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Strategies to Enhance Fertility in Dairy Cattle during Summer including Use of Cryopreservation of in Vitro Produced Embryos

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Title:
Strategies to Enhance Fertility in Dairy Cattle during Summer including Use of Cryopreservation of in Vitro Produced Embryos
Creator:
FRANCO, C. MOISES ( Author, Primary )
Copyright Date:
2008

Subjects

Subjects / Keywords:
Cattle ( jstor )
Dairy cattle ( jstor )
Embryos ( jstor )
Estrus ( jstor )
Heat stress disorders ( jstor )
Heifers ( jstor )
Insemination ( jstor )
Ovulation ( jstor )
Pregnancy ( jstor )
Pregnancy rate ( jstor )

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University of Florida
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University of Florida
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Copyright C. Moises Franco. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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7/24/2006
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445708168 ( OCLC )

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Full Text












STRATEGIES TO ENHANCE FERTILITY IN DAIRY CATTLE DURING SUMMER
INCLUDING USE OF CRYOPRESERVATION OF IN VITRO PRODUCED
EMBRYOS















By

C. MOISES FRANCO


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

UNIVERSITY OF FLORIDA


2006


































Copyright 2006

by

C. Moises Franco





























This professional achievement reflects the sacrifice and guidance of my family especially
that of my mother, Mercedes Y. Vaca El-Hage, who laid the foundations with strong
pillars in my life.

This dissertation is dedicated to my beloved son Talyn Izaak Franco Benton and
astonishing father Antonio Vicente Franco Monasterio (Jf) for their endless love, support
and most important, inspiration.






"EL HOMBRE SE AUTORREALIZA EN LA MISMA MEDIA EN QUE SE
COMPROMETE AL CUMPLIMIENTO DEL SENTIDO DE SU VIDA"

Victor Frankl
(1905-1997)
















ACKNOWLEDGMENTS

This thesis would not have been possible without the enthusiasm, knowledge,

guidance, tenacity, and, perhaps most importantly faith that I received from my academic

advisor, Peter J. Hansen. From the very first interview to the last queries on research

accomplishments and career plans, he was always eager to entertain my ideas in hope that

I fulfilled my dreams) and become successful. I was not sure I could handle an

undertaking of such a magnitude, but was able to thanks to his consistent effort and true

desire to keep me on track.

I would like to extend my sincere appreciation to my committee member Dr. Karen

Moore, for her insight and willingness to help me academically without fail and regards

to time. Despite having other maj or responsibilities, Dr. Carlos Risco was willing to help

whenever asked. I thank him for his assistance and especially for the desire to help me

learn to palpate. Thanks are also extended to Dr. Alvin Warnick for his advice and

suggestions for improving my research proj ects and academic training. I would also like

to thank Dr. Joel Yelich for his teaching, support, and enthusiasm while providing me

with ideas that can help me achieve my goals.

Special thanks are extended to my family for encouraging me to seek for myself a

demanding and meaningful education. This thesis could not have taken place without that

precious gift.

Most sincere appreciation is also due to to my colleague and friend Dr. Rocio M.

Rivera, whose willingness to assist me in my early stages as a master's student helped to









kindle my interest in this exploration. I would not have gotten this far if it was not for her

unique and excellent training doing IVF. Dr. Zvi Roth was an inspirational friend whose

passion for science was transmitted to me. He also expressed his kindness and love

towards my son. I also thank Dr. Joel Hernandez for his support, friendship, and

guidance.

Thanks are given to Maria B. Padua for her assistance with the completion of this

manuscript and Luis Augusto Castro e Paula. Their unconditional friendship and help at

any given time is sincerely appreciated. I am grateful to Dean Jousan for making the

time to proofread my writings throughout the years and for his assistance in various

research experiments. Special thanks go to Amber Brad for her personality and joy that

helped the lab be united. Best of all has been my colleague and friend Jeremy Block for

his patience, expertise and engaging conversations that helped develop in me new

dreams. In addition, he always remained motivated throughout my transfer experiments.

I also would like to thank Central Packing Co. management and personnel at

Center Hill, FL, for providing the ovaries used for various experiments and William

Rembert for his assistance in collecting these ovaries. Special thanks go to Mary Russell

and Elise Griffin, for their assistance at the University of Florida Dairy Research Unit. I

thank Luther White and Mark Saulter of Hilltop Dairy, R.D. Skelton and Mathew Steed

of Levy County Dairy, and Mauricio Franco and Faby Grisel of Sausalito Dairy for

cooperation and assistance with the proj ects. And last but not least, I would like to thank

Todd Bilby, Osiloam Gomez, Reinaldo Cooke, Patrick Thompson, Saban Tekin, and

Paolette Soto.




















TABLE OF CONTENTS


IM Le

ACKNOWLEDGMENT S .............. .................... iv

LIST OF TABLES ............_ ..... ..__ ..............ix...


LI ST OF FIGURE S .............. ...............x.....

AB STRAC T ................ .............. xi


CHAPTER

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


Infertility in Modern Dairy Cattle............... .. .... ..............
Causes for the Decline in Fertility in Dairy Cattle .............. ...............2.....
Milk Yield .............. ........ ...............
Milk yield and energy balance .............. ...............3.....
Milk yield and endocrine milieu .............. ...............5.....
Milk yield and heat stress ................. ...............6............ ...
Milk yield and diseases ........................ ...............9
Milk yield, estrus detection, and fertility .............. ...............10....
Changes in Herd Size as a Factor in Reduced Fertility ................. ................. .11
Inbreeding ................... ...... .. ...... ........ ... ..... ..........1
Strategies to Improve Fertility in Lactating Dairy Cattle .................. ............... .....12
Treatment with Bovine Somatotropin (bST) to Enhance Fertility ......................13
Treatment with GnRH to Delay Luteolysis............. ... .................1
Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus
Luteum ............_.. ......_ ...... ............... 17.
Induction of an Accessory Corpus Luteum .........._.._ ....._.. ........._.._....19
Progesterone Supplementation .............. ...............20....
Inhibition of Luteolysis .............. ...............21....
Nutritional Strategies............... ........ .........2
Fat feeding to improve energy balance .............. ...............22....
Admini strati on of anti oxi dants ................. ...............25........... ..
Crossbreeding ................. ...............26.......... ......
Embryo Transfer............... ............ ... ... .......2
Limitations to Optimal Pregnancy Rates Using IVP TET .............. ..............28
Cryopreservation of IVP Embryos ...........__......_ ....__ ................30
Summary and Obj ectives of the Thesis ................. ...............31........... ..











2 EFFECTIVENESS OF ADMINISTRATION OF GONADOTROPIN
RELEASING HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED
OVTULATION FOR INCREASING FERTILITY OF LACTATING DAIRY
COWS AND NON-LACTATING HEIFERS .............. ...............34....

Introducti on ................. ...............34.................
M materials and M ethods ............... .... ......... ........................3
Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation
in Heifers Subj ected to Timed Artifieial Insemination during Heat Stress .....3 5
Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................37
Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................3 8
Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination During Heat
Stress ................ ..... ..... ...... ...__ ..... ... .. .. .. ...........3
Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected
Estrus............... ...............40
Statistical Analysis .............. ...............40....
R e sults................ ........... .... ..... ..__ ... ... .. .. ... ... .........4
Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation
in Heifers Subj ected to Timed Artifieial Insemination During Heat Stress ....42
Experiment 2 GnRH administration at Day 11 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................42
Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination ....................43
Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation
in Lactating Cows Subj ected to Timed Artifieial Insemination During Heat
Stress ................ ..... ..... ...... ... .. .. .. ................4
Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected
E strus. ............... .... ............ .. ..... .. .............4
Overall Effectiveness of GnRH Treatment as Determined by Meta-Analysis....44
Discussion ........._..._. ...._ ... ...............44.....

3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED
EMBRYOS AND POST-TRANSFER ADMINISTRATION OF
GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF
HEAT- STRES SED DAIRY CATTLE .....__.....___ ..........._ ...........5

Introducti on ........... __... ... ._ ...............52...
M materials and M ethods .............. ..... .............. ...... .......5
Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred
D airy R ecipients............ ..... .... ..........................5
Experiment 2 Administration of GnRH on Day 11 after Anticipated
Ovulation in Lactating Recipients that Received an IVP Embryo ..................57
Statistical Analysis .............. ...............59....
Re sults................. ...............60._ ___.......












Experiment 1 Single or twin transfer of IVP embryos ................. ................60
Pregnancy and calving rates ............... ....._ ....._ ............6
Characteristics of gestation, parturition, and calves.. .................. .................61
Experiment 2 Administration of GnRH on Day 11 after Anticipated
Ovul ati on ........._..... ...._... ...............62....
Discussion ........._..... ...._... ...............62.....


4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B
TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED
BOVINE EMBRYO S PRODUCED IN VITRO ......____ ........ ...............72


Introducti on ............ .......__ ...............72...
M materials and M ethods .............. ...............73....

Embryo Production.................. .. .............7
Experimental Design and Embryo Manipulation ......____ ...... ...__...........74
Cryopreservation .............. ..... .. .............7
Thawing and Determination of Survival .............. ...............76....
Statistical Analysis .............. ...............76....
Re sults............ __.. ... ...._ ... ... .._ ... .......7
Effect of Hyaluronic Acid on Embryonic Development ..........._. ................... 77
Survival after Cryopreservation .............. ...............77....
Discussion ............ ..... .._ ...............78...


5 GENERAL DI SCU SSION ............_...... .__ ............... 2....


LIST OF REFERENCES ............_ .......__ ...............91...


BIOGRAPHICAL SKETCH ............_...... .__ ...............123...

















LIST OF TABLES


Table pg

2-1 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and ovulation synchronization protocol on pregnancy rates
of heifers during heat stress. ...._.._.._ ... ..._.__ ....___ ......._............49

2-2 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and season of insemination on pregnancy rates of lactating
cows subj ected to timed artificial insemination. ..........__......._ ..............50

2-3 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and season of insemination on pregnancy rates of lactating
cows subj ected to timed artificial insemination. ..........__......._ ..............50

2-4 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and Days in milk (<150 d vs > 150) at insemination on
pregnancy rates of lactating cows subj ected to timed artificial insemination
during heat stress. .........._ __..... ._ ...............51...

3-1 Effect of recipient type and number of embryos transferred per recipient on
pregnancy rates and losses. ............. ...............68.....

3-2 Effect of recipient type and number of embryos transferred per recipient on
characteristics of pregnancy and parturition. ................ .............. ........ .....69

3-3 Effect of recipient type and number of embryos transferred per recipient on
characteristics of calves born. ............. ...............70.....

4-1 Effect of hyaluronic acid added at day 5 after insemination on production of
blastocysts at day 7 and 8 after insemination. ............. ...............81.....

4-2 Effect of culture in hyaluronic acid and treatment with cytochalasin B on
survival after cryopreservation. ............. ...............81.....















LIST OF FIGURES


Finure pg

1-1 Rolling herd average (RHA, kg milk per lactation), calving interval (CI), and
services per conception (SPC) for 143 dairy herds continuously enrolled in the
Raleigh DHIA record system from 1970 to 1999. ............ .....................3

1-2 Temporal changes in first service pregnancy rate and annual average milk
production from high-producing Holstein-Friesian dairy herds in north-eastern
Spain. Data for pregnancy rate were recorded in the cool (October April
months) and warm season (May-September months). ........... ......................3

3-1 Maximum (open circles) and minimum (closed circles) daily air temperatures
and relative humidities (RH) during the experiments. .............. ....................7
















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

STRATEGIES TO ENHANCE FERTILITY IN DAIRY CATTLE DURING SUMMER
INCLUDING USE OF CRYOPRESERVATION OF IN VITRO PRODUCED
EMBRYOS


By

C. Moises Franco Vaca

May 2006

Chair: Peter J. Hansen
Major Department: Animal Sciences

There has been a precipitous decline in fertility of lactating dairy cows. In addition,

heat stress can further compromise fertility. The goals of this thesis were to 1) evaluate

strategies for enhancing fertility after artificial insemination using mid-cycle GnRH

treatment and 2) further develop embryo transfer using in vitro produced embryos as a

tool for increasing fertility. For the second obj ective, experiments tested whether

pregnancy rate could be improved by transfer of twin embryos and whether the

developmental competence of embryos after cryopreservation could be improved by

hyaluronic acid or cytochalasin B treatment.

A series of six experiments were conducted to test the efficacy of GnRH for

increasing fertility. Except for one experiment, in which GnRH administration at day 14

after insemination increased pregnancy rate, GnRH was without effect whether given at










day 11, 14 or 15 after insemination or at day 11 after anticipated ovulation in embryo

transfer recipients..

Neither unilateral transfer of two embryos nor administration of GnRH at Day 1 1

after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat

stress. Cytochalasin B treatment before freezing improved cryosurvival of bovine

embryos produced in vitro. In contrast, culture with hyaluronic acid was of minimal

benefit.

Taken together, GnRH treatment did not consistently increase pregnancy rates

when administered at Day 11-15 after insemination and is not recommended as a fertility-

enhancing treatment. Similarly, transfer of two embryos to the uterine horn ipsilateral to

the CL was not an effective method for increasing pregnancy rates in recipients. Transfer

of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B

since this molecule increased in vitro survival, and it remains to be tested whether

survival of IVP embryos after vitrification can be improved by cytochalasin B treatment.















CHAPTER 1
REVIEW OF LITERATURE

Infertility in Modern Dairy Cattle

Fertility is defined as the ability of a cyclic animal to establish pregnancy and is an

important economic trait that affects herd productivity in dairy cattle (Pecsok et al., 1994;

Plaizier et al., 1998). Unfortunately, there has been a decline in fertility in dairy cows

over the last 10-40 years. Fertility, whether traditionally measured as conception rate

(number of pregnant animals divided by the number of inseminated animals) or herd

pregnancy rate (number of pregnant animals divided by the number of animals eligible to

be bred), has declined in North America (Butler, 1998), Ireland (Roche, 2000), Spain

(L6pez-Gatius, 2003), and the United Kingdom (Royal et al., 2000). Other important

reproductive measurements have changed during this time as well, including increases in

days to first service, days to conception, and calving interval (de Vries and Risco, 2005).

The magnitude of these changes in reproductive function over time is illustrated for data

from herds in the United States (Figure 1-1) and northeastern Spain (Figure 1-2).

The incidence of infertility of dairy cows has been correlated with changes in dairy

cattle physiology and improvements in genetic progress, nutrition, and management

practices. This literature review will seek to identify physiological causes for this

decrease in fertility and describe efforts to improve fertility.









Causes for the Decline in Fertility in Dairy Cattle

Milk Yield

The Animal Improvement Programs Laboratory of the United States Department of

Agriculture (USDA) has estimated the genetic trend for milk yield with an average of 37

kg/yr during the 1960s, 79 kg/yr during the 1970s, 102 kg/yr during the 1980s, and 116

kg/yr for the period from 1990 to 1996 (http://aipl.arsusda.gov; Hansen, 2000). It has

long been known that fertility is reduced in lactating cows as compared to non-lactating

heifers (Ron et al., 1984; Nebel and McGilliard, 1993). Given that milk yield has

increased over time as fertility has declined, the possibility must be considered that the

increase in milk yield is one reason that has contributed to the decreased fertility in dairy

cattle.

There are indications that the genetic correlation between female fertility and milk

production is antagonistic (Kadarmideen et al., 2000; Royal et al., 2002). In contrast,

Mahanna et al. (1979) suggested that there was no negative genetic correlation between

milk yield and reproduction because there was no difference in fertility among heifers

with different genetic abilities for milk yield. There may be an environmental effect of

milk yield on fertility, however. As described by Lucy (2001), the increase in milk yield

over the period from 1970 has been associated with a corresponding decrease in fertility

as measured by increased services per conception and calving interval (Figure 1-1).

