Schemes utilizing GnRH and PGF2. to synchronize estrus have been the

forerunners of a more effective means to synchronize reproductive activity settle up the

basis for more recent protocol developed to synchronize ovulations. Recently, several

protocols to synchronize the time of ovulation and eliminate the need for estrous

detection have been developed; one of the first schemes implemented was the use of

progesterone-releasing devices implanted for 11 d with an injection of 5 mg estradiol

benzoate and 50 mg progesterone given at the time of insertion of the implant (Roche,

1975). Furthermore, when an injection of GnRH was given 30 h after removal of the

device, 8 of 12 cows ovulated by 60 h after removal. Subsequently, Peters et al. (1977)

developed a scheme to induce estrus in cattle with a palpable CL. Injection of PGF2. was

followed by an injection of 400 gtg estradiol benzoate 48 h later and insemination at 72 or

96 h after the PGF2.-injection. Because all cows had functional CL, the number of cows

and heifers expressing estrus was increased by 23 and 15 percentage units as compared

to PGF2. alone (for cows 90% vs 67%; for heifers 91 vs 76% estrous response).

Pregnancy rates to fixed timed Al were similar compared with the controls receiving only

a PGF2. injection.

Another protocol for timed insemination was developed by Thatcher and Chenault

(1976). Ovulation was induced by a GnRH injection 24-48 h after synchronization with

PGF2,. Inseminations at a fixed time at 15 h after GnRH resulted in fewer pregnancies

compared to cows receiving PGF2, alone and that were inseminated at detected estrus

(Thatcher and Chenault, 1976).

In another experiment, the effectiveness of a timed Al protocol which depended

upon 2-consecutive injections of PGF2, given 11 d apart and insemination at 77-80 h

after the last injection without any detection of estrus was compared to a similar system

where cows were bred upon detection of estrus (Lauderdale et al., 1981). There were no

differences in pregnancy rates between groups. Harsh environments might compromise

the efficiency of this timed Al protocol. When the two PGF2,-injection program of

Lauderdale et al. (1981) was implemented under heat-stress conditions (Lucy et al.,

1986), pregnancy rates per insemination were reduced from 51% in the control group

(bred at detection of estrus without PGF2.) to 23% in cows receiving 2 PGF2-injections.

However, herd pregnancy rates were similar with 88% for controls compared to 79% for

cows receiving 2 PGF2-injections.

Pursley et al. (1995) tested a novel protocol to synchronize ovulation and allow

fixed-time Al. In this protocol, a first injection of a GnRH-agonist was given at a random

stage of the estrous cycle (d 0), to induce ovulation of a dominant follicle and

synchronization of follicular growth. Seven days later (d 7) an injection of PGF2, was

given to induce luteolysis. Then, a second injection of a GnRH agonist was administered

48 h later (d 9) in cows or 24 h later (d 8) in heifers to induce ovulation. In this study

(Pursley et al., 1995), 18 of 20 cows responded to the first injection of GnRH by

ovulating a dominant follicle with an average diameter of 14.2 mm. A new follicular

TABLE 2-1. Effect of timed Al on pregnancy rates of lactating dairy cows and heifers.
Timed Al Pregnancy Ratea
Animal Controls Treatment
Controls Treated P < Ref1




cows GP-IDEd











heifers GP-IDE

heifers GP-IDE

heifers GP-IDE

heifers P-IDE



GPG-TI 30.5 %(128)

GPG-TI 26.5 % (85)











23 %(154)




48 % (177)

56% (98)

28% (78)

55% (22)

46% (22)

11% (22)


35.3% (85)

38% (156)



29 % (815)

14% (148)

25.8% (89)

45.5% (187)

53% (102)

35% (77)

50 % (14)

aPercent Pregnant=- number of pregnant/number bred and in parenthesis number of cows.
bRefererences: 1) Pursley et al., 1995; 2) Burke et al., 1996; 3) Stevenson et al., 1996; 4) Pursley
et al., 1997a; 5) Pursley et al., 1997b; 6) Vasconcelos et al., 1997; 7) de la Sota et al., 1997; 8)
Schmitt et al., 1996c; 9) Barros et al., 1997.
CGPG-TI=GnRH (d 0), PGF2. (d 7), GnRH (d 9), timed insemination 15-24 h after (d 10).
dGP-IDE=GnRH (d 0), PGF2, (d 7), and insemination at detected estrus.
P-IDE=PGF2 -injection and insemination at detected estrus.
fIDE=insemination at detected estrus.
gDay of PGF2a-injection modified to d 8 or 9 and second GnRH injection given at d 9.
hGPh-TI= GnRH (d 0), PGF2, (d 7), hCG (d 9), timed insemination 15-24 h after (d 10).
'Second GnRH injection at 30-36 h after PGF2z.
JTimed Al at 0 h after GnRH injection.
kLast injection (GnRH or hCG)24 h after PGF2,.
'NS= Non-significant statistically.
mExperiment performed during the summer.

-- 1
-- 1

-- 1

NS' 2

NS 3

-- 4

NS 5

-- 6

-- 6

0.01 7m

0.01 8

NS 8

NS 8

-- 4


wave emerged on average 2.5 d later (i.e., 2-4 d). All cows responded to the PGF2.-

injection given 7 d later, by regressing the CL. The size of the induced CL was less than

the size of the spontaneous ovulated CL (18 mm vs 28 mm, respectively). By the time of

the second GnRH injection (d 9), the average size of the ovulatory follicle was 13.9 mm

reaching an average peak size of 15.7 mm. All synchronized animals ovulated between

24-32 h after the second injection of GnRH.

Fertility to Synchronized Ovulation

Development of a timed artificial insemination (TAI) scheme based on

synchronized ovulation protocols (Pursley et al., 1995) offers several advantages as

proposed in a review by Thatcher et al. (1997). Among these are that TAI i) allows

insemination at a fixed time; ii) eliminates estrous detection; iii) offers an accurate

timing of insemination at the desired voluntary waiting period without compromising the

interval from calving to first service; iv) permits the synchronization of animals to be

utilized for embryo transfer; v) offers the alternative to control the time of first

insemination during the most economically appropriate period of the year and vi) allows

completion of first services before utilization of bovine growth hormone treatments.

Recent advances in ovulation control have made timed Al possible. In the first

report of its kind (Pursley et al., 1995), the TAI protocol included an injection of GnRH

on d 0 followed by injection of PGF2. on d 7, a second GnRH injection on d 9, and

insemination of all cows 15-24 h later (d 10). Ovulation was synchronized within a 6 h

period, from 26-32 h after the second injection of GnRH. However, most cows (12/20)

ovulated 28 h after the second injection of GnRH. The pregnancy rate to TAI in this

study was 55% when cows were bred at 48 h, 46% when bred at 24 h, and 11% when

bred at 0 h after the second injection of GnRH, switching injection of PGF2, at d 7

instead of d 9. Similar pregnancy rates (pregnant cows/total number of cows) for cows

receiving TAI (29%) was similar to cows bred to a detected estrus (30.5%) (Burke et al.,

1996). Even though pregnancy rates were similar, cows treated with TAI receive the first

Al at earlier stages postpartum and during a shorter period of time compared to controls

requiring estrous detection with sporadic use of PGF2a (Pursley et al., 1997a,b) or GnRH

followed by PGF2 7 d later (Burke et al., 1996).

Since the study of Pursley et al. (1995), various studies on use of timed Al have

been reported. Results are summarized in Table 2-1. Because the protocol (Pursley et al.

(1995) results in good synchrony of ovulation, pregnancy rates have generally been

similar to that for cows bred at estrus (Burke et al., 1996; Stevenson et al., 1996; Pursley

et al., 1997b). Nonetheless, because cows receiving TAI are bred more frequently or

earlier postpartum, overall reproductive function has often been improved by TAI. For

example, Pursley et al. (1997b) decreased the median days postpartum (PP) to Al from 83

d in the controls receiving PGF2. to 54 d in the TAI cows for the first synchrony

performed in the study. Median days postpartum to Al for subsequent synchronies were

128 d vs 96 d and 170 d vs 140 d for second and third synchrony, respectively. Similar

results were found by de la Sota et al. (1997) in a summer study, with an interval from

calving to first insemination of 91 d in the controls compared to 78 d in TAI-treated

cows. The interval from calving to conception for cows conceiving by a 120 d PP was

also reduced from 90 d in controls to 78 d in TAI-treated cows. Moreover, fertility per

insemination has been higher, or tended to be so, for TAI cows (Stevenson et al., 1996;

Pursley et al., 1997a; Vasconcelos et al., 1997; de la Sota et al., 1997).

Given effects of heat stress on estrous behavior (Gangwar et al., 1965; Monty and

Wolff, 1974), one might expect that the TAI protocol would be particularly effective

during the summer. de la Sota et al. (1997) observed that while only 18% of control cows

were bred within 91 d PP, this was increased to 100% within 59 d PP when TAI was

initiated by 50 d PP. Also, first service pregnancy rate was increased in the TAI group.

By 120 d PP, pregnancy rate for TAI cows was higher than for controls (27 vs 17%) and

this difference persisted for up to 365 d PP (Thatcher et al., 1997). TAI also reduced

interval from calving to first service (59 vs 91 d), and the interval from calving to

conception for cows pregnant by 120 d PP (78 vs 90 d). Due to its effect of significantly

increasing insemination rates, TAI also increased the number of services per conception

from 1.27 in the controls to 1.63 for the TAI cows. Thus, TAI during the summer

increased pregnancy rate by reducing the adverse effects of heat stress on estrous

detection, even though problems associated with poor fertility still remained.

Effectiveness of TAI in heifers has been controversial. Schmitt et al. (1996c)

compared pregnancy rate per insemination or first service for heifers receiving TAI vs

controls which received GnRH + PGF2. and which were bred at detected estrus. In one

experiment, pregnancy rate per insemination was lower for TAI compared with controls,

whereas in a second study pregnancy rates were similar for the two groups. Pursley et al.

(1997a) suggested that the TAI scheme was not satisfactory for implementation in heifers

because pregnancy rates were lower than for pregnancy rates of heifers allowed to have

three sequential injections of PGF2.. This conclusion was based on misinterpretation of

results. In particular, control cows were exposed to three sequential PGF2,-injections

every 14 d and had three opportunities to get bred compared with a single opportunity to

be bred for the TAI group. When pregnancy rates for the first breeding are evaluated,

differences in pregnancy rate between cows (23% control vs 38% TAI) and heifers

(28%controls vs 35% TAI) tended to disappear (Table 2-1).

Modifications of the conventional protocol (GnRH d 0; PGF2. d 7; GnRH d 9 and

TAI d 10) have been tested with variable results (Table 2-1). Delaying the PGF2.

injections to day 8 or 9 (Pursley et al, 1997a), tended to reduce pregnancy rates from 55%

with the usual 7 d PGF2a-injection to 46% when administered on d 8 and 11% (P< 0.01)

when given on d 9. Cows receiving a second injection of GnRH 30-36 h after PGF2a

(Pursley et al., 1997a) tended to have higher pregnancy rates as compared to the cows

receiving PGF2. and inseminated at detected estrus. Although, the effectiveness of such

a modification has not been compared to the conventional treatment. Vasconcelos et al.

(1997) compared pregnancy rates in TAI cows bred at the time of the second injection of

GnRH or 24 h later. Pregnancy rates were higher (P<0.05) for cows bred 24 h after

GnRH (35% vs 29%).

