Responses of bovine oocytes and preimplantation embryos to elevated temperature

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Responses of bovine oocytes and preimplantation embryos to elevated temperature
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Al-Katanani, Yaser Mohammad, 1969-

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RESPONSES OF BOVINE OOCYTES AND PREIMPLANTATION EMBRYOS TO
ELEVATED TEMPERATURE: POSSIBLE CAUSES OF EMBRYONIC LOSS AND
STRATEGIES TO IMPROVE FERTILITY UNDER HEAT STRESS CONDITIONS











iA
,.. ... .. --- 4


'By


YASER MOHAMMAD AL-KATANANI












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

UNIVERSITY OF FLORIDA


2001











This dissertation reflects the sacrifice, guidance and encouragement of many

people. This dissertation is dedicated to family:

My parents; Mohammad Kamal Al-Katanani (late) and Fatmeh Abdulaziz.

My brothers, Fathi (late), Fakhri, Ibrahim, Kamal, Yousef, Khaled, and Ismail.

My sisters, Fatheyeh, Halemeh, Amal, En'am, and Afaf.

All members of Al-Katanani family.

For their endless love and support throughout my academic career.














ACKNOWLEDGMENTS

The author would like to express deep thanks, appreciation and gratitude to Dr.

Peter J. Hansen, chairman of the supervisory committee, for his guidance, support,

training and encouragement throughout the program. Throughout the years, Dr. Hansen

has become more than a mentor and I can proudly call him a friend. I extend my

appreciation to my committee members: Dr. William W. Thatcher for his encouraging

words at some difficult times and for allowing me to use his laboratory to analyze

samples from the last project; Dr. Maarten Drost for his patience and active participation

in all the embryo transfer studies; Dr. Michael Fields and Dr. Kenneth Drury. I thank all

committee members for their insight and contribution to this dissertation.

I would like to extend my sincere thanks to collaborators at the University of

Florida and elsewhere; Dr. Dan Webb for providing the DHIA data; Dr. Ray Bucklin for

his help retrieving the weather data for Gainesville; and Dr. Joe West; University of

Georgia, for providing the weather data for Georgia; I also have great appreciation and

thanks to Dr. Jack Rutledge and Rick Monson from the University of Wisconsin for

providing the oocytes and the embryos for the embryo transfer experiments. I also thank

Central Packing-Co. management and personnel at Center Hill for providing the ovaries

used for various experiments. Special thanks go to John Gilliland and the farm personnel

at McArthur dairy for their help during the field trial. I also extend my thanks to Dale

Hissem and David Armstrong for their help with the experiments at the Dairy Research

Unit.








My sincere and special thanks are extended to current and previous fellow

graduate students in the laboratory for their help and contribution in the lab and in the

field. I owe special thanks to Dr. Carlos Arechiga, Dr. Morgan Peltier, Andrew

Majewski, Charles Krininger III, Jeremy Block, Dr. Fabiola-Paula Lopes, Rocio Rivera,

Saban Tekin, Dr. Paolete Soto, Olga Ocon and Heather Rosson. The experiments

described in this dissertation were done with a tremendous amount of help and labor from

all of them and could not have been completed without their help. I extend my thanks to

Marie-Joelle Thatcher and Oscar Hemrnandez for their help with the radioimmunoassay.

Last but not least, I want to extend thanks for my friends in the United States and

abroad for their friendship, help and support: Dr. Abdullah Alshankiti, Dr. Mohammad

Al-Widyan, Dr. Fahiem El-Borai, Dr. Ayden Guzeloglu, Dr. Metin Pancarci, Dr. Andres

Kowalski, Dr. Mario Binelli, Dr. Ricardo Mattos, Dr. Marcio Liboni, Ines Aviles; Dr.

Nasser Odetallah; Dr. Sami Kamal, Waleed Alatout; Ala' Samara; Basel Mujahed; Emad

Al-Far, Mohammad Al-Rashdan and Mohammad El-Mashaleh.















TABLE OF CONTENTS

Rage

ACKNOWLEDGMENTS ................................................................................................. iii

LIST OF TABLES................................................... ....... ix

LIST OF FIGURES.................................................... ............................................... xi

A BSTRA CT ......................................................... .................................................... xii

PR E FA C E ......................................................................................................................... xv

CHAPTERS

1 REVIEW OF LITERATURE .......................................................................................... 1

Effect of Heat Stress on Reproduction in Dairy Cattle ................................................... 1
Effect of Heat Stress on Estrus Detection............................................................... 2
Effect of Heat Stress on Pregnancy Rates Following Insemination ........................ 3
Effect of Heat Stress on Placental Growth.......................................................... 4
Association of Milk Yield and Fertility............................................ ..................... 5
Detrimental Effects of Heat Stress on Fertility........................................................... 6
Effect of Heat Stress on Oocyte Quality..................................................................... 6
Effect of Heat Stress on Embryo Development.................................................... 10
Effect of Heat Stress on Luteal Function .................................................................. 11
Effect of Heat Stress on the Reproductive Tract ................................................. 13
Breed Differences in Response to Heat Stress...................................................... 14
Mechanisms Involved in Embryonic Resistance to Heat............................................. 16
Relationship to Embryonic Genome Activation................................. ...................... 16
Role of Heat Shock Proteins and Induced Thermotolerance .................................... 19
Role of heat shock proteins in embryonic development....................................... 21
Ontogeny of induced thermotolerance.................................................................. 22
Apoptosis ..................................................................................................................... 23
Free Radicals and Antioxidants ............................................................................ 25
Strategies to Improve Conception Rate Under Heat Stress Conditions........................ 27
Cooling Cows to Improve Conception Rate .................................................. ...... 27
Long-term cooling of cows................................................................................... 27
Short-term cooling of cows.......................................................... ..................... 28
Improving Estrus Detection................................................................................... 30
Use of Timed Artificial Insemination ...................................... ................................. 31









Heat stress and timed artificial insemination..................................................... 34
Use of Embryo Transfer to Improve Pregnancy Rates............................................. 36
Cryopreservation of In Vitro Derived Embryos....................................................... 38

2 FACTORS AFFECTING SEASONAL VARIATION IN 90-DAY NONRETURN
RATE TO FIRST SERVICE IN LACTATING HOLSTEIN COWS IN A HOT
C L IM A T E ..................................................................................................................... 45

Introduction ................................................................. .................................................. 45
Materials and Methods .................................................................................................. 47
D ata ........................................................................................................................... 47
Analysis of Location and Milk Yield Effects..................... ...................................... 47
Analysis of the Relationship Between Meteorological Data and 90-d NRR............ 49
R results ........................................................................................................................... 5 1
Effect of Location on Seasonal Variation in 90-d NRR ........................................... 51
Effect of ME Milk Yield on Seasonal Variation in 90-d NRR................ ............. 51
Variation of 90-d NRR due to ME Milk Yield and Interval to First Service........... 51
Effect of Environmental Variables at Specific Days Relative to Insemination on 90-
d N R R ....................................................................................................................... 54
D discussion .................................................................. ................................................... 57

3 EFFECT OF SEASON AND EXPOSURE TO HEAT STRESS ON OOCYTE
COMPETENCE IN HOLSTEIN COWS...................................................................... 60

Introduction ...................................................................................................................60
Materials and Methods .................................................................................................. 61
M materials .................................................................................................................. 6 1
General Procedures for In Vitro Fertilization (IVF) ................................................. 62
Experiment 1 Effect of Season on Oocyte Competence of Holstein Cows............ 63
Experiment 2- Effect of the Magnitude of Heat Stress on Oocyte Quality............. 64
Animals and treatments ......................................................................................... 64
In vitro embryo production ................................................................................... 65
Determination of cell number ............................................................................... 66
Statistical Analyses............................................................................................. 67
R results ........................................................................................................................... 67
Experiment 1: Effect of Season on Oocytes Recovered from Abattoir ....................67
Experiment 2: Seasonal Variation and Effect of Magnitude of Heat Stress .... ....... 67
Discussion................................................................ 70

4 INDUCED THERMOTOLERANCE IN BOVINE TWO-CELL EMBRYOS AND
THE ROLE OF HEAT SHOCK PROTEIN 70 IN EMBRYONIC
DEVELOPMENT ........................................................................ ........................... 74

Introduction .................................................................................. ................................. 74
Materials and Methods .................................................................................................. 76
M materials ................................................................................................................... 76
In Vitro Production of Embryos ............................................................................... 77









Induction of Therm tolerance at the Two-Cell Stage............................................... 78
Role of HSP70i in Em bryonic Development............................................................ 80
Statistical Analysis .................................................................................................... 81
Results........................................................................................................................... 82
Induction of Therm tolerance at the Two-Cell Stage............................................... 82
Role of HSP70i in Embryonic Developm ent............................................................ 86
Discussion ..................................................................................................................... 89

5 PREGNANCY RATES FOLLOWING TIMED EMBRYO TRANSFER WITH FRESH
OR VITRIFIED IN VITRO PRODUCED EMBRYOS IN LACTATING DAIRY
COWS UNDER HEAT STRESS CONDITIONS ........................................................ 93

Introduction................................................................................................................... 93
M materials and M ethods.................................................................................................. 94
M materials ................................................................................................................... 94
Anim als..................................................................................................................... 95
Production of Vitrified Embryos.............................................................................. 97
Production of Fresh Embryos................................................................................... 99
Preparation of Embryos for Transfer........................................................................ 99
Embryo transfer .................................................................................................. 100
Statistical Analysis .................................................................................................. 101
Results ......................................................................................................................... 102
Comparison of TAI with TET-F and TET-V .......................................................... 102
Effect of BCS, ME and PPI Classes on Pregnancy Rate........................................ 104
Re-expansion of Embryos After Thawing........................... ................................... 107
Discussion................................................................................................................... 107

6 GENERAL DISCUSSION ................................ ................................................ .......... 110

APPENDICES

A SEASONAL VARIATION IN DEVELOPMENT OF IN VITRO PRODUCED
BOVINE EM BRYOS.................. '. .............................................................. . 121

Introduction ................................................................................................................. 121
M materials and M ethods ................................................................................................ 122
Results...................................................................................... .................................. 122
Discussion ................................................................................................................... 124

B EFFECT OF HEAT SHOCK AND CRYOPRESERVATION METHOD ON
SUBSEQUENT EMBRYO SURVIVAL UPON TRANSFER TO RECIPIENTS .... 125

Introduction................................................................................................................. 125
M materials and M ethods................................................................................................ 126
Anim als and Treatm ents ......................................................................................... 126
Embryo Transfer ...................................... ........................................................ ....... 127
Embryo Collection........................................................................................... ....... 128






viii


Results and Discussion ................................................................................................ 128

REFEREN CES ................................................................................................................ 131

BIO GRAPHICAL SKETCH ........................................................................................... 157














LIST OF TABLES


Table Page

1-1. Pregnancy rates following strategic cooling in lactating dairy cows ............................ 29

1-2. Pregnancy rate (PR) per first AI and cumulative pregnancy rate by 100 or 120 d
after insemination following OvSynch protocol or estrus synchronization and
insemination upon detected estrus in lactating dairy cows.................................. 33

1-3. Pregnancy rates (PR) per first AI and cumulative pregnancy rates by 90 or 120 d
after insemination following OvSynch protocol or estrus synchronization and
insemination upon detected estrus in lactating dairy cows under heat stress
conditions............................................................................................................. 35

2-1. Least squares means + SEM of 90 day non-return rate (90-d NRR) to first service as
affected by average daily temperature at day -10, 0 and +10 relative to
insemination in subsets of Holstein cows that did not experience average daily
temperatures > 25C either on the 10 days before or after the studied day.........56

3-1. Effect of season and degree of heat stress on oocyte competence ................................ 69

4-1. Embryonic development to the blastocyst stage following heat shock at the two-cell
stage ..................................................................................................................... 83

4-2. Induction ofthermotolerance in bovine embryos at the two-cell stage......................... 84

4-3. Ontogeny of induced thermotolerance in bovine embryos at the two-cell stage...........85

5-1. Pregnancy rate at day 45 following timed artificial insemination (TAI) or timed
embryo transfer using fresh (TET-F) or vitrified (TET-V) embryos................... 103

5-2. Effect of body condition score class, mature equivalent milk yield class and
postpartum interval class on pregnancy rates in cows following time artificial
insemination (TAI) or timed embryo transfer using fresh (TET-F) or vitrified
(TET-V ) em bryos................................................................................................. 105

5-3. Effect of milk yield on pregnancy rate after timed artificial insemination (TAI) or
timed embryo transfer using fresh (TET-F) or vitrified (TET-V) embryos.........106






x


B-1. Survival rate following transfer of fresh or frozen-thawed in vitro derived embryos..130














LIST OF FIGURES


Figure Page

2-1. Seasonal variation in 90-d non-return rate to first service in South Georgia, North
Florida and South Florida ..................................... ............................................. 52

2-2. Seasonal variation in 90-d non-return rate to first service as affected by ME milk
yield ................................................................................................................... 53

2-3. Regression coefficients for the relationship between ME milk yield and 90-d non-
return rate to first service (90-d NRR) as affected by month of first breeding .... 55

3-1. Effect of season on the percentage of Holstein oocytes that cleaved and developed
into blastocysts on d 8 after insemination .......................................................... 68

4-1. Proportion of two-cell embryos that developed into blastocysts on d 8 after
insemination following culture with anti-HSP70i or control mouse IgG,
antibody ............................................................................................................... 87

4-2. Proportion of two-cell embryos that developed into blastocysts on d 8 after
insemination following culture with anti-HSP70i or control mouse IgGi
antibody. ............................................................................................................. 88

6-1. Model depicting responses of bovine oocytes and early embryos to heat stress at
different days relative to fhe day of insemination (d 0) as determined by
experim ents in this dissertation............................................................................ 113

A-1. Development of bovine two-cell embryos to the blastocyst stage as affected by
month of oocyte collection and by exposure to heat shock during culture in
C R laa ................................................................................................................ 123















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

RESPONSES OF BOVINE OOCYTES AND PREIMPLANTATION EMBRYOS TO
ELEVATED TEMPERATURE: POSSIBLE CAUSES OF EMBRYONIC LOSS AND
STRATEGIES TO IMPROVE FERTILITY UNDER HEAT STRESS CONDITIONS
By

Yaser Mohammad Al-Katanani

December 2001

Chairman: Peter J. Hansen
Major Department: Animal Sciences

Pregnancy rates in lactating dairy cows per insemination are decreased during the

summer in hot climates. The deleterious effects of heat stress could be exerted on both

the developing oocyte and the preimplantation embryo. The goals of this dissertation

were 1) to determine effects of heat stress on oocyte competence, 2) to develop strategies

to protect the early embryo from the adverse effects of elevated temperatures and 3) to

develop strategies to improve fertility in lactating dairy cows under heat stress conditions.

An initial study was done to evaluate the magnitude of heat stress associated

infertility using Dairy Herd Improvement Association records on first service from 8124

Holstein cows from three locations. The decline in 90-d non return rate to first service

was of lower magnitude and shorter duration in South Georgia than in North or South

Florida. Also, the depression of 90-d non return rate was more pronounced in cows






xiii


producing more milk. In a second series of analyses, heat stress before and after breeding

as well as on the day of breeding was associated with low 90-d non return rate.

To determine effect of season and magnitude of heat stress on oocyte competence

in Holstein cows, two experiments were carried out where oocytes were collected and

subjected to in vitro maturation, fertilization and culture. In the first experiment, there

was a summer depression in the proportion of oocytes and cleaved embryos that

developed to the blastocyst stage. In the second experiment, non-lactating Holstein cows

were housed in one of three environments for 42 d before slaughter; two groups in

summer [heat stress (HS) or cool (C)] and one group in the winter (W). There was a

summer depression in oocyte quality in Holstein cows, and cooling cows for 42 d during

the summer did not alleviate this seasonal effect.

The 2-cell bovine embryo is very sensitive to heat shock but can also produce heat

shock protein 70 (HSP70) in response to elevated temperature. Accordingly, experiments

were conducted to test whether 2-cell embryos could be made to undergo induced

thermotolerance since this phenomenon is often dependent upon HSP70 synthesis.

Induced thermotolerance refers to the phenomena whereby exposure to a mild heat shock
t
makes cells more resistant to a subsequent, more severe heat shock. Regardless of the

conditions used, it was not possible to demonstrate induced thermotolerance in embryos

at the 2-cell stage. A role of HSP70 was indicated, however, since culture of embryos

with the inducible form of HSP70 antibody reduced the proportion of 2-cell embryos that

developed to blastocyst. Moreover, sensitivity to heat shock was not increased by

addition of HSP70 antibody to the culture medium.









A field trial was conducted to confirm beneficial effects of timed embryo transfer

on d 7 after anticipated ovulation as compared to timed artificial insemination (TAI) and

to determine the efficacy of vitrification as a method for embryo cryopreservation. Non-

pregnant, lactating Holstein cows in the summer were subjected to the Ovulation

Synchronization (OvSynch) protocol and were inseminated either on the day of

anticipated ovulation (TAI) or received a fresh or vitrified embryo on day 7 after

anticipated ovulation. Pregnancy rate following embryo transfer of fresh embryos was

higher than that after TAI and pregnancy rate following transfer of vitrified embryos was

no better than that following TAI.

Taken together, results suggested that oocyte quality in Holstein cows is reduced

during the summer. Lack ofthermotolerance in embryos at the 2-cell stage might explain

the sensitivity of embryos at this stage to elevated temperature. Finally, pregnancy rates

could be improved during the summer in hot climates by transferring fresh embryos to

recipients.










PREFACE

Pregnancy rates in lactating dairy cows are decreased significantly during the

summer under heat stress conditions (Ingraham et al., 1974; Badinga et al., 1985;

Cavestany et al., 1985; Putney et al., 1989a; Arechiga et al., 1998). This depression in

fertility is exacerbated by lactation because the high metabolic rates achieved by high

producing cows lower the upper critical temperature at which cows can regulate body

temperature (Berman et al., 1985). Heat stress also adversely affects reproduction in

cows by reducing the detection of estrus. The proportion of estrous cows not detected in

estrus by herdsmen was estimated to be about 80% during the summer months in Florida

compared to 66% in the cool months (Thatcher and Collier, 1986).

The adverse effects of elevated temperatures on fertility are likely exerted at

several levels including the uterus (Geisert et al., 1988a), corpus luteum (Howell et al.,

1994), oocyte (Baumgartner and Chrisman, 1987; Putney et al., 1989b; Edwards and

Hansen, 1996; 1997), and the preimplantation embryo (Putney et al., 1988a; Ealy et al.,

1993; Edwards and Hansen, 1997; Rivera and Hansen, 2001). Embryos acquire

resistance to elevated temperatures as they proceed in development (Dunlap and Vincent,

1971; Ealy et al., 1993; 1995; Edwards and Hansen; 1997; Arechiga and Hansen, 1998).