According to Nebel and McGilliard (1993) there was little or no association of increased

milk yield compromising fertility prior to the 1970s (Gaines, 1927; Boyd et al., 1954;

Currie, 1956; Smith and Legates, 1962) but adverse effects of milk yield have been

correlated with reduced fertility in studies conducted since 1975 (Spalding et al., 1975;









Laben et al., 1982; Fonseca et al., 1983; Stevenson et al., 1983; Hillers et al., 1984;

Wiggans et al., 1987; Faust et al., 1988).

Using a data set of Holstein, Jersey, and Guernsey cows, it was found that 0.014

more services per conception were required for each additional 100 kg of 120-d milk for

Holsteins and 0.028 services per conception for Jersey and Guernsey cows (Olds et al.,

1979). Similarly, cows with the highest milk yield had the lowest first service conception

rate (Faust et al., 1988) or 90-d non-return rate (Al-Katanani et al., 1999) and highest

number of services (Faust et al., 1988). Days to first insemination and days open also

increased linearly as milk yield increased in Jersey dairy cattle (Fonseca et al., 1983).

Expression of estrus at first postpartum ovulation is less likely in cows with higher

milk production (Westwood et al., 2002). Some studies (Nielen et al., 1989; Kinsel et al.,

1998), but not others (Deluyker et al., 1991), correlate the incidence of twins to milk

yield. Amount of milk yield, however, was not correlated to increased incidence of

multiple ovulations (L6pez-Gatius et al., 2005b), yet the incidence of double ovulations

and twinning rate has increased in modern dairy cattle (Wiltbank et al., 2000). Taken

together, the associations of milk yield with reduced duration of estrus, increased days to

first insemination, increased number of inseminations per conception, reduced first

service conception rates, and reduced progesterone levels post-ovulation compromise

herd fertility.

Milk yield and energy balance

One way in which milk yield could affect fertility is through effects on energy

balance. A critical phase exists in the period following calving when dry matter intake

does not meet the increased metabolic demands of lactation, and as a result, the animal









enters a state classified as "negative energy balance" (NEB). During the period ofNEB,

body reserves of fat and protein are mobilized (Bauman and Currie, 1980; Butler and

Smith, 1989). An animal under NEB tends to have low body condition score (BCS), and

both NEB and low BCS are associated with low fertility (O'Callaghan, 1999; Butler,

2000; Pryce et al., 2001; Pushpakumara et al., 2003).

Energy deficiency reduces or impairs gonadotropin secretion, and as an animal

reaches this state around parturition, gonadotropin secretion to support follicular

development and ovulation is compromised and reproductive problems (i.e., cystic

ovaries) associated with onset of ovarian activity become prevalent (Zulu et al., 2002ab).

Growth hormone stimulates insulin-like growth factor 1 (IGF-1) production by the liver

(Jones and Clemmons, 1995), but during NEB growth hormone receptors are

downregulated in a process referred to as "Growth Hormone Resistance" (Donaghy and

Baxter, 1996). As milk production increases during early lactation and the cow is under

NEB, the liver becomes refractory to growth hormone because growth hormone receptors

are decreased (Vicini et al., 1991), and this result in reduced plasma concentration of

IGF-1 (Pell et al., 1993).

Follicular growth is stimulated by IGF-1 (Webb et al., 2004) and reduced plasma

concentrations of this growth factor are observed in cows with high milk yield (Rose et

al., 2004) and together are highly correlated to delayed return to ovarian cyclicity (Taylor

et al., 2004). After calving, cows with IGF-I concentrations greater than 50 ng/ml at first

service were 5 times more likely to conceive than those with lower concentrations

(Taylor et al., 2004).









The fact that high-producing cows have greater energetic demands for lactation

does not necessarily mean that these cows have greater NEB or low BCS. Staples et al.

(1990) found that low-producing cows had lower dry matter intake and were at a greater

risk for failure to conceive due to anestrus and infertility than high-producing cows. It

was observed that the low-producing group, classified as non-responders, sustained milk

production from 28% of body tissue reserve vs 15.9 and 16.7% in the early responder and

late responder groups. This interaction was confirmed when low-producing cows had

lost the most body weight during the first 2 weeks of lactation and were in the greatest

energy deficit (Staples et al., 1990).

Milk yield and endocrine milieu

Cows displaying greater milk production often have higher dry matter intakes

(Staples et al., 1990; Hommeida et al., 2004), which has been demonstrated to decrease

circulating progesterone concentrations in lactating (Hommeida et al., 2004) and non-

lactating cows (Rabiee et al., 2001). Acute feeding reduced circulating progesterone by

25% in pregnant cows (Vasconcelos et al., 2003). Lucy and co-workers (1998) found

that circulating progesterone was lower in cattle genetically selected for high milk

production.

Sangsritavong et al. (2002) demonstrated that lactating cows have a much greater

steroid metabolism than non-lactating cows. As a result, lactating cows may have larger

luteal tissue volume on the ovary (Sartori et al., 2002; Sartori et al., 2004) yet experience

lower circulating progesterone and estradiol concentrations than heifers and dry cows (De

la Sota et al., 1993; Wolfenson et al., 2004). There is evidence that low progesterone









secretion can compromise fertility in dairy cattle (Mann and Lamming, 1999) and an

increase in progesterone secretion may facilitate embryonic development.

Progesterone provides nourishment for the concepts via induction of secretion of

proteins and other molecules from the endometrium (Garrett et al., 1988a). Low

peripheral concentrations of progesterone are also associated with increased luteinizing

hormone (LH) pulses (Ireland and Roche, 1982) that can stimulate luteolytic signals in

favor of pregnancy failure. Skarzynski and Okuda (1999) reported that blocking the

progesterone receptor with a progesterone antagonist (onapristone) increased

prostaglandin F2a (PGF2a) prOduction by bovine luteal cells harvested from mid-cycle

corpora lutea (CL) (Days 8-12). In addition, it was revealed that the bovine corpus

luteum (CL) does not undergo apoptosis until progesterone production has declined

(Juengel et al., 1993; Rueda et al., 1995).

Milk yield and heat stress

One reason why milk yield might decrease fertility of lactating cows is because it

increases their susceptibility to heat stress. Infertility is a particular problem during heat

stress (Ingraham et al., 1974; Putney et al., 1989b; Al-Katanani et al., 1999) and air

temperatures as low as 27oC can induce hyperthemia in lactating dairy cows (Berman et

al., 1985). Cows exposed to elevated temperatures to induce heat stress experienced

reduced pregnancy rates (Dunlap and Vincent, 1971) and increased embryonic mortality

(Putney et al., 1988ab; Ealy et al., 1993). On the other hand, provision of cooling in the

summer increased pregnancy rates as compared to non-cooled cows (Stott et al., 1972;

Roman-Ponce et al., 1981; Ealy et al., 1994).









The ability to regulate body temperature during heat stress is exacerbated by

lactation because of the excess heat production. The increase in body temperature in

response to heat stress is greater for lactating cows than heifers (Cole and Hansen, 1993)

and greater for high-producing cows than low-producing cows (Berman et al., 1985).

Data collected on fertility at first service from 8124 Holstein cows located in South

Georgia as well as North and South Florida support the idea that a high level of milk

production reduces fertility of lactating cows. When cows were grouped according to

mature equivalent milk yield, there was a milk yield class x month of breeding interaction

that resulted from the fact that the duration and magnitude of summer infertility increased

as milk yield increased (Al-Katanani et al., 1999).

Heat stress before, shortly after, and on the day of breeding is associated with

reduced fertility. Heat stress can compromise fertility throughout various reproductive

processes such as oocyte developmental competence (Picton et al., 1998; McNatty et al.,

1999) since the oocyte becomes sensitive to damage throughout the various stages of

follicular growth (Badinga et al., 1993). Indeed, follicular steroidogenesis, follicular

dynamics and altered concentrations of FSH and inhibin become altered in response to

heat stress (Badinga et al., 1994; Wolfenson et al., 1997; Roth et al., 2000). During heat

stress sperm can be damaged after insemination due to the generation of reactive oxygen

species (Ishii et al., 2005) and embryonic development can be compromised directly

(Monty et al., 1987). Not surprisingly the heat stress problem is multifactorial (Hansen et

al., 2001).

Heat stress of superovulated cows at day 1 after breeding reduced the proportion of

embryos that were blastocysts at day 8 after breeding, but heat stress on day 3, 5 or 7









after breeding did not affect subsequent embryonic development (Ealy et al., 1993).

Superovulated heifers experienced a high percentage of retarded embryos recovered on

day 7 after insemination after exposure to high temperature and humidity at the onset of

estrus for 10 h (Putney et al., 1989a). In another study heat stress was induced in

Holstein heifers by submitting them from day 1 to day 7 after estrus to 42oC for 7 h

(treatment) or 30oC for 16 h (control) and results obtained revealed more retarded

embryos with degenerate blastomeres on the day of recovery (20.7% vs. 51.5%,

respectively; Putney et al., 1988a).

One cause for the observed reduction in reproductive performance under heat stress

conditions is steroidogenic capacity and its effects on oocyte function (Roth et al., 2001;

Al-Katanani et al, 2002b; Roth and Hansen, 2004). Under heat stress, low estradiol

concentration in the follicular fluid of dominant follicles involves reduced aromatase

activity in the granulosa cells (Badinga et al., 1993) and reduced androstenedione

production by theca cells (Wolfenson et al., 1997). Although earlier studies were

inconsistent in demonstrating that plasma concentrations of estradiol are reduced under

heat stress (no change- Gwazdauskas et al., 1981; increase Rosenberg et al., 1982;

decrease Gwazdauskas et al., 1981), recent work points toward heat stress resulting in

lower estradiol concentrations in the follicular fluid (Badinga et al., 1993; Wolfenson et

al., 1995; Roth, 1998; Wilson et al., 1998ab).

Heat stress also has been reported to decrease (Rosenberg et al., 1982, Younas et

al., 1993; Howell et al., 1994), increase (Abilay et al., 1975; Roman-Ponce et al., 1981;

Trout et al., 1998), or have no effect (Wise et al., 1988; Wolfenson et al., 1995) on

peripheral concentrations of progesterone. Elevated temperatures in culture can directly









influence endometrium explants by increasing PGF2a Secretion (Putney et al., 1988c;

Malayer and Hansen, 1990) and from days 8-16 of pregnancy can reduce the size of the

embryo at day 17 (Biggers et al., 1987).

A retrospective survey involving 12,711 lactations from high-yielding dairy herds

in northeast Spain demonstrated that milk yield per cow increased from 1991-2000

(L6pez-Gatius, 2003; see Figure 2). For each 1000 kg increase in average milk yield in

the warm period, there was a decrease of 6% in pregnancy rate, and 7.6% in cyclicity,

and an increase of 8% in the incidence of inactive ovaries. During the cool period,

however, there was no change in fertility over time. Thus, the continual increase in milk

yield might have reduced fertility in Spain, at least, by exacerbating effects of heat stress.

Milk yield and diseases

Increased incidence of certain diseases has been associated with elevated milk

yield. High somatic cell score and clinical mastitis (Schukken et al., 1990; Barkema et

al., 1998; Chassagne et al., 1998; Fleischer et al., 2001); lameness (Green et al., 2002);

cystic ovarian disease (Fleischer et al., 2001; L6pez-Gatius et al., 2002); milk fever

(Fleischer et al., 2001); and acute metritis (Kelton et al., 1998) are all correlated with

milk yield.

Compared to non-mastitic herd-mates, high producing cows were at a greater risk

of developing clinical mastitis (Grahn et al., 2004). Number of days to conception,

artificial inseminations per conception and number of days to first artificial insemination

(AI) were significantly greater for cows with clinical mastitis (Barker et al., 1998), and

may affect embryonic survival when occurring after insemination (Soto et al., 2003).

According to Jousan et al., (2005) an elevated somatic cell count score among lactating









females influenced mid-to-late fetal loss (represented as occurring after day 70 to 90 of

gestation) and mastitis has been reported to affect pregnancy loss during the period of

embryonic (Chebel et al., 2004) and fetal development (Risco et al., 1999; Santos et al.,

2004a).

High yielding cows had an increased likelihood of becoming lame (Green et al.,

2002) and cows that had been treated for lameness had a negative influence on pregnancy

to Birst insemination and numbers of inseminations per service period (Petersson et al.,

2005). Similarly, non-lame cows were more likely to conceive at first service than lame

cows and lameness within the first 30 days after calving was associated with reduced

pregnancy rates at first AI and a higher number of services per conception (Hernandez et

al., 2001; Melendez et al., 2003). In a meta-analysis of several published papers, leg

problems were associated with an average increase of 12 days to conception (Fourichon

et al., 2000).

Cows that develop cysts remain infertile as long as this condition persists and early

spontaneous cyst recovery was negatively correlated with milk yield (L6pez-Gatius et al.,

2002). Similarly, elevated milk yield increased the risk of cows developing cysts (L6pez-

Gatius et al., 2002) and days from metritis occurrence to first AI is also correlated to

infertility (Loeffler et al., 1999). Milk yield in the current lactation is also correlated with

incidence of milk fever (Fleischer et al., 2001) and this disease reduces fertility (Chebel

et al., 2004).

Milk yield, estrus detection, and fertility

Milk yield may affect fertility indirectly by reducing the ability to accurately detect

estrus. An antagonistic relationship between increased milk production and days to first









visual estrus has already been reported. According to LC~pez et al. (2004), duration,

standing events, intensity (determined by the number of standing events per hour), and

standing time were reduced for high-producing cows as compared to low producers.

Similarly, Harrison et al. (1990) reported that elevated milk yield was correlated to a

longer period of estrus suppression. Westwood et al., (2002) indicated that high genetic

merit for milk yield influenced significantly the chance a cow showed weak signs of

estrus as compared to low milk producing cows.

Cows with elevated milk yield also had reduced circulating estradiol concentrations

on the day of estrus expression and shorter duration of estrus despite having larger

preovulatory follicle diameters (LC~pez et al., 2004).

Changes in Herd Size as a Factor in Reduced Fertility

Increased milk yield is not the only change in dairy farming over the last 50 years

and some of these other changes could also contribute to decreased fertility. One major

change has been the trend towards large farms. In a review, Lucy et al. (2001) cited data

from the USDA National Agricultural Statistics Services that nearly 30% of all dairy

farms in the United States have more than 500 cows. In addition, Stahl et al. (1999)

reported that the expansion of dairy herds comes in large part through the purchase of

first-lactation cows. Thus, as Lucy et al. (2001) pointed out, these more infertile

primiparous cows (Stahl et al., 1999) may have represented an increasingly larger

percentage of the herd as dairy herds have expanded over the last 10-40 years. The

importance of changes in herd size as a cause for infertility have been questioned by de

Vries and Risco (2005) who found no clear association with reproductive function.

Nevertheless, as the herd size is increased one would expect that the likelihood that

it becomes harder for accurately detecting estrus becomes a challenge because factors









associated with herd size such as the surface (concrete floor) on which the cow stands

will reduce the preponderance of cows displaying estrus activity (Britt et al., 1986;

O'Connor and Senger, 1997).