Replacement of the second injection of GnRH with hCG has been also tested in

heifers with satisfactory results. Pregnancy rates greater than 50 % have been obtained

(Schmitt et al., 1996c; Barros et al., 1997 Table 2-1), and these were similar to the


controls receiving GnRH, PGF2. and inseminated at detected estrus (IDE) (Schmitt et al.,


TAI has been effective with Zebu and Zebu cross cattle. When TAI was

performed on 14 Girolando heifers ( Zebu Gyr, 2 Holstein), a 50% pregnancy rate was

obtained (Barros et al., 1997). Another modification of this protocol has proven

successful with Zebu and Zebu crosses. Hence, the second injection of the GnRH-analog

was replaced by estradiol benzoate to induce release of LH and subsequent ovulation.

Fixed time insemination was at 36 h after the EB injection. Pregnancy rates were similar

to the conventional procedure with a range of 30 to 50% at fixed insemination, when the

cows treated were diagnosed with a CL. The purpose of the replacement of GnRH with

EB is to reduce cost of the procedure, especially in developing countries (Barros et al.,


Effects of Heat Stress on Reproductive Function of Cattle


The effect of heat stress on puberty in cattle is not well documented. One might

expect deleterious effects since heat stress can decrease growth rate (Collier et al., 1982a)

which is related to puberty (Davis et al., 1977). Additionally, when heifers of different

breeds were held in environmental chambers at 27 C (80 F) or 10 C (50 F) (Dale et al.,

1959a; 1959b), Brahman heifers exposed to 27 C had delayed puberty compared to 10 C.

In contrast, Shorthorn heifers experienced the opposite effect, and Santa Gertrudis heifers

did not show differences in the onset of puberty in response to heat.

Estrus Detection

Heat stress causes a reduction in both duration and intensity of estrus (Hall et al.,

1959; Gangwar et al., 1965; Madan and Johnson, 1973; Monty and Wolff, 1974; Abilay

et al., 1975; Gwazdauskas et al., 1981; Rodtian et al., 1994). The magnitude of effects of

heat stress on duration of estrus at different locations is summarized in table 2-2.

TABLE 2-2. Effects of elevated temperatures on duration of estrus in cows.
Duration of estrus
Non-heat Heat
Treatment stress Stress Reference
Cool vs hot climates' -17 12 Hall et al., 1959

February-May vs July-Septembe? 20 14 Gangwar et al., 1965

Climatic chamber, 18 C vs 24-35 C' 20 11 Gangwar et al., 1965
Climatic chamber, 18 C vs 34 C 17 12 Madan and Johnson, 1973

January-April vs July-September' 14 8 Monty and Wolf, 1974
January-April vs July-September' 14 8 Wolff and Monty, 1974
Climatic chamber, 18 C vs 34 C 17 13 Abilay et al., 1975
Climatic chamber, 21 C vs 32 C 21 16 Gwazdauskas et al., 1981
Summer, cooled vs non-cooled3 16 12 Wolfenson et al., 1988b
Summer, cooled vs non-cooled4 28 15 Lu et al., 1992
'Studies performed in Louisiana.
2Studies performed in Arizona.
3Study performed in Israel.
4Study performed in China.

It is controversial as to whether or not reduced expression of estrous behavior in

heat-stressed cows is associated with lower concentrations of plasma estradiol

(Gwazdauskas et al., 1981; Wise et al., 1988a; 1988b ). It is likely that reduced locomotor

activity during heat stress (Lotgering et al., 1985; Bell, 1987) is also a factor in a reduced

ovulation. In beef cattle, the number of estruses without ovulations increased during the

hot season (76% vs 24%) (Plasse et al., 1970). One possibility is that during heat stress,

problems with heat detection cause more cows to be bred that are not really in estrus.

Her et al. (1988) observed that access to cooling 1 d before expected estrus caused

increased signs of estrous behavior in Israeli-Holstein dairy cows (70 vs 45%). In another

study, it was observed that more cows exposed to fan-cooling for 21 d before estrous

synchronization with PGF2a, expressed estrus than did controls without access to fans

(71% vs 33%, respectively) (Younas et al., 1993). Similarly, a greater proportion of

Chinese Holstein cows exposed to cooling showed behavioral estrus compared to controls

experiencing hot temperatures (31 C at noon) (83% vs 67%, respectively) (Lu et al.,


Length of Estrous Cycle

Several studies have reported extended estrous cycle periods during the hot season

of the year (Stott and Williams, 1962; Labhsetwar et al., 1963; Gangwar et al., 1965,

Fuquay et al., 1981; Bond and McDowell, 1972; Monty and Wolff, 1974), or following

experimental heat stress (Wilson et al., 1996). In one study, the average length of the

estrous cycle was 20-21 days under cool climatic conditions (18 C), as compared to 21-25

days for cows exposed to control conditions of 23, 29 and 35 C (Gangwar et al., 1965).

In Mississippi, analysis of records from the University herd showed more short estrous

cycles (<15 d) and long estrous cycles (>30 d) during the summer than during other

climatic seasons (Fuquay et al., 1981). Madan and Johnson (1973) also observed longer

estrous cycle for cows at 33.5 C than for cows at 18 C (21.6 vs 19.5 days). Abilay et al.

(1975) obtained similar results. Lu et al. (1992), observed a reduced interval from second

PGF2,-injection to estrus (from 58 h to 37 h) in synchronized cows receiving cooling

from the beginning of synchronization (14 d) until 20 d after breeding.

However, heat stress might also exert carryover effects which are reflected at the

time of estrus and ovulation as a result of adverse effects on follicular recruitment,

selection and dominance leading to an oocyte of poor quality. Badinga et al. (1993)

reported that heat-stressed cows had smaller dominant follicles (14.5 vs 16.4 mm) with

less follicular fluid (1.1 vs 1.9 ml) than cooled cows. Subordinate follicles were bigger

(10.9 vs 7.9) and contained more fluid (0.4 vs 0.2 ml) in heat stressed cows compared to

cooled cows, possibly due to harmful effects of heat stress on the dominant follicle.

Similar results were observed when follicular development was monitored in heat-

stressed cows held under a shade structure during the months of April, June, August, and

November. In those cows, the first wave dominant follicle developed more slowly in

August than in April, June or November and the subordinate follicle tend to persist for

extended periods of time (Badinga et al., 1993). Wolfenson et al. (1995) observed a

suppression of plasma concentrations of estradiol and a tendency for reduced levels of

inhibin within d 7-10 of the estrous cycle, impairing follicular development and altering


the dominance of the first-wave follicle and the preovulatory follicle. Heat stress seemed

to hasten the decrease in size of the dominant follicle in the first wave, as well as the

emergence of the second dominant follicle.

Conception Rate

Several studies have described lower conception rates in environments

characterized by heat stress (Seath and Staples, 1941; Stott, 1961; Stott and Williams,

1962; Ingraham et al., 1974; Monty and Wolf, 1974; Thatcher et al., 1974; Gwazdauskas

et al., 1975; Vaught et al., 1977; Zakari et al., 1981; Badinga et al., 1985; Cavestani et al.,

1985; Fulkerson and Dickens, 1985; Weller and Ron, 1992; Lu et al., 1992; Ryan et al.,

1993; Farin et al., 1994). In Arizona, Stott and Williams (1962) reported conception

rates for dairy cows in a range of 17-36% during the hot season compared to 44-62%

during the cool season of the year. Similarly, Badinga et al. (1985) reported Florida

conception rates which ranged from 10-15% during the hot season and 40-50% during the

cool season of the year.

A negative correlation between uterine temperatures and pregnancy rates has been

reported. A rise in uterine temperature by 0.5 C above normal on the day of insemination

caused a 13% decline in the pregnancy rates of lactating Holstein cows exposed to heat

stress (Gwazdauskas et al., 1973). Additionally, other factors besides elevated

temperatures are also likely to be involved. For example, Ingraham et al. (1976) reported

a negative correlation between fertility and humidity as early as 11 d before breeding.


Experimental studies with climatic chambers to artificially mimic effects of heat

stress on animals have demonstrated adverse effects of elevated temperatures and

humidity on conception rate and early embryonic survival. When heifers were exposed to

a short heat stress period of 32 C for 72 h after breeding, 0% of the cows were pregnant

(0/23), compared to the control cows at 21 C in which 48% (12/25) were pregnant

(Dunlap and Vincent, 1971). The most sensitive period affecting the establishment of

pregnancy seems to be around the time of estrus or soon after. Putney et al. (1989b)

observed that superovulated cows exposed to heat stress for 10 h at the beginning of

estrus had lower percentages of normal embryos collected at d 7 after estrus. In addition,

heat stress reduced survival and development of embryos collected on d 8 from

superovulated cows exposed to heat stress on day 1, but embryos were less affected if

cows were exposed to heat stress on d 3, 5 or 7 (Ealy et al., 1993) suggesting

developmental stage differences in embryonic thermoresistance. In vitro exposure of

bovine embryos to heat shock has been more detrimental to two-cell embryos than

embryos at advanced stages of development (Ealy et al., 1995; Edwards and Hansen,


While embryos seem to be more susceptible at early stages of development, heat

stress experienced on d 8-16 of pregnancy has also been reported to compromise

embryonic development by reducing conceptus size (Biggers et al., 1987). Also, heat

stress on d 17 of pregnancy also increased uterine production of PGF2. in response to

oxytocin (Wolfenson et al., 1993). Therefore, early pregnancy losses due to heat stress

could also occur later in pregnancy, in part because of adverse effects of heat stress on

luteal function.

Embryonic mortality could be due to direct effects of hyperthermia on the embryo

or as a consequence of hyperthermia in the reproductive tract. Alliston and Ulberg

(1961), performed an experiment with reciprocal embryo transfer and demonstrate that

both embryos and the reproductive tract are compromised by heat stress, with more

severe effects occurring on the embryo. In such study, viability of the embryos from ewes

exposed to ambient temperatures at 32 C was lower than for ewes exposed to 21 C.

These findings have been reinforced by studies showing adverse effects of heat stress on

the quality of embryos (Boland and Gordon, 1989; Putney et al., 1988a; 1989b) obtained

from superovulated cows. Heat stress during the summer reduced the number of normal

and transferable embryos obtained by superovulation compared to winter (29.2 % vs 50

%, respectively) (Al-Nimer and Lubbadeh, 1995). With superovulated cows, lower

embryonic survival occurred when cows were exposed to heat stress from day 1 to 7 after

insemination as compared to controls (Putney et al., 1988a). Together, these results

suggest that preimplantation embryos are very susceptible to maternal heat stress.

Shade, fans, sprinklers, and misters, have been used to alleviate adverse effects of

heat stress and increase conception rates. Roman-Ponce et al. (1981) observed that black-

globe temperatures decreased from 36.7 C for cows without shade to 28.4 C for cows that

had access to shade. Accordingly, conception rate to all services was higher in cows

exposed to shade compared to cows not exposed to shade (38.2% vs 14.3%). Lu et al.

(1992), reported an increase in conception rates from 62.5 % in non-cooled cows to

90.0% in cows cooled through the use of sprinklers and forced ventilation. Similarly,

Thatcher (1974) obtained higher conception rates (39-40%) with cows cooled

continuously during the day by exposure to air conditioning than for cows with no

cooling or only partial night time exposure to air-conditioning (28%).