For example, exposure of superovulated dairy cows to elevated temperatures on d 1 after

insemination (i-e. 2-cell stage) decreased the proportion of live embryos recovered on d 8

while exposure to heat stress had no effect on embryonic development when applied on d

3, 5 and 7 after insemination (Ealy et al., 1993).

The overall objective of this dissertation was to develop information that can be

used to develop practical systems for overcoming effects of heat stress on fertility in








dairy cattle. There were three main objectives of this dissertation. The first objective

was to determine the effects of heat stress on oocyte competence. About 84-85 d are

required for the follicle to grow from the primordial stage to the preovulatory stage

(Picton et al., 1998; McNatty et al., 1999) and about half this time represents growth from

the early antral stage to the preovulatory stage (Lussier et al., 1987). Knowing the

window of oocyte sensitivity to elevated temperatures may be used for improving fertility

because it will allow guidance as to when cooling should be provided as well as types of

pharmacological manipulation that alter oocyte physiology. For example, one approach

to cooling cows is to use strategic cooling (Hansen, 1997) where cows are cooled at

certain times around the time of insemination. Pregnancy rates were improved in some

studies (Gauthier, 1983; Wise et al., 1988a; Ealy et al., 1994) while others did not find

any advantage of short term cooling (Stott and Wiersma, 1976; Her et al., 1988). Perhaps

heat stress is affecting oocytes during stages of development earlier than when cooling

was initiated in some studies.

To determine the effects of heat stress on oocyte competence, two approaches

were followed. First, a retrospective study was done using Dairy Herd Improvement

Association (DHIA) records for 8124 Holstein cows from three different locations (South

Georgia, North and South Florida) to evaluate effects of heat stress on certain days

relative to insemination on subsequent fertility estimated by 90-d non return rate (90-d

NRR). Further experiments were conducted to evaluate seasonal variation in oocyte

competence as determined by the ability of oocytes collected from Holstein cows to

become fertilized and give rise to blastocysts after in vitro maturation, fertilization and

culture. Additionally, an experiment was conducted to test whether oocyte quality in the






xvii


summer is affected by the magnitude of heat stress and whether cooling cows for 42 d

before oocyte collection will improve oocyte competence.

The second objective was to determine whether embryos at the 2-cell stage can

undergo induced thermotolerance and to test the role of the inducible form of heat shock

protein 70 (HSP70i) in embryonic development. Induced thermotolerance refers to the

process whereby exposure of cells to a mild heat shock makes them more resistant to

subsequent, more severe heat shock. It has been shown that bovine and murine embryos

can undergo induced thermotolerance (Ealy and Hansen, 1994; Arechiga et al., 1995).

This phenomenon has been correlated with heat shock protein 70 (HSP70) and

glutathione (Riabowol et al., 1988; Li et al., 1991; Arechiga et al., 1994; Arechiga et al.,

1995). Achieving induced thermotolerance at the 2-cell stage might lead to strategies to

decrease embryonic mortality. There is a reason to believe the 2-cell bovine embryo may

undergo induced thermotolerance. Although the embryonic genome in bovine embryos

does not become fully activated until the 8 to 16-cell stage (Memili and First, 2000),

some transcription has been reported at the 1-cell stage (Saeki et al., 1999; Memili and

First, 1999) and bovine embryos can synthesize HSP70 upon exposure to elevated

temperatures at the 2-cell stage (Edwards and Hansen, 1996; Edwards et al., 1997;

Chandolia et al., 1999).

Finally, a field trial was conducted to confirm the beneficial effects of timed

embryo transfer to bypass early embryonic mortality caused by heat stress and to evaluate

vitrification as a method for improving pregnancy rates after transfer of in vitro derived

embryos. Embryo transfer has been used to improve fertility in heat-stressed cows by

bypassing the adverse effects of heat stress on the oocyte and early embryo (Putney et al.,





xviii


1988a; 1989b; Ambrose et al., 1999; Drost et al., 1999). In addition to the intensive labor

and the need for high synchrony between donors and recipients, some reports indicated

that response to superovulation and the quality of embryos recovered is reduced during

the summer (Gordon et al., 1987; Putney et al., 1988c; Ryan et al., 1993). Embryo

cryopreservation and the use of in vitro produced embryos provide alternative sources of

embryos for practitioners. However, in vitro derived embryos did not survive freezing as

well as their in vivo counterparts (Leibo and Loskutoff, 1993; Pollard and Leibo, 1993;

1994; Wurth et al., 1994; Hasler et al., 1995; Drost et al., 1999). Recent advances in

estrus synchronization using hormones to synchronize ovulation tightly lead to the

development of the ovulation synchronization system (OvSynch) where cows could be

inseminated at a specific time without the need for estrus detection (timed artificial

insemination) or receive an embryo 7 d after the day of anticipated ovulation (timed

embryo transfer). In one study in Florida during the summer, pregnancy rates were

improved after timed embryo transfer using fresh in vitro derived embryos (Ambrose et

al., 1999). However, pregnancy rates did not differ between the timed embryo transfer

using frozen-thawed in vitro derived embryo and timed artificial insemination groups.













CHAPTER 1
REVIEW OF LITERATURE


Effect of Heat Stress on Reproduction in Dairy Cattle

Stress is defined as the magnitude of forces external to a system that act to

displace the system from the ground state (Yousef, 1984). One such stress for

homeothermic animals is heat stress. In dairy cattle, for example, ability of the dairy cow

to maintain core body temperature at 38.5C is compromised under heat stress

conditions. Rectal temperature of lactating dairy cows has been reported to range from

38.6C to 40.5C in Florida (Putney et al., 1989b; Wolfenson et al., 1995; Rivera and

Hansen, 2001). The increase in heat production associated with lactation exacerbates

regulation of body temperature so that lactating cows have higher body temperatures than

non-lactating cows (Cole and Hansen, 1993). The upper critical temperature in lactating

dairy cows at which hyperthermia occurs was reported to be as low as 27C (Berman et

al., 1985). Among the many consequences of heat stress on dairy cattle physiology is a

reduction in reproductive efficiency caused by decreased estrus detection (Thatcher and

Collier, 1986), increased embryonic mortality (Putney et al., 1988a; 1989a; Ealy et al.,

1993) and reduced fetal growth (Bonsma, 1949; Collier et al., 1982).

The mechanism by which heat stress decreases reproductive performance depends

on the magnitude of the hyperthermia experienced by dairy cows. The following sections

will present an overview of the adverse effects of heat stress on different aspects of

reproductive function including estrus detection, placental growth, oocyte quality and









early embryo mortality. Since early embryonic loss is a major cause of decreased

reproductive function during heat stress, and forms the topic of this dissertation, emphasis

will be placed on effects of heat stress on this aspect of reproductive function. Given the

importance of effects of heat stress on reproduction for overall economic efficiency of the

farm, possible strategies to improve fertility during heat stress will be presented as well.

Effect of Heat Stress on Estrus Detection

A primary cause for decreased reproductive function under heat stress conditions

is lower estrus detection rate. In a study conducted on a commercial dairy farm, Thatcher

and Collier (1986) reported that about 76- 82% of potential heats were undetected during

the months of June through September, while 44-65% of potential heats were missed

during October through May. Similarly, fewer cows were detected in estrus when the

temperature-humidity index was greater than 72 (Cartmill et al., 2001). Heat stress

causes a reduction in the duration (Abilay et al., 1975; Gwazdauskas et al., 1981;

Wolfenson et al., 1988a) and intensity (Gangwar et al., 1965) of estrus. Recently, Nebel

et al. (1997) used a remote mounting detection system and found a decrease in the

number of mounts in Holstein cows per estrus during the summer when compared to

winter.

It is controversial whether the reduced expression of estrus under heat stress

conditions is related to lower concentrations of plasma estradiol (Gwazdauskas et al.,

1981; Wise et'al., 1988a; 1988b; Badinga et al., 1993). In one study, Her et al. (1988)

reported more frequent silent ovulations or anestrus when cows were subjected to heat

stress. Reduced physical activity of cows during heat stress (Lotgering et al., 1985; Bell,

1987) might be another factor that reduces estrus detection









One approach to increase the proportion of cows detected in estrus is cooling at

different times relative to insemination. Cooling cows was associated with increased

estrus detection rate in some studies (Her et al., 1988; Wolfenson et al., 1988a; Younas et

al., 1993). On the other hand, Ealy et al. (1994) reported that cooling cows from 2 to 3 d

before breeding until d 5 to 6 after breeding did not significantly improve detection of

estrus compared to the non-cooled group although the proportion was higher for the

cooled cows (47% vs. 37%).

Effect of Heat Stress on Pregnancy Rates Following Insemination

Low pregnancy rates following insemination have been associated with heat stress

(Erb et al., 1940; Stott, 1961; Ingraham et al., 1974; Badinga et al., 1985; Cavestany et

al., 1985; Putney et al., 1989a, Ardchiga et al., 1998). Experimental application of heat

stress to cows reduced pregnancy rate (Dunlap and Vincent, 1971) and increased

embryonic mortality (Putney et al., 1988a; Ealy et al., 1993) whereas abatement of heat

stress during the summer increased pregnancy rate (Stott et al., 1972; Thatcher et al.,

1974; Roman-Ponce et al., 1977; Ealy et al., 1994).

The adverse effects of heat stress on fertility depend on the stage of development

when heat stress is applied. Ingrahamn et al. (1976) reported a negative correlation

between humidity on d 11 before insemination and subsequent fertility in Holstein cows.

Also, Dutt(1963) found that fertility in sheep was decreased when ewes were subjected

to heat stress 12 d before insemination. Moreover, Putney et al. (1989b) reported that

exposure of superovulated heifers to high temperature and humidity at the onset of estrus

for 10 h resulted in higher percentage of retarded embryos recovered on day 7 after

insemination. These results suggest that heat stress can adversely either affect follicular

development or that the oocyte can be disrupted by exposure to elevated temperatures.









Heat stress during early stages of development can affect subsequent survival of

embryos. In one study, exposure ofsuperovulated Holstein heifers to heat stress (30C

for 16 h and 42C for 7 h) from d 1 to d 7 after estrus resulted in an increase in the

proportion of retarded embryos with degenerate blastomeres recovered non-surgically on

d 7 (Putney et al., 1988a). While 51.5% of the embryos were normal in the control group

(20C), only 20.7% of the embryos were normal in the heat-stressed group. This

experiment suggests that embryos are sensitive to elevated temperatures during early

stages of development. Embryos seem to acquire resistance to elevated temperatures as

they proceed in development. Exposure of superovulated lactating dairy cows to heat

stress on d 1 after insemination reduced the percentage of live embryos recovered on day

7 while heat stress on d 3, 5 or 7 after insemination did not have an effect on subsequent

embryo survival (Ealy et al., 1993). The acquisition of thermal resistance as embryos

proceed in development has been reported in vitro as well. In particular, Edwards and

Hansen (1997) reported that fewer 2-cell bovine embryos developed to the blastocyst

stage upon exposure to elevated temperatures when compared to 4- to 8-cell embryos and

morulae. Although embryos become more resistant to heat stress as they develop, the

adverse effects of heat stress on fertility are not limited to early embryos. Exposure of

cows to elevated temperatures between d 8 and 16 of pregnancy resulted in lower embryo

size when embryos were recovered on d 17 (Biggers et al., 1987).

Effect of Heat Stress on Placental Growth

Placental function and subsequent milk production could be compromised during

the last third of pregnancy under heat stress conditions. Most of the fetal growth (Eley et

al., 1978) and mammary gland development (Byatte et al., 1994) occur during later stages









of pregnancy. During heat stress, blood flow increases to the peripheral tissues

(Alexander et al., 1987). As a consequence, exposure of pregnant sheep to elevated

temperatures resulted in decreased blood circulation to the uterus (Oakes et al., 1976;

Brown and Harrison, 1981) and placenta (Alexander et al., 1987). Thus, placental

hormone secretion (Collier et al., 1982; Malayer and Hansen, 1990a), size of the placenta

(Thatcher and Collier, 1986), placental weight (Head et al., 1981), total placental DNA

(Vatnick et al., 1991) and calf birth weight (Bonsma, 1949; Collier et al., 1982) can be

reduced under heat stress conditions.

A correlation between calf birth weight and subsequent milk production has been

reported (Chew et al., 1981; Collier et al., 1982). Such a result might suggest a decrease

in milk production after parturition when cows are exposed to heat stress during later

stages of pregnancy. Wolfenson et al. (1988b) found that cooling cows during the dry

period resulted in increased subsequent milk production. Collier et al. (1982).reported

similar effects that were not significant statistically.

Association of Milk Yield and Fertility

The increase in milk production experienced by dairy cows as a result of genetic

selection and improved management practices has been associated with a reduction in

pregnancy rates. In a study by Nebel and McGilliard (1993), pregnancy rates to first

service upon detected estrus declined from about 70% in heifers to about 52-38% in

lactating dairy cows as milk production increased. This data suggests that reproductive

performance is compromised by high levels of milk production. Also, analysis of a large

data set containing records between 1972 and 1996 in Kentucky indicated that an

increase in milk production that was associated with increased calving interval, days open

and services per conception (Silvia, 1998). In a recent review article by Lucy (2001),








other factors were cited that could compromise fertility of dairy cows (i.e. herd size,

estrus detection rate, disease incidence and postpartum energy balance).

One way in which high milk yield could exacerbate fertility is by increasing

animal susceptibility to heat stress. As milk yield increases, metabolic rate increases

(Berman and Morag, 1971) and, hence, thermoregulatory ability of cows with high milk

production is decreased (Berman et al., 1985). Under heat stress conditions in Florida,

conception rates were reported to be as low as 10-15% during the summer months

(Badinga et al., 1985). Similarly, the adverse effects of heat stress on reproduction could

be exacerbated by high milk yield. The evidence for this idea comes primarily from

comparison of lactating vs. non-lactating cows. The increase in body temperature upon

heat stress exposure is greater for lactating cows when compared to non-lactating cows

(Cole and Hansen, 1993). Also, the summer depression in fertility that occurs in lactating

cows in Florida was not observed in non-lactating heifers (Badinga et al., 1985).


Detrimental Effects of Heat Stress on Fertility

The deleterious effects of heat stress on fertility could be exerted on the

developing oocyte, early embryo and/or reproductive tract. The following sections will

review the evidence for effects on each of these systems and describe the mechanisms by

which heat stress decreases fertility.

Effect of Heat Stress on Oocyte Quality

Several researchers have evaluated the effect of season on oocyte competence as

determined by the ability ofoocytes to be fertilized and give rise to blastocysts in vitro.

In one study in Louisiana, Rocha et al. (1998) reported a higher proportion ofoocytes

that developed to the blastocyst stage during the cool months compared to hot months









when oocytes were collected from Holstein cows via ultrasound guided aspiration. In

another study using Holstein oocytes collected from an abattoir in Wisconsin (Rutledge et

al., 1999), there was a decrease in the proportion of oocytes developing to blastocysts

during July and August compared to other months. In Israel (Zeron et al., 2001), the

proportion of Holstein oocytes developing to blastocysts following chemical activation

was reduced during the summer months. Heat stress may disrupt oocyte quality by

disruption of follicular development or by directly altering oocyte function

Follicular growth refers to the development of the follicle from the primordial

stage (-35 grm) to the preovulatory stage. The estimated time for this process in cattle is

about 84-85 d (Picton et al., 1998; McNatty et al., 1999). Initiation of oocyte growth

from the primordial stage seems to be dependent on paracrine and autocrine factors rather

than endocrine factors (McNatty et al., 1999). One factor that has been identified to

initiate follicular growth is the kit ligand from the granulosa cells and the c-kit receptor

on the oocyte (Picton et al., 1998). However, a recent report (Reynaud et al., 2001) in

mice has shown no effect of kit receptor mutation on the initiation of folliculogenesis,

although oocyte growth and granulosa cell proliferation were decreased in the mutant

mice. It has been reported that receptors for FSH and LH were not expressed until the

follicle is above 2-4 mm in diameter (Gong et al., 1996). The time required for a follicle

to grow from the early antral stage to the preovulatory stage has been estimated to be

around 42 d (Lussier et al., 1987). Growth of the follicle is coincident with a high rate of

oocyte transcription and translation to produce proteins and RNA that are needed for

oocyte growth and also some RNAs that will be stored in the oocyte and used for early

embryonic development. In one study (Fair et al., 1997), it was found that oocytes do not








express transcriptional activity until the secondary and tertiary follicular stage.

Moreover, the RNA synthesis seems to be inactivated when the oocytes reach a diameter

of 110 gm (i.e. 2-3 mm follicle) (Fair et al., 1996; Hyttel et al., 1997). Also, Fair et al.

(1995) reported that 3H-uridine incorporation was higher in oocytes < 110 Pam when

compared to oocytes > 110 pm. These data suggest that certain insults during oocyte

growth and development might compromise oocyte quality and adversely affect

subsequent embryonic development. Consistent with this idea is the finding by Ingraham

et al. (1976) who reported a negative correlation between humidity on d 11 before

insemination and subsequent fertility in Holstein cows. Also, Dutt (1963) found that

exposure of ewes to a temperature of 90F on d 12 before insemination increased

subsequent embryonic death.

It has been reported (Badinga et al., 1993) that growth of the dominant follicle is

reduced during heat stress, which might cause incomplete dominance and increase

number of subordinate follicles (Wolfenson et al., 1995). Incomplete dominance could

result in ovulation of an aged follicle; such follicles contain oocytes with reduced fertility

(Mihm et al., 1999). This depression in oocyte quality during the summer might be due

in part to the fact that unlike other cells, the oocyte is transcriptionally inactive after

reaching about 110 pm, i.e., the 2-3 mm follicle (Hyttel et al., 1997) and does not

undergo increased synthesis of heat shock protein 70 in when exposed to heat shock

(Edwards and Hansen, 1997). Elevated temperature could conceivably have deleterious

effects on oocyte growth, protein synthesis or formation of transcripts required for

subsequent embryonic development. Indeed, low developmental competence of oocytes








was associated with shorter polyadenylation tail transcripts during oocyte maturation

(Brevini-Gandolfi et al., 1999).

Oocytes have been reported to be sensitive to elevated temperatures during the

final stages of maturation. Heat stress reduced the proportion of live embryos recovered

on day 7 after insemination when superovulated cows were exposed to high temperatures

for 10 h beginning at the onset ofestrus (Putney et al., 1989b). Research in mice has

shown that exposure ofoocytes to 35C for 12.5 h during maturation caused meiotic

arrest at metaphase II in 40% of the oocytes studied (Baumgartner and Chrisman, 1987).