Inbreeding

Inbreeding represents increased frequency of identical alleles at a gene locus and

the inbreeding percent is a measure for the genes of an individual that are identical by

descent (Wright, 1922; Falconer, 1981). It is generally considered that reproductive

function declines when inbreeding levels in a population rise above 6.25% (Hansen et al.,

2005). Increased degree of inbreeding as the result of use of AI could explain some of

the declines in fertility experienced by dairy cattle because inbreeding coefficients have

increased in all the maj or U. S. dairy breeds. Estimates of inbreeding in the U. S. dairy

population are near 5% currently (Short et al., 1992; Wiggans et al., 1995; Young et al.,

1996; Hansen, 2000; Wall et al., 2005) and increasing at a constant rate of about 0. 1% per

year for U. S. Holsteins (Hansen et al., 2005). At an average of 5%, it is likely that many

dairy cows have inbreeding coefficients above 6.25% (Hansen et al., 2005).

Thompson et al. (2000ab) found calving intervals to increase by 12 and 17 d for

Jersey and Holsteins cows, respectively, with levels of inbreeding >10%. Similarly,

inbreeding had pronounced negative effects on fertility at higher levels (10%) of

inbreeding (Wall et al., 2005). In another study, animals with an inbreeding coefficient

>9% had fewer transferable embryos following superovulation than animals with a lower

inbreeding coefficient (Alvarez et al., 2005).

Strategies to Improve Fertility in Lactating Dairy Cattle

Four general approaches to improve reproductive function in dairy cattle have

been developed. The first is to regulate the timing of ovulation using gonadotropin









releasing hormone (GnRH) and PGF2 O utilized in timed AI (TAI) programs. The

advantage of this approach is that this program maximizes the number of animals

inseminated and allows inseminations to be made at some pre-planned time to eliminate

the need for estrus detection. Pioneering studies (Thatcher et al., 1989; Twagiramungu et

al., 1992; Wolfenson et al., 1994) were able to synchronize estrus effectively, however,

subsequent studies at the University of Florida (Schmitt et al., 1996a) and University of

Wisconsin (Pursley et al., 1995) led to the development of the Ovsynch TAI program and

the demonstration that good pregnancy rates can be achieved (Thatcher et al., 2001;

Thatcher et al., 2002). Although this approach is an effective one and is widely used in

dairy herds, it involves regulation of events occurring before conception and is beyond

the scope of the present review. The second approach is to use information regarding the

hormonal basis for establishment of pregnancy and signaling between the maternal and

embryonic units during early pregnancy as the basis for pharmacological treatments to

improve embryonic survival. Failure of essential biochemical dialogue between the

concepts and the maternal unit undoubtedly contributes to embryonic mortality and

termination of pregnancy (Spencer et al., 1996; Spencer and Bazer, 2002). The third

approach has been to regulate the nutrition of the dairy cow to improve energy balance or

to provide specific nutrients that favor establishment and maintenance of pregnancy.

Finally, recent work has focused on use of embryo transfer to bypass early embryonic

death and perhaps coupled with crossbreeding may become an important alternative since

Holsteins have become more inbred (Hansen et al., 2005).

Treatment with Bovine Somatotropin (bST) to Enhance Fertility

Circulating concentrations of IGF-I, glucose, and cholesterol are reduced in

lactating animals (de la Sota et al., 1993; Beam and Butler 1997). Circulating









concentrations of IGF-I is influenced by nutrition (Adam et al., 1997) and closely related

to energy balance of the cow (Ginger et al., 1997; Beam and Butler, 1998; 1999). Present

in serum and in various tissues, IGF-I is produced mainly by the liver but other organs as

well (Murphy et al., 1987; Thissen et al., 1994). IGF-I regulates ovarian function in dairy

cattle (Breukink et al., 1998; Chase et al., 1998), is necessary for proper follicular

development in which a fully competent oocyte capable of inducing ovulation develops

(Lucy et al., 1992a), and is required for normal CL formation and function (Leeuwenberg

et al., 1996; Chase et al., 1998). Dairy cows that initiated estrous cyclicity during the

postpartum period had higher plasma IGF-I than anestrous cows (Thatcher et al., 1996),

cystic and inactive ovary or persistent CL cows (Zulu et al., 2002a).

Bovine somatotropin (bST) increases plasma concentrations of insulin, IGF-I, and

growth hormone (Bilby et al., 2004), perhaps by stimulating ovarian function especially

after IGF-1 plasma levels are reduced in lactating animals (de la Sota et al., 1993). In

addition, inj section of bST stimulates concepts growth by day 17 of pregnancy (Bilby et

al., 2004). Additional studies provided evidence that bST can improve pregnancy rates in

lactating cows (Moreira et al., 2000b; Morales-Roura et al., 2001; Santos et al., 2004b).

Superovulated donor cows that received bST treatment experienced reduced number of

unfertilized oocytes, increased number of embryos that developed to the blastocyst stage,

and increased number of transferable embryos (Moreira et al., 2002). Collectively, these

studies indicate that critical thresholds of GH and IGF-I concentrations are needed to

stimulate reproductive performance (Bilby et al., 2004).

Treatment with GnRH to Delay Luteolysis

The estrous cycle is characterized by 2, 3, and sometimes 4 waves of follicular

growth (Sirois and Fortune, 1988; Ginther et al., 1996). During the second half of the









luteal phase, development of an estrogenic follicle facilitates the luteolytic process via

secretion of estradiol. Non-pregnant cows have higher peripheral concentrations of

estradiol on days 16 and 18 after breeding compared to pregnant animals (Ahmad et al.,

1997). Thatcher et al. (1991) examined the largest and second largest follicles present on

day 17 after estrus in pregnant and cyclic dairy cows. In the cyclic cows, the largest

follicle had greater aromatase activity and contained more estradiol and less progesterone

in the follicular fluid than the second largest follicle. These relationships were reversed

in pregnant animals, which indicated an earlier recruitment of the third wave of follicular

development in the pregnant animal associated with delayed luteolysis and higher

pregnancy rates. That these follicles play an important role in luteolysis was shown by

Villa-Godey et al. (1985), who reported that electrocautery to destroy large follicles was

associated with an extension of the estrous cycle.

Estradiol is now known to be one of three hormones that control uterine secretion

of PGF2,, With progesterone and oxytocin also being involved. Pulsatile release of PGF2,

from the luminal epithelium of the endometrium is stimulated via oxytocin (Roberts and

McCracken, 1976; Silvia and Taylor, 1989; Milvae and Hansel, 1980). Progesterone and

estradiol regulate this process because estradiol induces formation of oxytocin receptors

(Silvia and Taylor, 1989; Zingg et al., 1995; Robinson et al., 2001) after progesterone

exposure (Ginther, 1970; Garrett et al., 1988b; Lafrance and Goff, 1988). While

progesterone initially suppresses PGF2, Secretion by blocking oxytocin receptors during

the early and mid-luteal phase of the estrous cycle, the endometrium becomes responsive

to oxytocin and progesterone receptors become down regulated as the estrous cycle

progresses (Lafrance and Goff, 1988; Spencer and Bazer, 1995).










Delaying luteolysis might improve pregnancy rate by allowing embryos more time

to produce sufficient quantities of interferon-z (IFN- z). Eliminating or decreasing

estradiol production from the dominant follicle during the critical period of early

pregnancy could be one strategy to improve pregnancy establishment (Thatcher et al.,

2000; Binelli et al., 2001). One approach for doing this is to use GnRH to regulate

follicular function.

Gonadotropin releasing hormone is a decapeptide that plays a central role in

regulating reproductive processes. Release of GnRH from the hypothalamus occurs in a

pulsatile fashion and can be regulated by various internal and external signals.

Hypothalamic GnRH is synthesized in cell bodies of neurosecretory neurons, and is

transported to and released from the median eminence into the hypothalamic-

hypophyseal portal system (Loucopoulos and Ferin, 1984). GnRH has its primary effects

at the pituitary gonadotrope and stimulates the pulsatile release of the gonadotropins

luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the peripheral

circulation (Chenault et al., 1990). Two potential gonadotropin responsive tissues within

the ovary are the CL and the follicle. LH release induces ovulation or luteinization of

large ovarian follicles present at the time of treatment (Thatcher and Chenault, 1976).

One strategy tested for increasing pregnancy rate is to inject GnRH or GnRH

analogues at day 11-14 after estrus to increase progesterone secretion (Willard et al.,

2003) and delay luteolysis (Macmillan and Thatcher, 1991), thereby increasing the

chance for an embryo to initiate its own antiluteolytic mechanism. Inj section of GnRH at

this time can lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and









Lamming, 1995a) in an action that likely involves luteinization of the dominant follicle

(Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994).

Improvement of fertility has been seen by administration of GnRH or its analogues

at day 1 1-14 in nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows

(Macmillan et al., 1986; Lajili et al., 1991; Sheldon and Dobson, 1993; Drew and Peters,

1994; Willard et al., 2003; L6pez-Gatius et al., 2005a). In contrast to these positive

results, there was no favorable effect of similar treatments of GnRH or GnRH analogues

on pregnancy rates in other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al.,

1994; Bartolome et al., 2005). In a meta-analysis of published results, Peters et al. (2000)

concluded that the overall effect of GnRH administration between day 11 and 14 after

anticipated ovulation was positive but that results were not consistent between studies.

Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus
Luteum

As mentioned earlier, high-yielding dairy cows are more likely to have lower

circulating concentrations of progesterone throughout the estrous cycle than cows with

lower milk yields because of increased rate of progesterone catabolism (Lucy et al., 1998;

Vasconcelos et al., 1999). Given the importance of progesterone concentration for

embryonic survival (Man and Lamming, 2001), efforts have been made to increase

progesterone secretion in cows. One possible effect of mid-cycle treatment with GnRH is

to increase progesterone secretion (Schmitt et al., 1996b; Willard et al., 2003). Another

approach for increasing progesterone concentrations has been to regulate the size of the

preovulatory follicle to affect subsequent CL function.

Optimum differentiation and growth rate of the CL varies according to the duration

and amplitude of the ovulatory LH surge such that inhibition of LH release preceding the










preovulatory surge of LH resulted in development of a smaller CL in diameter (Quintal-

Franco et al., 1999). Induced ovulation of small follicles resulted in a smaller CL and

reduced secretion of progesterone than when a larger follicle ovulated (Vasconcelos et

al., 2001). In another study (Perry et al, 2005), regression analysis indicated that

pregnancy rate for cows with induced ovulation with an ovulating follicle of 14.5 mm

was higher than for cows ovulating follicles <10.3 mm in diameter. It was further

revealed that 39% of cows that lost their pregnancy had ovulatory follicles <11 mm in

diameter. Among cows that ovulated spontaneously, however, pregnancy rates at day 27

and 68 were independent of ovulatory follicle size (Perry et al., 2005). In contrast to this

result, Vasconcelos et al (1999) found that the group of cows ovulating larger follicles

had lower pregnancy rates on day 28 and 98 after AI and higher pregnancy loss between

these times.

Administration of GnRH just prior to or at the time of the LH surge causes an

amplified preovulatory surge ofLH (Lucy and Stevenson, 1986; Yoshioka et al., 2001).

Inj section of GnRH at or near the time of estrus increased the proportion of large luteal

cells in the CL on day 10 of the estrous cycle (Mee et al., 1993), peripheral progesterone

concentrations during the first 7 days of the estrous cycle (Lucy and Stevenson, 1986),

and increased pregnancy rates in repeat breeding cows (Stevenson et al., 1990; Mee et al.,

1993).

Ullah et al. (1996) observed that GnRH treatment at estrus in dairy cows improved

pregnancy rates and increased peripheral progesterone concentration. Conversely, GnRH

administered to lactating dairy cows at the time of AI did not affect pregnancy rates

(Ryan et al., 1994). Similarly, Mee et al. (1990) concluded that GnRH treatment at 1 h or









12 to 16 h after first detected estrus did not improve pregnancy rates at first service. Mee

et al. (1990) mentioned that 16 studies in the literature suggest an overall advantage in

pregnancy rate of 6 percentage points (53 vs. 59%) or an 1 1% improvement for cows

receiving GnRH treatment at the time of AI or up to 6 h preceding AI.

Induction of an Accessory Corpus Luteum

Progesterone concentrations following ovulation have been positively correlated to

volume of uterine secretions (Garrett et al., 1988a), concepts development (Garrett et

al., 1988a; Mann et al., 1996), the embryos ability to secrete IFN-z (Kerbler et al., 1997;

Mann et al., 1998), embryo viability for subsequent survival (Stronge et al., 2005), and

perhaps most importantly conception rates (Hansel, 1981; Fonseca et al., 1983; Shilton et

al., 1990; Larson et al., 1997). One possible approach to increasing progesterone

secretion has been to induce formation of an accessory CL by administering GnRH or

hCG, LH or their analogues at a time when the first wave dominant follicle is present

after ovulation (metestrus) (Rajamahendran and Sianangama, 1992; Schmitt et al., 1996b;

Santos et al., 2001). Santos et al. (2001) reported that hCG treatment on d 5 of a

synchronized estrous cycle induced an accessory CL in 86.2% of treated cows, increased

plasma progesterone by 5 ng/ml, and increased conception rates on day 28 from 38.7% to

45.8% and on day 90 of pregnancy from 3 1.9% to 38.4%. Lactating dairy cows treated

with GnRH on d 5 (Willard et al., 2003) and hCG on day 7 (Raj amahendran and

Sianangama, 1992) or day 4 in heifers (Breuel et al., 1989) reported successful accessory

CL formation and an increase in conception rates and pregnancy rate.

Besides stimulating luteal tissue formation, treatment of cows to induce ovulation

of the first wave dominant follicle with GnRH or GnRH analogues also reprograms

follicular growth to increase the proportion of estrous cycles composed of three follicular









waves as compared to two waves (Diaz et al., 1998). Such an effect could reduce the

probability that a large, highly estrogenic follicle is present during the critical period of

pregnancy recognition. Compared to animals with two-wave cycles, Holstein cows

(Townson et al., 2002) and beef cows (Ahmad et al., 1997) with a three-wave cycle had

higher conception rates and a longer luteal phase (Ginther et al., 1989).

Progesterone Supplementation

The ability of the concepts to secrete IFN-z is related to its developmental

progress and progesterone concentration of the pregnant female (Mann et al., 1999). Low

progesterone concentration in plasma as early as day 6 after insemination has been

implicated as a contributing factor for cows failing to conceive (Bulman and Lamming,

1978; Lukaszewska and Hansel, 1980; Kimura et al., 1987; Lamming and Darwash,

1995; Inskeep, 1995; Mann and Lamming, 1999; Hommeida et al., 2004). Enhanced

luteolytic signals also result from suboptimal progesterone concentrations after

insemination (Mann and Lamming, 1995b). Another approach to increase fertility of

lactating dairy cows has been to directly supplement cows with progesterone. A meta-

analysis of 17 studies revealed that progesterone supplementation after insemination

produced an overall improvement in conception rate of 5% and that the timing of

progesterone supplementation was a critical factor (Mann and Lamming, 1999). One

study revealed depressed conception rates when controlled internal drug releasing

(CIDR) devices containing progesterone were inserted in heifers on day 1 or day 2

following estrus (Van Cleef et al., 1989). In contrast, injection of progesterone (100 mg)

on day 1, 2, 3, and 4 of pregnancy advanced development of conceptuses to 14 days of

gestation in beef cows (Garrett et al., 1988a). These conceptuses had increased length

and secreted a greater array of proteins into medium following a 24 hour culture. When










progesterone supplementation was initiated beginning at day 10 of pregnancy, Macmillan

et al. (1991) found a slight decrease in pregnancy rate (-2.7%), Sreenan and Diskin,

(1983) obtained a small increase (4.3%), and Robinson et al. (1989) obtained a large

increase (29.3%) in pregnancy rate. Villarroel et al. (2004) found that first and second

lactation repeat-breeder Holstein cows were 3.26 times more likely to become pregnant

when cows received progesterone releasing intravaginal device (PRID 1.55g of

progesterone) on day 5 through 19 post-AI.