Late Pregnancy

Heat stress in late pregnancy could be quite detrimental considering that about

60% of fetal development (Eley et al., 1978) and most of mammary gland development

(Thatcher and Roman-Ponce, 1980) occurs during the last third of pregnancy. During

periods of heat stress, there is circulation of the blood flow into peripheral body tissues

to promote heat loss into the environment. As a consequence, in pregnant cows there is a

reduction in the circulating blood going to the uterus (Oakes et al., 1976; Brown and

Harrison, 1981), and placenta (Alexander et al., 1987) that leads to a reduction in the

amount of hormones circulating through the placenta (Collier et al., 1982b; Malayer and

Hansen, 1990b), and reduced placental weight (Head et al., 1981), total placental DNA

(Vatnick et al., 1991), placental size (Thatcher and Collier, 1986; Sultan et al., 1987) and

calf birth weight (Bonsma et al., 1949; Collier et al., 1982b; Sultan et al., 1987;

Wolfenson et al., 1988a).

Birth weight of calves has been correlated with milk yield for the succeeding

lactation (Chew et al., 1981; Collier et al., 1982b). Such an effect could result because of

altered secretion of placental hormones that participate in manmogenesis and

lactogenesis. Wolfenson et al. (1988a) found a 3.5 kg/d increase in milk yield of the

subsequent lactation by cooling heat-stressed cows during the dry period although no

significant effect of cooling during the dry period on subsequent milk yield was found by

Collier et al. (1982b).

Postpartum Anovulatory Period

Prolonged periods of postpartum anestrus influence negatively the efficiency of

production in cattle (Short et al., 1990). Effects of heat stress on the duration of the

postpartum anovulatory period have not been extensively studied. There is some

evidence that prepartum heat stress may exert adverse effects on reestablishment of

estrous cycles postpartum. Intervals from calving to conception were extended when

cows were exposed to heat stress for 60 days before parturition as compared to cows

exposed to heat stress for 30 days before parturition (Moore et al., 1992). Lewis et al.

(1984) found no difference in the intervals from calving to first estrus, interval from

calving to conception and services per conception between controls cows or cows

exposed to heat stress during the last third of gestation. However, prepartum heat stress

was associated with increased circulating concentrations of prostaglandins (PGFM)

postpartum and longer intervals to uterine involution.

Mechanisms by which Heat Stress Reduces Conception Rates in Cattle

Heat stress compromises embryonic development by disrupting the physiological

and biochemical events responsible for the establishment and maintenance of pregnancy.

The following sections will include some of the processes that can be altered by heat

stress and which result in decreased conception rates.

Detrimental Effects of Elevated Temperature on Sperm

The process of spermatogenesis in bulls is severely compromised by exposure to

elevated temperatures (Casady et al., 1953; Rhynes and Ewing, 1973). Heating the

scrotum of bulls for as little as 1 h reduced viability of ejaculated sperm (Austin et al.,

1961; Gerona and Sikes, 1970). Spermatogenesis ordinarily takes place at a temperature

about 4 C lower than core body temperature. Elevations in body temperature due to heat

stress likely increase testicular temperature and, as a result, semen quality and sperm

motility are reduced (Patrick et al., 1954; Johnson et al., 1963; Kelly et al., 1963;

Meyerhoeffer et al., 1985; Parkinson, 1987; Ax, 1987).

Heat stress compromises spermatogenesis by affecting spermatocytes, spermatids

and, sometimes, B spermatogonia (De Alba and Riera, 1966; Amann and Schanbacher,

1983). Given the dynamics of the spermatogenic cycle, detrimental effects of heat stress

are demonstrable in semen approximately 2 weeks after exposure to heat stress

(Wettemann and Desjardins, 1979; Wettemann et al., 1976; 1979; Cameron and

Blackshaw, 1980; Meyerhoeffer et al., 1985). Also, since spermatogenesis requires 45 to

61 days (Waites and Setchell, 1969; Amann and Schanbacher, 1983), adverse effects on

sperm can persist for 7 to 10 weeks after exposure to heat stress (Meyerhoeffer et al.,


Detrimental effects of heat stress on spermatozoal production in bulls can be

completely avoided by collecting and freezing semen from bulls not exposed to heat

stress and breeding cows by artificial insemination. However, it is possible that sperm

may be damaged by being deposited and transported along the reproductive tract of a

hyperthermic female exposed to heat stress. Howarth et al. (1965) capacitated sperm in

the uterus of heat-stressed female rabbits and then used the semen for insemination of

females remaining at 21 C and mated with vasectomized males 10 h before insemination.

There was no difference in fertilization rate between females bred with heat-stressed

sperm (100%) as compared to sperm capacitated in non heat-stressed females (84%).

However the number of conceptuses per implantation site at d 12 post coitum was lower

(37% vs 72%, respectively) for females bred with heat-stressed sperm compared to

females bred with control sperm. Moreover, sperm contribute microtubules to the oocyte

that form the centriole in the first cleavage division (Sutovsky et al., 1996) and it is

possible that heat stress disrupts development by damaging these proteins. Similarly,

Burfening and Ulberg (1968) found that exposure of rabbit sperm cultured at 40 C for 3 h

before insemination did not affect fertilization rate (95% heat-shock vs 100% control) but

reduced embryonic survival by 9 d post coitum as compared to females inseminated with

sperm maintained at 38 C before insemination (75% vs 53%, respectively).

In cattle, effects of heat shock on ejaculated sperm have been variable.

Monterroso et al., (1995) reported that exposure of sperm to 41 to 42 C had little effect on

sperm functions, such as motility, presence of acrosomes, DNA integrity or fertilizing

ability. In contrast, elevated temperatures might compromise the fertilization process.

Lenz et al. (1983) observed higher sperm penetration (53 vs 40%) in oocytes when in

vitro fertilization took place at 39 C compared to 41 C. Such an effect could involve

either sperm or oocytes.

Detrimental Effects of Elevated Temperatures on Oocytes

Oocytes can also be compromised by elevated temperatures. Such effects could

involve direct disruption of oocyte function or changes in follicular development that lead

to alterations in oocyte quality.

In mice, Baumgartner and Chrisman (1987) observed that heat stress caused

retention of the first polar body during metaphase II. In another study, Fiorenza and

Mangia (1992) observed that heat stress caused appearance of chromosomal defects in

unfertilized oocytes. Exposure of preovulatory mouse oocytes to an elevated temperature

of 43 C for 20-40 min inhibited meiosis resumption and blocked protein synthesis (Curci

et al., 1987).

Bovine oocytes exposed to an in vitro heat shock of 41 C showed inhibition of

meiotic maturation when the subsequent fertilization rate was evaluated (Lenz et al.,

1983). Exposure of bovine oocytes to a 41 C heat shock during the first 12 h of in vitro

maturation decreased development to the blastocyst stage from 35% to 18% in controls at

39 C (Edwards and Hansen, 1997). Exposure to 42 C caused a reduction in oocyte

protein synthesis and this effect was exacerbated if oocytes were denuded as compared to

those with intact cumulus (Edwards and Hansen, 1996).


In another study, Putney et al. (1989b) found that superovulated heifers exposed to

heat stress in the period between the onset of estrus and artificial insemination had

reduced number of normal embryos recovered at day 7 after estrus.

The sensitivity of oocytes to heat shock might be due, at least in part, to their

inability to synthesize new heat shock proteins. In both mice, (Curci et al., 1987) and

cattle (Edwards and Hansen, 1996; Edwards et al., 1997) heat shock failed to increase

synthesis of HSP70. Moreover, microinjection of unfertilized mouse oocytes with

HSP70 mRNA increased resistance to lethal heat shocks of 42 and 43 C (Hendrey and

Kola, 1991).

Heat stress may also compromise oocyte quality by affecting follicular

development. Badinga et al. (1993) found that heat stress affected follicular selection and

dominance and compromised the quality of ovarian follicles. Cows with access to shade

had larger dominant follicles with more fluid than unshaded cows. Perhaps, less

dominant follicles result in the ovulation of a more persistent or aged follicle which has

an oocyte of lowered quality. Persistent dominant follicles have resulted in reduced

pregnancy rates in several studies (Savio et al., 1993b; Wehrman et al., 1993; Xu et al.,

1996). Wolfenson et al. (1995) observed that heat stress impairs follicle development by

hastening the decrease in size of the first-wave dominant follicle, as well as the

emergence of the second dominant (preovulatory) follicle, suggesting that heat stress

caused persistence of the dominant follicle and reduced quality oocytes.

Detrimental Effects of Elevated Temperatures on Preimplantation Embryos

The early embryo is very susceptible to elevated temperature. As compared to

embryos exposed to 38 C, exposure of fertilized 1-cell rabbit embryos to 40 C increased

postimplantation embryonic mortality after transfer to pseudopregnant females (Alliston

et al., 1965). Exposure to elevated temperature has also been reported to reduce viability

and subsequent development of cultured embryos in mice (Gwazdauskas et al., 1992;

Ar6chiga et al., 1994; Ealy and Hansen, 1994) and cattle (Ealy et al., 1992; Ealy et al.,

1995; Edwards and Hansen, 1997).

Embryonic sensitivity to heat shock decreases as embryonic development

progresses (Dutt, 1963; Tompkins et al., 1967; Ealy et al., 1993; Table 2-3). This is true

for cattle as well as for some other species. Dunlap and Vincent (1971) observed that

exposure of cows to 32 C for 72 h immediately following breeding reduced pregnancy

rate. Similarly, Putney et al. (1988a) found that the percent of normal embryos recovered

from superovulated heifers that were heat-stressed from day 1 to 7 post insemination was

lower than for animals maintained in a thermoneutral environment (20.7 % vs 51.5 %).

Ealy et al. (1993) found that maternal stress had more detrimental effects on embryonic

development when the stress occurred on day-1 post estrus than at later times (Table 2-3).

Acquisition of thermoresistance as embryos progress in development has also been

demonstrated with cultured embryos. Exposure of bovine embryos to a heat shock of 41

C for 3 h reduced development to blastocyst in 2-cell embryos but not in morulae (Ealy

et al., 1995). Similarly, exposure of embryos to a heat shock of 41 C for 12 h severely

reduced development to the blastocyst stage compared to controls (39 C), for 2-cell

embryos (0 vs 26%), less severe for 4- to 8-cell stage embryos (10% vs 25%) and no

effect for morulae (42% vs 37%) (Edwards and Hansen, 1997).

TABLE 2-3. Effect of maternal heat stress at different stages of pregnancy on
embryonic survival of preimplantation embryos from different species!
Temperature Day of Embryonic
Species treatment (C) pregnancy viability (%) Reference













Cattle control
heat stress
heat stress
heat stress
heat stress
aAdapted from Monterroso (1995).

Dutt, 1963

Tompkins et al., 1967

Ealy et al., 1993


Ealy et al. (1993) suggested that embryos acquire increase thermoresistance after

day 1 or 2 after fertilization. Therefore, embryos transferred to recipients at day 7-8

would have higher thermoresistance than earlier stage embryos, and pregnancy rates to

embryo transfer in summer might be higher than pregnancy rates to AL. Indeed this has

been observed in cows provided with fresh but not frozen/thawed embryos (Putney et al.,

1989a; Drost et al., 1994; Ambrose et al., 1988). Moreover, the reproductive function of

embryo-transfer donors and recipient cows was not affected by seasonal variation in

commercial embryo transfer unit (Putney et al., 1988d).

Given the fact that heat stress seems to exert its greatest effect in early pregnancy;

it would be expected that providing cooling during early pregnancy would increase

pregnancy rate during periods of heat stress. Such concept has been called strategic

cooling (Hansen et al., 1997). In fact, cooling cows around the time of estrus and after

insemination increased pregnancy in some studies while having no effect in another

(Table 2-4).