In another study, Fiorenza and Mangia (1992), found that exposure of mouse oocytes

during maturation to a temperature of 38.5-40C blocked oocytes at metaphase I and

increased the incidence of chromosome morphological abnormalities. Moreover, protein

synthesis was reduced and resumption of meiosis was inhibited when preovulatory mouse

oocytes were exposed to elevated temperature (Curci et al., 1987). Similar findings have

been reported in bovine oocytes where exposure of oocytes to 41C during maturation

reduced the number ofoocytes that progressed to metaphase II (Lenz et al., 1983). Also,

the proportion ofoocytes that developed to blastocyst was reduced from 35% to 18%

when oocytes were exposed to 41C during the first 12 h of maturation in vitro (Edwards

and Hansen, 1997). In another experiment, exposure ofoocytes during the first 12 h of

maturation to 41C or 42C reduced protein synthesis by oocytes (Edwards and Hansen,

1996). Moreover, the percentage decrease in protein synthesis was more pronounced in

denuded oocytes compared to oocytes with the cumulus intact when oocytes were

exposed to 42C during the first 12 h of maturation (Edwards and Hansen, 1996). This

might suggest a protective role of the cumulus cells during oocyte maturation.









Effect of Heat Stress on Embryo Development

Embryonic development and subsequent survival are compromised when early

embryos are exposed to elevated temperatures. Data in cattle (Ealy et al., 1992; 1995;

Edwards and Hansen 1997; Rivera and Hansen, 2001), sheep (Dutt, 1963), and mice

(Gwazdauskas et al., 1992; Arechiga et al., 1994; Ealy and Hansen, 1994) have indicated

that exposure of early embryos to elevated temperatures reduced viability and embryo

development. The findings of these experiments suggest that early embryos may lack

some protective mechanisms and or molecules that are required for protection against the

adverse effects of elevated temperatures.

As mentioned previously, embryos acquire resistance to the deleterious effects of

heat stress as they advance in development. Exposure of superovulated lactating cows to

elevated temperatures at d 1 after breeding when embryos are at the 1- to 2-cell stage

(Ealy et al., 1993) reduced viability of embryos when recovered on d 8 after breeding.

Similarly, Dunlap and Vincent (1971) reported that exposure of beef heifers to high

temperature (32C) for 72 h immediately after breeding resulted in 0% conception rate

compared to 48% in the control cows kept at 21C. However, heat stress on d 3, 5 or 7

after breeding did not affect subsequent embryonic development or survival on d 8 (Ealy

et al., 1993). Similar results have been reported in vitro. Edwards and Hansen (1997)

found a reduction in the proportion of 2-cell embryos that developed to the blastocyst

stage following heat shock. However, in the same study, embryos at the 4- to 8-cell stage

were more resistant to heat shock. Furthermore, embryos at the morula stage were not

affected by heat shock. Similarly, Ealy et al. (1995) found that exposure of bovine

embryos at the 2-cell stage to elevated temperature reduced the proportion of 2- cell








embryos that developed to the blastocyst stage but has no effect on subsequent embryonic

when embryos at the morula stage were exposed to heat shock. In mice, Ardchiga and

Hansen (1998) found that the proportion of embryos that developed to the blastocyst

stage was reduced when heat shock (41C for 1 h) was applied at the 2-cell stage as

compared with embryos at the 4-cell stage and morula stage.

The deleterious effect of heat stress might be exerted on the embryo or on the

microenvironment where the embryo resides during early development. Results of a

reciprocal embryo transfer study (Alliston and Ulberg, 1961) found that the adverse

effects of elevated temperatures were exerted on both the embryo and the reproductive

tract, with more deleterious effects on the embryo itself. In this study, animals were kept

either at thermoneutral temperature (21 C) or were heat stressed (32C). Embryos were

recovered on d 3 after insemination and transferred to recipients also kept either at 21 or

32C using a reciprocal embryo transfer scheme. Embryo survival for animals

maintained at 21 C was 56%. However, only 9.5% of embryos survived when donors

were maintained at 32C and recipients at 21C. Embryo survival was 24% when donors

were kept at 21C and recipients at 32C.

Effect of Heat Stress on Luteal Function

Embryonic development might adversely be affected by heat stress because of a

disruption in luteal function. In one experiment, Biggers et al. (1987) found that

exposure of cows to a temperature of 37C for 12 h followed by 33C for the remainder

of the day between d 8 and 16 of pregnancy reduced the size of the embryos and luteal

tissue weight recovered on d 17. This decrease in the embryo size might compromise

embryonic ability to produce interferon-r (IFN-x) which is necessary for maternal








recognition of pregnancy and prevention of luteolysis. In one study, Geisert et al.

(1988b) found that the bovine embryo must be around 15 mm in length on d 15 to 17 of

pregnancy in order to produce IFN-T. Exposure of d 17 bovine embryos to heat shock

(43C for 18 h) in vitro decreased IFN-T secretion by 72% (Putney et al., 1988b). In

contrast, exposure of cows to heat stress from d 8 to 16 of pregnancy did not affect IFN-r

secretion when embryos were recovered on d 17 and cultured in vitro (Geisert et al.,

1988a).

Heat stress may also cause an increase in prostaglandin F2. (PGF2a) secretion that

leads to luteolysis of the corpus luteum. Indeed, Wolfenson et al. (1993) reported an

increase in uterine production of PGF2a in response to oxytocin on d 17 under heat stress

conditions. Moreover, exposure ofendometrium collected on d 17 of the estrous cycle to

elevated temperatures in vitro increased synthesis ofPGF2a (Malayer et al., 1988, Putney

et al., 1989c; Malayer and Hansen, 1990a).

Results regarding the effects of heat stress on the peripheral concentrations of

progesterone during the luteal phase of the estrous cycle are not consistent. In some

reports, heat stress has been found to increase peripheral progesterone concentration

(Abilay et al., 1975; Roman-Ponce et al., 1981; Trout et al., 1998). However, other

reports found a decrease (Rosenberg et al., 1982, Younas et al., 1993, Howell et al.,

1994) or no effect (Wise et al., 1988a, Wolfenson et al., 1995) of heat stress on peripheral

progesterone concentrations. In cases where an increase in progesterone concentrations

was found might be due to a possible acute heat stress effects on adrenal secretion of

progesterone (Thatcher, 1974). In contrast, low progesterone concentrations during heat

stress were associated with reduced blood flow and morphological changes to the corpus








luteum (CL) as reported in hyperthermic rabbits (Wolfenson and Blum, 1988). In a

recent review article (Wolfenson et al., 2000), the author suggested that the discrepancy

regarding effects of heat stress on progesterone may reflect the difference between the

acute and chronic heat stress. In particular, acute heat stress causes an increase and

chronic causes a decrease in progesterone concentrations.

Effect of Heat Stress on the Reproductive Tract

Establishment of pregnancy depends on close communication between the mother

and the developing embryo. Heat stress and the hyperthermia associated with it can

compromise the function of the oviduct and the uterus. The results of reciprocal embryo

transfer study by Alliston and Ulberg (1961) suggested that the adverse effects of heat

stress were exerted on the embryo as well as on the reproductive tract with more

pronounced effect on the embryo itself

Heat stress could act on the reproductive tract by altering protein secretion and

hormone function. In one study, Malayer et al. (1988) reported a reduction in the

secretion of several proteins when endometrial explants were exposed to heat shock

(43C) compared to controls (39C) for 24 h. However, this effect was dependent upon

the day of the estrous cycle. The effects of heat shock were more pronounced in tissues

collected on d 0 or 2 of the cycle compared to d 5 or 8. Moreover, protein secretion of 7

polypeptides was reduced in uterine tissues collected from the uterine horn ipsilateral to

the corpus luteum but not from tissues contralateral to the corpus luteum. Thus, there

may be a local effect of ovulation at least at the beginning of the cycle (i. e. d 0 and 2).

Exposure of endometrial explants to elevated temperatures (42 or 43C) in vitro caused

an increase in the synthesis of heat shock proteins 72 (HSP72) and heat shock protein 90








(HSP90) (Malayer et al., 1988; Malayer and Hansen, 1990a; 1990b). Since these proteins

are associated with the estrogen and progesterone receptor complex (Smith and Toft,

1993; Johnson et al., 1996, Nair et al., 1996, Sabbah et al., 1996), the increase in those

proteins following heat shock could conceivably affect steroid receptor function.

One of the consequences of heat stress is the redistribution of blood flow from the

visceral organs to the periphery (Roman-Ponce et al., 1978; Alexander et al., 1987),

which might cause a decrease in the perfusion of nutrients and hormones (i. e.

progesterone and estradiol) to the endometrium and oviduct. The results regarding the

effect of heat stress on peripheral estradiol concentrations are not consistent. A recent

report (Wolfenson et al., 1995) reported an increase in peripheral concentrations of

estradiol-17p between d 1 and 4 of the estrous cycle and a reduction from d 4 through 8.

Also, Wilson et al. (1998a; 1998b) reported a decrease in peripheral concentrations of

estradiol-17p during d 11 through 21 of the estrous cycle. However, Roman-Ponce et al.

(1981) and Wise et al. (1988a, 1988b) reported an increase in the concentration of

estradiol-177p as a result of heat stress.

Breed Differences in Response to Heat Stress
t
The ability of cows to regulate their body temperature is critical for their fertility.

In one study, Gwazdauskas et al. (1973) reported an association between uterine

temperature and fertility. Tropically-adapted breeds have lower body temperature under

heat stress conditions (Finch, 1986; Hammond et al., 1996; 1998). Moreover,

reproductive performance for Zebu cows in tropical and subtropical environments is

higher than for European-type cows (Peacock et al., 1971; 1977). Rocha et al. (1998)

reported a depression in oocyte quality following in vitro maturation, fertilization and








culture when oocytes were collected from non-lactating Holstein and crossbred Angus

cows in Louisiana. In another study, there was a summer depression in the proportion of

oocytes that developed to the blastocyst stage in Wisconsin (Rutledge et al., 1999) when

oocytes were collected from an abattoir that slaughters primarily Holstein cows.

In Israel, Zeron et al. (2001) found that development of oocytes collected from

Holstein cows at an abattoir following chemical activation was reduced during the

summer. In contrast, no seasonal variation was seen in a study in Florida using oocytes

recovered at an abattoir (Rivera et al., 2000). In the latter study, oocytes were from both

beef and dairy cows (-75% feedlot and crossbred cattle; 25% Holstein) and the lack of

seasonal variation may reflect a preponderance of cows that have superior

thermoregulatory mechanisms (for example, Bos indicus breeds, Finch, 1986). Similarly,

no seasonal variation in in vitro fertilization performance was found in oocytes collected

from Brahman cows (Rocha et al., 1998).

Differences between tropically-adapted breeds and non-adapted breeds in

response to elevated temperatures have been found to occur at the cellular level also. In

one study, Malayer and Hansen (1990b) found that exposure of oviducts to heat shock

(43 C) caused a depression in protein synthesis from both oviducts collected from

Brahman cows. In contrast, the depression in protein synthesis due to heat shock in

Holstein cows was only seen in the oviduct ipsilateral to corpus luteum. In another study,

Paula-Lopes et al. (2001) found that the deleterious effect of heat shock (41C for 6 h) on

subsequent embryo development was more pronounced for embryos from Holstein and

Angus cows when compared to embryos from Brahman cows. It is likely that embryos

from Brahman cows have more effective mechanisms to stabilize cellular function in








response to heat shock. One possibility is that synthesis of heat shock proteins and

antioxidant content is different between breeds since those molecules have been

associated with thermal resistance of embryos. However, Kamwanja et al. (1994) found

no difference in HSP70 synthesis when lymphocytes from Angus, Brahman and Senepol

heifers were exposed to elevated temperatures.


Mechanisms Involved in Embryonic Resistance to Heat

Embryos become more resistant to deleterious effects of elevated temperatures as

they proceed in development. The following sections will discuss possible association of

embryonic resistance to heat shock with genomic activation. In addition, the possible

role of heat shock proteins and other thermoprotective molecules will be addressed.

Relationship to Embryonic Genome Activation

Early embryonic development depends on maternal mRNA stored in the oocyte

since the embryonic genome is transcriptionally inactive for a period after fertilization.

Embryonic genome activation (EGA) refers to the transition from control by maternally-

inherited transcripts to the production of transcripts that are of embryonic origin. This

process is associated with gradual degradation of maternally-inherited transcripts. The

sensitivity of early embryos to stress (Ealy et al., 1992; 1995; Edwards and Hansen,

1997; Rivera and Hansen, 2001) might be due to the fact that the embryonic genome is

largely suppressed during early cleavage stages. Thus, the range of cellular adaptive

responses utilized by the early embryo is likely to be reduced as compared to embryos

that have completed embryonic genome activation.

The timing for activation of the embryonic genome is a species-specific process.

In the mouse, while a minor round of transcription has been reported at the 1-cell stage,








the genome is not fully activated until the 2-cell stage (Matsumoto et al., 1994; Schultz et

al., 1995). Mouse embryos cultured with a-amanitin, an inhibitor ofRNA polymerase II

(Bensaude et al., 1983; Conover et al., 1991), do not develop beyond the 2-cell stage

(Rambhatla and Latham, 1995). Also, the appearance of zygotic transcripts in mouse

embryos was not detected until the 2-cell stage (Davis and Schultz, 1997; Aoki et al.,

1997). These data suggest that the major genomic activation occur at the 2-cell stage in

mouse embryos. In cattle, some transcription can occur as early as the 1-cell stage (Saeki

et al., 1999; Memili and First, 1999). Also, bovine embryos have been shown to undergo

transcription at the 2-cell stage and synthesize the heat-inducible form of heat shock

protein 70 (HSP70i) after heat shock (Edwards and Hansen, 1996; Edwards et al., 1997;

Chandolia et al., 1999). The embryonic genome is not fully activated until the 8-16-cell

stage (Barnes and First, 1991; Jones and First, 1995; De Sousa et al., 1998). However,

bovine embryos cultured with a-amanitin were blocked at the 8-16 cell stage (Barnes and

First, 1991).

In the marine and bovine embryo, alterations in chromatin structure have been

associated with the onset of genomic activation (Shultz et al., 1995; Thompson et al.,

1995; Memili and First, 1999; Ma et al., 2001). It is proposed that a more loose

chromatin structure at the time of DNA replication allows transcription factors to have

access to their binding sequences in the DNA and initiate transcription (Wolffe, 1994;

Davis and Schultz, 1997). In bovine embryos, Memili and First (1999) reported a

dramatic decrease in transcriptional and translational activity in embryos at the 1- and 2-

cell stage labeled with 3H incorporation in the presence of the DNA replication inhibitor

aphidicolin. Similar results were reported in mouse embryos (Ma et al., 2001).








Moreover, genomic activation has been associated with a decrease in the translation

initiation factor (elF-lA), and aphidicolin prevents this decline at the late 2-cell stage in

mouse embryos (Davis et al., 1996).

There is evidence that the degree of histone acetylation may also be involved in

genomic activation. Histones become hyperacetylated from the 1- to the 2-cell stage in

the mouse (Schultz et al., 1995) at a time coincident with transcriptional activity.

Treatment of mouse embryos at the 2- and 4-cell stage with trapoxin to induce histone

hyperacetylation enhanced incorporation ofBrUTP (Aoki et al., 1997). On the other

hand, inhibition ofhistone deacetylase in bovine embryos at the 1- and 2-cell stage using

tricostatin A resulted in transcriptional activity that was not different from the control

embryos (Memili and First, 1999). In addition, Segev et al. (2001) found that early

bovine embryos express histone deacetylase 1, 2 and 3.

The role of cell-cycle factors in genomic activation in bovine embryos was tested

by Jones and First (1995). It was proposed that cell cycle events regulate transcription

through phosphorylation of the large subunit of the RNA polymerase II. Indeed, RNA

polymerase II was present in the nonphosphorylated inactive form at the 2-cell stage and

the phosphorylated form at the 8-cell 'stage. Moreover, maternal stores of the cell cycle

control protein CDC25 were present until the 8-cell stage; the first embryonic expression

ofcdc25 was seen at the late 8-cell stage. Also, overexpression ofcdc25 at the 4- and 8-

cell stage suppressed transcription and shortened cell cycle to a length similar to this

found at the 2-cell stage. Based on these findings, Jones and First (1995) speculated that

the high levels of CDC25 protein during early stages of development (i.e. 2-cell stage)

keep RNA polymerase II in an inactive form. As the embryo uses those factors,








repression of the C-terminal domain kinases is relieved and the RNA polymerase II

becomes activated via phoshorylation.

Role of Heat Shock Proteins and Induced Thermotolerance

Cells and embryos have evolved a homeostatic adaptive mechanism to respond to

different insults in their microenvironment by producing a set of proteins called heat

shock proteins. This phenomenon was first characterized in Drosophila where exposure

of cells to heat shock resulted in a puffing pattern on polytene salivary gland

chromosomes (Ritossa, 1962) and synthesis of a distinct set of proteins termed heat shock

proteins (Tissieres et al., 1974).

Six families of heat shock proteins (HSP) have been characterized. Five of these

are named according to molecular weight and include HSP1 10, HSP90, HSP70, HSP60,

and HSP27. The sixth one, Ubiquitin is an 8.5 kDa protein involved in labeling

proteins for destruction by proteolysis (Kochevar et al., 1991; Nover and Scharf, 1991).

Many heat shock proteins are produced in the absence of heat shock and serve as

molecular chaperones by interacting with other proteins to keep them in a stable

conformation (Hendrick and Harti, 1993). Thus, heat shock proteins are involved in

steroid receptor action, endocytosis, transfer of proteins across the mitochondria, and

folding of nascent proteins during biosynthesis.

Like other heat shock proteins, members of the HSP70 family play a role in cell

function in the absence of stress as well as in protecting cells from damage associated

with heat shock and other cellular insults. There are about 6 to 7 members of the HSP70

genes in mice and human (Georgopoulos and Welch, 1993). Constitutively expressed

genes for HSP70 are called collectively heat shock cognate 70 (HSC70). While produced

in the absence of heat shock, expression increases upon heat shock (Welch et al., 1989).









A second type of HSP70 family are the heat-inducible genes, which are present in low

amounts in unstressed cells while expression increased greatly upon exposure to elevated

temperatures and other stresses (Welch et al., 1989). The third type of the HSP70 family

is regulated by glucose rather than by heat shock. The HSP70 family members are weak

ATPases that are involved in post-translational modification of proteins (Georgopoulos

and Welch, 1993; Lund, 1995), transport of proteins into or through intracellular

membranes and as ATPases causing disassembly ofclatherin-coated vesicles (Pelham,

1984; Nover and Scharf, 1991; Mager and DeKruijff, 1995).

Upon heat shock, there is an increase in synthesis of HSP70. The expression of

hsp70 gene is regulated by heat shock factors (HSF) that bind to DNA sequences called

heat shock response elements (HSE) at the promoter region of the targeted hsp70 gene.