Inhibition of Luteolysis

The maintenance of a functional CL depends directly upon the intensity of

embryonic signals that attenuates endometrial secretion of PGF2a. Pregnancy fails if an

embryo does not produce sufficient amounts of IFN-z or if production is delayed until

after the critical time-period between days 14 and 17 when the luteolysis would otherwise

occur.

Intrauterine infusions of recombinant bovine IFN-z from days 14 to 24 of the

estrous cycle increased lifespan of the CL and duration of the estrous cycle (Meyer et al.,

1995). Further studies with a large number of cows needs to test whether this treatment

increases pregnancy rates. Co-transfer of embryonic vesicles to increase trophoblastic

signals has been reported to increase pregnancy rates in embryo transfer recipients

(Heyman et al., 1987). Administration of IFN-a by intramuscular injection, which can

also block luteolysis, decreased pregnancy rates in heifers (Barros et al., 1992) because

IFN-a has several adverse actions such as causing hyperthermia (Newton et al., 1990).

Administration of a prostanoid synthesis inhibitor could suppress the luteolytic

stimulus in early pregnancy. Injection of flunixin meglumine (a prostaglandin synthesis

inhibitor) neutralized oxytocin-induced PGF2a TeleaSe, reduced the frequency of short










cycles, and increased pregnancy rate from 33.3% in oxytocin challenged cows to 80% in

oxytocin treated cows that received a flunixin meglumine inj section (Lemaster et al.,

1999). In another study, effects of flunixin meglumine on pregnancy rate were farm or

location dependent (Purcell et al., 2005). Together, these results suggest that certain

conceptuses are unable to inhibit uterine PGF2a Secretion and that reducing prostaglandin

synthesis and stimulating IFN-z secretion could improve pregnancy rates.

Nutritional Strategies

Dairy cows reach peak production on average within the first 4 to 6 weeks after

parturition. Unfortunately, feed and energy intake do not reach maximum levels until

approximately 10 12 weeks postpartum. The end result is a lactating cow with

insufficient nutritional requirements that enters a NEB status.

As mentioned before, energy balance is defined as the difference between energy

gain from feed intake minus the energy expenditure associated with maintenance of

physiological function, growth, and milk production (Staples et al., 1990). Several

studies have reported that negative energy status impaired reproductive performance

(Butler and Smith, 1989; Jorritsma et al., 2000). Different nutritional strategies to

improve energy balance or alter nutrient delivery to improve reproductive function are

described in this section.

Fat feeding to improve energy balance

Fats are glyceride esters of fatty acids that can have a direct effect on the

transcription of genes that encode proteins that are essential to reproductive events

(Mattos et al. 2000). Dietary fats typically increase concentrations of circulating

cholesterol, the precursor of progesterone (Grummer and Carroll, 1991). Ruminants fed









supplemental fat often have a slight increase in blood progesterone concentrations [see

Staples et al. (1998) for review]. Hawkins et al. (1995) suggested that the increase seen

in circulating progesterone when cows are fed supplemental fat was from a reduced rate

of clearance of progesterone rather than an increase in progesterone synthesis. Fat

supplementation has also been shown to stimulate programmed growth of a preovulatory

follicle (Lucy et al., 1993), total number of follicles (Lucy et al., 1991ab; Wehrman et al.,

1991; Thomas and Williams, 1996; Beam and Butler, 1997; Lammoglia, 1997), and size

of preovulatory follicles (Lucy et al., 1990, 1991a, 1993; Beam and Butler, 1997; Oldick

et al., 1997).

Garcia-Boj alil et al. (1998) reported that accumulated plasma progesterone from 0

to 50 days in milk (DIM) was greater, pregnancy rates improved, and energy status did

not change when cows were fed diets of 2.2% calcium salts of fatty acids (CSFA)

compared to non fat-supplemented cows. Similarly, Scott et al. (1995) fed CSFA at 0 or

450 g/d from 1 to 180 or 200 DIM and reported a tendency for CSFA to increase the

proportion of cows exhibiting standing estrus (71.4% vs. 65.6) and a reduction in the

proportion of cows with inactive ovaries.

Other studies have also found a beneficial effect of feeding supplemental fats on

fertility of lactating cows (Erickson et al., 1992; Sklan et al., 1994) while some studies

have found no beneficial effect. Although fertility results are inconsistent when cows

were evaluated after being fed supplemental fat, Staples et al. (1998) suggested that

positive effects (17 percentage unit improvement) are more often reported. When first AI

service and conception or pregnancy rate data was examined, ten studies (Schneider et

al., 1988; Bruckental et al., 1989; Sklan et al., 1989; Armstrong et al., 1990; Ferguson et









al., 1990; Sklan et al., 1991; Garcia-Bojalil, 1993; Scott et al., 1995; Burke et al., 1996;

Son et al., 1996) report an improvement (P < 0. 10) while two studies (Erickson et al.,

1992; Sklan et al., 1994) revealed a strong negative influence accompanied by a large

increase in milk production. Among studies that reported an improvement (Armstrong et

al., 1990; Ferguson et al., 1990; Sklan et al., 1991), a reduced number of services per

conception by feeding a fat supplemented diet occurred as well.

Dietary fats could favor reproductive processes through actions related to energy

balance or through specific actions of individual fatty acids on tissue function. Mattos et

al (2000) has suggested that altered uterine and ovarian function can be mediated through

specific fatty acid precursors in the diet to allow increased steroid and/or eicosanoid

secretion. There are many examples of effects of feeding diets high in specific fatty

acids. Linoleic acid supplemented in the diet prepartum can stimulate arachidonic acid

synthesis and lead to higher concentrations of the series 2 prostaglandins (Thatcher et al.,

1994). It is speculated that linolenic acid may compete with arachidonic acid for binding

sites of a key enzyme, cyclooxygenase 2 (PGHS-2), which is necessary for the synthesis

of PGF2a (Mattos et al., 2000; 2004).

Supplementation of the diet with Hish meal has been reported to reduce uterine

PGF2a Secretion of lactating dairy cows (Thatcher et al., 1997). Fish meal contains

relatively high concentrations of two polyunsaturated fatty acids of the n-3 family, EPA

(eicosapentaenoic acid) and DHA (docosahexaenoic acid). Concentrations of EPA and

DHA in Hish oil have been reported to be 10.8 and 1 1.1% of total fatty acids (Donovan et

al., 2000). EPA and DHA can inhibit secretion of PGFza in different cell culture systems

(Levine and Worth, 1984; Achard et al., 1997) including bovine endometrial cells










(Mattos et al., 2001). Using fish meal to replace soybean meal as a source of protein,

Bruckental et al. (1989) and Armstrong et al. (1990) reported higher pregnancy and

conception rates. These results suggest that high concentrations of EPA and DHA in the

diet can reduce PGF2a endometrial secretion and aid in establishment of pregnancy rates.

Administration of antioxidants

Reactive oxygen species are a possible source of infertility because ovarian

steroidogenic tissue (Carlson et al., 1993; Margolin et al., 1992), spermatozoa (Rivlin et

al., 2004), and preimplantation embryos (Fujitani et al., 1997) become compromised as a

consequence of free radical damage. Vitamin E (i.e., a-tocopherol) and p-carotene are

maj or antioxidants present in plasma membranes of cells (Wang and Quinn, 1999; 2000).

Treatment of cows with vitamin E and selenium can increase the rate of uterine

involution in cows with metritis (Harrison et al., 1986) and improve fertilization rates in

ewes (Segerson and Ganapathy, 1980) and cows (Segerson et al., 1977). In general,

however, treatment of lactating cows with vitamin E alone, through feeding or inj section,

had little or no benefits on postpartum cows (Kappel et al., 1984; Stowe et al., 1988;

Arechiga et al., 1998a; Paula-Lopes et al., 2003).

p-carotene is another cellular antioxidant and is thought to be present at the interior

of membranes or lipoproteins (Niki et al., 1995). Cows fed diets deficient in p-carotene

had lower amounts of progesterone in the CL (Ahlswede and Lotthammer, 1978). In

spite of this, its effect on fertility is controversial. Some authors report benefits of

feeding supplemental p-carotene (Ahlswede and Lotthammer, 1978; Rakes et al., 1985;

Arechiga et al., 1998b) whereas others do not (Wang et al., 1982; Akordor et al., 1986).

There was no strong relationship between serum concentrations of p-carotene and fertility









in dairy cattle (Gossen et al., 2004; Gossen and Hoedemaker, 2005). Injection of vitamin

A, a metabolite of p-carotene, resulted in an increase in the number of recovered

blastocysts from superovulated cows (Shaw et al., 1995).

Crossbreeding

Two bulls (Chief and Elevation) make up about 30% of the gene pool of U. S.

Holsteins (Hansen et al., 2005). As mentioned previously, inbreeding coefficients are

rising in American dairy cattle (Short et al., 1992; Wiggans et al., 1995; Young et al.,

1996; Hansen, 2000; Wall et al., 2005) and there is some evidence that this has

contributed to the decline in fertility seen in dairy cattle (Thompson et al., 2000ab;

Alvarez et al., 2005; Wall et al., 2005). Crossbreeding represents a strategy for

preventing effects of inbreeding especially if the milk yield of crossbreds can approach

that of Holstein cattle.

A study in Canada revealed that some groups of crossbred cattle were equivalent to

Holstein controls in lifetime net profit (McAllister et al., 1994). Hansen et al. (2005)

conducted a study using seven large dairies in California to compare characteristics of

several crossbred animals (Normande-Holstein, Montebeliarde-Hol stein, and

Scandinavian Red-Holstein) versus Holsteins. Milk production as well as fat and protein

production during the first 150 DIM among first lactation cows was not significantly

different among breed types. Holsteins produced an average of 29.9 kg, followed by

Scandinavian Red-Holstein with 29.7 kg, Montebeliarde-Hol stein with 28.8 kg, and

Normande-Holstein with 26.5 kg. Calving difficulty and stillbirths were reduced in

crossbred animals. Survival rates indicate that purebred animals left these dairies sooner.

The first service conception rate was 22% for Holsteins compared to 30 35% for

crossbreds. There were also significantly fewer days open for crossbred cows. Thus,










crossbreeding offers some promise for enhancing fertility. One unanswered question is

the optimal type of mating scheme for the crossbred animals themselves and whether the

resultant loss of heterosis in the F2 animals will reduce any advantage over purebred

cows .

Embryo Transfer

The concept of using embryo transfer (ET) as a tool to increase pregnancy rates is

based on the observation that disruptive events such as anovulation, ovulation of oocytes

with low developmental competence, compromised oviductal transport or uterine

environment, and insemination errors or damaged spermatozoa all occur before the time

when embryos are ordinarily transferred (day 6 8 after estrus) (Hansen and Block,

2004). Selection of morula and blastocyst stage embryos for transfer offers the chance to

avoid pregnancy failure associated with the early stages of embryonic development (day

0 8 after estrus).

It has been proposed that during absence of heat stress, pregnancy rates following

embryo transfer as compared to AI in lactating cows are not optimal (Putney et al.,

1989b; Drost et al., 1994; Ambrose et al., 1997). However, ET may become a more

effective strategy to increase pregnancy rates as compared to AI in lactating cows during

periods of heat stress, and the magnitude of the increased temperature does not seem to

influence overall success following transfer (Hansen and Arechiga, 1999). As embryos

advance in their development, the effects of elevated temperatures become less

significant because embryos become more resistant to the deleterious effects of elevated

temperatures (Ealy et al., 1992; Ealy and Hansen, 1994; Ealy et al., 1995; Edwards and

Hansen, 1997; Rivera and Hansen, 2001). As a result, pregnancy rates following ET









during heat stress are higher than pregnancy rates to AI (Putney et al., 1989b; Ambrose et

al., 1999; Al-Katanani et al., 2002a) although not in the absence of heat stress.

One potential constraint for embryo transfer in lactating cows is the short duration

of estrus and lack of intense mounting activity seen in dairy cows (Dransfield et al.,

1998). This phenomenon is exacerbated by heat stress (Nebel et al., 1997) and will limit

the number of embryos transferred in lactating cows in a program that is dependent upon

estrus detection. The first report of a timed embryo transfer (TET) protocol, where

ovulation was synchronized using an Ovsynch protocol, was by Ambrose et al. (1999)

who evaluated the efficiency of TET using either fresh or frozen-thawed in vitro

produced (IVP) embryos and TAI under heat stress conditions. Pregnancy rates in cows

that received a fresh IVP embryo were higher compared to cows in the TAI group.

Limitations to Optimal Pregnancy Rates Using IVP TET

For ET to replace AI on a wide scale in commercial herds ET must become an

economical breeding alternative and embryos must be inexpensive to produce (Hansen

and Block et al., 2004). Superovulation provides the best source of embryos while the

most likely inexpensive source of embryos will be produced from slaughterhouse oocytes

by IVP since superovulation is costly and requires intensive management and careful

synchronization of the donor cows.

Although embryos produced using IVP systems are relatively inexpensive as

compared to embryos produced by superovulation, pregnancy rates achieved following

transfer of an IVP embryo are often less than what is obtained following transfer of an

embryo produced by superovulation. For example, Hasler (2003) reported a 36.7%

pregnancy rate for in vitro derived embryos vs. 54.8% for in vivo embryos. The reason

for the poor survival of IVP embryos is not known. However, IVP embryos are different









from in vivo embryo in terms of morphology (Massip et al., 1995; Crosier et al., 2001;

Rizos et al., 2002), gene expression (Bertolini et al., 2002a; Lazzari et al., 2002;

Lonergan et al., 2003), metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b)

and chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). One or more of

these alterations likely contributes to the poor embryo survival after transfer. Calves born

as the result of in vitro production are also more likely to experience developmental

defects (Hasler et al., 2003; Farin et al., 2006).

One possible strategy for increasing pregnancy rates is to transfer two embryos into

the uterine horn ipsilateral to the CL. This approach is based on the idea that the

likelihood is increased that the cow receives at least one embryo competent for sustained

development. In addition, the transfer of two embryos into the ipsilateral uterine horn to

the CL is likely to increase the amounts of IFN-z and other embryo-derived signaling

molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer

of embryonic vesicles to increase trophoblastic signals has been reported to increase

pregnancy rates in ET recipients (Heyman et al., 1987).

In a recent study, there was a tendency for higher calving rates for recipients that

received two embryos in the uterine horn ipsilateral to the CL as compared to recipients

that received one embryo (Bertolini et al., 2002a). The requirement for the antiluteolytic

signal in cattle to be locally administered (del Campo et al., 1977, 1983) means that one

should expect pregnancy rates to be higher in cows that received two embryos in the

same uterine horn (unilateral transfer) than for cows that received two embryos

distributed in both uterine horns (bilateral transfer). The opposite was true for heifers

(Anderson et al., 1979). In other studies, transfer of embryos to create two pregnancies in









the uterine horn ipsilateral to the CL has produced a similar pregnancy rate as bilateral

twins and single pregnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or

reduced pregnancy rate as compared to bilateral transfer (Rowson et al., 1971).