Detrimental Effects of Heat Stress on Luteal Function

Severe heat stress may compromise pregnancy at more advanced stages of

gestation. In one study, heat stress from d 8 to 16 of pregnancy caused a reduction in

conceptus size and luteal tissue weights (Biggers et al., 1987). Perhaps, retardation of

embryonic growth as a consequence of elevated temperature compromises the ability of

embryos to produce sufficient interferon-l: (IFN-t) or other cellular products necessary for

pregnancy recognition. Geisert et al. (1988) determined that bovine embryos must reach


approximately 15 mm in length (15-17 days) in order to produce (IFN-tr). Heat shock of

of cultured day 17 embryos also reduced (IFN-T) secretion (Putney et al., 1988c). In

contrast, there was no effect of maternal heat stress through day 8 to 16 on ability of

embryos to release (IFN-T) when they were removed from cows and subsequently

cultured (Geisert et al., 1988). Heat stress may also increase PGF, release and promote

luteolysis. Exposure of cultured endometrium from d 17 of the estrous cycle increased

PGF2. synthesis (Malayer et al., 1988; Putney et al., 1989c; Malayer and Hansen, 1990a).

Also, heat stress increased oxitocin-induced uterine PGF2. secretion on d 17 of the cycle

in lactating dairy cattle (Wolfenson et al., 1993).

There is some evidence that secretion of progesterone, the hormone responsible

for maintenance of the uterus during pregnancy is altered in response to heat stress.

TABLE 2-4. Effects of strategic cooling around on pregnancy rates of cows in hot
Strategic Pregnancy rate (%)b
Location Cooling Treatment Controls Cooled Ref
Arizona 4-6 d after estrus 22% (13/61) 33% (19/63) 1
Arizona 8-16 d after PGF2. 17% (3/18) 29% (10/35) 2
Guadeloupe 12 d, until d 10 after A I 13% (2/15) 53% (8/15) 3

Israel 10 d, from 1 d before estrus 36% (8/22) 31% (9/29) 4
Florida 8 d after PGF2. 6% (2/32) 16% (8/50) 5
aAdapted from Hansen et al., 1997.
bPregnancy rate= proportion of cows pregnant that were inseminated, and, in parentheses,
number of cows pregnant/number bred.
cReferences: 1) Stott and Wiersma, 1976; 2) Wise et al., 1988b; 3) Gauthier D., 1983; 4)
Her et al., 1988; 5) Ealy et al., 1994.

However the specific effects of progesterone have been highly variable. Several studies

have reported increased concentrations of progesterone in cycling cows exposed to heat

stress (Abilay et al., 1975; Vaught et al., 1977; Roman-Ponce et al., 1981; Trout et al.,

1998). However, others found that heat stress decreased progesterone concentrations

(Robertshaw and Whittow, 1967; Folman et al., 1983; Younas et al., 1993), and yet other

studies found no effect of heat stress (Gwazdauskas et al., 1981; Rosemberger et al.,

1982; Biggers et al., 1987; Wise et al., 1988a;1988b). Such a discrepancy among studies

could be due to changes in water intake and utilization in response to heat stress, or an

alternative explanation could be variation in redistribution of blood flow upon heat stress.

Effects of Elevated Temperature on Oviduct and Uterus

A study by Alliston and Ulberg (1961) suggested that heat stress exerts effects on

both the embryo and the reproductive tract and that effects on the embryo are more

detrimental to subsequent pregnancy than are effects on the reproductive tract. In this

study, embryo donors were maintained at 21 or 32 C. Embryos were recovered at d 3 of

pregnancy and transferred to recipients that were subsequently maintained at 21 or 32 C.

Embryo survival rate was 56% when both donors and recipients were at 21 C, 24% when

donors were maintained at 21 C and recipients at 32 C, and 9.5% for donors maintained at

32 C and recipients at 21 C.

Heat stress could alter function of the oviduct and uterus, by affecting secretion

of hormones regulating reproductive tract function, or by direct effects of elevated

temperature on cells of the reproductive tract.

Malayer et al. (1988) found that secretion of polypeptides from bovine

endometrial explants was disrupted by exposure to elevated temperature. In particular, at

43 C, secretion of 7 polypeptides was reduced in tissue from the uterine horn ipsilateral to

the corpus luteum but not in tissue from the contralateral horn. Subsequently, differences

between Brahman and Holstein cows in response of oviductal and endometrial explants to

heat shock (39 vs 43 C) were examined (Malayer and Hansen, 1990a). Heat shock

increased secretion of macromolecules in both oviducts of Brahmans but depressed

secretion in oviducts ipsilateral to the side of ovulation of Holsteins. Heat shock proteins

(HSP) of 72 kDa and 90 kDa were found in the endometrium, and their concentrations

were increased by heat (Malayer et al., 1988; Malayer and Hansen, 1990a; 1990b).

Heat stress may also affect function of the reproductive tract by affecting blood

flow. Increases in uterine blood flow induced by estradiol were reduced by heat stress in

dairy cattle (Roman-Ponce et al., 197&b) and sheep (Roman-Ponce et al., 1978a). Such

reduction in uterine blood flow may elevate uterine temperature and affect availability of

water, electrolytes, nutrients, hormones and growth factors to the uterus.

Mechanisms to Protect Cells from Elevated Temperatures

There are direct effects of heat shock on function of embryos (Ealy et al., 1992;

1995; Ealy and Hansen, 1994; Arrchiga et al., 1994; 1995; Edwards and Hansen, 1997;

Edwards et al., 1997). The magnitude of these effects on embryos depends upon stage of

development and it is likely that effects of elevated temperature depend upon the

biochemical mechanisms present in cells to limit the effects of heat shock. At least two


general systems for thermoprotection exist; heat shock proteins, which stabilize protein

structure, and antioxidants which limit effects of increased levels of free radicals induced

by heat shock.

Protective Effects of Heat Shock Proteins

HSP have been implicated in the phenomenon known as induced thermotolerance,

in which cells that are briefly exposed to a sublethal heat shock become resistant to a

subsequent, more severe heat shock (Li and Werb, 1982; Li and Mak, 1989; Duncan and

Hershey, 1989; Maytin et al., 1990; Nover et al., 1991; Nover and Scharf, 1991).

Associated with exposure to sublethal heat shock is the new synthesis of proteins with

Mrs 27,000, 68,000, 70,000 and 96,000 two to four hours after heat shock (Li and Mak,

1985; Landry et al., 1989; Lavoie et al., 1993).

Heat shock proteins are identified according to their molecular weight. Thus,

HSP27 is a 27,000 Mr protein and HSP70 is a 70,000 Mr protein. HSP90 is one of the

most abundant heat shock proteins, being about 1 % of total intracellular protein in the

cytoplasm of normal cells (Nover and Scharf, 1991). Amounts of HSP90 increase in

response to heat shock (Moore et al., 1987; Yamazaki et al., 1989). Like many HSPs,

HSP90 is a molecular chaperone that exerts its function by binding to other proteins and

causing modifications in their structure and function. Among its actions, HSP90 causes

changes in protein phosphorylation (Duncan and Hershey, 1987) and inhibits translation

by activating the kinase that inhibits eIF-2a (Matts and Hurst, 1989; Rose et al., 1989a;

1989b). HSP90 is also part of the steroid receptor complex; upon steroid binding,


HSP90 becomes dissociated and the receptor binds to steroid response elements on DNA

(Baulieu, 1987; Baulieu E and Catelli, 1989; Pratt, 1992).

The HSP70 family is represented by two major classes of proteins induced by

heat shock. The first class, HSP68 in mouse (Hunt and Calderwood, 1990) and HSP72 in

humans (Welch et al., 1989), is present at very low levels in unstressed cells; production

increases dramatically in cells stressed by heat or chemical shock (Welch et al., 1989).

The second class of HSP70, often called HSC70, is constitutively expressed at high

levels in the absence of heat. This protein has been referred to as HSC70 in mouse

(Giebel et al., 1988) and HSP73 in humans (Welch et al., 1989). While these proteins are

synthesized in large amounts by unstressed cells, synthesis also increases after heat shock

(Welch et al., 1989).

The distribution of HSP70 proteins in cells changes during heat shock. In

unstressed cells, HSP70 is associated with the cytoskeleton but these proteins migrate to

the nucleus (Velazquez et al., 1980; Velazquez and Lindquist, 1984; Welch et al., 1989;

Nover and Scharf, 1991; Lazlo et al., 1992), especially in the nucleolus. Later in heat

shock, HSP70 becomes associated with polysomes and ribosomes (Welch et al., 1989).

The HSP70 family of proteins have been shown to be involved in folding of

proteins, insertion of proteins into or through intracellular membranes and as ATPases

causing disassembly of clathrin-coated vesicles (Pelham, 1984; Nover and Scharf, 1991;

Wynn et al., 1994; Mager and DeKruijff, 1995). It has been postulated that during heat

shock, HSP70 interacts with other proteins to affect their folding, assembly, reorientation


and relocation so as to prevent denaturation or help repair damaged proteins (Welch et al.,

1989; Nover and Scharf, 1991).

Several lines of evidence indicate that HSP70 can confer thermotolerance.

Riabowol et al. (1988) demonstrated that injection of antibodies to HSP72 and HSP73

increase susceptibility of fibroblasts after exposure to heat shock. L929 cells have greater

resistance to heat shock than LS cells and also a higher capacity to synthesize large

amounts of HSP70 (Margulis et al., 1987). Also, injection of HSP70 mRNA made mouse

oocytes more resistant to heat shock (Hendrey and Kola, 1991) while microinjection of

human HSC70 mRNA caused thermotolerance in fibroblasts (Li et al., 1991).

Transfection of mouse L cells or monkey COS cells with an HSP70 gene reduced

deleterious effects of temperature on nucleolar morphology and export of preribosomes

(Pelham, 1984).

HSP27 is a protein also increased by heat shock (Mirkes et al., 1996). One of its

fuctions is to prevent protein aggregation and promote functional refolding of denatured

proteins (Jakob et al., 1993; Ciocca et al., 1993). Heat shock resistance was also

conferred by expression of the human HSP27 gene in rodent cells (Landry et al., 1989)

and accumulation of the HSP27 in CHO cells induces thermoresistance (Lavoie et al.,


Control of Gene Expression

Heat shock gene expression is regulated by sequence elements (i.e., HSE for heat

shock elements). Heat shock gene transcription is stimulated by the binding of the


transcription factor known as HSF (heat shock factors, HSF1 and HSF2) to the heat shock

elements (HSE) located upstream of the TATA box. There are at least three HSF's:

HSFI, HSF2 and HSF3 (Rabindran et al., 1991; Scheutz et al., 1991; Nakai and

Morimoto, 1993). Only HSF-l responds to heat and other physiological stresses (Baler et

al., 1993; Sarge et al., 1993; Fawcett et al., 1994; Fiorenza et al., 1995). In non shocked

cells, HSF is present in an inactive form unable to bind to the HSE. Exposure to heat

shock induces the activation of HSF and allows modification into a form that binds to

HSE and activates transcription (Morimoto et al., 1992). The primary signal to induce

expression of heat shock protein genes are damaged proteins (Ananthan et al., 1986). In

the absence of heat shock, HSF are associated with HSP. Therefore, in response to heat

shock, denaturation and malfolding of proteins increase the presence of protein substrates

that compete with HSF for association with HSP70. As a consequence of this heat shock

or other stressors, removal of the negative regulatory activity on HSF DNA-binding is

initiated and HSP dissociates to refold damaged proteins (Clos et al., 1990). That allows

phosphorylation which causes trimerization of HSF which become able to bind to the

HSE (Nover, 1991; Westwood et al., 1991). Thus, HSF binds DNA and acquires

transcriptional activity, leading to transcription, and HSP synthesis (Mager and De

Kruijff, 1995).