There are at least three HSF: HSF1, HSF2, and HSF3 (Rabindran et al., 1991; Scheutz et

al., 1991; Nakai and Morimoto, 1993; Pirkkala et al., 2001). HSF3 is exclusively avian

(Tanabe et al., 1998). Only HSF1 and HSF3 are activated upon heat shock or other

stresses (Baler et al., 1993; Fawcett et al., 1994; Fiorenza et al., 1995; Pirkkala et al.,

2001) while HSF2 is involved in gene expression regulation in the absence of heat shock

(Sistonen et al., 1994). The HSFs are associated with HSP70 in the cytosol in the

absence of heat shock. Upon exposure to stress, HSP70 dissociates from HSF and binds

hydrophobic motifs that are exposed upon denaturation or damage of proteins (Ananthan

et al., 1986). That allows phosphorylation and trimerization of HSF1 which then

translocates to the nucleus and bind to HSE (Nover, 1991; Westwood et al., 1991;

Morimoto et al., 1996) to activate transcription of hsp70.








Cells can be made more resistant to heat shock by increasing HSP70. In one

study, microinjection of HSP70 mRNA (Hendrey and Kola, 1991) increased survival of

mouse oocytes to heat shock. In contrast, Riabowol et al. (1988) demonstrated that

fibroblasts were more susceptible to heat shock after injection of HSP70 antibodies.

HSP70 is believed to protect cells from the damaging effects of heat shock by

maintaining protein structure, refolding denatured proteins, stabilizing the cytoskeleton

and helping to preserve ribosomal function (Mizzen and Welch, 1988; Georgopoulos and

Welch, 1993; Liang and MacRae, 1997).

Role of heat shock proteins in embryonic development

Heat shock proteins are important for early embryonic development. In mouse

embryos, transcripts for heat shock protein 70 are among the first of embryonic origin to

be produced (Bensaude et al., 1983; Conover et al., 1991; Shultz et al., 1995).

Additionally, culture of mouse embryos with antibodies specific for HSP60, HSP70 or

HSP90 reduced subsequent embryonic development (Neuer et al., 1998). In this study,

the proportion of embryos that hatched by d 5 decreased from 68% in the control group to

29% when anti-HSP60 was used, 28% with the anti-HSP70 and 57% when embryos were

cultured with HSP90 antibody. Als6, transfection of mouse embryos at d 2.5 after coital

plug with antisense oligonucleotides complementary to the stress-inducible form of

HSP70 mRNA reduced embryonic development to the blastocyst stage (Dix et al., 1998).

While 90% of the control embryos developed to blastocysts, only 30% of the treated

embryos became blastocysts when transfected with 5 pLM of HSP70 antisense, and 0%

became blastocysts when the higher concentration (10 p.M) was used. In bovine

embryos, administration of anti-HSP70 to the culture medium at 50 pg/ml from d 3 until









d 9 after insemination reduced the proportion of cleaved oocytes that became blastocysts

to 7.7% compared to 17.3% in the control (Matwee et al., 2001). However, development

to the blastocyst stage was not affected when the concentration of the antibody was

reduced to 0.1 [ig/ml. These data suggest a role for HSP70 in embryonic development as

early as d 3 after insemination.

Ontogeny of induced thermotolerance

Induced thermotolerance refers to the phenomena whereby exposure of cells to a

mild heat shock makes them more resistant to subsequent, more severe heat shock

(Nover, 1991). Heat shock protein 70 has been implicated in induction of

thermotolerance (Sato et al., 1996; Gabai et al., 2000). Indeed, microinjection of HSP70

antibodies increased the sensitivity of fibroblasts to heat shock (Riabowol et al., 1988).

Also, administration of mRNA for the constitutive form of HSP70 makes fibroblasts

more resistant to heat shock and induced thermotolerance (Li et al., 1991).

Induced thermotolerance has been shown to occur in both murine (Muller et al.,

1985; Ealy and Hansen, 1994; Arechiga et al., 1995) as well as bovine embryos at the

blastocyst stage (Ealy and Hansen, 1994). Induction of thermotolerance has been shown

to depend on the stage of development when embryos were exposed to different

treatments. While exposure of mouse embryos to a mild heat shock at the 1-cell (Muller

et al., 1985) or 2-cell stage (Arechiga and Hansen, 1998) did not make embryos more

resistant to a subsequent severe heat shock, it was possible to induce thermotolerance in

mouse embryos at the 8-cell, morula and the blastocyst stages of development (Muller et

al., 1985; Ealy and Hansen, 1994; Arechiga et al., 1995, Arechiga and Hansen, 1998).









Similarly, it was not possible to induce thermotolerance in the in vitro derived bovine

embryos on d 3 or d 4 after insemination (Jyh-Chemg et al., 1999), but exposure of

bovine embryos at the blastocyst stage to a mild heat shock made them more resistant to a

subsequent severe heat shock (Ealy and Hansen, 1994).

The response of the embryo to induced thermotolerance treatments was affected

by the culture medium. Ealy et al. (1994) reported that mouse embryos cultured in

serum-free medium (morula and blastocyst stage) did not undergo induced

thermotolerance although embryos cultured with serum are capable of induced

thermotolerance. The effects of serum seems independent of HSP70 synthesis because

mouse embryos cultured in serum-free medium were able to synthesize HSP70 upon

exposure of embryos to heat shock (Edwards et al., 1995). Also, a temperature of 40C

for 1 h was sufficient to induce thermotolerance in bovine embryos at the blastocyst stage

(Ealy and Hansen, 1994) even though this temperature did not cause an increase in

HSP70 in another study (Edwards et al., 1995). In other cells also, induction of

thermotolerance occurred independently from the synthesis of heat shock proteins

(Kampinga et al., 1992; Borrelli et al., 1996). Taken together, embryonic synthesis of

HSP70 is not the only limiting factor that determines the ability of embryos to undergo

induced thermotolerance and other unclassified molecules can also be involved.

Apoptosis

Apoptosis is a form of cell death that results in the removal of damaged or

abnormal cells without induction of an inflammatory response. Morphologically, it is

distinguished by DNA degradation caused by endonucleases which cleave the DNA

between nucleosomes and give rise to 180-200 bp DNA fragments (Wyllie et al., 1980;









Arends and Wyllie, 1991). Those DNA fragments can be detected in situ using terminal

deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) procedure

(Gavrieli et al., 1992).

Apoptosis has been reported in mouse (Brison and Schultz, 1997), human (Hardy,

1989; Yang et al., 1998), and cow embryos (Paula-Lopes and Hansen, 2000; Matwee et

al., 2000; 2001). In one study, Matwee et al. (2000) did not detect apoptotic cells in

bovine embryos using TUNEL procedure at early stages of development (zygote, 2- and

3-7 cell embryos). There was, however, an increase in the proportion of apoptotic cells

as embryos proceeded in development. Failure to detect apoptosis before the 8-16 cell

stage suggests that apoptosis is developmentally regulated and may be associated with the

major round of genome activation which occur at the 8-16 cell stage in bovine embryos

(Barnes and First, 1991; Jones and First, 1995; De Sousa et al., 1998). Perhaps, apoptosis

depends on some mechanisms that are transcriptionally regulated and dependent upon

full embryonic genome activation.

Heat shock induces apoptosis in cells by activating sphingomyelinase that cleaves

sphingomyelin in the cell membrane to generate ceramide as a second messenger.

Ceramide activates protein kinase /c-jun N-terminal kinase through a series ofkinases

that leads to apoptosis (Veheij et al., 1996). Heat shock protein 70 has been reported to

block apoptosis (Mosser et al., 1997; McMillan et al., 1998; Samali et al., 1999) by

blocking the release of cytochrome c from mitochondria (Mosser et al., 2000) and

SAPK/JNK activation (Mosser et al., 1997).

Exposure of bovine embryos to elevated temperatures was associated with

increased apoptosis. Paula-Lopes and Hansen (2000) found that exposure of bovine








embryos on d 5 after fertilization (> 16-cells) to 40 and 41C for different times (3, 6, 9

and 12 h) increased the proportion ofblastomeres undergoing apoptosis. In another

experiment, it was possible to block apoptosis if embryos were exposed to a mild heat

shock of 40C for 80 min prior to exposure to 41C for 9 h (Paula-Lopes and Hansen,

2000). The temperature used (40C for 80 min) has been shown earlier (Ealy and

Hansen, 1994) to induce thermotolerance in bovine embryos at the blastocyst stage.

Perhaps previous exposure of embryos to a mild heat shock caused an increase in HSP70

synthesis and blocked heat-induced apoptosis.

Free Radicals and Antioxidants

Production of reduced oxygen species during heat shock is one of the causes for

disruption of cellular function associated with this stress (Loven, 1988). Heat shock can

cause an increase in activity of enzymes that produce free radicals including

cyclooxygenase (Malayer et al., 1990) and xanthine oxidase (Skibba et al., 1989).

Production of free radicals and other reactive oxygen molecules can be detrimental by

causing oxidation of DNA, proteins and lipids. Antioxidants such as vitamin E and P-

carotene maintain the integrity ofphospholipids in cell membranes by interacting with

free radicals and preventing damage caused by oxidative damage and peroxidation.

Glutathione in the cytoplasm has a role as a free-radical sink by donating an electron to

reduce reactive oxygen species. Moreover, glutathione has been implicated in induced

thermotolerance. Depletion of glutathione increased sensitivity of cells to heat shock

(Mitchell et al., 1983; Russo et al., 1984; Will et al., 1999) and administration of

extracellular antioxidants conferred thermoprotection in CHO cells (Kapiszewska and

Hopwood, 1988) and lymphocytes (Malayer et al., 1992). Enzymes such as superoxide









dismutase and glutathione peroxidase also contribute to cells antioxidant defense by

catalyzing the reduction of free radical species (Di Mascio et al., 1991).

Embryos possess some of these protective mechanisms against reactive oxygen

species. In the mouse, embryonic glutathione content declines after fertilization until a

nadir is reached at the blastocyst stage (Gardiner and Reed, 1994). However, mouse

embryos were able to synthesize glutathione as early as the 2-cell stage (Gardiner and

Reed, 1995a). In cattle, the glutathione content of the embryo decreased from the 1-cell

to the 2- to 8-cell stage and then start increasing again at the 9- to 16-cell stage of

development (Lim et al., 1996). Moreover, the cow embryo through the morula stage

possesses transcripts for cupper-zinc superoxide dismutase, catalase, and glutathione

peroxidase (Harvey et al., 1995).

There is evidence that glutathione is important for embryonic development.

Inhibition of glutathione synthesis caused a reduction in embryo development in the

murine (Gardiner and Reed, 1995b; Ar6chiga and Hansen, 1998) and bovine (Takahashi

et al., 1993). Moreover, oocyte competence was reduced when the synthesis of

glutathione was inhibited (Edwards and Hansen, 1997). Also, supplementation of culture

medium with reduced glutathione (GSH) improved embryonic development in mice

(Legge and Sellens, 1991; Gardiner and Reed, 1994) and cattle (Luvoni et al., 1996).

Antioxidants may also play a role in protecting embryos against heat shock.

Inhibitory effects of heat shock on embryonic development were reduced when

antioxidants including glutathione, vitamin E and taurine were added to the culture

medium in both bovine (Ealy et al., 1992) and murine embryos (Malayer et al., 1992;

Arechiga et al., 1994; 1995). Resistance of mouse embryos at the morula stage to heat








shock was increased when glutathione synthesis was stimulated by addition of S-

adenosyl methionine to culture medium (Ar6chiga et al., 1995). In the same study,

embryonic content of glutathione tended to decrease following heat shock, suggesting a

role for glutathione in eliminating free radicals that are generated by heat shock.


Strategies to Improve Conception Rate Under Heat Stress Conditions

Several reproductive management programs have been practiced to improve

reproductive performance of lactating dairy cows under heat stress conditions. Those

include cooling cows to alleviate the adverse effects of hyperthermia on the oocyte and

early embryo, estrus synchronization to reduce effects of heat stress on estrus detection,

and embryo transfer to bypass the effects of heat stress on the oocyte and early embryo.

The following sections will discuss some of these strategies and their impact on

reproductive performance of dairy cows.

Cooling Cows to Improve Conception Rate

Long-term cooling of cows

The best cooling strategy is one that allows for long-term continued cooling. This

is so because heat stress can affect both the oocyte (Putney et al., 1989b; Edwards and

Hansen, 1996; 1997; Rocha et al., 1998; Zeron et al., 2001) and the embryo (Alliston and

Ulberg, 1961; Putney et al., 1988a; Ealy et al., 1993). The estimated time for an oocyte

to grow from the primordial follicle to the preovulatory stage is about 84-85 d (Picton et

al., 1998; McNatty et al., 1999). In one study, Thatcher et al. (1974) found that cooling

cows in Florida for 79 or 148 days in an air-conditioned environment during the day or

continuously improved conception rates when compared to cows kept without shade or

cows that were cooled at night only. In another study, Roman-Ponce et al. (1977)









reported that providing shade for lactating dairy cows during the summer in Florida

resulted in conception rates of 44.4% compared to 25.3% in cows without shade.

Wolfenson et al. (1988a) showed that conception rates in Israel were restored in the

summer by frequent cooling of cows for 150 days postpartum (59% in the cooled vs. 17%

in the non cooled group). Cows were cooled by sequential wetting (30 sec) and forced

ventilation (4.5 min) for 7.5 h periods at intervals of 2 h between 0730 and 1830.

Short-term cooling of cows

The concept of cooling cows during the time when oocytes and early embryos are

most sensitive to heat stress has been referred to as strategic cooling (Hansen, 1997). It

depends on the synchronization of the estrous cycle and providing cooling for a limited

number of days around the time of insemination. Some improvement has been reported

following provision of short-term cooling in some studies (Gauthier et al., 1983; Wise et

al., 1988a; Ealy et al., 1994) while others (Stott and Wiersma, 1976; Her et al., 1988) did

not find an advantage of such cooling. A summary of these studies is illustrated in Table

1-1.

Failure to restore conception rates in some studies might suggest that heat stress

might be affecting the cow before or'after the time cooling was applied. The estimated

time for the oocyte to grow from the early antral stage to the preovulatory stage is about

42 days (Lussier et al., 1987) and about 84-85 d to grow from the primordial stage to the

preovulatory siage (Picton et al., 1998; McNatty et al., 1999). Perhaps, heat stress during

this period of time compromised oocyte quality and subsequent embryonic development.

Moreover, heat stress can affect embryo quality at later stages of development. Exposure

of cows to elevated temperatures between d 8 and 16 of pregnancy resulted in lower

embryo size when embryos were recovered on d 17 (Biggers et al., 1987).









Table 1-1. Pregnancy rates following strategic cooling in lactating dairy cowsa

Pregnancy rate (%)b

Location Cooling period Control Cooled P values Ref.e


Arizona


4-6.5 d, beginning


at estrus


Guadeloupe


12 d, until d 10


after estrus


10 d, until -d 8


after estrus


Arizona


Florida


8 or 16 d after
prostaglandin
8 d after
prostaglandin


13/61(22%)


2/15(13%)


8/22 (36%)


3/18(17%)


19/63 (30%)


8/15 (53%)


9/29(31%)


10/35 (29%)


2/32 (6%) 8/50 (16%)


aAdapted from Hansen (1997).
bPregnancy rate=number of cows pregnant/number of cows inseminated, and, in
parentheses pregnancy rate.
NS=Not significant
dp=0.02 by ANOVA but not significant by CATMOD.
eReferences: 1) Stott and Wiersma (1976) 2) Gauthier (1983) 3) Her et al. (1988) 4) Wise
et al. (1988a) 5) Ealy et al. (1994).


Israel


0.05


0.05









Cooling cows around the time of estrus has also been used to improve estrus

detection. In one study in Israel (Her et al., 1988), cooling cows for one day before

expected estrus until d 8 after estrus was associated with an increase in the proportion of

cows with detected standing behavior (70%) when compared to non-cooled cows (45%).

Moreover, anestrus and silent ovulations were less frequent in cooled cows. In another

study in Florida in the summer, Ealy et al. (1994) reported non-significant increase in

percentage of cows detected in estrus when cooling was initiated from 2 to 3 d before

expected estrus until 5 to 6 after insemination. The proportion of cows detected in estrus

was 47% in the cooled group compared to 37% in the cows provided with shade only.

Improving Estrus Detection

Accurate detection of estrus and timing of insemination is a challenge under heat

stress condition because the duration (Monty and Wolff, 1974; Abilay et al., 1975;

Wolfenson et al., 1988a) and intensity (Gangwar et al., 1965) of estrus is decreased. The

most common method to detect cows in estrus includes visual observation, exposure to

teaser bulls or other cows or a combination of both. Also, the use of tail head paint along

with different synchronization schemes has been reported to improve estrus detection

rates in cows (Macmillan et al., 1988).

One approach for improving estrus detection is the use ofpedometry and pressure

sensing radiotelemetric systems (Dransfield et al., 1998; Nebel et al., 2000). Both of

these systems ihave been reported to increase efficiency ofestrus detection. The

pedometry system is based on the idea that physical activity of cows increases at estrus

(Vamrner et al., 1994; Amrney et al., 1994). In a review by Lehrer and colleagues (1992), it

was stated that 70-80% of cows in estrus were detected using the pedometer system. The

HeatWatch system is a commercial system based on radiotelemetry to recording









mounting events (Nebel et al., 2000). A pressure sensor is placed on the tail head of the

cow and is activated by weight of the mounting cow. The sensor transmits a radiowave

transmission signal that includes cow identification, date, time and duration of the

mounting event. This system has not been tested under heat stress conditions. However,

In one study (Jobst et al., 2000), there were differences in pregnancy rates per first AI

between herds using visual observation and those with the HeatWatch system.

However, cumulative pregnancy rates at 120 d postpartum were higher for the visual

observation herds compared to herds using HeatWatch system (56% vs. 46.3%, P <

0.05).

Use of Timed Artificial Insemination

To overcome the problem of estrus detection, a system called OvSynch has been

developed to pharmacologically synchronize ovulation so as to achieve acceptable

pregnancy rates upon timed artificial insemination (TAI). The OvSynch protocol is

based on the use of gonadotropin releasing hormone (GnRH) and prostaglandin F2,

(PGF2a). The first study describing the OvSynch protocol was performed by Pursley et

al. (1995). The system consisted of an injection of GnRH (100 jlg) on d 0 of the

protocol, PGF2a on d 7, a second injection of GnRH on d 9 (100 pig) and insemination

performed at 16-24 h later. The first injection of GnRH induces a surge of luteinizing

hormone (LH) and follicle stimulating hormone (FSH) (Chenault et al., 1990). The LH

surge will cause ovulation or luteinization of any large follicles on the ovaries

(Macmillan and Thatcher, 1991) and thereby allow the synchronized emergence of a new

follicular wave. The prostaglandin F2l injection given 7 d later causes regression of the

CL that was formed after the first GnRH injection as well as regression of pre-existing









CL. Regression of the CL results in decreased progesterone levels within 48 h allowing

for the final maturation of the dominant follicle that emerged as a result of the first GnRH

injection 7 d earlier. The second GnRH injection theoretically induces ovulation of the

dominant follicle within 28 h. In the study by Pursley et al. (1995), ovaries were

monitored daily by trans-rectal ultrasonography starting 5 d before treatment until

ovulation. Formation of a CL was observed in 18/20 cows after the first GnRH injection.