Cryopreservation of IVP Embryos

An additional limitation to the widespread use of IVP embryos in cattle is their

poor survival following cryopreservation. Hasler et al. (1995), Ambrose et al., (1999) and

Al-Katanani et al. (2002a) indicated that IVP embryos do not survive freezing as well as

embryos produced in vivo based on pregnancy rates following transfer as compared to

non-frozen embryos. In vitro survival rates following thawing (Pollard and Leibo, 1993;

Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al.,

1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et

al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a) are consistently lower for IVP

embryos as compared to embryos produced in vivo by superovulation.

Among the metabolic changes associated with IVP embryos linked to poor

freezability is an increase in lipid content (Abe et al., 1999; Rizos et al., 2002).

Mechanical delipidation (Tominaga et al., 2000; Diez et al., 2001) and addition of

inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can improve embryo

survival following cryopreservation. Hatching rates were higher for delipidated embryos

compared to controls when day 7 blastocysts were frozen (Murakami et al., 1998), but

pregnancy rates after the transfer of delipidated embryos was 10.5% compared to 22% for

control embryos (Diez et al., 2001). Although delipidated embryos can survive freezing

conditions when tested in vitro, special consideration must be taken since these embryos

do not reflect higher pregnancies and remain less viable than control embryos.









Manipulating the cryopreservation process to minimize damage to the embryo has

also been considered. Of most promise are procedures based on vitrification, which is

defined as "the solidification of a solution (glass formation) brought about not by

crystallization but by extreme elevation in viscosity during cooling" (Fahy et al., 1984).

Vitrification depends on rapid cooling and thawing of embryos while using high

concentrations of cryoprotectants associated with elevated cooling rates (~2500oC/min,

Palasz and Mapletoft, 1996). Although vitrification does not eliminate toxic effects of

cryoprotectants and osmotic damage, the rapid cooling has been reported to decrease

chilling injury and prevent damage associated with high lipid content (Dobrinsky, 1996;

Martino et al., 1996ab). In vitro survival rates following the thawing of vitrified IVP

embryos was either equal (Van-Wagtendonk et al., 1995) or superior to embryos frozen

conventionally (Dinnyes et al., 1995; Agca et al., 1998; O'Kearney-Flynn et al., 1998).

Sensitivity of in vivo derived embryos to cryopreservation is much less and the

complex environment where the embryo develops is key. It has been reported that

embryos cultured in the sheep oviduct (26%) compared to synthetic oviductal fluid in

culture systems (7%) were better able to tolerate freezing conditions. Embryos cultured

in Buffalo rat liver cells or oviductal cells were more resistant to freezing as well as

compared to embryos not subj ected to co-culture (Massip et al., 1993; Leibo and

Loskutoff, 1993; Tervit et al., 1994).

Summary and Objectives of the Thesis

There has been a precipitous decline in fertility of dairy cows over the last 10-40

years and heat stress is associated with infertility in lactating dairy cows. To characterize

events associated with infertility is important and the purpose of the present series of

experiments described in this thesis was to evaluate strategies that help overcome








reproductive failure. Improving reproductive function in dairy cattle is of maj or interest

and experiments were designed to 1) evaluate strategies for enhancing fertility after AI

using GnRH treatment and 2) further develop ET using IVP embryos as a tool for

increasing fertility by testing whether pregnancy rate could be improved by transfer of

twin embryos and whether the developmental competence of embryos after

cryopreservation could be improved.


4.0



o
3.0 -,



2.0 O


1.5 u


-15


-14.5 f


14 m
I




1.5 ~



2000


1980


1990


Year

Figure 1-1. Rolling herd average (RHA, kg milk per lactation), calving interval (CI), and
services per conception (SPC) for 143 dairy herds continuously enrolled in the
Raleigh DHIA record system from 1970 to 1999 (Lucy, 2001).


9000



8000 .



7000 -



6000



5000
1970













50 s 10500
Cool season
S451 10000

S40-
-9500
co Warm Season Milk Yield
c 35-T
"~ 9000 a,
S30 -1
e 8500 =
o 25- >

a, 20 -1 -8000

.5~~~ 15. 7500


Figure 1-2. Temporal changes in first service pregnancy rate and annual average milk
production from high-producing Holstein-Friesian dairy herds in north-eastern
Spain. Data for pregnancy rate were recorded in the cool (October April
months) and warm season (May-September months). Data were drawn by P.J.
Hansen (unpublished) based on data of Lopez Gatius (2003).















CHAPTER 2
EFFECTIVENESS OF ADMINISTRATION OF GONADOTROPIN RELEASING
HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED OVTULATION FOR
INCREASING FERTILITY OF LACTATINTG DAIRY COWS AND NON-
LACTATINTG HEIFERS

Introduction

One of the approaches proposed to improve fertility in cattle is administration of

GnRH or GnRH analogues at day 1 1-15 after estrus. Inj section of GnRH at this time can

lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and Lamming, 1995a)

in an action that likely involves luteinization of the dominant follicle (Thatcher et al.,

1989; Rettmer et al., 1992a; Ryan et al., 1994). In some cases, extended estrous cycle

length (Lynch et al., 1999) and increased progesterone secretion also results (Rettmer et

al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). Improvement

of fertility has been seen by administration of GnRH or its analogues at day 1 1-14 in

nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows (Macmillan et

al., 1986; Lajili et al., 1991; Sheldon et al., 1993; Drew and Peters, 1994; Willard et al.,

2003; L6pez-Gatius et al., 2005a). In contrast to these positive results, there was no

favorable effect of similar treatments of GnRH or GnRH analogues on pregnancy rates in

other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al., 1994; Bartolome et

al., 2005). In a meta-analysis of published results, Peters et al. (2000) concluded that the

overall effect of GnRH administration betweendDay 11 and 14 after anticipated ovulation

was positive, but that results were not consistent between studies.









It is possible that GnRH treatment is more effective at increasing pregnancy rate

per insemination during periods of heat stress than in cool weather because circulating

concentrations of progesterone can be reduced in cows subj ected to heat stress

(Wolfenson et al., 2000). In addition, the anti-luteolytic process may be compromised

because heat stress can decrease growth of the filamentous stage concepts (Biggers et

al., 1987) and increase uterine prostaglandin-Fza Secretion from the uterus (Wolfenson et

al., 1993). Beneficial effects of GnRH treatment at day 1 1-12 after insemination on

fertility have been observed in lactating dairy cows during heat stress (Willard et al.,

2003; L6pez-Gatius et al., 2005a). The purpose of the present series of experiments was

to evaluate the effectiveness of GnRH treatment at either day 11, 14 or 15 after

anticipated ovulation for improving fertility of lactating cows and heifers and determine

whether the beneficial effect of GnRH was greater during summer than winter.

Materials and Methods

Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in
Heifers Subjected to Timed Artificial Insemination during Heat Stress

The experiment was conducted at a commercial dairy located in Trenton, Florida

(29037' N 82o49' W) from July to September, 2003 using 149 Holstein heifers. The

heifers ranged in age from 13-23 mo (mean=539 d, SD=76) and ranged in weight from

316 to 448 kg (mean=360 kg, SD=32). Heifers were maintained on grass pasture with

supplemental grass hay. Heifers were randomly allocated to one of four treatments in a 2

x 2 factorial design with main effects of timing of insemination (protocol A vs B) and

treatment (vehicle vs GnRH). The experiment was replicated twice with between 70 and

79 heifers per replicate. Heifers were subj ected to timed artificial insemination (TAI)

based on a protocol published previously (Martinez et al., 2002ab). On Day -10 of the









protocol (Day 0 equals the day of anticipated ovulation), heifers received 100 Cpg (i.m.) of

GnRH (Fertagyl, equivalent to 50 pug /ml gonadorelin diaecetate tetrahydrate; (Intervet

Inc. Millsboro, DE) and an unused intravaginal progesterone-releasing device insert

(EAZI-BREED CIDR" insert, 1.38 g of progesterone, Pfizer Animal Health, New York,

NY, USA). At Day -3, CIDR devices were removed and 25 mg (i.m.) of prostaglandin

Fz, (PGF2,; 5 ml Lutalyse", Pfizer Animal Health, New York, NY, USA) was

administered. A second 100 Cpg GnRH injection was given 48 h after CIDR withdrawal

(Day -1). Regardless of estrus behavior, heifers in protocol A were inseminated 24 h

after the second GnRH injection (d 0) and heifers in protocol B were inseminated at the

same time as the second GnRH injection (d -1). Two individuals conducted all

inseminations and semen from one sire was used for all heifers. Heifers from each

synchronization treatment protocol were randomly allocated to receive either 100 Cpg of

GnRH, (i.m.) or an equivalent volume (2 ml) of vehicle (9 mg/ml of benzyl alcohol and

7.47 mg/ml of sodium chloride in water) at Day 11 after anticipated ovulation.

On the day of insemination and on Day 11 after anticipated ovulation, a 10-ml

blood sample was collected via coccygeal or jugular venipuncture into heparinized tubes

(Becton Dickinson, Franklin Lakes, NJ) to measure the proportion of heifers successfully

synchronized. An animal was considered synchronized if progesterone concentrations

were lower than 1 ng/ml on the day of insemination and greater than 1 ng/ml on Day 11

after anticipated ovulation. A third blood sample was collected in a subset of 76 heifers

at Day 15 after anticipated ovulation (i.e., 4 d after the inj section of GnRH or vehicle) to

determine the effect of GnRH treatment on serum concentrations of progesterone.

Pregnancy was diagnosed by palpation per rectum at Day 44-51 after insemination.









Blood samples were stored on ice (~2-4 h) until centrifugation at 2,000 x g for 20

min at 4 oC to obtain plasma. Plasma was stored at -20 oC until assayed for progesterone

concentrations using a progesterone radioimmunoassay kit (Coat-a-Count@; Diagnostic

Products Corp., Los Angeles, CA). The sensitivity of the assay was 0.1 ng/ml and the

intrassay and interassay CV were each 6%.

Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

This study took place at the University of Florida Dairy Research Unit (Hague,

Florida; 29046' N 82o25' W). A total of 244 primiparous and multiparous lactating

Holstein cows housed in freestall barns equipped with a fan-and-sprinkler system were

used. Cows were fed a total mixed ration (TMR) to meet or exceed requirements

recommended for lactating dairy cows, were milked three times a day, and received

bovine somatotropin (Posilac, Monsanto Corp., St. Louis, MO) according to

manufacturer' s recommendation. Cows were subj ected to the OvSynch TAI program

(Schmitt et al., 1996a; Pursley et al., 1998); 100 Cpg (i.m.) GnRH (Fertagyl equivalent to

50 Cpg /ml gonadorelin diaecetate tetrahydrate, Intervet, Millsboro, DE) was inj ected at

Day 0 of the protocol, 25 mg (i.m.) PGF2, (5 ml of Lutalyse", Pfizer Animal Health, New

York, NY, USA) was given at Day 7, 100 Cpg (i.m.), GnRH was again injected, i.m., at

Day 9, and cows were inseminated 16 h later (the day of anticipated ovulation). At the

time of insemination (from January September, 2004), 244 cows were between 76 and

594 days in milk (DIM; mean= 176, SD= 114). Multiple individuals conducted

inseminations (n=7) and multiple AI sires were used (n=45).

Cows were randomly assigned within pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium









chloride in water) at Day 11 after anticipated ovulation (i.e., 11 d after insemination).

Rectal temperature was recorded in a subset of cows (n=134) on the afternoon of Day 1 1

after TAI at 1500 1600 h. Pregnancy was diagnosed by rectal palpation at ~Day 46

after insemination.

Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

This study was conducted at two different locations using lactating Holsteins.

Farm 1 was the University of Florida Dairy Research Unit at Hague, Florida while farm 2

was a commercial dairy in Chiefland, Florida (29030' N 82o52' W). Cows from farm 1

(n=307) were inseminated from February November 2004 and cows in farm 2 (n=170)

were inseminated from June October 2004. At both farms, primiparous and multiparous

cows were used. At farm 1, 307 cows were TAI between 76 590 DIM (mean= 187,

SD= 102). Multiple individuals conducted inseminations (n=7) and multiple AI sires

were used (n=42). At farm 2, 170 cows were used for first service after calving using

seven different sires and one inseminator. The TAI protocol was designed to achieve

insemination at 60 + 3 d in milk. Cows in both farms were housed in freestall barns

equipped with fans and sprinklers, were fed a TMR, were milked three times a day, and

received Posilac@ (Monstanto, St. Louis, MO) according to manufacturer' s directions.

Cows in farm 1 were subjected to an OvSynch protocol as described for

Experiment 2. Cows for farm 2 were subj ected to a TAI protocol that incorporated a pre-

synchronization with PGF2, (Moreira et al., 2001) and the CIDR-Synch ovulation

synchronization protocol (Portaluppi and Stevenson, 2005). Cows received two

inj sections of 25 mg PGF2, (i.m.) (Lutalyse) 14 d apart starting on Day 21-27 DIM.

Twelve days after the second PGF2, inj section, a timed ovulation synchronization protocol









was initiated. Cows received 100 Cpg (i.m.) GnRH (2 ml of Cystorelin ; Merial Limited,

Iselin, NJ, USA) and an unused EAZI-BREED CIDR" intravaginal progesterone-

releasing device insert. Seven days later, CIDR devices were removed and 25 mg (i.m.)

PGF2, waS given. Cows received a second 100 Cpg (i.m.) injection of GnRH at 72 h after

CIDR withdrawal. Estrus was detected using tail chalk or KaMar estrus detection

patches (KAMAR Inc., Steamboat Springs, CO, USA). Cows observed in estrus at 24 or

48 h after CIDR removal were inseminated at estrus. Cows not observed in estrus were

inseminated at 72 h after CIDR withdrawal. Ovulation was anticipated to occur 72 h

after CIDR withdrawal. All animals received the GnRH inj section at 72 h regardless of

estrus behavior. Cows were also randomly assigned within pair to receive either 100 Cpg

(i.m.) GnRH (2 ml of Cystorelin ; Merial Limited, Iselin, NJ, USA), or vehicle (as for

experiment 2) at 14 d after anticipated ovulation. Pregnancy was diagnosed by rectal

palpation at ~Day 45 after insemination.

Rectal temperature was recorded in a subset of 100 cows in Farm 1 and 39 cows in

Farm 2 at 1500 h of Day 14 after anticipated ovulation.

Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination During Heat Stress

This study took place at the University of Florida Dairy Research Unit with

inseminations in April to June, 2005. A total of 137 primiparous and multiparous

lactating Holstein cows ranging in DIM from 78 to 566 d (mean= 185, SD= 110) were

subj ected to an OvSynch protocol as described for Experiment 2. Multiple individuals

conducted inseminations (n=4) and multiple AI sires were used (n=22).

Cows were randomly assigned within pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium









chloride in water) at Day 14 after anticipated ovulation (i.e., 14 d after insemination).

Pregnancy was diagnosed by rectal palpation at ~Day 46 after insemination.

Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus

This study took place at a commercial dairy in Chiefland, Florida. A total of 296

primiparous and multiparous lactating Holstein cows inseminated at detected estrus were

used. Cows were inseminated from April August, 2005. At the time of insemination,

cows were between 51 and 235 DIM (mean= 122, SD= 40).

Estrus was detected using tail chalk or KaMar estrus detection patches (KAMAR

Inc., Steamboat Springs, CO, USA). Estrus detection patches were visually monitored

twice (morning and afternoon) daily by the inseminator. When cows were first diagnosed

in estrus in the afternoon, insemination was performed the next morning. When estrus

was first detected in the morning, cows were inseminated at that time. Cows were bred

by one inseminator and 3 1 different sires used. Every other day of the experiment, cows

were selected to receive injections at Day 14 or 15 after insemination. Within each day,

cows were randomly assigned within a pair to receive 100 Cpg (i.m.) GnRH or an

equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

chloride in water). Pregnancy was diagnosed by rectal palpation at ~Day 45 after

insemination.