Other studies by Burdon et al. (1987) with human HeLa cells suggested that there

is a connection between lipid peroxidation and activation of heat shock protein gene

activation since HSP synthesis increased in the presence of SOD inhibitors. Similarly,

other studies suggest interrelationships between antioxidants and HSP synthesis for


thermoprotection. Depletion of the antioxidant glutathione inhibited the development of

thermotolerance and decreased HSP in CHO cells (Russo et al., 1984). A more recent

study (Rokutan et al., 1996), reported that GSH depletion impaired transcriptional

activation of heat shock genes in gastric mucosal cells of guinea pigs.

Ontogeny of Heat Shock Protein Synthesis in the Embryo

Some HSP70 is present in mouse (Hendrey and Kola, 1991) and cow oocytes

(Edwards and Hansen, 1997). However, heat-inducibility does not occur (Hendrey and

Kola, 1991; Edwards and Hansen, 1997). In the mouse, germinal vesicle breakdown is

followed by a decrease in the constitutive and induced forms of HSP70 (Curci et al.,

1987; Curci et al., 1991). The mRNA for HSP70 (Manejwala et al., 1991) is also


In the mouse, HSP70 is produced constitutively during initiation of transcription

at the 2-cell stage and is considered one of the first major products of zygotic gene

activation (Bensaude et al., 1983: Manejwala et al., 1991; Christians et al., 1995).

Transcription begins as early as the 1-cell stage, and continues throughout the early 2-cell

stage; HSP70 gene expression is repressed before the completion of the second round of

DNA replication (Christians et al., 1995). Expression of HSP70 during this time is

controlled by progressive maturation of chromatin structure (Thompson et al., 1995).

Heat inducibility of HSP70 does not occur until later in development. Several studies

reported that increased synthesis of HSP70 in response to heat shock does not occur until

the morula or blastocyst stages (Wittig et al., 1983; Morange et al., 1984; Muller et al.,

1985; Hahnel et al., 1986). However, heat-induced synthesis of HSP70 was found to

occur as early as the 8-cell stage in the mouse embryo in one study (Edwards et al., 1995)

and at the 16-32-cell stage in another study (Bevilacqua et al., 1995). Differences

between studies could be due to type of heat shock used (40 C for Edwards vs 43 C or

higher for other studies), or the fact that Bevilacqua et al. (1995) used a gene construct to

study HSP expression. Mezger et al. (1994) found that mouse oocytes, one-cell embryos

and two-cell embryos responded to heat shock by an increase in heat shock element-

binding activity; at the four-cell stage, no binding activity was induced by heat shock.

In the cow, heat-induced HSP70 synthesis can occur early in embryonic

development. Edwards et al. (1997) found that exposure of bovine embryos to heat shock

induced synthesis of HSP68 (HSP70) as early as the 2-cell stage of development.

Constitutive forms of HSP70 (HSC70 and HSC71) were synthesized at all stages of

development but heat-induced synthesis was seen only until the expanded blastocyst

stage. Induction of HSP68 was a-amanitin independent at the 2-cell stage but was

blocked by a-amanitin as early as the 4-cell stage, suggesting increased HSP68

translation at the 2-cell stage and increased HSP68 transcription at the 4-cell stage.

Ontogeny of Thermotolerance in the Early Embryo

As for many other cultured cells, preimplantation mouse (Muller et al., 1985;

Ardchiga et al., 1995) and bovine (Ealy and Hansen, 1994) embryos are capable of

undergoing induced thermotolerance. This is a response whereby exposure to a mild

heat shock makes cells more resistant to a subsequent more severe heat shock (Nover,


1991). There is evidence that thermotolerance depends upon the stage of development of

the embryo. In one study, Muller et al. (1985) found induced thermotolerance in murine

blastocysts but not in one-cell embryos. Ealy and Hansen (1994) observed that the

ontogeny of induced thermotolerance depended upon culture conditions. Induction of

thermotolerance was first apparent at the 8-cell stage when embryos were collected at the

2 to 4-cell stage and then allowed to develop in vitro. In contrast, when embryos were

developed in vivo and collected at the desired stage, thermotolerance was not induced

until the blastocyst stage of development. In the study by Ealy et al. (1994), serum

supplementation was critical for induced thermotolerance since this phenomenon was not

observed when embryos were placed in medium without serum supplements.

Acquisition of the ability to undergo heat-induced synthesis of heat shock proteins

does not seem to be the only limiting factor determining the timing of embryonic

resistance by itself. While bovine two-cell embryos were able to undergo synthesis of

HSP70 by heat-shock induction (Edwards and Hansen, 1996), development of these

embryos was severely compromised in response to a heat shock of 41 C for 12 h

(Edwards et al., 1997). Moreover, thermotolerance expression has been determined to

occur independently of HSP synthesis (Kampinga et al., 1992; Borrelli et al., 1996).

Borrelli et al. (1996) observed activation of the heat shock transcription factor by

thermotolerance induction using 43 C for 12 min) but not an increase in steady-state

levels of HSP70-mRNA. Levels of HSP27, HSP70, and HSP90 remain constant. Lack

of concordance between ontogeny of HSP70 synthesis and induced thermotolerance

suggest that some other factors besides HSP synthesis could be involved in

thermotolerance of embryos.

Heat Shock and Free Radicals

Adverse effects of heat shock may be caused, at least in part, by an increased

generation of free radicals. Free radicals are species that have one or more unpaired

electrons. Free radicals are produced as byproducts of normal oxidative metabolism and

are critical for the operation of a wide spectrum of biological processes (Freeman and

Crapo, 1982). An equilibrium between the generation and dissipation of free radicals

must exist, or otherwise increased levels of free radicals may alter cellular function.

Therefore, free radical molecules may be responsible for adverse effects of elevated

temperature on cells if these are unable to eliminate them.

Superoxide anion (.02-) radical is the most abundant reduced oxygen species

produced in cells. It is produced from various enzymatic reactions including reduction of

02 during electron transport in the mitochondria, from several oxidases via lipooxygenase

in peroxisomes, from cyclooxygenases in membranes, and from oxidation of xanthine in

the cytoplasm (Loven, 1988; Allen, 1991). Superoxide radicals undergo dismutation to

hydrogen peroxide through actions of superoxide dismutase. Hydrogen peroxide itself

can directly oxidize biologically important molecules and may also create the highly

reactive hydroxyl radical (.OH) during metal-catalyzed decomposition (Freeman and

Crapo, 1982; Halliwell and Gutteridge, 1984; Gutteridge, 1986; Halliwell, 1987).


Hydrogen peroxide is removed intracellularly by conversion to water through actions of

glutathione peroxidase in cytoplasm and catalase in peroxisomes.

One of the most damaging effects on cells caused by free radicals is lipid

peroxidation (Halliwell and Gutteridge, 1984; Ellis, 1990; Wells and Winn, 1996). This

phenomenon occurs in membranes or free fatty acids when any species has sufficient

reactivity to remove a hydrogen atom, thereby causing an unpaired electron on the carbon

atom. The carbon radical of polyunsaturated fatty acids tends to be stabilized by

molecular rearrangement to form a conjugated diene, which rapidly reacts with 02 to

generate a hydroperoxyl radical, which then causes a chain reaction to remove hydrogen

atoms from other lipid molecules (Wefers and Sies, 1983; Halliwell and Gutteridge,

1984; Ellis, 1990). Such chain reactions during lipid peroxidation can result in

generation of additional free radical molecules (Allen, 1991) and production of toxic

molecules such as alcohols, ketones, and aldehydes that can disrupt the cytoskeleton and

mitochondria to cause cross-linking of proteins, lipids and nucleic acids (Younes and

Siegers, 1984; Machlin and Bendich, 1987; Loven, 1988) and membrane disruption

(Kuzuya et al., 1991).

Protective enzymes and non-protein sulfhydryls in plasma are among the targets

for free radical attack. Since these reactions can modify proteins, one major alteration

caused by oxidant species is enzyme inactivation (Wefers and Sies, 1983; Halliwell and

Gutteridge, 1984; Machlin and Bendich, 1987) and an increase in disulfide bond

formation in membrane proteins (Rybczynska et al., 1993).


Loven (1988) has proposed that oxidized species may be responsible for cellular

dysfunction and altered differentiation caused by heat shock. Several pieces of evidence

support this hypothesis since oxygen consumption increases in hyperthermic cells

(Mitchell and Russo, 1983; Allen, 1991) and exposure to heat shock stimulates free-

radical producing enzymes such as xanthine oxidase (Skibba and Stadnicka, 1986; Skibba

et al., 1989b), urate oxidase (Skibba et al., 1986) and cyclooxygenase (Malayer et al.,

1990). Moreover, heat shock of perfused liver caused increased release of

hydroperoxides (Skibba et al., 1986; Skibba et al., 1989b) and some other oxidative

molecules produced during oxidative metabolism such as diaziquone that induce DNA

fragmentation and cross-linking (Szmigiero and Kohn, 1984; Szmigiero et al., 1984).

Other pieces of evidence supporting Loven's theory are those related to the

protective roles of antioxidants during hyperthermia. Continuous heat shock treatment of

postimplantation rat embryo cells at 42.5 C or acute exposure at 43 C or 45.5 C resulted

in rapid elevations of GSH to 120-200% compared to controls (Mirkes, 1987). GSH

increased more rapidly at 45.5 C than at 43 C. Depletion of GSH by selective inhibitors

such as diethylmaleate or D,L-buthionine-[S,R]-sulfoximine (BSO) increases the

susceptibility of cells to heat shock (Mitchell et al., 1983; Russo et al., 1984; Shrieve et

al., 1986; Roizin-Towle et al., 1986; Harris et al., 1991). Omar and Lanks (1987)

observed in normal and simian virus 40-transformed mouse embryo cells that induction

of thermotolerance (caused by exposing cells to a mild heat shock) is associated with high

antioxidant enzyme levels (SOD, catalase, glutathione peroxidase). Heat shock by itself

increased GSH concentrations in CHO cells, whereas heat shock combined with an acid

environment (pH=6.7) induced sensitivity to elevated temperatures and caused a

depletion in GSH levels (Freeman et al., 1985). This depletion has also been shown for

other antioxidants such as vitamin E, SOD, catalase and dimethyl sulfoxide (Yoshikawa

et al., 1993). Kandasamy et al. (1993) observed that administration of SOD and GSHPx

attenuated radiation induced by hyperthermia. Moreover, exogenous administration of

antioxidants enabled lymphocytes (Malayer et al., 1992) and preimplantation embryos

(Malayer et al., 1992; Ealy et al., 1992; Ardchiga et al., 1994; 1995) to be more resistant

to elevated temperatures.

However, other studies do not support the concept that GSH is essential for

thermotolerance. Roizin-Towle et al. (1986) reported that thiol depletion by

diethylmaleate and BSO had no effect on the response of hamster cells to an acute (45 C)

or chronic (42.5) hyperthermia. Konnings and Peninga (1985) found that there was no

increase of GSH observed in Ehrlich ascites tumor cells after exposure to 44 C. Ealy et

al. (1995), has also found no protective effects of GSH on early embryos.

Evidence that Free Radicals are Involved in Effects of Heat on Embryos

Fujitani et al. (1997) found reduced development of embryos to blastocyst stage

upon generation of oxygen radicals by 2,2'-azobis (2-amidinopropane) dihydrochloride

(AAPH). Exposure of embryos to heat shock decreased embryonic levels of the

intracellular antioxidant glutathione in mouse embryos (Ardchiga et al., 1995). In

addition, effects of heat shock on cultured embryos have been reduced by antioxidant

supplementation (Ealy et al 1992; 1995; Ardchiga et al., 1994; 1995).