Furthermore, a new follicular wave was initiated in 20/20 cows following the GnRH

treatment, and regression of the CL occurred in 20/20 cows after the PGF2, injection.

Moreover, ovulation occurred within 24 to 36 h in all cows in the experiment.

Since this initial report, several experiments have been conducted to evaluate and

compare the OvSynch protocol to other synchronization protocols in which cows were

inseminated at detected estrus. Other estrus synchronization systems evaluated included

a single injection of PGF2a, two injections of PGF2, 14 d apart, and GnRH injection

followed 7 d later by PGF2a (Select Synch). Representative results of some experiments

are summarized in Table 1-2. In general, pregnancy rates per first AI in these studies

were similar between cows subjected to OvSynch and cows inseminated at detected

estrus (Burke et al., 1996; Pursley et al., 1997a; 1997b; Stevenson et al., 1999; Jobst et

al., 2000). Moreover, the cumulative pregnancy rates at 120 d post partum (Burke et al.,

1996; Stevenson et al., 1999; Jobst et al., 2000) were not different between the OvSynch

and control groups. However, in one study (Pursley et al., 1997b), pregnancy rates at 100

d post partum were significantly higher in the OvSynch group compared to controls (35%

vs. 53%). In this study, the median days to first AI was 54 d in cows subjected to

OvSynch compared to 83 d in the control group. Moreover, days open were lower for











Table 1-2. Pregnancy rate (PR) per first AI and cumulative pregnancy rate by 100 or 120
d after insemination following OvSynch protocol or estrus synchronization
and insemination upon detected estrus in lactating dairy cows

Control PR per AI2 Cumulative PR3
Reference' Group Control OvSynch Control OvSynch

1 GnRH+PGF4 30.5% (128) 29.0% (171) 58.8% 56.2
2 3xPGF+TAI5 38.9% (154) 37.8% (156) NA NA8
3 PGF6 37.0%(166) 39.0%(167) 35.0% 53.0%*
4 GnRH+PGF4 26.8%(112) 35.6%(115) 70.8% 74.0%
5 3xPGF7 33.2%(187) 30.1% (209) 53.1% 50.6%
'References: 1) Burke et al. (1996) 2) Pursley et al. (1997a) 3) Pursley et al. (1997b) 4)
Stevenson et al. (1999) 5) Jobst et al. (2000).
2Pregnancy rate per AI=number of cows pregnant/number of cows inseminated. In
parentheses, shown the number of cows.
Cumulative pregnancy rate by 100 (reference 3) or 120 d (references 1, 4 and 5) post
Vartum=number of cows pregnant/number of cows submitted to the protocol.
GnRH followed 7 d later by PGF2a and cows inseminated at estrus.
5One PGF2, injections and cows inseminated at estrus. Cows not observed in estrus were
given two injections of PGF2a and TAI at 72 to 80 h after the third PGF2a injection.
Cows were given one PGF2a and inseminated at detected estrus.
7Two PGF2a injections 14 d apart and cows were AI at estrus. Cows not observed in
estrus were given a third PGF2, 14 d later and inseminated at detected estrus.
8NA=not available
*P < 0.001









cows in the OvSynch groups compared to control (99 vs. 118).

One factor that might affect response to the OvSynch protocol is the stage of the

estrous cycle when the program is initiated. Indeed, Moriera et al. (2000) tested this

hypothesis and concluded that the ideal time to initiate the OvSynch protocol is when

cows are between d 5 and 10 of the cycle. This time of the cycle coincides with the

presence of a responsive follicle that will ovulate in response to the first injection of

GnRH and the presence of a mature responsive corpus luteum 7 d later that will respond

to the prostaglandin injection. This finding was tested in a field trial (Moreira et al.,

2001) where cows were presynchronized by two injections of prostaglandin F2a (PGF2a)

14 d apart. Cows were given the first injection of GnRH 12 d after the second PGF2a

when cows that responded to the presynchronization were expected to be between d 5 and

10 of the cycle. Based on plasma progesterone and after excluding anestrous cows,

pregnancy rates per Al at 74 d post insemination were higher for cows that were

presynchronized compared to non-presynchronized cows (46.9% vs. 34.4%). Therefore,

presynchronization increased pregnancy rate to first service by increasing the proportion

of cows that are at an ideal stage of the cycle.
I
Heat stress and timed artificial insemination

Given the negative effects of heat stress on estrous behavior, duration and

intensity (Gangwar et al., 1965; Monty and Wolff, 1974; Abilay et al., 1975; Wolfenson

et al., 1988a), ifwas hypothesized that the use of OvSynch protocol under heat stress

conditions could improve pregnancy rates in lactating dairy cows during the summer by

increasing the proportion of cows inseminated. Several studies have been conducted to

evaluate the efficacy of the OvSynch system under heat stress conditions. Results are

summarized in Table 1-3. In general, pregnancy rates per Al were similar between cows








Table 1-3. Pregnancy rates (PR) per first AI and cumulative pregnancy rates by 90 or
120 d after insemination following OvSynch protocol or estrus
synchronization and insemination upon detected estrus in lactating dairy cows
under heat stress conditions.

Control PR per AI2 Cumulative PR3
Reference' Group Control OvSynch Control OvSynch

I PGF4 4.8% (156) 13.9% (148)* 16.5% 27%"
2
experiment I IDE5 12.5% (184) 13.6% (169) 9.8% 16.6%**
experiment 2 IDE5 8.6% (35) 11.4% (35) 14.3% 34.3%*
3 AI at estrus 24% (65) 33.0% (118) NA NA
4 GnRH+PGF6 22.6% (218) 16.4% (207) NA NA
'References:1) De la Sota et al. (1998) 2) Ardchiga et al. (1998) 3) Momcilovic et al.
(1998) 4) Cartmill et al. (2001).
2 Pregnancy rate per AI=number of cows pregnant/number of cows inseminated. Shown
in parentheses are the number of cows.
3Cumulative pregnancy rate by 90 (reference 2) or 120 d (reference 1) post
partum=number of cows pregnant/number of cows submitted to the protocol.
Cows were given one PGF2, and inseminated at detected estrus.
5Cows were inseminated at detected estrus (IDE).
6GnRH followed 7 d later by PGF2, and cows inseminated at estrus.
P=0.055
**P < 0.05.
1P < 0.01.









subjected to the OvSynch protocol compared to controls under heat stress conditions

(Arechiga et al., 1998; Momcilovic et al., 1998; Cartmill et al., 2001). The exception is

for the study conducted by De la Sota et al. (1998) where pregnancy rates per Al were

significantly higher in the OvSynch group compared to control. Also, higher pregnancy

rates per Al were found in the OvSynch group in the study by Momcilovic et al. (1998),

but it was not statistically significant. While effects on pregnancy rate per Al are

equivocal, a clear benefit of TAI on overall pregnancy rates is apparent. By 90 to 120

days postpartum, cumulative pregnancy rates in two studies (De la Sota et al., 1998;

Arechiga et al., 1998) were significantly improved in the OvSynch group compared to

controls. In the De la Sota study (1998), cows subjected to OvSynch had fewer days

open (77 vs. 90; P < 0.05), shorter interval to first service (58 vs. 91; P < 0.05) and more

services per conception (1.63 vs. 1.27). Thus, OvSynch can be used to improve overall

reproductive performance under heat stress conditions because it eliminates the need for

estrus detection and increase the number of cows inseminated. However, the use of the

OvSynch does not generally improve embryonic survival since it does not alter events

causing heat-induced embryonic death.

Use of Embryo Transfer to Improve Pregnancy Rates

As discussed before, bovine embryos become more resistant to elevated

temperatures as they proceed in development (Dunlap and Vincent, 1971; Ealy et al.,

1993; 1995; Edwards and Hansen, 1997). This fact has lead to the idea of using embryo

transfer on d 7 of the estrous cycle to improve fertility by bypassing the adverse effects of

heat stress on embryos during the first 7 d following insemination. Moreover, since only

embryos at the morula or blastocyst stage are typically transferred, embryo transfer can

be used to bypass the deleterious effects of heat stress on the oocyte (Zeron et al., 2001).








There are data to suggest this approach is effective. For example, while

pregnancy rates following artificial insemination dropped to about 10-15% during the

summer months in Florida (Badinga et al., 1985), there was no significant effect of

ambient air temperature on pregnancy rates following embryo transfer when a data set of

19,936 non-surgical embryo transfer for the southern United States was analyzed (Putney

et al., 1988c). Moreover, Putney et al. (1989a) conducted a study to compare pregnancy

rates following embryo transfer or artificial insemination under heat stress conditions. In

this study, heifers were used as embryo donors since it has been reported that fertility of

non-lactating animals is less affected by heat stress (Badinga et al., 1985). Pregnancy

rates were significantly higher in the cows receiving a fresh embryo compared to cows

bred via artificial insemination (29.2% vs. 13.5%; P < 0.001).

Superovulation provides an on-farm tool to produce embryos. However, the

response to superovulation and embryo quality can be compromised under heat stress

conditions (Ryan et al., 1993; Gordon et al., 1987; Monty and Racowsky, 1987; Putney et

al., 1988c). Moreover, superovulation and embryo transfer is costly and require intensive

management of the donor cows and careful synchronization with recipients. An

alternative, potentially more practical procedure involves cryopreservation of embryos

during the cool time of the year and transfer to heat-stressed recipients as needed.

Another approach is to use in vitro derived embryos. This procedure is relatively

inexpensive ani-could be used to produce large number of embryos within a short time

for transfer to synchronized recipients. Also, those embryos could be cryopreserved and

transferred to recipients when needed. The effectiveness of using in vitro derived and








cryopreserved embryos to improve pregnancy rates in the summer was tested in two

studies in Florida (Drost et al., 1999; Ambrose et al., 1999).

In one study, Drost et al. (1999) evaluated pregnancy rates during the summer

following artificial insemination (Al), embryo transfer of a frozen-thawed, in vivo

produced embryo following superovulation of donors (ET-DON), and transfer of a

frozen-thawed in vitro derived embryo (ET-IVF). Pregnancy rates at d 42 were

significantly higher (P < 0.05) in the ET-DON group (35.4%) compared to Al and ET-

IVF (21.4% and 18.8% respectively). Thus, the transfer of frozen-thawed in vivo

embryos improved pregnancy rates during summer, but the transfer of a frozen-thawed in

vitro embryo was no better than artificial insemination. The development of the

OvSynch system to tightly synchronize ovulation has led to the potential for timed

embryo transfer (TET) whereby embryos are transferred without the need for estrus

detection. Ambrose et al. (1999) evaluated the efficiency of timed embryo transfer using

either fresh or frozen-thawed in vitro derived embryos under heat stress conditions.

Pregnancy rates in cows that received a fresh embryo (TET-fresh) were higher (P < 0.05)

compared to cows in the TAI group and cows that received a frozen-thawed in vitro

derived embryo (TET-frozen). The least squares means were 17.5 3.0, 6.7 3.2 and

6.1 3.8 for the TET-fresh, TAI and TET-frozen groups, respectively. Again, pregnancy

rates were improved by embryo transfer when the embryo was fresh but not when

cryopreserved.

Cryopreservation of In Vitro Derived Embryos

The fact that the studies of Drost et al. (1999) and Ambrose et al. (1999) indicated

that in vitro derived embryos did not survive freezing was not a phenomenon unique to








heat stress. Rather, in vitro produced embryos do not survive cryopreservation as well as

their in vivo counterparts in the absence of heat stress (Pollard and Leibo, 1993; Leibo

and Loskutoff, 1993; Pollard and Leibo, 1994; Wurth et al., 1994; Hasler et al., 1995). In

one study, Hasler et al. (1995) reported a pregnancy rate of 42% following the transfer of

in vitro derived frozen-thawed embryo compared to 56% and 67% for cows receiving an

in vitro derived fresh or an in vivo produced, frozen-thawed embryo, respectively.

Sensitivity of in vitro derived embryos to cryopreservation is likely due to

biochemical and morphological differences between embryos produced in vitro vs. those

that develop in vivo (Massip et al., 1995; Wright and Ellington, 1995; Thompson, 1997).

The microenvironment of the embryo affects subsequent composition and development.

In vivo embryos develop in a complex, dynamic environment that involves

communication between the embryo and the reproductive tract. On the other hand, in

vitro production systems occur in a static or semi-static environment where the embryo

has limited ability to change that environment. In addition, while procedures to alter in

vitro maturation, fertilization and culture are in place, these systems may not be optimal

for practices associated with these processes. Also, the oocytes used for in vitro

production may not be at the correct hiaturation status for optimal development (Sirard,

2001). There is abundant evidence that embryos produced in vitro differ

morphologically, biochemically and ultrastructurally from in vivo-derived embryos. For

example, in vitro-produced embryos have lower total cell number and inner cell mass to

trophectoderm ratio (Iwasaki et al., 1990). Also, in vitro embryos have reduced cell to

cell coupling which is an indication of gap junction communication. It is determined by

microinjection of a dye (i. e. fluorescein) into a single blastomere and detecting dye








diffusion to other blastomeres (Prather and First, 1993; Plante and King, 1994; Boni et

al., 1999). At the biochemical level, in vitro embryos have lower glucose oxidation

(Thompson et al., 1991) and amino acid uptake (Partridge et al., 1996). However, there

was no difference between in vivo and in vitro embryos in ATP production, oxygen and

glucose uptake (Thompson et al., 1996a, 1996b). Ultrastructurally, in vitro blastocysts

have lower volume density of the mitochondria and increased volume density of lipids

compared to in vivo blastocysts (Crosier et al., 2001). Furthermore, Leibo and Loskutoff

(1993) estimated that, as compared to in vivo derived embryos, in vitro embryos have

lower buoyant density. This density depends on the ratio of lipid to proteins. This

observation has lead to propose that the high sensitivity of in vitro derived embryos to

freezing might be due in part to their high lipid content (Pollard and Leibo, 1993; Leibo

and Loskutoff, 1993).

Attempts have been made to improve survival of in vitro embryos to freezing by

altering the culture medium in which embryos develop. It has been reported that

embryos cultured in Buffalo rat liver (BRL) cells and oviductal cells were more resistant

to freezing conditions (Massip et al., 1993; Leibo and Loskutoff, 1993, Tervit et al.,

1994). In one experiment, the highest re-expansion (55-82%) and hatching rates (41-

54%) were obtained when embryos were cultured with oviductal epithelial cells

compared to hatching rate of 6% in conditioned modified medium and 19-40% re-

expansion when-embryos were cultured in medium 199 and fetal calf serum (Massip et

al., 1993). In another experiment in sheep, Tervit et al. (1994) found that 26% of

embryos cultured in oviducts were graded good after freezing and thawing compared to

7.0% when embryos were cultured in synthetic oviductal fluid.








Given the changes in lipid content in in vitro produced embryos (Pollard and

Leibo, 1993; Leibo and Loskutoff, 1993), efforts have been made to improve freezability

by reducing lipid content. The approach has been centrifugation for cytoplasmic lipid

polarization followed by lipid removal using microsurgery (Murakami et al., 1998; Diez

et al., 2001). In both studies, lipid removal at the 1-cell stage did not have detrimental

effect on subsequent embryonic development to the 8-cell (Murakami et al., 1998) or to

the blastocyst stage (Diez et al., 2001). In the first study, when embryos at the 8-cell

stage were frozen and thawed, the proportion of embryos that developed to the blastocyst

stage was higher in the delipidated embryos compared to controls (47.8 vs. 24.2%; P <

0.01). In the experiment of Diez et al. (2001), hatching rate was higher for delipidated

embryos compared to controls when d 7 blastocysts were frozen-thawed and cultured for

3 d (45.2 vs. 22.4%, P < 0.02). In another experiment by Diez et al. (2001), pregnancy

rates after transfer of a frozen-thawed delipidated embryo were only 10.5% compared to

22.2% for the controls. Thus, while lipid removal improved embryonic development in

vitro after freezing and thawing, it caused lower pregnancy rates after transfer to

recipients.

Attempts have also been made to improve freezability of in vitro derived embryos

by manipulation of the cryopreservation process. The main goal of the cryopreservation

process is to minimize damage that could happen to the embryo during cryopreservation.

This damage in-ludes ice crystal formation, osmotic injury, toxic effects of

cryoprotective agents, zona and embryo fracture (Massip et al., 1995; Dobrinsky, 1996;

Kasai, 1996; Saha et al.; 1996). A successful freezing protocol depends on cooling

embryos to sub-zero temperatures with minimal damage.








The first successful freezing of mouse embryos was established by Whittingham

and coworkers (1972). One year later, the first calf was born following the transfer of a

frozen/thawed embryo (Wilmut and Rowson, 1973). Those observations formed the

basis for development of the embryo cryopreservation protocols that are widely used

commercially by bovine practitioners. This technique is based on provision of a

cryoprotectant that dehydrates the cell and prevents ice crystal formation when cells are

cooled to subzero temperatures (Queenan et al., 1995). The cryoprotectant used is

usually glycerol or the less toxic ethylene glycol (Kasai et al., 1992). Embryos are

typically loaded into 0.25 ml straws in a column of the cyoprotectant between two

columns of a non-permeating molecule (i.e. sucrose). Straws are then placed in a

programmable machine and cooled slowly to -7C. During cooling, the formation of

extracellular ice creates an osmotic gradient that favors the efflux of intracellular water.

Intracellular dehydration during cooling lessens ice crystal formation and freezing injury.

At about -5 to -8C, the column of fluid is subjected to a second step called seeding

which is done manually by touching the fluid column by forceps that is cooled in liquid

nitrogen. Seeding involves introduction of a seed crystal that causes rapid formation of

ice crystals in the medium. After seeding, a holding time of about 5 min allows for

intracellular water to equilibrate with extracellular water through exosmosis (Friedler et

al., 1988). Cooling is then continued at a rate of 0.3-0.6C/min until the temperature is

-35C. Embryos are held for about 10 min before being plunged in liquid nitrogen.

Vitrification is another approach for cryopreservation that depends on rapid

cooling and thawing of embryos. Vitrification means 'the solidification of a solution

(glass formation) brought about not by crystallization but by extreme elevation in








viscosity during cooling' (Fahy et al., 1984). The basis for this technique is to use high

concentrations of cryoprotectants associated with rapid cooling rates (-2500C/min,

Palasz and Mapletoft, 1996). The high concentration of cryoprotectant prevents

extracellular ice crystal formation and the damage associated with ice crystals.

Furthermore, the rapid cooling associated with vitrification has been reported to decrease

chilling injury and to prevent damage associated with high lipid content (Dobrinsky,

1996; Martino et al., 1996a; 1996b).

Vitrification does not eliminate toxic effects ofcryoprotectants and osmotic

damage, however. Several attempts have been made to reduce the toxic effect of

cryoprotectants. Toxicity of cryoprotectants depends on their interaction with the

intracellular components of the cell (Dobrinsky, 1996) and their permeating properties.