Statistical Analysis

Data on pregnancy rate were analyzed by logistic regression with the LOGISTIC

and GENMOD procedures of SAS (SAS for Windows, Release 8.02; SAS Inst., Inc.,

Cary, NC). For the LOGISTIC procedure, a backward stepwise logistic model was used.

Variables were continuously removed from the model by the Wald statistic criterion if

the significance was greater than 0.20. The Wald X2 statistic was used to determine the










significance of each main effect that remained in the reduced model. The adjusted odds

ratio (AOR) estimates and the 95% Wald confidence intervals from logistic regression

were obtained for each variable that remained in the final statistical model following the

backward elimination. Data were also analyzed by PROC GENMOD and P values for

significant treatment effects are reported from this analysis. The full mathematical model

for experiment 1 included main effects of inseminator, treatment, protocol, replicate,

replicate x protocol, replicate x treatment, replicate x inseminator, protocol x treatment,

protocol x inseminator, treatment x inseminator. The full mathematical model for

experiment 2 included the effects of season of insemination (January to March vs April to

September), treatment, and season x treatment. For experiment 3, the full mathematical

model included the effects of farm, treatment, season of insemination (warm vs cool

season; farm 1 = October to March vs April to September; farm 2 = June to September vs

October to November), and season x treatment, season x farm, and treatment x farm. In

addition, a subset of data composed of cows from farm 2 only was analyzed where the

additional factor of estrus detection (yes or no) was included in the model. For

experiment 4, the full mathematical model included the effects of treatment, month of

insemination, parity (1 vs others), sire, DIM at insemination class (<150 d vs > 150 d),

parity x treatment, DIM class x treatment and month x treatment. For experiment 5, the

full mathematical model included the effects of treatment, season of insemination (April

and May vs June to August), parity (1 vs > 1), number of services (1, 2 and >2), DIM at

insemination class (<150 d vs > 150 d) and interactions of main effects with treatment.

Since interactions were not significant, data were reanalyzed with main effects only.









Data on rectal temperatures were analyzed by least-squares analysis of variance

using the GLM procedure of SAS. The model included effects of season (Exp.2) or

season, farm and farm x season (Exp. 3).

A meta-analysis was performed using Mantel-Haenszel procedures available using

software downloaded from http://www.pitt.edu/~superl/lecture/lecl 171/index.htm.

Three analyses were performed using all experiments, experiments with GnRH

treatment at Day 11, and experiments with GnRH treatment at Day 14 or 15.

Results

Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in
Heifers Subjected to Timed Artificial Insemination During Heat Stress

Based on progesterone concentrations measured at insemination and at Day 11 after

anticipated ovulation, estrous cycles of 137/149 (92%) of the heifers were successfully

synchronized. Pregnancy rate was not significantly affected by GnRH treatment or

insemination protocol. This is true whether all heifers were considered (Table 1) or only

those successfully synchronized (results not shown). There was also no effect (P > 0.10)

of GnRH treatment at Day 11 on concentrations of plasma progesterone on Day 15.

Values were 3.5 & 0.19 ng/ml for heifers receiving vehicle and 3.6 & 0.19 ng/ml for

heifers receiving GnRH.

Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

Treatment with GnRH did not significantly (P > 0.10) affect pregnancy rate per

insemination (Table 2). This was true for inseminations in both cool seasons (January to

March) and warm season (April to September) (results not shown). There was also no

significant difference in pregnancy rate between seasons.









Rectal temperatures were higher (P < 0.001) for cows in the warm season (least-

squares means + SEM; 39.3 + 0.07 oC) than for cows in the cool season (38.9 + 0.07 oC).

Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination

Inj section of GnRH increased pregnancy rates at both farms (treatment, P < 0.02;

treatment x farm, non-significant) (Table 3). While pregnancy rates were lower in

summer than winter (P < 0.05), the effect of GnRH was apparent in both seasons and the

season x treatment interaction was not significant.

Cows in farm 2 were monitored for estrus. No cows were seen in estrus at 24 h

after PGF2a, 4.7% (8/171) were detected in estrus at 48 h, 32.2% (55/171) at 72 h, and

63.1% (108/171) were not detected in estrus. Cows in estrus at 48 h were inseminated at

that time while other cows (those seen in estrus at 72 h and those not seen in estrus) were

inseminated at 72 h. There was an estrus detection class (detected in estrus vs not

detected) x treatment interaction (P < 0.03) on pregnancy rate per insemination that

reflected the fact that GnRH was effective at increasing pregnancy rate for those cows

displaying estrus [3/29 (10%) for control and 12/34 (3 5%) for GnRH[ but had no effect

for those cows not displaying estrus [7/54 (13%) for control and 4/54 (8%) for GnRH].

Rectal temperatures were higher (P < 0.01) for cows in the warm season (least-

s uares means + SEM: 39.4 + 0.06 oC) than for cows in the cool season (39. 1 + 0. 11 oC)

and hi her (P < 0.001) for farm 2 (39.5 + 0.10 oC) than for farm 1 (39.1 + 0.07 oC but

there was no farm x season interaction.

Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in
Lactating Cows Subjected to Timed Artificial Insemination During Heat Stress

Treatment with GnRH did not significantly affect pregnancy rate (Table 4).

Pregnancy rate was higher (P<0.02) for cows inseminated at or before 150 DIM (30.3%,









20/66) than for cows inseminated after 150 DIM (12.7%, 9/71). There were no other

significant main effects or interactions of GnRH treatment with other effects.

Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus

Overall, pregnancy rate was higher (P<0.0001) for cows inseminated in April and

May (55/171, 32.2%) than for animals inseminated in June, July or August (12/125,

9.6%). There were, however, no other significant main effects or interactions of GnRH

treatment with other effects. Pregnancy rates were 25.6% (32/125) for cows receiving

vehicle at day 14 or 15, 20.7% (19/92) for cows receiving GnRH at Day 14, and 20.3%

(16/79) for cows receiving GnRH at Day 15.

Overall Effectiveness of GnRH Treatment as Determined by Meta-Analysis

When data from multiple experiments were considered together by meta-analysis,

there was no significant effect of GnRH on pregnancy rate. This was the case when all

experiments were considered (odds ratio=0.97; 95% CI=0.63, 1.50), or whether

experiments with GnRH treatment on Day 11 (odds ratio=0.87; 95% CI=0.50, 1.50) or

Day 14 or 15 (odds ratio=1.06; 95% CI=0.68, 1.65) were considered separately.

Discussion

Overall, there was no significant effect of GnRH treatment on pregnancy rate. In

particular, GnRH treatment at Day 11 after anticipated ovulation did not improve

pregnancy rate of heifers or lactating cows in any experiment, whether animals were

exposed to heat stress or not. Moreover, GnRH did not consistently improve fertility

when given at Day 14 after anticipated ovulation or at Days 14 or 15 after insemination.

In one experiment (experiment 3), administration of GnRH at Day 14 after anticipated

ovulation in cows subjected to TAI increased pregnancy rate of lactating cows in









summer and winter at two locations. However, this positive effect could not be replicated

either in lactating cows subj ected to TAI or for cows inseminated at standing estrus.

The variability in response to GnRH is reminiscent of the results of the meta-

analysis of published studies performed by Peters et al. (2000) in which inconsistency

between studies was noted. Variability in results could reflect either error in estimates of

treatment effects because of small numbers of experimental units or variability in

biological responses to GnRH. The number of animals used for the present studies varied

and could have been too small in some studies to detect significant differences or have

lead to sampling errors that obscured the magnitude or direction of the treatment

differences. However, meta-analysis of the entire data set, involving 1303 cows,

indicated that there was no overall effect of GnRH.

It is also possible that herds differ between each other or over time in the

predominant biological response to GnRH treatment. Presumably, beneficial effects of

GnRH post-insemination on fertility are related to its actions to cause LH release.

Treatment with GnRH at Day 1 1-15 of the estrous cycle can decrease function of the

dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994; Mann

and Lamming, 1995a) and increase progesterone secretion (Rettmer et al., 1992a;

Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). The reduction in

estradiol-17P secretion caused by GnRH should delay luteolysis and conceivably allow a

slowly-developing concepts additional time to initiate secretion of interferon-z. Low

progesterone secretion may also compromise fertility in dairy cattle (Mann and

Lamming, 1999; Lucy, 2001) and an increase in progesterone secretion caused by GnRH

may facilitate embryonic development. Whether a herd responds to GnRH by undergoing









follicular changes may depend upon the characteristics of follicular growth because a

follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001).

Perhaps, herds that do not respond to GnRH with an increase in fertility are herds where

many cows have lower follicular growth or follicular wave characteristics that do not

result in sufficient follicular development at the time of inj section.

One example of the potential importance of follicular dynamics in determining

responses to GnRH is the expected response to GnRH treatment at Day 11 after

anticipated ovulation. In the current studies, injection of GnRH at Day 11 after

anticipated ovulation did not increase pregnancy rates in either lactating Holstein cows or

nulliparous heifers. For lactating cows, the absence of an effect of GnRH at Day 11 was

seen in both summer and winter. This result, which agrees with other studies in which

inj section of GnRH at Day 11 does not affect fertility (Stevenson et al., 1993; Jubb et al.,

1990), is in contrast to other studies indicating that GnRH treatment at Day 11 can

increase fertility of heifers (Rettmer et al., 1992b) and lactating cows (Sheldon and

Dobson, 1993; Willard et al., 2003). One factor that could influence the effectiveness of

GnRH treatment at Day 11 is the number of follicular waves that an individual animal

expresses. Animals with estrous cycles characterized by three follicular waves have

larger second-wave dominant follicles at Day 11 of the estrous cycle than animals with

two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) and thus the

preponderance of cycle type (two-wave vs three-wave) within a herd may determine

effectiveness of GnRH treatment at Day 11. There is variation from study to study in the

relative frequency of three-wave vs two-wave cycles, at least among Holstein heifers

(Ginther et al., 1989; Knopf et al., 1989; Rajamahendran et al., 1991; Gong et al., 1993),










and this variation is evidence for herd-to-herd variation in frequency of follicular wave

patterns.

Even in animals with three-wave follicular cycles, Day 11 would appear to not be

an optimal time of the estrous cycle for using GnRH to cause luteinization because the

second-wave dominant follicle is smaller at Day 11 than at 14-15 in heifers (Ginther et

al., 1989; Ko et al., 1991) and lactating cows (Ko et al., 1991). Results from a limited

number of cows in Experiment 3 suggested that the effectiveness of GnRH at Day 14

after anticipated ovulation depends upon whether cows are detected in estrus.

Presumably, ovulation occurred on average sooner for cows in estrus at 48 and 72 h after

prostaglandin than for cows not detected in estrus (which contains cows that had not

initiated estrus by 72 h as well as some cows in which estrus occurred by 72 h but was

not detected). Among those detected in estrus, GnRH injection improved fertility from

10.3% to 35.3%. Among animals not detected in estrus, however, there was no

difference in pregnancy rate between animals treated with vehicle (13.0%) or GnRH

(7.6%). It is likely that GnRH did not affect pregnancy rate in the cows not detected in

estrus because this group included cows that were anovulatory at insemination or that

were not successfully synchronized; GnRH would be unlikely to increase pregnancy rate

in these animals.

It was hypothesized that beneficial effects of GnRH would be greater during heat

stress because this condition can decrease growth of the filamentous stage concepts

(Biggers et al., 1987), increase uterine prostaglandin Fza Secretion from the uterus

(Wolfenson et al., 1993) and reduce circulating concentrations of progesterone

(Wolfenson et al., 2000). Beneficial effects of GnRH treatment at Day 11-12 after









insemination on fertility have been observed in lactating dairy cows during heat stress

(Willard et al., 2003; L6pez-Gatius et al., 2005a). There was no evidence, however, that

GnRH was more effective during the summer. In particular, the increase in pregnancy

rate caused by inj section of GnRH at Day 14 during experiment 3 was similar for cows

inseminated in summer and winter. In other experiments conducted during the summer,

GnRH was without beneficial effect.

In experiment 1, there were no differences in pregnancy rates for Holstein heifers

inseminated either at second GnRH inj section (24.4%) or 24 after GnRH (19.8%). This

result is similar to results of Pursley et al. (1998) who reported little difference in

pregnancy rates and no differences in calving rates between lactating cows inseminated at

0, 8, 16, or 24 h after the second GnRH inj section of the OvSynch regimen. The

pregnancy rates achieved with heifers in experiment 1 were low compared to other

studies in which heifers received a similar ovulation synchronization program (Martinez

et al., 2002ab). The low fertility was not a result of delayed puberty or unresponsiveness

to the synchronization protocol because 92% of the heifers had both low progesterone

concentrations during the expected periovulatory period and high progesterone

concentrations at the predicted luteal phase of the cycle. It is possible that some of these

heifers classified as synchronized experienced short estrous cycles (Schmitt et al., 1996b;

Moreira et al., 2000a). The experiment was conducted during the summer and it is also

possible that heat stress reduced fertility. Although fertility in Holstein heifers does not

always decline during the summer (Ron et al., 1984; Badinga et al., 1985), there is one

report (Donovan et al., 2003) that heifers from a dairy farm in north central Florida

inseminated in summer were more than four times less likely to become pregnant to first





































GnRH Treatment
GnRH 20/78 25.6 1.29 0.59 -2.83 0.41
Vehicle 14/71 19.7


Protocol 4
B 20/79 25.3 1.34 0.61 -2.95 0.41
A 14/70 20.0


insemination than heifers inseminated during the rest of the year. It is also possible that

the one sire used to inseminate all heifers was not a fertile bull.

In conclusion, inj section of GnRH at Day 11-15 after anticipated ovulation or

insemination did not consistently increase pregnancy rates in heifers or lactating cows.

The fact that GnRH administration was effective in one study indicates that such a

treatment may be useful for increasing pregnancy rate in some herds or situations. More

work will be required to describe factors that could identify which groups of cows would

be most likely to benefit from GnRH treatment.

Table 2-1. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and ovulation synchronization protocol on pregnancy
rates of heifers during heat stress.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2


Data represent the number of females pregnant at Day 44-5 1 after insemination / total number
of females inseminated.
2 Derived from PROC GENMOD.
3 Wald chi-square statistic =0.54 (N.S).
4 Wald chi-square statistic = 0.40 (N.S.)










Table 2-2. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 11 after
anticipated ovulation and season of insemination on pregnancy rates of
lactating cows subj ected to timed artificial insemination.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 26/121 21.5 0.66 0.37 -1.18 0.16
Vehicle 36/123 29.3

Season 4
January March 30/103 29.1 1.38 0.77 -2.48 0.27
April September 32/141 22.7
'Data represent the number of females pregnant at ~d 45 after insemination / total number of
females inseminated.
SDerived from PROC GENMOD.
2Wald chi-square statistic =1.50 (N.S).
4 Wald chi-square statistic = 1.38 (N.S.)

Table 2-3. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and season of insemination on pregnancy rates of
lactating cows subj ected to timed artificial insemination.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 49/241 20.3 1.76 1.07 -2.89 0.02
Vehicle 3 0/23 6 12.7

Season 4
Oct, Nov, Feb,
40/187 21.4 1.76 1.08 -2.87 0.02
March
May September 39/290 13.5
Data represent the number of females pregnant at ~Day 45 after insemination / total number of
females inseminated.
SDerived from PROC GENMOD.
SWald chi-square statistic =4.94 (P=0.026).
4 Wald chi-square statistic = 5.12 (P=0.024)









Table 2-4. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald
confidence intervals (CI) for effect of GnRH administration at Day 14 after
anticipated ovulation and Days in milk (<150 d vs > 150) at insemination on
pregnancy rates of lactating cows subj ected to timed artificial insemination
during heat stress.