Malayer et al. (1992) observed that provision of the antioxidant, taurine, increased

survival of murine embryos exposed to a heat shock of 42 C. In another study, Ealy et al.

(1992) also observed a beneficial effect of taurine in bovine embryos exposed to 42 C.

Provision of GSH, a stronger antioxidant, increased viability and development to the

blastocyst stage to a greater extent than taurine in bovine embryos exposed to a 42 C

(Ealy et al., 1992). Ealy et al. (1992) found no beneficial effect of culturing bovine

embryos with antioxidants at 38.5 C on viability or subsequent development to the

blastocyst stage. Additionally, provision of glutathione, taurine and glutathione ester did

not alleviate the effects of heat shock on development of 2-cell embryos (Ealy et al.,

1995). In contrast, other studies have shown that various antioxidants (SOD, GSH, DTT,

thioredoxin, taurine, etc.) increased viability and development to blastocyst in murine

(Nasr-Esfahani et al., 1990b; Legge and Sellens, 1991; Goto et al., 1992; Nasr-Esfahani

and Johnson, 1992b; Dumoulin et al., 1992; Ardchiga et al., 1994; 1995), rabbit (Li et al.,

1993) and bovine (Takahashi et al., 1993) embryos exposed to temperatures considered


Protective Effects of Antioxidants

Increased generation of free radical molecules can disrupt and damage DNA,

proteins, carbohydrates and lipids. This damage is known as oxidative stress, and many

biological compounds play a role in preventing it. Among these are enzymatic systems,

such as superoxide dismutase, glutathione peroxidase, catalases, etc., that enzymatically

remove free radicals. Additionally, various molecules act as sinks for free radical

metabolism. These molecules react with free radicals and remove them from the cell.

These include glutathione, vitamins E and C, P-carotene and other carotenoids, taurine,

and hypotaurine. In this review, emphasis will be placed on three major antioxidants:

glutathione, a major water-soluble antioxidant, vitamin E, the lipid-soluble antioxidants

that protects membranes from lipid peroxidation, and P-carotene, a lipid-soluble

antioxidant that is an effective deactivator of singlet oxygen and free radicals.


The tripeptide glutathione (y-glu-cys-gly) is present intracellularly at high

concentrations. Intracellular concentrations of GSH are also relatively high in mouse

oocytes (Nasr-Esfahani and Johnson, 1992a) but levels decline after fertilization. Two

enzymatic step processes are involved in GSH synthesis. First, y-glutamylcysteine

synthetase catalyses the synthesis of y-glutamylcysteine, which then is converted to

glutathione by the action of glutathione synthetase through addition of glycine (Ellis,

1990). GSH synthesis can be blocked at the first enzymatic step by blocking y-

glutamylcysteine synthetase through the action of DL-buthionine-[S,R]-sulfoximine

which is a selective inhibitor of glutathione (Griffith and Meister, 1979a; 1979b).

GSH is oxidized and converted to glutathione disulfide (GSSG) that is formed by

a disulfide bond between two y-glu-cys-gly chains (Floh6 and Guinzler, 1976). Some

GSH is resynthesized by reduction of GSSG catalyzed by glutathione reductase.

However, a portion of GSSG is lost from the cell because GSSG can readily cross cellular

membranes (Allen et al., 1985). In contrast, GSH cannot easily traverse cellular

membranes (Jensen and Meister, 1983). Rather, GSH enters cells after cleavage by y-

glutamyltranspeptidase and is then resynthesized inside the cell. Thus, supplementation

of GSH in cultured cells is not quite effective because only causes a small rise in

intracellular levels of GSH (Jensen and Meister, 1983).

GSH reacts with free radicals and lipid peroxides to removed them and regulate

their levels (Arias and Jakoby, 1976; Kosower, 1976). Free radicals react with GSH and

reduce disulfide bonds in proteins (Kosower, 1976; Meister, 1985a; 1985b). For example,

glutathione peroxidase and glutathione transferase use GSH as a hydrogen donor to

reduce hydroperoxides to alcohols. Glutathione peroxidase requires selenium as a

cofactor. Therefore, selenium status is related to antioxidant status (Rotruck et al., 1973;

Smith et al., 1974) and selenium deficiency causes a variety of disorders including

increased fragility of erythrocytes, muscle fiber degeneration (white muscle disease),

encephalopathies and reproductive disruption involving spermatogenesis and ovarian

function (Van Vleet, 1987). Disulfide bonds are reduced by the action of glutathione

dehydrogenases (Ellis, 1990; Di Mascio et al., 1991) to maintain cellular proteins in their

reduced and active state (Fariss et al., 1984).

BSO treatment caused inhibition or reduction in thermoprotection response in

hamster fibroblasts (Mitchell et al., 1983; Russo et al., 1984; Shrieve et al., 1986) and rat

postimplantation embryos (Harris et al., 1991). Also induced thermotolerance in mouse

morulae was abolished upon BSO treatment (Ar6chiga et al., 1995).

Cleavage stage embryos seem to have limited capacity to synthesize GSH and

are susceptible to GSH-depletions (Gardiner and Reed, 1995a). Levels of GSH at


different stages of development have been characterized in murine embryos (Gardiner and

Reed, 1994) and pig oocytes (Yoshida et al., 1993). Levels of GSH are relatively high in

oocytes and 2-cell embryos and start to decline after fertilization decreasing about 10-fold

from the 2-cell stage to the blastocyst stage. The presence of an oxidative agent (tertiary

butyl hydroperoxide; tBH ) decreased GSH levels by 75% in the 2-cell embryos but only

25% in blastocysts (Gardiner and Reed, 1994) indicating that GSH status changes

dramatically during development. Embryos collected early on day 3 of development

(approximately 48 h pdp) were unable to recover GSH levels, whereas, embryos collected

late on day 3 of development recover GSH levels within 5 h (Gardiner and Reed, 1995b).

There are several lines of evidence that glutathione is involved in resistance to

heat shock. Depletion of GSH with sulfoximines caused increased temperature

sensitivity of murine mammary adenocarcinoma cells (Jones and Douple, 1990).

Furthermore, intracellular concentrations of GSH were increased by heat shock in

hamster fibroblasts (Mitchell et al., 1983), mouse fibroblasts and Ehrlich tumor cells

(Konings and Penninga, 1985), CHO cells (Issels et al., 1985), and rat postimplantation

embryos (Harris et al., 1991). Thus, GSH may be increased as a thermoprotective

mechanism in response to heat shock. In addition, GSH prevented heat-stress induced

plasma membrane blebbling of CHO cells (Kapiszewska and Hopwood, 1988) and

inhibition of preimplantation development in mouse (Arechiga et al., 1995) and bovine

embryos (Edwards and Hansen, 1997). Intracellular injection of GSSG made CHO cells

thermotolerant (Lumpkin et al., 1988).

While these studies provide strong evidence that GSH plays a critical role in

thermotolerance, the role of GSH could vary between cell types since Roizin-Towle et al.

(1986) found that GSH depletion increased thermosensitivity of lung carcinoma cells

whereas it had no effect on thermal resistance of hamster fibroblasts. Moreover,

Anderstam and Harms-Ringdahl (1986) reported that, even though BSO depleted GSH

levels, thermotolerance was still induced in C3H mammary carcinomas. Also, Freeman

et al. (1990) found no thermoprotective effects of a chronic elevation in the levels of GSH

in CHO cells.

Neurons are deficient in glutathione and exhibit accelerated induction of mRNA

and protein for HSP32 (heme oxygenase-1) in rat brain cells (Ewing et al., 1992). One

study found that HSP70 synthesis is lessened by treatment of Chinese hamster V79 cells

with BSO, the GSH-synthesis inhibitor (Mitchell and Russo, 1983; Russo et al., 1984).

In another study, (Harris et al., 1991) observed that BSO-treated preimplantation embryos

produced HSP70 in response to heat. Glutathione depletion has impaired transcriptional

activation of heat shock genes in primary cultures of guinea pig gastric mucosal cells

(Rokutan et al., 1996).

Vitamin E

Vitamin E is a generic term for all tocopherol and tocotrienol derivatives that

function as the major lipid-soluble antioxidant of cells (Scott, 1980; Ullrey, 1981). A

major antioxidant function of vitamin E is to assure low levels of peroxides to maintain

membrane integrity and prevent oxidative damage and peroxidation of membrane


phospholipids by reactions of hydrogen peroxide, "O2- or .OH with unsaturated fatty acids

(Mc Cay and King, 1980; Molenaar et al., 1980; Ullrey, 1981; Halliwell and Gutteridge,

1984; Gutteridge, 1986). Vitamin E is involved in synthesis of coenzyme Q and various

vitamins also (Ullrey, 1972; Scott et al., 1980). Vitamin E participates in sulfur amino

acid metabolism by preventing fatty hydroperoxidation (Scott et al., 1980).

There are interrelationships between GSH and vitamin E. Depletion of

intracellular GSH in rat hepatocytes resulted in reduced levels of vitamin E and protein

thiols and increased lipid peroxidation (Pascoe et al., 1987). Treatment of human blood

platelets with diamide, a thiol oxidizing agent, decreased GSH and vitamin E and

increased lipooxygenase activity (Calzada et al., 1991). In fact, GSH helps prevent

depletion of vitamin E during peroxidative reactions because reactions in which GSH

participate spare vitamin E (Di Mascio et al., 1991). Also, when vitamin E levels are

insufficient, there is increased consumption of GSH (Golden and Ramdath, 1987).

Involvement of vitamin E alone or in combination with other antioxidant

molecules at the cellular level may protect embryos from elevated temperatures. Some

studies have related vitamin E function with elevated temperatures and increased

production of free radical molecules protecting from cell injury (Reed et al., 1987).

Duthie et al. (1991 ) observed that vitamin E decreased peroxidative events associated

with malignant hyperthermia in pigs. Also, absence of vitamin E caused increased

damage of gastric mucosa of pigs (Duthie et al., 1991). These lesions were diminished by

treatment with other antioxidant enzymes such as Cu/Zn-SOD and catalase (Yoshikawa et

al., 1989). Vitamin E increased survival of embryos mouse embryos exposed to heat


shock (Ar~chiga et al., 1994). However, there was no beneficial effect of administration

of vitamin E at the time of breeding on pregnancy rates of heat-stressed cows (Ealy et al.,


Effects of selenium, vitamin E and their combination on fertility of cows have

been variable (Segerson et al., 1977; Arrchiga et al., 1994; Gwazdauskas et al.,, 1979;

Schingoethe et al., 1982; Kappel et al., 1984; Hidiroglou et al., 1987; Stowe et al., 1988).

Differences between studies in the amount of vitamin E and/or selenium administered,

the period of administration, and nutritional status of the experimental animals with

respect to vitamin E and selenium intake could explain some of these differential results.

Injection of vitamin E and/or selenium can enhance neutrophil function (Eicher et

al., 1994; Kanno et al., 1996; Ndiweni and Finch, 1996; Politis et al., 1995; 1996) and

could promote removal of microorganisms, uterine tissue remodeling and involution.