In one study, Kasai (1994) tested the toxicity of 5 different cryoprotectants on mouse

embryos and found that ethylene glycol and glycerol were the least toxic compared to

dimethyl sulfoxide (DMSO), propylene glycol and acetamide. Another approach is to

increase the pre-equilibration steps to achieve equilibrium between the intracellular and

extracellular components to lead to shrinkage of the embryo and the resultant

concentration of intracytoplasmic components (Mahmoudzadeh et al., 1995). Inclusion

of non-permeating saccharides also can reduce the osmotic shock probably by decreasing

the amount ofintracellular cryoprotectant. For example, sucrose (Kasai et al., 1990) or

trehalose (Yoshihno et al., 1993) has been found to reduce toxicity (Kasai et al., 1990).

The efficacy of cryopreservation of in vitro derived embryos by vitrification as

compared to slow freezing has been tested in several studies. As evaluated by re-

expansion and hatching of embryos following thawing, in vitro survival rate for





44


vitrification was either equal (Van-Wagtendonk et al., 1995) or higher (Mahmoudzadeh

et al., 1994; Dinnyes et al., 1995; Reinders et al., 1995; Agca et al., 1998a; O'Kearney-

Flynn et al., 1998) than for conventionally-frozen embryos. Also, pregnancy rates

following transfer to recipients were equal (Van-Wagtendonk et al., 1994; O'Kearney-

Flynn et al., 1998) or higher for vitrified embryos compared to embryos preserved via

conventional freezing (Reinders et al., 1995; Agca et al., 1998a).













CHAPTER 2
FACTORS AFFECTING SEASONAL VARIATION IN 90-DAY NONRETURN RATE
TO FIRST SERVICE IN LACTATING HOLSTEIN COWS IN A HOT CLIMATE

Introduction

Fertility in dairy cows is depressed during the summer months in warm areas of

the world (Hansen, 1997). This phenomenon is caused primarily by heat stress because

experimental application of heat stress to cows reduced pregnancy rate (Dunlap and

Vincent, 1971), and increased embryonic mortality (Putney et al., 1988a; Ealy et al.,

1993) whereas abatement of heat stress during the summer increased pregnancy rate

(Stott et al., 1972; Thatcher et al., 1974; Roman-Ponce et al., 1977; Ealy et al., 1994).

The magnitude of the seasonal depression in fertility is influenced by environmental

factors that define the magnitude of heat stress and internal factors of the cow that

determine the ability to regulate body temperature. Environmental factors such as air

temperature, humidity, etc. are determined in large part by geographical location as well

as characteristics of animal housing., One of the features of the cow that determines

thermoregulatory ability is milk yield, which affects metabolic rate and the magnitude of

hyperthermia experienced during heat stress (Berman et al., 1985). However, while there

is a greater reduction in fertility during the summer for lactating cows than for non-

lactating heifers (Badinga et al.; 1985), it is not known whether heat stress causes a more

severe depression in fertility for cows producing higher milk yields.

The deleterious effect of heat stress on embryonic development depends on the

day relative to ovulation at which cows are subjected to heat stress. Exposure of cows to








high air temperature and humidity for 10 h beginning at the onset of estrus increased the

incidence of retarded embryos recovered on d 7 of pregnancy (Putney et al., 1989b).

Thus, reproductive events in the preovulatory period are susceptible to disruption by heat

stress in a way that leads to altered embryonic development. Thereafter, embryos appear

to acquire some thermal resistance as development proceeds because exposure of

superovulated lactating Holsteins to heat stress on d 1 after estrus decreased viability and

development of embryos recovered at d 8, but heat stress had no effect if applied on d 3,

5 or 7 after insemination (Ealy et al., 1993). Similar observations have been noted in

embryos exposed to heat shock in culture (Edwards and Hansen, 1997). It is not known

whether heat stress before ovulation can compromise subsequent fertility. Such effects

are possible because heat stress can alter follicular development (Wolfenson et al., 1995)

and the oocyte can be disrupted by exposure to elevated temperature (Edwards and

Hansen, 1996). Ingraham et al. (1976) reported a negative correlation between humidity

on d 11 before insemination and subsequent fertility in Holstein cow. Also, research in

sheep (Dutt, 1963) indicated that heat stress 12 d before estrus reduced subsequent

fertility.

The present study had two major objectives. The first objective was to utilize

records compiled by DHIA to determine whether the seasonal variation in 90 day non-

return rate to first service (90-d NRR) of lactating dairy cows in South Georgia and

Florida is more pronounced in the southern part of the two-state region and to test

whether this variation is affected by milk yield. A second objective was to use DHIA

data to evaluate whether there is an association between heat stress at specific days

relative to breeding with subsequent fertility.








Materials and Methods

Data

The study utilized DHIA breeding records for 8124 Holstein cows in 17 herds

from South Georgia [n=7 herds located within 45-170 km from Tifton (31 28' N 83 32'

W); 943 cows], North Florida [n=5 herds located within 27-62 km from Gainesville (29

38' N 82 20' W); 4878 cows], and South Florida, [n=5 herds located in Okeechobee

County (27 16' N 80 46' W); 2303 cows] during the period of January 1994 until May,

1996. Records for each cow were for one lactation. Furthermore, the data were

screened to include only those cows that had at least one service, were lactating, and had

an interval to first service greater than 35 d and less than 150 d. The 90-d NRR to first

service was used to estimate pregnancy rate. Cows were considered pregnant to first

service if they were not observed in estrus or rebred within 90 d from first breeding date.

Weather data were obtained for South Georgia (the meteorological station in

Tifton) and North Florida (the agricultural weather station in Gainesville). Data included

daily minimum and maximum temperature, minimum and maximum relative humidity,

wind speed, and solar radiation.

Analysis of Location and Milk Yield Effects

The effects of location and ME milk yield on 90-d NRR to first service were

analyzed by least-squares analysis of variance using the General Linear Models

procedure ofSAS (SAS, 1989). Data were analyzed as one data set. In addition, subsets

of data were analyzed to compare North Florida to South Florida and South Georgia to

North Florida. For location effects on 90-d NRR, the model included main effects of

location, herd(location), and month of first service and interactions between main effects.









Interval to first service was used as a covariate. Data were analyzed initially with all

interactions in the model and then reanalyzed after removing non-significant interactions

and higher order (3 and 4 way) interactions that did not solve. In initial analyses, year

was included in the model. Levels of significance for effects of interest were similar

whether year was included or not. Data in one year (1996) were not distributed across all

months and, as a result, least-squares means gave distorted results. Accordingly, year

was not included in analyses reported in this chapter.

In the second analysis, cows were grouped according to mature equivalent (ME)

milk yield [1 < 4536 kg, n=123 cows; 2=4536-9072 kg, n=3833; 3 > 9072 kg, n=4168],

and data analyzed by least-squares analysis of variance as described above. Main effects

in the model were location, herd(location), month of first service and ME milk yield and

interactions between main effects. Interval to first service was used as a covariate.

Regression analysis was carried out to determine whether the relationship

between ME milk yield and 90-d NRR varied with month of first service. Initial analysis

indicated the R2 for the model with only the linear effect of milk yield included as a

continuous variable was similar to the R2 for the model that also included quadratic and

cubic effects of milk yield. Therefore, only the linear effect was included for subsequent

analysis. Data were analyzed by least-squares analysis of variance with 90-d NRR as the

dependent variable and with location, herd(location), month of first service, and location

x month of first service as independent class effects and with ME milk yield as a

continuous independent effect. Data were then reanalyzed with the same model except

with the term milk yield x month replacing milk yield. Heterogeneity of regression was

determined by calculating whether there was a significant reduction in residual sums of








squares caused by using milk yield x month of first service in the model instead of milk

yield (Wilcox et al., 1990).

Analysis of the Relationship Between Meteorological Data and 90-d NRR

Weather data were obtained for two locations (South Georgia and North Florida).

Data included daily minimum and maximum temperature, minimum and maximum

relative humidity, wind speed, and solar radiation. Average daily temperature (Tav) was

calculated as the average of the daily minimum and maximum dry bulb temperature.

Canonical discriminant analysis was carried out using the discriminant analysis

procedures of SAS (SAS, 1989) to derive linear combinations of the weather variables

that had the highest possible multiple correlation with 90-d NRR. This procedure derives

canonical variables, which are linear combinations of the quantitative variables (different

combinations of weather variables in this data set) that summarize between class

variation in much the same way that principal components summarize total variation.

This procedure allowed determination of the combination of weather variables on the day

of breeding that gave the best prediction of 90-d NRR.

The third analysis was performed to evaluate the relationship between weather

variables at various times relative to first insemination on 90-d NRR to first service. The

goal was 1) to confirm prior observations that heat stress on the day of insemination

causes reduced fertility (Putney et al., 1989b) and 2) to test whether heat stress during the

period of folli-llar growth preceding ovulation as well as during the period of embryonic

development is associated with reduced fertility. Canonical discriminant analysis

indicated that Tav on the day of insemination was as good a predictor of subsequent 90-d

NRR as more complex combination of weather variables. Therefore, only Tav was related

to 90-d NRR. Three days were chosen for analysis: d 0 (i. e., the day of insemination), d








-10 (i. e., 10 d before insemination; to determine effects of heat stress during follicular

growth on subsequent fertility) and d 10 (i. e., 10 d after insemination; to determine

effects of heat stress on the developing embryo). For each day examined, each cow was

classified according to the Ta, she was exposed to on that day and placed in one of six

temperature categories (< 6C, 7 to 10C, 11 to 15C, 16 to 20C, 21 to 25C, > 25C).

Analysis of variance was then performed to determine effects of temperature category on

90-d NRR to first service. The model included main effects of the temperature class on

the day of interest, location, and herd(location). Orthogonal contrasts were performed to

separate temperature effects into individual degree-of-freedom comparisons.

One problem with interpreting such an analysis is that environmental

temperatures on one day are highly correlated with temperatures on preceding and

ensuing days. Thus, a relationship between high Tav on any given day with fertility may

not represent an effect of heat stress on that day but rather reflect effects of heat stress on

other days. To reduce this problem, the data set was reduced before analysis as follows.

For analysis of effects of Tav on d -10, only cows exposed to Tav less than 25C on d -9 to

0 were analyzed. Thus, effects of high daily air temperature on d -10 likely represent

effects of heat stress on d -10 or before but not on days closer to ovulation because only

cows being exposed to cool temperatures on d -9 to 0 were used for the analysis. For

analysis of effects at d 0, only cows having Tav less than 25C on d -10 to -1 were

analyzed. Effects of high daily air temperature on d 0 are thus not likely to represent

effects of heat stress before estrus. For analysis of effects on d 10, the subset of cows

analyzed were those cows being exposed to Tav,, < 25C on d 0 to 9.








Results

Effect of Location on Seasonal Variation in 90-d NRR

There was a location x month of first service interaction affecting 90-d NRR to

first service (Figure 2-1; P < 0.001). Interaction of location x month of first

service occurred when comparing North Florida to South Florida only (P < 0.05) and

South Georgia to North Florida only (P < 0.05). These interactions resulted because,

while the 90-d NRR declined during warm months in all locations, the decline was of

greater magnitude and occurred for a longer period in South Florida than in North Florida

and for North Florida than in South Georgia. For example, the least-squares means for

90-d NRR in July was 18.9 7.2%, 7.0 2.8% and 4.7 3.7% for South Georgia, North

Florida and South Florida, respectively. The number of months where least squares

means for 90 day NRR were < 20% was 2 for South Georgia (June, July), 5 for North

Florida (May-September), and 7 for South Florida (April to October).

Effect of ME Milk Yield on Seasonal Variation in 90-d NRR

There was a milk yield x month of first service interaction (P=0.06) when cows

were grouped according to ME milk yield (1 < 4536 kg, n=123 cows; 2=4536-9072 kg,

n=3833; and 3 > 9072 kg, n=4168; Figure 2-2). As milk yield class increased, the

magnitude of the summer depression in 90-d NRR to first service was of greater

magnitude and lasted for more months. For example, the 90-d NRR in July was 44.9,

13.5 and 5.3% for classes 1,2 and 3, respectively. The number of months where 90-d

NRR was < 20% was 0, 3 and 6 months for classes 1, 2 and 3, respectively.

Variation of 90-d NRR Due to ME Milk Yield and Interval to First Service

Regression coefficients for the relationship between 90-d NRR and ME milk yield










100


Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec


Month of first service


Figure 2-1.


Seasonal variation in 90-d non-return rate to first service in South Georgia
(*), North Florida (0) and South Florida (A). Data are least-squares means
+ SEM adjusted for interval to first service.









100
90-
o 80
0-6- 70
Co,
L_
C..: 60-.



" 40
0
-o
c- 30-

6, 2i0
0) 10

0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Month of first service


Figure 2-2. Seasonal variation in 90-d non-return rate to first service as affected by ME
milk yield. Results represent least-squares means SEM adjusted for
interval to first service when cows were grouped according to milk yield (*<
4536 kg; 0 4536-9072 kg; A >9072).









by month of first insemination are shown in Figure 2-3. An increase in milk yield was

associated with a depression in 90-d NRR. The magnitude of the depression was affected

by month (heterogeneity of regression; P < 0.005). This effect was not significant if data

for the month of April were eliminated from the analysis although there was a trend for

the magnitude of the change in 90-d NRR per change in ME milk yield to be greater in

warm months.

Effect of Environmental Variables at Specific Days Relative to Insemination on 90-d
NRR

To determine effects of heat stress at d 10 before insemination (d -10), a subset of

cows were analyzed which experienced Tav < 25C from d -9 before insemination until

insemination (d 0). As shown in Table 2-1, the 90-d NRR decreased as Ta, on d -10

increased. Orthogonal contrasts indicated that 90-d NRR for cows having Tav > 20C on

d -10 (i.e., temperature classes 5 and 6) was less (P < 0.001) than 90-d NRR for cows

with Ta,,v < 20C (i.e., temperature classes 1-4) on d -10 (36.5 vs. 60.1%). For the

analysis of temperature effects on d 10 after insemination (d +10), we analyzed a subset

of cows that experienced Tav< 25C from the day of insemination (d 0) until d 9 after

insemination. The 90-d NRR decreased as temperature on d +10 increased (Table 2-1).

Orthogonal contrasts indicated that 90-d NRR for cows having Tav > 20C on d 10 after

insemination was less (P < 0.001) than for cows with Tav < 20C on day +10 (41.1 vs.

56.9%). To determine the effect of Tav on the day of insemination on subsequent fertility,

a subset of cows which experienced Tav < 25C from d 10 until d 1 before insemination

was analyzed. As shown in Table 2-1, 90-d NRR decreased as Tavon d 0 increased.

Orthogonal contrasts indicated that 90-d NRR for cows having Tav > 20C on the day of



















-3-

-4

-5)


-9-

-10-


-11


Jan Feb Mar


Figure 2-3.


Apr May Jun Jul Aug Sep Oct Nov Dec

Month of first service


Regression coefficients for the relationship between ME milk yield and 90-d
non-return rate to first service (90-d NRR) as affected by month of first
breeding.












Table 2-1. Least squares means + SEM of 90 day non-return rate (90-d NRR) to first
service as affected by average daily temperature at day -10, 0 and +10 relative
to insemination in subsets of Holstein cows that did not experience average
daily temperatures > 25C either on the 10 days before or after the studied
day.


Days
relative to
insemination
-10


Days relative
to insemination
where T< 25C
-9 to 0


Temperature
class on the
day of
interest (C)
<6


7to 10

11 to 15

16 to 20

21 to 25

>25


-10 to -1


7 to 10

11 to 15

16 to 20

21 to 25

,>25


0 to 9


55.5 2.5


503


61.3 2.1 904

53.9 2.0 1029


37.6 2.5
35.4 6.5


65.1 2.6

56.5 2.6


501

58


490


57.02.1 974


59.6 2.0


1034


38.5 2.6 478

44.2 9.2 58


64.4 2.3 707


55.0 2.5


51.9 2.1 917
56.3 2.0 1006


47.0 2.5


>25 35.18.4


90-d NRR
69.5 2.5


Number
of cows
513


P value
< 20C
vs. > 20C
0.001


0.001


0.001


7to 10

11 to 15

16 to 20

21 to 25


504









insemination was less (P < 0.001) than that for cows having Tay < 20C on day 0 (41.4 vs.

59.6%).


Discussion

Results obtained from analysis of data of cows inseminated in South Georgia and

Florida indicated that the magnitude of the summer decline in 90-d NRR depends upon

location and ME milk yield. In particular, summer infertility is exacerbated as cows are

located further south and as milk yield increased. It is to be expected that geographical

location would affect the magnitude of summer infertility but it is notable that such

effects were seen over relatively short distances. In particular, cows in North Florida

were more affected by season than cows in South Georgia although they were only ~200

km south. Analysis of the data also suggest that heat stress may act at several

physiological time points to disrupt establishment of pregnancy, including before

ovulation, on the day of insemination and after embryonic development has proceeded.

The measure of fertility used in this study (90-d NRR) overestimates pregnancy

rate because any cow having no breeding record 90-d from the first insemination is

classified as pregnant. In this way, some non-pregnant cows with silent or undetected

estrus will be classified as pregnant. In fact, the depression in pregnancy rate in the

summer is likely to be of even greater magnitude than indicated by 90-d NRR because of

the increased frequency of unobserved estrus in summer (Thatcher and Collier, 1986).

Possible differences in culling rate between seasons could also lead to seasonal bias in the

accuracy of 90-d NRR.

The increase in milk yield experienced by dairy cows in the last 25 years has been

coincident with a reduction in pregnancy rate (Silvia, 1998). There are likely many








reasons for this decline including alterations in follicular function (Bilby et al., 1998) and

endocrine differences between Holstein cows selected for milk yield and control cows

(Lucy et al., 1998; Royal et al., 2000). The present results indicate that high milk yield

exacerbates the effects of heat stress on fertility. In fact, the decline in 90-d NRR was

greater for cows with high milk yield than cows with low milk yield. Although not

significant, the relationship between milk yield and 90-d NRR tended to be larger during

the summer months of the year. The major reason why high milk yield exacerbates

effects of heat stress on reproduction is likely related to the increased metabolic rate and

decreased thermoregulatory ability for cows with high milk yield. In animals producing

above 24 kg/day, and cows exposed to temperatures within the range of 10 to 24C, rectal

temperature increased 0.02C for each kilogram fat-corrected milk production (Berman et

al., 1985).