Pregnancy rate
Proportion % AOR 95% Wald CI P-value 2
GnRH Treatment
GnRH 11/73 15.1 0.43 0.18 -1.04 0.05
Vehicle 18/64 28.1


Days in milk at
insemination
< 150 d 20/66 30.3 3.11 1.27 7.62 0.02
> 150 d 9/71 12.7


SData represent the number of females pregnant at
number of females inseminated.
2 Derived from PROC GENMOD.
3 Wald chi-square statistic =3.55 (P=0.060).
4 Wald chi-square statistic = 6.12 (P=0.013)


-Day 45 after insemination / total















CHAPTER 3
EFFECT OF TRANSFER OF ONE OR TWO INT VITRO-PRODUCED EMBRYOS
AND POST-TRANSFER ADMINISTRATION OF GONADOTROPIN RELEASING
HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE

Introduction

The in vitro produced (IVP) embryo is different from the embryo produced in vivo

in terms of morphology (Iwasaki et al., 1992; Massip et al., 1995; Crosier et al., 2001),

gene expression (Bertolini et al., 2002a; Lazzari et al., 2002; Lonergan et al., 2003),

metabolism (Khurana et al., 2000b), and incidence of chromosomal abnormalities

(Iwasaki et al., 1992; Viuff et al., 2000). Not surprisingly, pregnancy rates achieved

following transfer of an IVP embryo are often less than what is obtained following

transfer of an embryo produced by superovulation and calves born as the result of in vitro

production are more likely to experience developmental defects (Hasler et al., 2003).

Problems associated with the transfer of IVP embryos have limited the realization of the

potential of these embryos for enhancing genetic improvement and reproductive

performance of lactating dairy cattle (Rutledge, 2001; Hansen and Block et al., 2004).

One method that might be useful for increasing pregnancy rates in dairy cattle

recipients that receive an IVP embryo is to transfer two embryos into the uterine horn

ipsilateral to the CL. Such a treatment might increase pregnancy rate because the

likelihood is increased that the cow receives at least one embryo competent for sustained

development. In addition, the transfer of two embryos into the ipsilateral uterine horn is

likely to increase the amounts of interferon-z and other embryonic signaling molecules in

the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of










embryonic vesicles to increase trophoblastic signals has been reported to increase

pregnancy rates in embryo transfer recipients (Heyman et al., 1987). For the current

experiment, both embryos were transferred into the uterine horn ipsilateral to the CL

because of the requirement for the antiluteolytic signal in cattle to be locally administered

(Del Campo et al., 1977; 1983). In a recent study with a small number of transfers (n=10

to 28 recipients), there was a tendency for higher calving rate for recipients that received

two embryos in the uterine horn ipsilateral to the CL as compared to recipients that

received one embryo (Bertolini et al., 2002b). Anderson et al. (1979) found a tendency

for pregnancy rates to be higher in cows that received two embryos in the same uterine

horn (unilateral transfer) than for cows that received two embryos distributed in both

uterine horns (bilateral transfer); the opposite was true for heifers. In other studies,

transfer of embryos to create two pregnancies in the uterine horn ipsilateral to the CL has

produced a similar pregnancy rate as bilateral twins and single pregnancies (Sreenan and

Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to

bilateral transfer (Rowson et al., 1971).

Another treatment that has potential for increasing pregnancy rates in embryo

transfer recipients is inj section of GnRH at Day 11 after the anticipated day of ovulation.

Such a treatment was shown to increase pregnancy rates in heat-stressed, lactating cows

following insemination (Sheldon and Dobson, 1993; Willard et al., 2003) and embryo

transfer (Block et al., 2003). Treatment with GnRH or its analogues at Day 11 tol2 of

the estrous cycle has been reported to increase progesterone secretion (Ryan et al., 1994;

Willard et al., 2003) and inhibit function of the dominant follicle (Savio et al., 1990;

Ryan et al., 1994) to possibly delay luteolysis.










The purpose of the current pair of experiments was to examine the effectiveness of

unilateral transfer of twin embryos and treatment with GnRH at Day 11 after the

anticipated day of ovulation for increasing pregnancy rates in dairy cattle recipients that

received IVP embryos. Experiments were performed during periods of heat stress

because embryo transfer offers benefits as a method for increasing pregnancy rate as

compared to AI in females subj ected to heat stress (Rutledge, 2001).

Materials and Methods

Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred Dairy
Recipients

The experiment was conducted at a commercial dairy located in Santa Cruz,

Bolivia (17048' S, 63"10' W) from November December, 2004. Data on minimum and

maximum air temperatures during the experiment collected by Servicio Nacional de

Meteorologia e Hidrologia (http://www.senamhi. gov.bo/meteorologia/) for Santa Cruz

are presented in Figure 1. Females receiving embryos included 32 virgin crossbred

heifers sired by Simmental, Gyr, or Brown Swiss bulls and Holstein or Holstein

crossbred dams and 26 lactating, crossbred cows with the proportion of Holstein varying

from 1/2 to 15/16. The heifers ranged in age from 363 to 2070 d (mean = 850 d and

median = 664 d; SD = 421 d) and ranged in weight from 247 to 430 kg (mean = 310 kg

and median = 288 kg; SD = 52.3 kg). Animals were maintained on grass pasture until

two weeks prior to the start of the synchronization program when they also received a

supplement of 6 kg/head/d of spent brewers' grain. The cows ranged in age from 820 to

4075 d (mean = 2083 d and median = 1670 d; SD = 986 d), were maintained on grass

pasture, and received 11 kg of brewers' grains and 2 kg of a soybean-based concentrate

mixture before each milking. Cows were milked two times per day and ranged from 110









to 417 d in milk (mean =190 d and median = 170 d; SD = 75 d). Milk yield per day

across all days of lactation ranged from 5.9 to 21.1 kg/d (mean = 12.5 kg/d and median =

12.6 kg/d; SD = 3.8 kg/d).

Recipients were synchronized for timed embryo transfer using a modified OvSynch

protocol (Portaluppi and Stevenson, 2005) with the inclusion of a controlled intravaginal

drug releasing device (EAZI-BREED CIDR" insert, 1.38 g of progesterone, Pfizer

Animal Health, New York, NY, USA). On Day -10 (Day 0 equals the day of anticipated

ovulation), females received 100 Cpg (i.m.) of GnRH (1 ml of Profertil@; Tortuga Cia.

Zootecnica Agraria, Sho Paulo, Brazil) and an intravaginal progesterone-releasing device

insert that had been used one time previously. On Day -3, CIDR devices were removed

and females received 150 Cpg (i.m.) of PGFz, (2 ml of Prostaglandina Tortuga, Tortuga

Cia. Zootecnica Agraria). On Day 0, 100 Cpg (i.m.) of GnRH was administered.

Behavioral symptoms of estrus were monitored about 5 times each day for 3 d following

CIDR removal and PGF2, inj section. On Day 6 after anticipated ovulation, all females,

including those not seen in estrus, were examined per rectum for the presence of a CL

using an Aloka 210 ultrasound unit equipped with a 5 MHz linear array probe (Aloka,

Wallingford, CT, USA). A group of females having a CL (n=32 heifers and n=26 cows)

were randomly selected within recipient type (heifers or cows) to receive one (n=31

females) or two (n=27 females) embryos on Day 7 after anticipated ovulation. For

embryo transfer, an epidural block of 5 ml of lidocaine hydrochloride (2% w/v; Sparhawk

Laboratories Inc., Lenexa, KS, USA) was administered to each recipient, and one or two

IVP embryos were deposited into the uterine horn ipsilateral to the ovary containing the

CL. One technician conducted all transfers.









A total of 85 blastocysts (72 at Day 7 after insemination and 13 and Day 8 after

insemination) were transferred in this experiment. Of these, six were produced by

Transova (Sioux City, IA, USA) using Holstein oocytes and a Holstein sire and were

cultured in Synthetic Oviductal Fluid (SOF) medium. Embryos were shipped overnight

in a portable incubator to Gainesville, FL, USA on Day 4 after insemination. Embryos

were transferred to fresh microdrops of a modified SOF (Fischer-Brown et al., 2002)

prepared by Specialty Media (Phillipsburg, NJ, USA) and cultured at 38.5oC in a

humidified atmosphere of 5% 02 and 5% (v/v) CO2 (balance N2). The remainder were

produced using oocytes obtained from ovaries of a variety of breeds collected at a local

abattoir located at a travel distance of approximately 1.5 h from the Gainesville

laboratory. Procedures, reagents, and media formulation for oocyte maturation,

fertilization, and embryo culture were as previously described (Roth and Hansen, 2005)

with some modifications. Cumulus-oocyte complexes were matured for approximately

22 h at 38.50C in an atmosphere of 5% (v/v) CO2 in humidified air and then inseminated

with a cocktail of Percoll-purified spermatozoa from three different bulls of various

breeds. At 8 12 h post-insemination (hpi), putative zygotes were denuded of cumulus

cells by suspension in Hepes-TALP medium (Caisson, Rexburg, ID, USA) containing

1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexed in a

microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in

50 Cll microdrops of KSOM-BE2 (Soto et al., 2003) (Caisson, Rexburgh, ID, USA) at

38.50C in an atmosphere 5% (v/v) CO2 in air.

Regardless of method of production, embryos greater than 16 cells in appearance

were collected at 1300 h on Day 6 or 7. Embryos were placed in groups of 21 to 65 into









2 ml cryogenic vials (Nalge Company, Rochester, NY, USA) filled to the top with

KSOM-BE2 that was pre-warmed and equilibrated in 5% (v/v) CO2 in air. Embryos

produced by Transova were kept separately from those produced using ovaries from the

local abattoir. Vials containing embryos were placed in a portable incubator (Minitube of

America, Verona, WI, USA) that had been pre-warmed to 390C for 24 h prior to use.

Embryos were shipped by air and arrived at Santa Cruz de la Sierra, Bolivia, at 1100 h

the next day (Day 7 or 8 after in vitro insemination) and transported by ground to the

farm.

Embryos were transferred over a time span from 1300 h and 2000 h. One or two

embryos were loaded into 0.25 cc straws in Hepes-TALP (Caisson) containing 10% (v/v)

bovine steer serum (Pel-Freez, Rogers, AR, USA) and 100 CLM 2-mercaptoethanol

(Sigma-Aldrich, St. Louis, MO, USA). Embryos were transferred to recipients that were

palpated the day before and had a detectable CL. Recipients were randomly assigned to

receive one or two embryos, and all embryos were transferred into the ipsilateral horn to

the CL. Pregnancy diagnosis was performed by rectal palpation at Day 64 and 127 post-

transfer, and the number of fetuses was recorded on Day 127. Data collected at calving

included length of gestation (with the day of transfer being considered Day 7 of

gestation), occurrence of dystocia (defined as needing assistance), sex, weight and

viability of each calf, and occurrence of retained placenta (failure of the placenta to be

expelled within 12 h after calving). Calf survival until Day 7 of age was also recorded.

Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in
Lactating Recipients that Received an IVP Embryo

This study took place at a commercial dairy located in Bell, FL, USA (290 45' N

82o 51' W) from June to October, 2004. Data on minimum and maximum air









temperatures and average relative humidity collected by the Florida Automated Weather

Service (http:.//fawn.ifas.ufl_ edu) for Alachua, FL, USA are presented in Figure 1. A total

of 87 multiparous, lactating Holstein cows in late lactation were used as recipients. Cows

were fed a total mixed ration to meet or exceed requirements recommended for lactating

dairy cows, milked three times a day, and received bovine somatotropin (Posilac",

500 mg sometribove zinc, Monsanto, St. Louis, MO, USA) according to manufacturer' s

directions. Cows were housed in a dry lot with access to a permanent shade structure

without fans or sprinklers and with access to a cooling pond.

Cows were prepared for embryo transfer in groups of 6 to 18; a total of 10

replicates were completed. To synchronize recipients for timed embryo transfer, cows

received 100 Cpg (i.m.) of GnRH (2 ml of Cystorelin ; Merial Limited, Iselin, NJ, USA),

on Day -10; 25 mg (i.m.) of PGF2,, On Day -3; and 100 Cpg (i.m.) of GnRH, on Day 0

(i.e., the day of anticipated ovulation). On Day 7 after anticipated ovulation, all cows

were palpated per rectum for the presence of a CL. Cows that had a palpable CL received

an epidural block of 5 ml of lidocaine (2%, w/v), and a single embryo was transferred to

the uterine horn ipsilateral to the ovary containing the CL. Recipients were randomly

assigned to receive 100 Cpg (i.m) of GnRH or vehicle (9 mg/ml of benzyl alcohol and 7.47

mg/ml of sodium chloride in water) on Day 11 after anticipated ovulation.

The embryos used for transfer were produced in the Gainesville laboratory using

oocytes of various breeds and a pool of semen from three bulls of various breeds as

described for Experiment 1. A different pool of semen was used for each replicate.

Presumptive zygotes were cultured in groups of ~30 in 50 Cll microdrops of modified

SOF (Fischer-Brown et al., 2002) containing 100 ng/ml of insulin-like growth factor-1









(Upstate Biotechnology, Lake Placid, NY, USA). Embryos were cultured at 38.50C in a

humidified atmosphere of 5% (v/v) 02 and 5% (v/v) CO2 with the balance N2. On Day 7

after insemination, blastocysts were harvested and transported to the farm in 2 ml

cryogenic vials (20 to 25 embryos/tube) filled to the top with pre-warmed Hepes-TALP.

Tubes containing embryos were placed in a portable incubator (Minitube of America,

Verona, WI, USA) that had been pre-warmed to 390C for 24 h prior to use. Embryos

were transported to the farm and loaded in 0.25 cc straws prior to transfer into recipients.

Pregnancy was diagnosed by rectal palpation at Day 45 to 53 after anticipated ovulation.

Statistical Analysis

Categorical data were analyzed by logistic regression using the LOGISTIC

procedure of SAS for Windows (Version 9, SAS Institute Inc., Cary, NC, USA) with a

backward stepwise logistic model. Variables were continuously removed from the model

by the Wald statistic criterion if the significance was greater than 0.2. The full statistical

model for Experiment 1 included treatment (one embryo or two embryos), parity (cows

vs heifers), estrus (observed in estrus vs not observed) and treatment x parity on

pregnancy rate, pregnancy loss, calving rate, calf mortality and twinning rate. The only

variable in the final mathematical model for Experiment 2 was GnRH treatment as other

effects (replicate and replicate x treatment) were not significant. The adjusted odds ratio

estimates and the 95% Wald confidence intervals (CI) from logistic regression were

obtained for each variable that remained in the final statistical model following the

backward elimination. Data were also analyzed with the GENMOD procedure of SAS to

determine the significance of each effect that remained in the reduced model; P values for

logistic regression analyses reported in the tables are derived from these analyses. Data

for gestation length and calf birth weight were analyzed by analysis of variance using









Proc GLM. The full statistical model included the effects of treatment, parity and

treatment x parity. The X2 test was used to determine whether the sex ratio of calves

differed from the expected 1:1 ratio.