Prepartum treatment with selenium and vitamin E has been reported to hasten uterine

involution in cows with metritis (Harrison et al., 1986). Treatment with vitamin E and

selenium also increased fertilization rate in cattle (Segerson et al., 1977) and sheep

(Segerson and Ganapathy, 1981). This effect could reflect an action at the cellular level

to regulate free radical generation in the ovary (Harrison and Conrad, 1984), sperm

(Alvarez and Storey, 1989), ovulated oocyte or embryo. Thus, selenium and vitamin E

could act at the level of the ovary, uterus, gamete or developing embryo to enhance

pregnancy rate. Higher levels of vitamin E have been found in the corpus luteum,

compared with muscle or liver tissue (Velasquez-Pereira et al., 1997, personal



P-carotene is a plant derived antioxidant that is also a precursor to vitamin A. It is

more lipophilic than a-tocopherol and is assumed to be present at the interior of

membranes or lipoproteins, which enables it to scavenge free radicals within the

lipophilic compartment more efficiently than does a-tocopherol (Niki et al., 1995). 3-

carotene participates inactivating reactive chemical species, such as singlet oxygen and

free radicals that would otherwise initiate reactions such as lipid peroxidation; it is a very

effective quencher of singlet oxygen (Burton and Ingold, 1984)

P-carotene was first identified in bovine corpus luteum as early as 1913, (Escher,

1913). Lotthammer (1978) proposed that P-carotene might have specific and unique

functions in reproduction. Sklan (1983) found an increase in the amount of n-carotene

being converted in the corpus luteum of cattle immediately after ovulation, suggesting

that n-carotene have a function in the production of progesterone. Graves-Hoagland et al.

(1988), found that progesterone production in vitro by luteal cells was reduced in

response to P-carotene supplementation. Cows fed diets deficient in P-carotene had lower

amounts of progesterone in the corpus luteum (Schultz et al., 1974; Ahlswede and

Lotthammer, 1978). Recently, it has been reported that P-carotene may inhibit adverse

effects of free radicals on the enzymatic activity of adrenal P450scc (Youngs et al., 1995).

Effects of supplemental feeding of P-carotene on cattle fertility remains

controversial. Several authors have found beneficial responses of P-carotene on

reproductive function of cows (Ahlswede and Lotthammer, 1978; Lotthammer et al.,


1978; Meyer et al., 1975; Ascarelli et al., 1985; Rakes et al., 1985), whereas, others have

not (Wang et al., 1982; Bindas et al., 1984a,b; Akordor, et al., 1986) and there is one

report that supplemental P-carotene had an adverse effect on fertility (Folman et al.,

1987). One reason for such discrepancies could be the variation among studies in number

of cows, level of antioxidant supplementation, environment and management practices at

each farm.

However, antioxidant properties of P-carotene might be exerted in other cells as

well by reducing excessive levels of free radicals. An additional effect of P-carotene

might be expected through its metabolism to vitamin A, because supplementation of

vitamin A increased embryonic survival in pigs (Whaley et al., 1997) and development of

superovulated bovine embryos to the blastocyst stage (Shaw et al., 1995). Such effect is

expected to be greater under hot and humid environments because elevated temperatures

may increase free radicals at the cellular level.

Additionally, several other studies have reported a role of vitamin A or P-carotene

for promoting udder health and reducing somatic cell counts (Daniel et al., 1991; Chew,

1993). P-carotene may also have a beneficial role increasing milk yield in lactating dairy




Approximately 1-2% of metabolized oxygen is converted to a reactive oxygen

species (Fulbert and Cals, 1992) and several biochemical systems exist in cells and

extracellular fluid to remove these molecules. Antioxidant systems include molecules

such as P-carotene and vitamin E which act as membrane antioxidants to maintain the

integrity of phospholipids against oxidative damage and peroxidation (McCay and King,

1980; Di Mascio et al., 1991; Dargel, 1992). Various enzymes also remove free radicals.

Among these, glutathione peroxidase is a selenium-dependent enzyme that utilizes

electrons from glutathione and other thiols to convert peroxides to water (Flohd and

Gdnzler, 1976).

Increased generation of free radicals may overwhelm antioxidant defense

mechanisms and compromise cellular function (Freeman and Crapo, 1982; Youn et al.,

1991; Dargel, 1992; Fulbert and Cals, 1992). Production of free radicals could represent

a source of infertility because ovarian steroidogenic tissue (Margolin et al., 1990; Carlson

et al., 1993;Young et al., 1995), spermatozoa (Aitken, 1994) and preimplantation

embryos (Fujitani et al., 1997) are sensitive to free radical damage. In some studies,

administration of P-carotene (Bindas et al., 1984b; Ascarelli et al., 1985; Bonomi et al.,

1994) or vitamin E and selenium (Segerson et al., 1977; Ardchiga et al., 1994) improved

fertility of dairy cattle, while, in other studies, providing high amounts of these

antioxidants were without measurable beneficial effects on fertility (Gwazdauskas et al.,

1979; Schingoethe et al., 1982; Kappel et al., 1984; Bindas et al., 1984a; Rakes et al.,

1985; Akordor et al., 1986; Folman et al., 1987; Hidiroglou et al., 1987; Stowe et al.,


There were two objectives of the present series of studies. The first was to

determine whether n-carotene administered at days -6, -3 and 0 relative to breeding could

increase pregnancy rates of lactating dairy cows exposed to heat stress. We hypothesized

that n-carotene activity might be important during the preovulatory period since injection

of vitamin A, a metabolite of n-carotene, increased development of bovine embryos to the

blastocyst stage (Shaw et al., 1995). Moreover, it was reasoned that antioxidant therapy

would be beneficial particularly in heat-stressed cows because exposure of cells and

tissues to elevated temperature can increase free radical production (Loven, 1988) and

because of evidence in lactating dairy cows that heat stress can lead to a reduction in

blood cell concentrations of the antioxidant glutathione (Trout et al., 1998). The second

objective was to determine whether a single intramuscular injection of vitamin E and

selenium at 30 d after parturition would enhance postpartum reproductive function of

lactating dairy cows in a temperate climate. Previously (Ardchiga et al., 1994), it was

found that injection of vitamin E and selenium at 21 d before expected parturition

reduced services per conception of lactating cows. This effect could, in part, reflect

effects of vitamin E and selenium on uterine health associated with parturition because

injection of vitamin E and selenium can reduce incidence of retained fetal membranes

(Harrison et al., 1984; Eger et al., 1985; Ardchiga et al., 1994). By administering vitamin

E and selenium at 30 d postpartum, it was possible to determine whether or not there is an

enhancement in fertility independent of periparturient changes in uterine and placental


Materials and Methods

Plasma Concentrations of .-Carotene in Cows Injected with [-Carotene

A preliminary experiment was conducted to determine the time trends for plasma

concentrations of P-carotene and selected metabolites in cows receiving i.m. injections of

P-carotene. The experiment was conducted at the University of Florida Dairy Research

Unit (Hague, Florida) in July and August with ten lactating Holstein cows (70-147 d

postpartum) fed a diet similar to that described for Experiment 2, farm 3. Cows were

assigned randomly to treatment with either P-carotene (Lucarotin, BASF, Parsippany,

NJ) (n=4) or physiological saline solution (0.9%, w/v) as a control (n=6). Cows were

injected at 0800 h at days 0, 3 and 6 with either 800 mg: P-carotene or an equivalent

volume of saline (20 ml). Blood samples were collected into heparinized tubes from

coccygeal vessels at days -6, -3, 0 (before injection at 0800 h and 12 h later) 1, 3, 4, 6, 7,

12, and 19 d relative to time of first injection. Plasma samples were analyzed for P3-

carotene, retinol, and retinyl palmitate using high pressure liquid chromatography as

described by Njeru et al. (1992) and Brocas et al. (1998).

Effect of D-Carotene on Fertility of Lactating Dairy Cows

To determine the effect of P-carotene on fertility, an experiment was conducted on

three dairy farms in North Florida (Bell and Hague, Florida) from July-September, 1994

(i.e., during a period of heat stress). Cows were fed the total mixed ration formulated at

each farm composed (dry basis) of 1.5 Mcal/kg Net Energy for Lactation (NFL, 17.4 %

crude protein (CP), 26.4% acid-detergent fiber (ADF), 49.7% neutral-detergent fiber

(NDF), 0.92% Ca, 0.43% P, 0.32% Mg, 2.22% K, 0.56% Na (farm 1); 1.5 Mcal/kg NEL,

18.7 % CP, 24.8% ADF, 45.3% NDF, 0.94% Ca, 0.44% P, 0.37% Mg, 1.12% K, 0.45%

Na (farm 2) and 1.5 Mcal/kg NEL, 16.4 % CP, 26.6 % ADF, 42.4% NDF, 0.70% Ca,

0.46% P, 0.34% Mg, 1.35% K, 0.49% Na (farm 3) (diets analyzed chemically at

Northeast DHIA Forage Laboratory, Ithaca, NY). The dietary content of retinyl

palmitate, n-carotene and retinol was evaluated in a composite sample of 4 weekly

samples of the total mixed rations of each farm. Spectrophotometric analyses were

performed by ABC Research Laboratories at Gainesville, FL using methods 941.15 and

960.45 of the Official Methods of the American Organization of Agricultural Chemists

(AOAC, 1995). Dietary content of P-carotene and retinol were 5124 and 10733 IU/kg

(farm 1); 7731 and 9285 IU/kg (farm 2); and 4101 and 10501 IU/kg (farm 3).

Every two weeks, cows were identified that were either 1) unbred and past the

voluntary waiting period for breeding (40 d postpartum) or 2) bred previously but

subsequently diagnosed nonpregnant. Cows were assigned randomly to control or P-

carotene treatments. Once assigned to treatment, cows received an i.m. injection of 800

mg P-carotene or saline at day 0 (i.e., day -6 before expected estrus). At day 3 (day -3

before expected estrus), cows were injected with 25 mg prostaglandin F2. (Lutalyse,

Upjohn Co. Kalamazoo, MI) and were given a second injection of P-carotene or saline.

When cows were detected in estrus, they were bred via artificial insemination according

to the AM-PM rule (i.e., bred in the afternoon if observed in estrus in morning and bred

in morning if observed in estrus the previous afternoon) and were injected with either 3-

carotene or saline at breeding. Some cows came into estrus and were bred by herdsmen

before receiving all three injections. These were removed from the experiment.

Cows that did not respond to Lutalyse and were not bred subsequently were used

in the experiment again two weeks after initial injection of P-carotene or control. The

cows received the same treatment (P-carotene or placebo) as they initially received.

There were a total of 88 (control) and 77 (P-carotene) observations from 68 (control) and

59 (P-carotene) cows. Pregnancy rates for cows not returning to estrus were evaluated

55-90 d after breeding.

Effect of Injection of Vitamin E and Selenium on Reproductive Function

This experiment utilized 186 lactating dairy cows from a commercial dairy

located in Central Mexico in the vicinity of Coacalco, Estado de Mdxico (1955' latitude

and 9909' longitude), at an elevation of 2200 m above sea level. The region has a humid

and temperate climate with long and moderate summers (annual mean temperature varies

from 12 to 18 C). Cows were used only if the uterus was diagnosed as healthy at 30 d

postpartum by per-rectal palpation (i.e., absence of pyometra and endometritis). Cows

were assigned randomly to either a control group that was not injected or to a group that

received a single intramuscular injection of 10 ml MU-SE (Schering-Plough Animal

Health Corp., Kenilworth, N.J.) at day 30 3 postpartum. The amount of MU-SE

administered is equivalent to 500 mg vitamin E as DL-a -tocopheryl acetate (680 IU) and

109.5 mg of sodium selenite (equivalent to 50 mg selenium).

Estrus detection was performed visually nearly continuously throughout the day.

All cows were bred by one person via artificial insemination after a voluntary waiting

period of 50 d postpartum. Artificial insemination was performed according to the AM-

PM rule. Pregnancy was determined by palpation per rectum at 45 to 60 d after service.