Discriminant analysis was used to predict which combination of the weather

variables available (average daily temperature, minimum and maximum relative

humidity, wind speed and solar radiation) on the day of insemination gave the highest

prediction of 90-d NRR. The fact that the average of daily minimum and maximum

temperature on the day of breeding was as good a predictor of fertility as various

combinations of weather variables does not mean that factors such as humidity or solar

radiation are unimportant to the thermal biology of cattle but rather that limitations in the

nature of data (for example, data were collected at a central weather station rather than on

site where cattle were located; also housing differ between herds) limited the predictive

power of weather variables. Nonetheless, analysis of the relationship between average

temperature on various days and subsequent 90-d NRR revealed some potential









biological effects of heat stress on embryo loss. In particular, 90-d NRR was reduced

when heat stress (i. e., Ta, > 20C) occurred on d -10 before breeding or at d 10 after

breeding. One problem with interpreting relationships between temperature at specific

times with fertility is that there is high correlation between temperature on a certain day

with subsequent and previous days. To reduce this problem, the data set was reduced to

subsets of cows that were exposed to cooler temperatures on the 10 d before or after the

day of interest. For example, to evaluate effects of air temperature on d 10 before

breeding, only those cows having average daily temperatures less than 25C on d -9 to 0

relative to insemination were analyzed. Thus, the relationship between air temperature

on d -10 and fertility is less likely to reflect heat stress on d -9 to 0.

Earlier, Dutt (1963) reported that heat stress at 12 d before breeding reduced

fertility in ewes, suggesting heat stress could compromise oocyte function. One way

such an effect could be mediated is through alterations in follicular function as reported

elsewhere (Badinga et al., 1993; Wolfenson et al., 1995). Ealy et al. (1993) and Edwards

and Hansen (1997) reported that embryos become more resistant to heat stress as

development proceeds and so the relationship between heat stress at d 10 after breeding

and 90-d NRR was somewhat unexpected. However, Biggers et al. (1987) reported that

heat stress from d 8-16 of pregnancy reduced embryo weight at d 17. Also, heat stress

can affect endometrial prostaglandin secretion (Putney et al., 1989c) and conceivably

lead to luteolysis and embryonic loss. At a practical level, finding that the physiological

window for heat stress effects is so large means efforts to improve fertility in summer by

cooling cows for a limited number of days will not be completely successful, as has been

indeed proven to be the case (Ealy et al., 1994).













CHAPTER 3
EFFECT OF SEASON AND EXPOSURE TO HEAT STRESS ON OOCYTE
COMPETENCE IN HOLSTEIN COWS


Introduction

Heat stress before insemination has been associated with decreased fertility in

cattle (chapter 2). These findings are consistent with previous reports in cattle (Ingraham

et al., 1974; Putney et al., 1989b) and sheep (Dutt, 1963). Some of this infertility may

reflect damage to the developing oocyte. Indeed, there are two reports indicating that

oocyte competence, as determined by developmental rate following in vitro fertilization

(IVF), is lower in summer than winter (Rocha et al., 1998; Rutledge et al., 1999). There

are several potential mechanisms by which heat stress could compromise oocytes. Heat

stress has been reported to alter follicular development by reducing steroid hormone

production (Wolfenson et al., 1997; Wilson et al., 1998a) and these changes in follicular

steroid concentration could disrupt oocyte growth. In addition, heat stress reduces

growth of the dominant follicle (Badinga et al., 1993) and causes incomplete dominance

so that there is increased growth of subordinate follicles (Wolfenson et al., 1995).

Incomplete dominance could result in ovulation of an aged follicle; such follicles contain

oocytes with reduced competence (Mihm et al., 1999).

Finally, the hyperthermia coincident with heat stress in cows could also directly

inhibit oocyte function. Unlike other cells, the oocyte is transcriptionally inactive after

reaching about 110 pm (i. e. 2-3 mm follicle; Hyttel et al., 1997) and does not undergo

increased synthesis of heat shock protein 70 in response to heat shock (Edwards and








Hansen, 1997). Elevated temperature could conceivably have deleterious effects on

oocyte growth, protein synthesis or formation of transcripts required for subsequent

embryonic development.

The objectives of the current study were to evaluate the effect of season on oocyte

competence in Holstein cows and to test whether oocyte quality in the summer is affected

by the magnitude of heat stress.


Materials and Methods

Materials

Modified Tyrode's solutions were obtained from Cell and Molecular

Technologies (Lavallete, NJ) to prepare HEPES-Tyrode's Albumin Lactate Pyruvate

(TALP), IVF-TALP and Sperm-TALP using recipes described by Parrish et al. (1986).

Bovine serum albumin (BSA) Fraction V, essentially fatty acid free BSA (EFAF-BSA),

and 4', 6'-diamidino-2-phenylindole (DAPI) were purchased from Sigma Chemical

Company (St. Louis, MO). Bovine steer serum and heat-treated fetal calf serum were

purchased from Pel-Freez (Rogers, AR) and Atlanta Biologicals (Norcross, GA),

respectively. Percoll was obtained from Amersham Pharmacia Biotech (Uppsala,

Sweden). Follicle stimulating hormone (FSH) was Folltropin'V from Vetrepharm

Canada Inc. (London, Ontario). Gonadotropin-releasing hormone (GnRH) was

Cystorelin from Rhone Merieux, New York, NY. Prostaglandin F2, (PGF2a) was

Lutalyse from Pharmacia/Animal Health (Kalamazoo, MI).

Frozen semen from various Holstein bulls was donated by Select Sires Inc.

(Rocky Mount, Virginia). Oocyte collection medium was TCM-199 with Hank's salts

without phenol red supplemented with 2% (v/v) bovine steer serum, 0.04 U heparin/ml,








100 U/ml penicillin-G, 0.1 mg/ml streptomycin, and an additional 1 mM glutamine.

Oocyte maturation medium was TCM-199 with Earle's salts supplemented with 10%

(v/v) bovine steer serum, 2 ptg/ml estradiol 17-P, 20 utg/ml FSH, 0.2 mM sodium

pyruvate, 50 ug/ml gentamicin and an additional 1 mM glutamine. Potassium Simplex

Optimized Medium (KSOM) was obtained from Cell and Molecular Technologies

(Lavallete, NJ). The KSOM, which contains 1 mg/ml BSA, was modified on the day of

use by adding 2-3 mg/ml EFAF-BSA, 2.5 jtg/ml gentamicin, essential amino acids (basal

medium eagle) and non-essential amino acids (minimal essential medium) purchased

from Sigma.

General Procedures for In Vitro Fertilization (IVF)

Ovaries were obtained at slaughter and transported to the laboratory in saline

solution [0.9% (w/v) NaCI containing 100 U/ml penicillin-G and 100 j.g/ml

streptomycin] at room temperature. Upon arrival at the laboratory, ovaries were washed

with pre-warmed saline solution. Cumulus oocyte complexes (COCs) were collected

from 2 to 5 mm follicles by slicing the surface of each ovary and agitation in a beaker

containing oocyte collection medium. The COCs were cultured in groups of 10 in pre-

equilibrated 50 ul drops of oocyte maturation medium covered with mineral oil. The

COCs were allowed to mature for 22 to 23 h at 38.5C in an atmosphere of 5% (v/v) CO2

in humidified air. After maturation, COCs were washed once in HEPES-TALP and

transferred to 600 ul wells of IVF-TALP in groups of -30 per well. Frozen-thawed

sperm were purified by Percoll gradient centrifugation as described by Parrish et al.

(1986). The pellet was collected, placed in a 15 ml conical tube containing 10 ml SP-

TALP and centrifuged at 1000 x g for 5 min. The supernatant was removed and the









viable sperm were re-suspended in IVF-TALP to achieve an approximate concentration

of 25 million spermatozoa per ml. Oocytes were fertilized by adding 25 pil sperm

suspension and 25 il PHE [0.5 mM penicillamine, 0.25 mM hypotaurine, and 25 gM

epinephrine in 0.9% (w/v) NaCI] to each 600 pl well. After 8 tolO hat 38.5C and 5%

(v/v) CO2 in humidified air, presumptive zygotes were removed from the fertilization

wells and denuded of the cumulus cells by vortexing in ~50 pI of HEPES-TALP for 5

min in a 2 ml microcentrifuge tube. Putative zygotes were washed twice in HEPES-

TALP and cultured in pre-equilibrated 50 pI drops of modified KSOM overlaid with

mineral oil. Heat-treated fetal calf serum [10%(v/v)] was added to each drop on d 5 after

insemination (day of insemination is d 0). Development to blastocysts and cleavage rates

were recorded on d 8 after insemination.

Experiment 1 Effect of Season on Oocyte Competence of Holstein Cows

This experiment was conducted over a period of one year starting from March,

2000 until February, 2001. There were 18 replicates conducted. Replicates performed

from April through September (n=10) were considered the warm season and replicates

performed from October through March (n=8) represented the cool season. Ovaries were

obtained from slaughtered Holstein cows at a commercial abattoir in Central Florida and

transported to the laboratory for processing as described earlier. After maturation, COCs

were fertilized using two straws of frozen semen from one Holstein bull (a different bull

was used for each replicate; when possible each bull was used once in the warm season

and once in the cool season). Development to the blastocyst stage and cleavage rate were

recorded on d 8 after insemination.








Experiment 2- Effect of the Magnitude of Heat Stress on Oocvte Quality

Animals and treatments

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

in Hague (29 38' N 82 20' W) with a total of 40 non-lactating Holstein cows. The

experiment was conducted during two seasons (summer, June to September, 2000, and

winter, December, 2000 to February, 2001). In addition, cows in summer were randomly

assigned to two environments [heat stress (HS) and cool (C) groups]. Before entering the

study, cows were located in a pasture with access to shade cloth and shade trees. Thus,

regardless of treatment group, cows in summer were exposed to heat stress before the

study began.

During the experimental period cows in the HS group in summer were housed in a

lot where the only structure present was shade cloth. The C group in summer was housed

in a free-stall barn with fans and foggers to alleviate heat stress. The winter (W) group of

cows was housed in the same lot that was used for the HS cows in summer. Cows were

maintained in the respective facilities for a period of 42 d before slaughter. This time

was chosen because it represents the time required for a follicle to grow from the early

antral stage until the preovulatory stage (Lussier et al., 1987). All groups were fed the

same basal diet. Rectal temperature, respiration rate, body surface temperature and black

globe temperature were recorded at 1600 h twice weekly throughout the experiment.

Cows irrthe summer were assigned to groups and were slaughtered on d 18 to 19

of the estrous cycle over a period of 7 weeks (two cows from each group each week).

Cows in the winter were slaughtered on d 18 to 19 of the estrous cycle in three groups

(n=4 per group). To assure that two cows per group in summer and 4 cows in winter

were slaughtered on the same day for each replicate, twice as many cows as needed for









each group were subjected to estrous synchronization. Estrus was synchronized 14 d

after cows entered their respective facilities by an i.m. injection of 100 Pg GnRH,

followed 7 d later by an i.m. injection of 25 mg PGF2 Estrus was determined by visual

detection and tail head paint score (Macmillan et al., 1988). Cows were palpated per

rectum 8 to 9 d after the prostaglandin injection to insure the presence of the corpus

luteum (CL). Two cows (summer) or four cows (winter) with palpable CL from each

group were slaughtered on d 18 to 19 of the cycle at the Meat Processing Facility at the

Department of Animal Sciences. Semen from a different bull was used to inseminate

oocytes each week. Bulls used in the summer were also used in the winter.

In vitro embryo production

After slaughter, ovaries were obtained and washed with saline solution. For each

cow, the oocytes from the largest follicle (i.e., the presumed preovulatory follicle) and the

second largest follicle were aspirated separately in oocyte collection medium using a

syringe attached to an 18-gauge needle. Recovered oocytes were washed once in oocyte

collection medium and cultured individually in a pre-equilibrated 10 pI drop of oocyte

maturation medium overlaid with mineral oil. Additional COCs were collected from

each pair of ovaries for each cow by slicing the surface. The COCs were cultured in

groups of 10 in pre-equilibrated 50 pil drops of oocyte maturation medium covered with

mineral oil. The COCs were allowed to mature for 22 to 23 h at 38.5C in an atmosphere

of 5% (v/v) CO2 in humidified air.

After maturation, individual oocytes were washed once with HEPES-TALP and

transferred to a 10 pl drop of IVF-TALP covered with mineral oil. Oocytes were

fertilized by adding 2 pl sperm suspension and 2 pl PHE. Depending on the number of








oocytes recovered per cow, grouped oocytes from each cow were fertilized separately in

groups of-10 to 30 in 600 pl IVF-TALP as described earlier. After 8 to 10 h, individual

oocytes were removed from the fertilization medium, washed twice with HEPES-TALP

and transferred to a 10 pl drop of modified KSOM medium covered with mineral oil. On

d 5 after insemination, 1 1il of fetal calf serum was added to each drop. For the grouped

oocytes, presumptive zygotes were removed from the fertilization wells, and denuded of

the cumulus cells as described earlier. Denuded putative zygotes were washed twice in

HEPES-TALP and cultured in pre-equilibrated 25 pl drops of modified KSOM.

Embryos were assigned to drops so that similar number of embryos per drop could be

achieved between treatments. Heat-treated fetal calf serum [10%(v/v)] was added to each

drop on d 5 after insemination. Cleavage rates and proportions of oocytes and cleaved

embryos developing to blastocysts were recorded on d 8 after insemination.

Determination of cell number

On d 8, embryos at the blastocyst stage were washed three times in 100 PI drops

ofPBS (pH=7.4) containing 1 mg/ml polyvinylpyrrolidone (PBS-PVP), and then fixed in

100 pl drops ofparaformaldehyde [4% (w/v)] for 1 h at room temperature. Embryos

were washed three times in 100 pl drops of PBS-PVP before being transferred to a poly-

L-lysine coated slide. Embryos were dried on the slide at room temperature for 24 h and

stored in a slide box at room temperature for later determination of total cell number.

Embryos were incubated with PBS-PVP solution containing 0.0001% (w/v) DAPI for 10

min at room temperature and then washed in PBS-PVP. Antifade solution [ProLong

Antifade Kit; (Molecular Probes)] was added and the slide was mounted with coverslip.

Total cell number was determined using an epiflourescence microscope.








Statistical Analyses

Data were analyzed by least squares analysis of variance using the General Linear

Models Procedures ofSAS (SAS, 1989). Percentage data were subjected to an arcsin

transformation before analysis to adjust for non-normality of percentage data. For

experiment 1, the main effect in the model was season with a total of 10 observations in

the warm season and 8 in the cool season. For experiment 2, the mathematical model

included the main effect of group and cow (group). Cow was considered a random

variable and cow within group was used as the error term for group. Dependent variables

were percent blastocyst at d 8, cleavage rate and rectal temperature. Group effects were

partitioned into individual degree of freedom contrasts to test winter (W) vs. summer (HS

and C) and HS vs. C.


Results

Experiment I Effect of Season on Oocytes Recovered from Abattoir

Cleavage rate of bovine oocytes during the warm season (April to September) was

slightly higher (P < 0.001) than the cool season (October to March) (Figure 3-1).

However, the proportion of oocytes and cleaved embryos that developed to blastocysts on

d 8 after insemination was lower (P < 0.001) during the warm season (April to

September) when compared to the cool season (October to March) (Figure 3-1).

Experiment 2 Seasonal Variation and Effect of Magnitude of Heat Stress

Rectal temperatures of cows in the summer (HS and C) were higher (P < 0.001)

than winter (W) and were lower for C than HS (P < 0.001, Table 3-1). For oocytes

cultured in groups, the number of oocytes recovered per cow was similar among all

groups in cleavage rate. However, the proportion of oocytes that developed to
















Oocytes cleaved
*


Oocytes to
blastocyst


Cleaved embryos
to blastocyst


So00


Season



Figure 3-1. Effect of season on the percentage of oocytes that cleaved and developed into
blastocysts on d 8 after insemination. Results are least-squares means
SEM of 10 replicates during warm season (April to September) and 8
replicates during cool season (October to March).
Significant difference between the two seasons (P < 0.001)


100-

90 -

80

70-


0\ '











Table 3-1. Effect of season and degree of heat stress on oocyte competence


Variable He;
Number of cows
Rectal temperature (C)
Grouped oocyte culture
Oocytes collected/cow
Cleavage rate (oocytes
fertilized/total oocytes)

Blastocyst on d 8 (%)
(from total oocytes)

Blastocyst on d 8 (%)
(from cleaved embryos)

Total cell number

Single oocyte culture

Number of oocytes
collected from largest
follicles

Cleavage rate

Cell number (d 8)

Blastocyst on d.8 (%)


at Stress (HS)
14
39.8 0.1a


19.3 2.9
88.3 2.2


LSM SEM
Cool (C)
14
39.2 0.1b


25.9 2.9
88.9 2.0


11.4 3.7a 10.3 3.3a


12.5 4.2a


68.7 8.9


58.3 14.3

2.6 1.2

0


11.4 3.8a


75.3 12.0


66.7 20.9

3.7 1.6

0


a, b Superscripts represent least squares mean that differ (P < 0.001) as determined by
orthogonal contrasts (HS and C vs. W; and HS vs. C).


Winter (W)
12
38.7 0.1c


25.1 3.2
88.4 2.2


29.9 3.7b


34.3 4.2b


82.4 6.4



4



75.0 25.6

6.5 2.0

0








blastocysts at d 8 was lower (P < 0.001) for summer than winter; there was no difference

between HS and C. The same differences were seen when development was expressed as

the proportion of cleaved embryos that developed to blastocysts. There was no statistical

difference in blastocyst cell number between HS, C and W groups.

Recovery of oocytes from the largest and second largest follicles was incomplete.

Only 22 of 40 cows yielded an oocyte from the largest follicle and 8 of 40 yielded an

oocyte from the second-largest follicle. None of the oocytes cultured individually

developed to blastocysts. For oocytes from the largest follicle, there was no significant

difference in cleavage rate or embryo cell number between groups (Table 3-1).

Nonetheless, the trends for development (embryo cell number on d 8) ofoocytes from the

largest follicle paralleled results from the pooled oocytes, with development tending to be

greater for oocytes collected in winter.


Discussion

Results of the current experiments indicate that oocyte competence in Holstein

cattle located in a warm climate declines during summer. Even though cleavage rate was

not reduced during warm weather, zygotes formed during the summer had reduced

competence to develop to the blastocyst stage. The finding that cooling cows during the

summer for 42 d before the anticipated day of oocyte collection did not alleviate the

summer depression in oocyte quality suggests that either 1) the degree of cooling was not

sufficient to prevent the adverse effects of heat stress; 2) heat stress damaged the oocyte

earlier in development than when cooling was initiated; or 3) seasonal effects represent

factors other than heat stress.








The finding that there is a summer-time reduction in ability ofoocytes from

Holstein cows to develop in culture following fertilization is consistent with previous

reports using oocytes recovered from non-lactating Holstein and crossbred Angus cows

in Louisiana (Rocha et al., 1998) and oocytes recovered from an abattoir in Wisconsin

(Rutledge et al., 1999). In Israel (Zeron et al., 2001), development of oocytes collected

from Holstein cows at an abattoir following chemical activation was reduced during the

summer. In contrast, no seasonal variation was seen in another study in Florida using

oocytes recovered at an abattoir (Rivera et al., 2000). In the latter study, oocytes were

from both beef and dairy cows and the lack of seasonal variation may reflect a

preponderance of cows that have superior thermoregulatory mechanisms (for example,

Bos indicus breeds; Finch, 1986) and/or embryos that are more tolerant to elevated

temperatures (Paula-Lopes et al., 2001). Similarly, no seasonal variation in IVF

performance was found in oocytes collected from Brahman cows (Rocha et al., 1998).

Cooling cows for 42 d before collection of oocytes did not improve embryonic

development in the summer. Such a result could be interpreted to mean that heat stress is

not the factor responsible for seasonal variation. Interestingly, the percentage of oocytes

that developed to blastocyst in the summer was lower in experiment 2, using non-

lactating cows, than for experiment 1, in which presumably many of the cows were

lactating at the-time of slaughter. Such a result is surprising because effects of heat stress

on fertility are lover for non-lactating cows (Badinga et al., 1985). Caution in comparing

absolute differences between experiments is in order, however. The fact that oocytes in

experiment 1 were collected at an abattoir means that location where slaughtered cows








were purchased (some from cooler environments) as well as variation in physiological

status could obscure effects of heat stress.

If in fact, the seasonal variation in oocyte quality seen in these experiments is

caused by heat stress, there are two other possible explanations for the failure of cooling

to improve oocyte quality in experiment 2. First, although lower than for HS cows, rectal

temperature of the cooled group was still elevated (39.2C vs. 38.7C for cows in winter).

A temperature rise of0.5C above basal body temperature has been associated with

decreased pregnancy rate (Gwazdauskas et al., 1973). Another possibility is that heat

stress acted to damage the oocyte earlier in development than when cooling was initiated.

Cooling, which was for 42 d before slaughter, encompassed the 42 d in which the follicle

grows from the antral to the preovulatory stage (Lussier et al., 1987). However, primary

follicles begin growth about 84 to 85 d before ovulation (Picton et al., 1998; McNatty et

al., 1999) and heat stress early in follicular growth could have compromised oocyte

quality in both summer groups.

There was no reduction in cleavage rate ofoocytes during the warm season. This

finding is consistent with previous reports (Rocha et al., 1998; Rutledge et al., 1999;

Rivera et al., 2000), and suggested that the cellular mechanisms involved in oocyte

maturation, sperm binding and fertilization are not compromised by heat stress.

However, the ability of zygotes to develop to blastocysts was reduced during the summer.

Thus, some conironent of the embryo that was formed from the oocyte was likely

damaged by heat stress. There is evidence that heat stress can alter phospholipid

composition of oocytes (Zeron et al., 2001). Exposure of cultured oocytes to heat shock

can reduce protein synthesis in mice (Curci et al., 1987) and cattle (Edwards and Hansen,








1997). Oocytes from small follicles also have reduced developmental potential

(Hendriksen et al., 2000). Given that heat stress has been reported to increase the number

of small follicles (Badinga et al., 1993; Trout et al., 1998), it is possible that oocytes

recovered in summer came from follicles of average smaller size. However, not all

reports indicate more small follicles during heat stress (Wilson et al., 1998a). It may also

be that heat stress reduces synthesis and storage of transcripts required to support early

embryonic development (Memili and First, 2000).

In conclusion, results indicate oocyte competence in Holstein cattle located in a

warm climate declines during summer. Such a result suggests that damage to the oocyte

is one cause for reduced fertility during the summer in warm climates. Moreover,

seasonal variation in oocyte competence can lead to reduced performance of procedures

to produce in vitro-derived embryos using oocytes recovered from abattoir ovaries.

Because cooling cows during the summer for 42 d before the anticipated day of oocyte

collection did not alleviate the summer depression in oocyte quality, it is possible that

heat stress can damage the oocyte during the period preceding antral follicle formation or

that seasonal effects represent factors other than heat stress.













CHAPTER 4
INDUCED THERMOTOLERANCE IN BOVINE TWO-CELL EMBRYOS AND THE
ROLE OF HEAT SHOCK PROTEIN 70 IN EMBRYONIC DEVELOPMENT

Introduction

The early preimplantation embryo has only limited ability to respond to stress.

Thus, several perturbations in the embryo's microenvironment, including heat shock

(Edwards and Hansen, 1997; Rivera and Hansen, 2001), acid pH (Narasimhan et al.,

1996), ethanol (Su et al., 1998) and nitric oxide (Kim et al., 1997) can be more

deleterious to subsequent development when occurring during the first few cleavage

stages than during later stages. One reason for the heightened sensitivity of the early

embryo to stress may be the fact that the embryonic genome is largely suppressed during

early cleavage stages. In cattle, while some transcription can occur as early as the 1 cell-

stage (Saeki et al., 1999), the embryonic genome is not fully activated until the 8- to 16-

cell stage (Barnes and First, 1991; Jones and First, 1995; De Souza et al., 1998). Thus,

the range of cellular adaptive responses utilized by the early embryo likely is reduced as

compared to embryos that have completed embryonic genome activation.

Recently, it has been shown that bovine embryos at the 2-cell stage can undergo

transcription and synthesize the heat-inducible form of heat shock protein 70 (HSP70i)

after heat shock (Edwards and Hansen, 1996; Edwards et al., 1997; Chandolia et al.,

1999). Constitutive and inducible forms of HSP70 play an important role in maintaining

cellular function during heat shock by acting as molecular chaperones to stabilize or

refold proteins damaged by heat stress (Morimoto et al., 1996) and blocking apoptosis








(Mosser et al., 1997; McMillan et al., 1998; Samali et al., 1999). Provision ofantisense

mRNA against two heat inducible HSP70 molecules (HSP70-1 and HSP70-3) made

mouse preimplantation embryos more susceptible to arsenic (Dix et al., 1998) while

injection of HSP70 mRNA into mouse oocytes increased resistance to heat shock

(Hendrey and Kola, 1991).

One of the phenomena in which HSP70 has been implicated is induced

thermotolerance. This term refers to the process whereby cells are made more resistant to

a severe heat shock by prior exposure to a mild heat shock. Induced thermotolerance can

occur in bovine and murine embryos (Ealy and Hansen, 1994; Ardchiga et al., 1995).

Microinjection of antibodies to HSP70 increased the sensitivity of fibroblasts to heat

shock (Riabowol et al., 1988), while microinjection of mRNA for the constitutive form of

heat shock protein 70 (HSC70) induced thermotolerance in fibroblasts (Li et al., 1991).

The current series of experiments was designed to understand the role of induced

thermotolerance and HSP70i in development of bovine embryos. The first objective was

to test whether 2-cell bovine embryos could undergo induced thermotolerance. While

induced thermotolerance has been described for bovine blastocysts (Ealy and Hansen,

1994), it is not known how soon during development the bovine embryo develops the

capacity for induced thermotolerance. Given the capacity of the 2-cell embryo for

synthesis of HSP70 in response to heat shock (Edwards and Hansen, 1996, Edwards et

al., 1997), it is possible that induced thermotolerance can occur in these embryos. If so,

such a finding would indicate that cellular responses to stress in 2-cell embryos are highly

developed despite the large-scale repression of gene expression at this stage of

development. In addition, it might be possible to identify strategies for decreasing








embryonic mortality in cattle during heat stress through manipulation of induced

thermotolerance responses. The second objective was to evaluate the role of the heat-

inducible form of HSP70 in development of bovine embryos in the absence and presence

of heat shock. Since administration of antibody for HSP70 into culture medium has been

reported to reduce preimplantation embryonic development in mice (Neuer et al., 1998),

the approach was to determine whether addition of antibody to HSP70i to culture

medium reduced development in the absence and presence of heat shock.


Materials and Methods

Materials

All chemicals were purchased from Sigma Chemical Company (St. Louis, MO)

unless otherwise mentioned. Follicle stimulating hormone (FSH) was Folltropin-V

from Vetrepharm Canada Inc. (London, Ontario). Bovine steer serum and heat-treated

fetal calf serum were purchased from Pel-Freez (Rogers, AR) and Atlanta Biologicals

(Norcross, GA), respectively. Modified Tyrode's solutions were obtained from Cell and

Molecular Technologies (Lavallete, NJ) to prepare HEPES-Tyrode's Albumin Lactate

Pyruvate (TALP), IVF-TALP and Sperm-TALP were prepared using recipes described

by Parrish et al. (1986). Percoll was obtained from Amersham Pharmacia Biotech

(Uppsala, Sweden). A mouse monoclonal antibody to the inducible form of HSP70 (SPA

810) was purchased from StresGen Biotechnologies (Victoria, B.C., Canada). This

antibody is specific for the inducible form of HSP70 and does not cross react with the

constitutive form of HSP70. Also, this antibody has been shown to react with bovine

HSP70. Mouse IgG monoclonal antibody was obtained from US Biological

(Swampscott, MA).









Oocyte collection medium was TCM-199 with Hank's salts without phenol red

supplemented with 2% (v/v) bovine steer serum, 0.04 U heparin/ml, 100 U/ml penicillin-

G, 0.1 mg/ml streptomycin, and an additional 1 mM glutamine. Oocyte maturation

medium was TCM-199 with Earle's salts supplemented with 10% (v/v) bovine steer

serum, 2 jig/ml estradiol 17-P, 20 jtg/ml FSH, 0.2 mM sodium pyruvate, 50 jtg/ml

gentamicin and an additional 1 mM glutamine. Embryos were cultured in CRlaa that

was prepared as described by Rosenkrans et al. (1993).

In Vitro Production of Embryos

Ovaries obtained from cows of a variety of beef and dairy breeds were obtained at

a commercial abattoir in Central Florida and transported to the laboratory in saline

solution containing 100 U/ml penicillin-G and 100 tg/ml streptomycin at room

temperature. Upon arrival to the laboratory, ovaries were washed with pre-warmed saline

solution. Cumulus oocyte complexes (COCs) were collected by slicing the surface of

each ovary. The COCs were cultured in groups of 10 in pre-equilibrated 50 jl drops of

oocyte maturation medium covered with mineral oil. The COCs were allowed to mature

for 22-23 h at 38.5C in an atmosphere of 5% (v/v) CO2 in humidified air. After
I
maturation, COCs were washed once in HEPES-TALP and transferred in groups of ~30

oocytes to 600 gl of IVF-TALP. Frozen-thawed sperm from three different bulls were

pooled and then purified by Percoll gradient centrifugation as described by Parrish et al.

(1986). The pellet was collected, placed in a 15 ml conical tube containing 10 ml SP-

TALP and centrifuged at 1000 x g for 5 min. The supernatant was removed and the

viable sperm were re-suspended in IVF-TALP to achieve an approximate concentration

of 25 million spermatozoa per ml.









Oocytes were fertilized by adding 25 pl sperm suspension and 25 P1 PHE [0.5

mM penicillamine, 0.25 mM hypotaurine, and 25 pM epinephrine in 0.9% (w/v) NaCI] to

each 600 p1 of IVF-TALP well. After 8-10 h at 38.5C and 5% (v/v) CO2 in humidified

air, presumptive zygotes were removed from the fertilization wells and denuded of the

cumulus cells by vortexing in -50 p1 of HEPES-TALP for 5 min in a 2 ml

microcentrifuge tube. Putative zygotes were washed twice in HEPES-TALP and cultured

in groups of 25 to 30 embryos in pre-equilibrated 50 ill drops of CRlaa. Heat-treated

fetal calf serum (10% v/v) was added to each drop on d 5 after insemination (day of

insemination is d 0).

Induction of Thermotolerance at the two-Cell Stage

A total of three experiments were performed to determine whether induced

thermotolerance occurs in 2-cell embryos. In each experiment, the CO2 percentage was

adjusted when temperatures higher than 38.5C were used to prevent pH changes due to

decreased gas solubility at higher temperatures (Rivera and Hansen, 2001). In particular,

the CO2 percentage was 6.0% for 40C, 7.0% for 41C and 8.0% for 42C.

In experiment 1, putative zygotes were placed in microdrops of CRl aa after

fertilization. Beginning at 28-32 h post insemination, when -50% of embryos were at the

2-cell stage, microdrops containing embryos were exposed to one of four treatments. The

four treatments imposed were 38.5C for 7.5 h (control), 40C for I h and 38.5C for 6.5

h (mild heat shock), 38.5C for 3 h and 41C for 4.5 h (severe heat shock); or 40C for 1

h, 38.5C for 2 h, and 41C for 4.5 h (induced thermotolerance). These temperatures

were chosen because exposure to 40C for 1 h followed by 38.5C for 2 h has been

shown to induce thermotolerance in bovine blastocysts (Ealy and Hansen, 1994).









Treatments are referred to as 38.5/38.5C; 40/38.5C; 38.5/41C, and 40/41C. After the

treatment periods, all embryos were placed at 38.5C and 5% CO2. Fetal calf serum

(10% v/v) was added at d 5 and the proportion of embryos becoming blastocysts at d 8

was recorded. The experiment was replicated on 5 occasions with a total of 178-221

embryos per treatment.

For experiment 2, two temperature treatments were tested for ability to induce

thermotolerance; 41C or 42C for 80 min. The latter temperature has been shown to

induce HSP70 synthesis in bovine 2-cell embryos (Chandolia et al., 1999). Embryos at

the 2-cell stage (-28-32 h post insemination) were separated from other embryos and

oocytes, placed into a new microdrop of CRlaa (10-12 embryos/drop) and assigned to

one of six treatments as follows: 1) exposure to 38.5C for 15 h and 20 min at

38.5/38.5C; control; 2) 41C for 80 min (41/38.5C, a mild heat shock); 3) 42C for 80

min (42/38.5C, another mild heat shock); 4) 38.5 C for 3 h and 20 min followed by

41C for 12 h (38.5/41C; a severe heat shock; 5) 41C for 80 min, 38.5C for 2 h and

41C for 12 h (41/41C; an induced thermotolerance treatment); and 6) 42C for 80 min,

38.5C for 2 h and 41C for 12 h (42/41C; another induced thermotolerance treatment).

At the end of each treatment, all embryos were returned to culture at 38.5C for the

remainder of the experiment. Fetal calf serum (10% v/v) was added on d 5 after

insemination and the proportion of 2-cell embryos that developed to blastocyst was

recorded on d 8 after insemination. The experiment was replicated 3 times with a total of

40-58 embryos per treatment.

In experiment 3,2-cell embryos were separated from other embryos and oocytes

at 28-32 h post insemination, and placed in a new drop of CRlaa and assigned to one of









three treatments: 1) exposure to 38.5C for 9 h and 20 min (38.5/38.5C; control); 2) 38.5

C for 3 h and 20 min followed by 41C for 6 h (38.5/41C; a severe heat shock); and 3)

41C for 80 min, 38.5C for 2 h and 41C for 6 h (41/41C; induced thermotolerance).

Embryos were cultured at 38.5C and 5% CO2 after the treatment period. Fetal calf

serum (10% v/v) was added on d 5 after insemination. The proportion of 2-cell embryos

that developed to blastocyst was recorded on d 8 after insemination. The experiment was

replicated 21 times with a total of 249-285 embryos per treatment.

Role of HSP70i in Embryonic Development

Experiments 4 and 5 were conducted to determine whether the presence of a

monoclonal antibody against HSP70i blocks development at 38.5C and reduces ability

of embryos to survive heat shock. For each experiment, putative zygotes were denuded

of cumulus cells and were placed in 45 pl microdrops of CRlaa. Either 5 Pl of phosphate

buffer saline containing 1 mg/ml anti HSP70i or mouse IgGi were added to each drop

(final concentration of immunoglobulin=100 pg/ml). At 28-32 h post insemination,

embryos that were not at the 2-cell stage were removed from the microdrops. The 2-cell

embryos were retained in the drops and were then exposed to one of the following

treatments: 1) 38.5C for 9 h and 20min (38.5/38.5C; control); 2) 38.5C for 3 h and 20

min followed by 41C for 6 h (38.5/41C; severe heat shock); and 3) 41C for 80 min,

38.5C for 2 hi and 41C for 6 h (41/41C; induced thermotolerance). At the end of

treatment, all embryos were washed three times in CRIaa and then transferred to a new

CRlaa drop without antibody or IgG1 and cultured at 38.5C and 5% CO2. On d 5 after

insemination, 5 pl (10% v/v) of fetal calf serum was added to each drop. The proportion

of 2-cell embryos that developed to blastocyst was recorded on d 8 after insemination.









The experiment was replicated 6 times with 74-83 embryos per treatment. Experiment 5

was conducted similarly except that the severe heat shock was of a shorter duration (41C

for 3 h instead of 6 h). The experiment was replicated 10 times with 102-111 embryos

per treatment.

Statistical Analysis

Data were analyzed by least squares analysis of variance using the General Linear

Models (GLM) procedures of SAS (SAS, 1989). Percentage data were transformed using

the arcsin transformation before analysis. For induced thermotolerance experiments, the

mathematical model included the main effects of replicate, treatment (mild and severe

heat shock) and their interaction. An interaction between the mild (40C, 41C, or 42C)

and severe (41C for 4.5, 6 or 12 h) heat shock was an indication of induced

thermotolerance. Data for experiments 4 and 5 were analyzed using a mathematical

model that included the main effect of replicate, temperature treatment (38.5/38.5C,

38/41 C and 41/41 C), presence or absence of antibody and the interaction of

temperature treatment and antibody. In addition, a subset of data containing results from

both experiments 4 and 5 for embryos cultured at 38.5C was also subjected to statistical

analysis to test the overall effect ofahti-HSP70i on development in the absence of heat

shock (n=16 replicates). The mathematical model included the main effect of replicate

and antibody (anti-HSP70i vs. IgGI) on the proportion of 2-cell embryos that developed

to blastocyst.









Results

Induction of Thermotolerance at the Two-Cell Stage

A series of experiments was conducted to determine whether thermotolerance

could be achieved in bovine embryos at the 2-cell stage. In experiment 1 (Table 4-1), it

was tested whether prior exposure of embryos to 40C for 1 h (i.e., a temperature shown

earlier to induce thermotolerance in bovine blastocysts; Ealy and Hansen, 1994) would

make embryos more resistant to a more severe heat shock of41C of 4.5 h. Exposure of

embryos to 40C for 1 h did not affect subsequent development. However, embryonic

development was slightly reduced when embryos were exposed to 41C for 4.5 h;

previous exposure to 40C for 1 h did not improve embryonic development for embryos

exposed to 41C.

For experiment 2 (Table 4-2), the proportion of 2-cell embryos that developed to

blastocysts was lower (P < 0.05) when embryos were exposed to 42C for 80 min. This

temperature treatment was earlier shown to induce synthesis of HSP70i in bovine

embryos at the 2-cell stage (Edwards and Hansen, 1996). A milder heat shock of41C

for 80 min did not have a significant effect on development to the blastocyst stage. None

of the 2-cell embryos exposed to a severe heat shock of 41 C for 12 h developed to the

blastocyst stage and this was true even for embryos that were exposed to either 41 or

42C for 80 mrin before exposure to severe heat shock. Results of experiment 3 are

shown in Table 4-3. Exposure of embryos to 41C for 6 h decreased (P < 0.05) the

proportion of 2-cell embryos developing to blastocyst. However, prior exposure to 41 C

for 80 min did not reduce effects of41C for 6 h.