Cows were milked twice a day. During each milking, the cows were fed 2 kg of

alfalfa hay. Afterwards, cows were given ad libitum access to fresh chopped alfalfa or

alfalfa hay, and were fed -28 kg/cow of a mixed concentrate ration based on corn silage

and sorghum that had a calculated composition (dry basis) of 1.67 Mcal/kg NEL, 19.1%

CP, 30.7% NDF and 17.2% ADF. The ration for the cows was formulated to provide

supplemental vitamin E (500 IU/cow/d) and selenium (0.3 ppm/cow/d).

Statistical Analysis

All data were analyzed by least squares analysis of variance using the General

Linear Models Procedure of SAS (SAS, 1989). The mathematical model used to analyze

data for experiment I included main effects of treatment, cow(treatment) and day. For


experiment 2, main effects were treatment, farm, and replicate (i.e., each two week period

in which treatments were initiated). The data for proportion of cows responding to

Lutalyse included some cows used on more than one occasion. For this variable, data

were analyzed two ways: with and without cow(treatment) in the model. For experiment

3, main effects were treatment and parity (parity 1 vs others). Analyses were performed

with all interactions in the model and then reanalyzed after removing interactions that

were non-significant. Effect of treatment on services per conception in Experiment 3

were also analyzed by CATMOD (SAS, 1989) with categories for services per conception

of 1, 2, 3 and 4-5.


Plasma Concentrations of 1-Carotene in Cows Injected with P-Carotene

Intramuscular injection of 800 mg P-carotene increased plasma concentrations of

P-carotene from 2.7 + 0.38 [ig/ml before injection to 5.55 + 0.38 gtg/ml at 24 h after

injection (Figure 3-1). The first, second and third injections of P-carotene resulted in

slightly higher elevations in plasma P-carotene concentrations. Concentrations remained

elevated until the end of the sampling period (i.e., 19 d after the first P-carotene injection

and 13 d after the last injection). Retinyl palmitate concentrations were also increased

from 0.40 + 0.07 gig/ml before injection to 1.08 + 0.07 [tg/ml at 24 h after injection

(Figure 3-1). Concentrations remained elevated until the end of the sampling period.

-6 -3 0

3 6 9 12 15 18

Time (d)

Figure 3-1. Plasma concentrations of n-carotene, retinyl palmitate and retinol from heat-
stressed dairy cows injected, i.m., with 800 mg n-carotene (@) or saline (0) at days 0, 3
and 6 (indicated by arrows). Concentrations of 3-carotene were affected by treatment
(P<0.O01), time (P<0.001) and treatment x time (P palmitate were affected by treatment (P<0.01), time (P<0.001) and treatment x time

There was no difference in retinol concentrations between treated and control cows

(Figure 3-1).

Effect of P-Carotene on Fertility of Lactating Dairy Cows

There was no effect of P-carotene on the proportion of cows that were detected in

estrus after PGF2a treatment, the timing of estrus relative to injection of PGF2. or on the

proportion of animals that conceived to the insemination following PGF2, (Table 3-1).

TABLE 3-1. Effect of administration of P-carotene on estrous synchronization, interval
to estrus, variation of interval to estrus and pregnancy rate.
Control P-carotene

Estrus within 96 h after PGF2, (%)a.e 46.6 + 5.4 (41/88) 49.4 + 5.7 (38/77)
Interval, PGF2, to estrus (d) be 2.83 +0.16 2.90+ 0.16
S.D., interval to estrus (d)C'e 1.03 1.07
Pregnancy rate (%)d,e 24.4 + 6.7 (10/41) 21.6 7.0 (8/37)f
a Least square means + SEM and, in parentheses, number of animals responding/number
of animals injected with Lutalyse (Note some cows were subjected to more than one
Lutalyse injection regimen).
bLeast-squares means + SEM.
Standard deviation.
dLeast squares means + SEM and, in parentheses, number of animals pregnant/number of
animals inseminated at PGF2.-induced estrus.
'All differences between control and P-carotene were non-significant (P>0. 10).
'One cow did not receive the 3 P-carotene injections.

Effect of Injection of Vitamin E and Selenium on Reproductive Function

There was no effect of injection of vitamin E and selenium on interval from

calving to first breeding or pregnancy rate at first breeding (Table 3-2). However, among


cows not pregnant to first service, pregnancy rate at second service was higher (P=0.07)

for cows injected with vitamin E and selenium. Similarly, there were fewer services per

conception (P<0.05) for cows receiving vitamin E and selenium; interval from calving to

conception was less for these cows also. The distribution of services per conception was

affected by treatment (P<0.05) because fewer cows injected with vitamin E and selenium

required four-to-five services to achieve pregnancy (Figure 3-2).

TABLE 3-2. Effect of postpartum administration of Vitamin E and selenium on
reproductive function of lactating dairy cows.
Control Vit. E/selenium
Interval,calving to first service (d) a 63.3 + 2.7 61.7 + 2.5
Pregnancy rate at first service (%)b 46.1 + 5.3 (41/89) 45.4 + 5.1 (44/97)
Pregnancy rate at second service (%)b 52.1 + 7.0 (25/48) 69.8 + 6.6 (37/53)t
Services/conception a 2.0 + 0.11 1.7 + 0.10"
Interval, calving to conception (d)a 98.1 + 4.5 84.6 + 4.3*
aLeast square means + SEM.
b Data represent least-squares means + SEM and, in parentheses, number of animals
pregnant/number of animals bred.
t P=0.07




0 1 2 3 4 5

Services per conception

Figure 3-2. Frequency distribution of services per conception for control cows (black
bars) and cows treated with vitamin E and selenium (open bars). The distribution of
services per conception were affected (P<0.05) by treatment.


It was hypothesized that three injections of P-carotene (at 6 and 3 d before

anticipated estrus and at insemination) would increase pregnancy rate in heat-stressed

cows because the P-carotene would 1) eliminate some of the increased free radicals

produced as a result of heat stress (Loven, 1988; Trout et al., 1998) and 2) increase

concentrations of vitamin A, which has been shown to increase embryonic development

in superovulated cows (Shaw et al., 1995). The injection scheme used was sufficient to

elevate plasma concentrations of P-carotene and retinyl palmitate for at least 13 d after

the day of the last injection. The peak concentration of p-carotene achieved averaged

7.44 gtg/ml, which is similar to concentrations achieved in a study in which feeding of [3-

carotene for at least 90 d increased pregnancy rates by 120 d postpartum under heat-stress

conditions (Chapter 4). Nonetheless, injection of P-carotene did not improve fertility.

There are several possible reasons for this. First, it is possible that the sensitivity of early

embryos to elevated temperature is resistant to antioxidant therapy. While morulae were

made more resistant to heat shock in culture by incubation with glutathione or taurine

(Ealy et al., 1992), no such thermoprotective effect was seen from 2-cell embryos (Ealy et

al., 1995). It may also be that injections of P-carotene did not elevate concentrations of

P-carotene in the oviduct where the embryo resided. Recently, it was found that dietary

supplementation of heat-stressed cows with P-carotene had no effect on pregnancy rate of

heat-stressed cows at first service but did increase pregnancy rate at 120 d postpartum if

cows received P-carotene for at least 90 d (Chapter 4). One such explanation for these

findings is that prolonged exposure to u-carotene was required to sufficiently elevate

concentrations of n-carotene in the oviduct and uterus. It may also be possible, as recent

data suggest (Trout et al., 1998), that the major depletion of antioxidants during heat

stress is in the cytosol and not in the membrane where p-carotene is most active. Finally,

it is possible that, as reported by Shaw et al. (1995), administration of vitamin A can

increase embryonic development but that either 1) these effects are exerted on embryos

from superovulated cows only or 2) the increase in retinyl palmitate achieved in the

present study was not of sufficient magnitude to mimic the effect seen by Shaw et al.


In contrast, the combined administration of vitamin E and selenium at day 30

postpartum increased fertility of lactating dairy cows that were not under heat stress

situations. In particular, injection of vitamin E and selenium increased fertility in cows

receiving two or more inseminations to achieve pregnancy. A single prepartum injection

of vitamin E and selenium also increased fertility (Ardchiga et al., 1994). In this earlier

study, it was thought that vitamin E and selenium may have been increasing fertility by

increasing uterine health because the incidence of retained fetal membranes also was

lowered. Effects on uterine health may have been less in the current study because cows

with uterine disorders at day 30 were excluded from the experiment and because injection

of vitamin E and selenium was delayed until 30 d after parturition. Nonetheless, given

the likelihood of subclinical infections, some beneficial effects of vitamin E and selenium

on fertility could be a consequence of improved uterine health. Administration of vitamin

E and/or selenium can enhance neutrophil function (Eicher et al., 1994; Ndiweni and

Finch, 1996; Politis et al., 1995; 1996). Perhaps, increased neutrophil activity promotes

removal of microorganisms, uterine tissue remodeling and involution. Prepartum

treatment with selenium and vitamin E has been reported to hasten uterine involution in

cows with metritis (Harrison et al., 1986). Treatment with vitamin E and selenium might

also increase oocyte quality or sperm survival since treatment with vitamin E and

selenium increased fertilization rate in cattle (Segerson et al., 1977) and sheep (Segerson

and Ganapathy, 1981).

It is not surprising that a single injection of vitamin E and selenium could cause a

positive effect on reproductive function several weeks after the injection because

administration of these molecules has long-term effects in cattle. Intramuscular injection

of vitamin E caused elevated amounts in serum for at least 28 d (Charmley et al., 1992)

whereas injection of selenium increased concentrations of selenium in whole blood and

serum for 28 d and whole blood-glutathione peroxidase activity for at least 84 d (Maas et

al., 1993). Injections of vitamin E and selenium 3 and 1.5 weeks before calving increased

erythrocyte GSH peroxidase in dairy cows during the first 12 weeks of lactation (Lacetera

et al., 1996).

Effects of selenium, vitamin E or their combination on fertility have been variable

with some reports of an increase in fertility (Segerson et al., 1977; Ardchiga et al., 1994)

and some reports of no effect (Gwasdauskas et al., 1979; Schingoethe et al., 1982; Kappel

et al., 1984; Hidiroglou et al., 1987; Stowe et al., 1988). Differences between studies in

the amount of vitamin E and/or selenium administered, the period of administration, and

nutritional status of the experimental animals with respect to vitamin E and selenium


intake could explain some of these differential results. Given the effectiveness of vitamin

E and selenium as administered in the present study, and the possible importance of

antioxidant status for heat-stressed cows (Chapter 4; Trout et al. 1998), it will be

important to determine if injection of vitamin E and selenium increases fertility of heat-

stressed cows.



Heat stress drastically reduces pregnancy rates in dairy cows. In Florida,

conception rates decline from 40 to 50% during cool months to less than 10% during the

hot months of the year (Badinga et al., 1985). In addition to effects on embryonic

mortality (Putney et al., 1989b; Ealy et al., 1993), heat stress reduces duration and

intensity of behavioral estrus (Gangwar et al., 1965; Madan and Johnson, 1973; Abilay et

al., 1975) so that a smaller proportion of cows is detected in estrus during heat-stress

conditions (Thatcher and Collier, 1986). The major strategy to reduce the effects of heat

stress on reproduction has been to alter the environment of the cow through the use of

shade, fans, or evaporative cooling (Bucklin et al., 1991). However, this approach has

not eliminated all problems associated with heat stress. For example, for dairy herds in

Arizona, the interval from calving to conception during summer remained 24 to 67 d

longer than did the interval during winter even though during summer cows were

maintained in barns with evaporative coolers (King et al., 1988). As a result, additional

reproductive strategies are needed to counteract the adverse effects of heat stress.