Results

Experiment 1 Single or twin transfer of IVP embryos

Pregnancy and calving rates

Data are summarized in Table 1. At Day 64 of gestation, the pregnancy rate tended

to be higher (P=0.07) for cows than for heifers. While there were no significant effects

of number of embryos transferred or parity x number transferred, heifers that received

two embryos tended to have lower pregnancy rates than those that received a single

embryo (20% for two embryos vs 41% for one embryo) while there was no difference in

pregnancy rate due to number of embryos transferred to cows (50% for two embryos vs

57% for one embryo).

Pregnancy losses between Day 64 and 127 occurred in one group only cows

receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but decreased

to 17% at Day 127. There was no difference in pregnancy rates at Day 127 between

cows and heifers, but recipients that received two embryos had lower pregnancy rates

(17% for cows and 20% for heifers) than recipients that received one embryo (57% for

cows and 41% for heifers, P < 0.03).

Pregnancy loss after Day 127 occurred in one female only. In particular, a cow

receiving a single embryo gave birth to a stillborn calf at 251 d of gestation. Like for

pregnancy rate at Day 127, there was no difference in calving rate between cows and

heifers, but recipients that received two embryos had lower calving rates (17% for cows









and. 20% for heifers) than recipients that received one embryo (50% for cows and 41%

for heifers, P < 0.03).

Estrus was detected at 24, 48 or 72 h after prostaglandin inj section in 21/32 heifers

(8 at 24 h after injection and 13 at 48 h) and 19/26 cows (1 at 24 h after injection, 14 at

48 h and 4 at 72 h). While not statistically different (P=0. 11), there was a tendency for

pregnancy rates to be lower for animals not detected in estrus. For example, pregnancy

rates at Day 127 for animals receiving one embryo was 55% (11/20) for animals in estrus

vs 36% (4/11) for animals not observed in estrus. Pregnancy rates at Day 127 for animals

receiving two embryos were 25% (5/20) for animals in estrus vs 0% (0/7) for animals not

observed in estrus.

Characteristics of gestation, parturition, and calves

Gestation length was affected by recipient type x number of embryos transferred

(P<0.05; Table 2). For cows, gestation length was slightly longer for those receiving one

embryo as compared to those receiving two embryos while the opposite was true for

heifers. Two of 5 females calving that received two embryos produced twin calves. There

was no significant effect of recipient type or number of embryos transferred on dystocia

or incidence of retained placenta (Table 2). Sex ratio (including the one stillborn calf)

was in favor of males with 15 males compared to 7 female calves born (68% male; Table

3). This ratio tended to be different from the expected 1:1 ratio (P<0. 10).

While there were no significant differences, there was a tendency for calf mortality

at birth to be greater for heifers receiving two embryos than for other groups (Table 3).

None of the cows lost their calf at birth and only 1 of 7 heifers receiving a single embryo

experienced calf death at birth. In contrast, 2 of 3 heifers receiving two embryos

experienced calf loss. One heifer had twin fetuses and both were born dead as a result of










complications with calving. Another heifer gave birth to a single calf that was born dead

as a result of complications with calving. The calf from the third heifer was born alive.

All calves born alive were alive 7 d later.

Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation

Administration of GnRH at Day 11 after anticipated ovulation had no effect

(P>0.10) on pregnancy rates. Recipients treated with GnRH had a pregnancy rate of

17.8% (8/45) while those recipients that received placebo had a pregnancy rate of 16.7%

(7/42). The odds ratio was 1.08 with 95% Wald confidence interval of 0.23 and 3.30.

Discussion

The purpose of the experiments described here was to examine two strategies for

increasing pregnancy rates in heat-stressed dairy recipients that receive an IVP embryo.

Neither approach, transferring two embryos into the uterine horn ipsilateral to the CL or

inj section of GnRH at Day 11 after anticipated ovulation, increased pregnancy rates.

Results of Experiment 1 indicated that the transfer of two embryos into recipients

led to pregnancy loss and that such loss occurred earlier for heifers than for cows. There

was a distinct difference in pregnancy rate between heifers that received one or two

embryos as early as Day 64 of gestation. Among cows, in contrast, there were no

differences in pregnancy rate at this stage of gestation between recipients that received

one or two embryos. By Day 127, however, cows that received two embryos experienced

substantial mid-to-late fetal loss and pregnancy rate and subsequent calving rate was

lower for this group than for cows that received a single embryo.

The most likely explanation for the increased frequency of pregnancy loss in

recipients receiving two embryos is uterine crowding, with the effects of crowding

occurring sooner in gestation for nulliparous animals than for multiparous animals.









Similar results were obtained in another study (Anderson et al., 1979). In that study,

calving rates and twinning rates were similar for cow recipients regardless of whether

twin transfers were performed via bilateral or unilateral placement. For heifers, in

contrast, calving rate and twinning rate was lower for unilateral twin transfers than for

bilateral transfers. Using heifers, Rowson et al. (1971) also found lower embryonic

survival rates and twinning rates for recipients of unilateral twin transfers than for

recipients of bilateral transfers.

It is evident, however, that uterine capacity can vary between herds of cattle. Thus,

there were no differences in pregnancy success between recipients of twin embryos

placed unilaterally or bilaterally for heifers (Sreenan and Diskin 1989; Reichenbach et al.,

1992) or cows (Sreenan and Diskin 1989). Similarly, embryonic survival rate for beef

cows selected for twinning was similar for those having unilateral or bilateral multiple

ovulations (Echternkamp et al., 1990). In lactating dairy cows, in contrast, the likelihood

of a twin pregnancy resulting from multiple ovulation going to term was higher if

ovulations occurred bilaterally than if unilateral ovulations occurred (L6pez-Gatius et al.,

2005b). Perhaps, identification of the biological processes controlling uterine capacity

will lead to new approaches for increasing the efficacy of producing twins in cattle.

In an earlier study, administration of GnRH at Day 11 after anticipated ovulation

tended to increase pregnancy and calving rates in lactating Holstein recipients (Block et

al., 2003). The management of these cows was similar to those in Experiment 2. In both

studies, recipients were exposed to heat stress and received an IVP embryo using a timed

embryo transfer protocol. Effectiveness of treatment with GnRH or its analogues at 1 1

tol2 d after estrus for inseminated cows has yielded variable results, as some reports









indicated a positive effect (Sheldon and Dobson, 1993; Willard et al., 2003) while others

indicated no effect (Ryan et al., 1994). One factor that could influence the effectiveness

of GnRH treatment at Day 11 is the number of follicular waves that a female experiences

during an estrous cycle. Females with estrous cycles characterized by three follicular

waves have larger second-wave dominant follicles at Day 11 than females with two-wave

cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991). Given that a follicle must

reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001), the

preponderance of cycle type (two-wave vs three-wave) within a herd may determine

effectiveness of GnRH treatment at Day 11. Finally, it remains possible that failure to

observe an effect of GnRH treatment was because the number of animals per group was

low. The pitfalls associated with interpretation of experiments with low numbers has

been discussed (Amann, 2005) and could be responsible for the variation in results for

trials to test effects of GnRH on pregnancy rates in embryo transfer recipients.

Estrus is difficult to detect in lactating dairy cows because of the short duration of

estrus and the large proportion of cows that do not display intense mounting activity

(Dransfield et al., 1998). This problem, which is exacerbated by heat stress (Thatcher et

al., 1986), makes embryo transfer in lactating cows inefficient if recipient selection is

based solely on estrus detection. The first report of a timed embryo transfer protocol,

where ovulation was synchronized using an OvSynch protocol, was by Ambrose et al.

(1999). The suitability of timed embryo transfer as a method for preparing recipients was

demonstrated in Experiment 1 because calving rates were 50 and 41% for cow and heifer

recipients that received a single embryo, respectively. Similarly, using beef recipients, a

pregnancy rate of 49% was achieved using timed embryo transfer (Bo et al., 2002). In










contrast, pregnancy rate at Day 45 of gestation in Experiment 2 was only 17%. Low

pregnancy rates have been reported in other studies with timed embryo transfer using

lactating, heat-stressed recipients with pregnancy rates at ~ 45 d of gestation following

timed embryo transfer ranging from 11 26% (Ambrose et al., 1999; Al-Katanani et al.,

2002a; Block et al., 2003). The reason for the differences in pregnancy rates between

Experiment 1 and 2 cannot be deduced because of the large number of variables between

studies including nutrition, housing, level of milk yield, stage of lactation, breed,

synchronization protocol, and embryo culture protocol.

Despite the effectiveness of timed embryo transfer, there was a tendency for

pregnancy rates in Experiment 1 to be higher for those recipients detected in estrus. Most

of the animals not detected in estrus likely ovulated after the last GnRH inj section because

embryos were only transferred to recipients with a detectable CL. Nonetheless, some

cows in this group probably were not synchronized with respect to predicted ovulation

time.

Transfer of IVP embryos has been associated with large calf syndrome, increased

rates of fetal loss, sex ratio skewed towards the male and increased rate of dystocia and

calf mortality (see Hasler et al., 2000; Hansen and Block, 2004; Farin et al., 2004 for

review). There are also reports of prolonged gestation length (Kruip and den Dass, 1997;

Rerat et al., 2005). In Experiment 1, most characteristics of the fetus and calf that were

measured in females receiving one embryo were within normal ranges including

gestation length, rates of fetal loss, calf birth weight, and calf survival at birth and within

the first 7 d of age. The incidence of dystocia among females receiving one calf was 21%

and it is difficult to determine whether this value is high because of the particular mating









combinations used (embryos of diverse genotypes transferred into females of several

different genotypes). In a study with Holsteins bred by artificial insemination, the

frequency of difficult births ranged from 6 to 18% (Dj emali et al., 1987).

The one abnormality identified was a skewed sex ratio with 68% of the calves

being male. While previous work suggests that the altered sex ratio among IVP embryos

is due to toxic effects of concentrations of glucose in excess of 1 mM on female embryos

(Kimura et al., 2005), the concentration of glucose in the medium used for culture here

(KSOM-BE2) contains only 0.2 mM glucose (Soto et al., 2003). Others have found a

tendency for male embryos to become blastocysts sooner in development when cultured

in KSOM than female embryos (Nedambale et al., 2004b). Differences in sex ratio have

been seen as early as between the eight-cell and morula stages of development (Block et

al., 2003). While it is possible that selection of most embryos for transport done on Day

6 after insemination exacerbated the skewed sex ratio, Block et al. (2003) reported that

64% of calves born as a result of transfer of IVP embryos cultured in modified KSOM

were male even though embryos were harvested for transfer on Day 8 after insemination.

In conclusion, results indicate that unilateral transfer of two embryos to increase

pregnancy rate is unwarranted. The fact that fetal loss occurred sooner for heifers than

cows points out the importance of uterine capacity as a limiting factor for maintenance of

fetal development of two conceptuses. There was also no evidence that GnRH treatment

at Day 11 after anticipated ovulation improves pregnancy rate. Finally, the suitability of

timed embryo transfer as a method for preparing recipients for transfer was evident by the

high pregnancy and calving rates achieved with crossbred females that received a single

embryo. Additional research is warranted to reduce incidence of skewed sex ratio.









While sexed semen could be used to control sex ratio (Wilson et al., 2005), it is likely

that the underlying biological causes of altered sex ratio affect other aspects of embryo

physiology also.












Table 3-1. Effect of recipient type and number of embryos transferred per recipient on pregnancy rates and losses.
Pregnancy loss Pregnancy loss
Pregnancy rate, d Pregnancy rate, d
Reipen tpe64ofesatinb between Day 64 and between Day 127
127fgetaton" 127 of gestation Calving rate" and calving
Latain cw8/14 (57%) 8/14 (57%) 0/8 (0%) 7/14 (50%)1/(3%
single embryo
Lactating cow -
6/12 (50%) 2/12 (17%) 4/6 (66%) 2/12 (17%) 0/2 (0%)
two embryos
Nulliparous heifer -
7/17 (41%) 7/17 (41%) 0/7 (0%) 7/17 (41%) 0/7 (0%)
single embryo
Nulipros eier 3/15 (20%) 3/15 (20%) 0/3 (0%) 3/15 (20%) 0/3 (0%)>
two embryos
a Data are the proportion of animals pregnant of those that received embryos and, in parentheses, the percent pregnant.
b Logistic regression indicated effect of recipient type (P=0.07). The odds ratio estimate was 0.38 (heifer/cow) (95% Wald CI = 0. 13,
1.14; Wald Chi-Square statistic = 2.96, P=0.08).
" Logistic regression indicated an effect of number of embryos transferred (P<0.03). The odds ratio estimate was 4. 13 (one
embryo/two embryos) with a 95% Wald CI of 1.243, 13.690. Wald Chi-Square statistic 5.36; P<0.03).
d Data are the proportion of pregnant recipients at Day 64 that lost their pregnancy by Day 127 of gestation and, in parentheses, the
percent pregnancy loss.
e Data are the proportion of animals that calved of those that received embryos and, in parentheses, the percent pregnant.
* Logistic regression indicated an effect of number of embryos transferred (P<0.03). The odds ratio estimate was 3.62 (one
embryo/two embryos) with a 95% Wald CI of 1.090, 12.047. Wald Chi-Square statistic 4.41; P<0.04).
g Data are the proportion of pregnant recipients at Day 127 that lost their pregnancy before calving and, in parentheses, the percent
pregnancy loss.
h One cow expelled a stillborn calf at 251 d of gestation.









Table 3-2. Effect of recipient type and number of embryos transferred per recipient on
characteristics of pregnancy and parturition.
Gestation Twin Retained
Recipient type length, da pregnancieSb Dystociac placentad


)

)

)

)


Lactating cow -
282 + 3 0/7 (0%) 2/7 (29%) 4/7 (57%
single embryo
Lactating cow -
274 + 5 1/2 (50%) 0/2 (0%) 1/2 (50%
two embryos
Nulliparous heifer -
276 + 3 0/7 (0%) 1/7 (14%) 5/7 (71%
single embryo
Nulipfos eier 284 + 4 1/3 (33%) 1/3 (33%) 2/3 (67%
two emb~ryos-
aData are least-squares means + SEM. Gestation length was affected by recipient type x
number of embryos transferred (P<0.05).
b Data are the proportion of pregnancies in which twin calves were born and, in
parentheses, the percent pregnant. Logistic regression indicated an effect of number of
embryos transferred (P<0.02).
" Data are the proportion of pregnancies in which dystocia was recorded at birth and, in
parentheses, the percent cows experiencing dystocia.
d Data are the proportion of cows calving that experienced retained placenta and, in
parentheses, the percent cows experiencing retained placenta.









Table 3-3. Effect of recipient type and number of embryos transferred per recipient on
characteristics of calves born.
Sex ratio Calf birth Calf mortality
aMF) -egt kb Calf mortality
Recipient type at birth" today7
aged
Lacatig cw -5:3e 34 + 3 0/7 (0%) 0/7 (0%)
single embryo
Lactating cow -
2:1 25 + 5 0/3 (0%) 0/3 (0%)
two embryos
Nulipaoushei4: 3 26 +3 1/7 (14%)' 0/6 (0%)
single embryo
Nullipru heifer -
~ ~p~~s~~4:0 25 + 3/4 (75%)' 0/1 (0%)
a The overall sex ratio of 15 male and 7 females tended to be different (P<0. 10) than the
expected 1:1 ratio.
b Data are least-s uares means + SEM.
" Data are the proportion of calves that were bomn dead and, in parentheses, the percent bomn
dead.
d Data are the proportion of calves born alive that died before d 7 of live and, in parentheses,
the percent death before Day 7.
e Data includes the stillborn calf at 251 d of gestation
'One calf was stillborn from a cow not experiencing dystocia.
g One heifer had twin fetuses and both were born dead as a result of complications with
calving. The other two heifers gave birth to a single calf. One calf was born alive and the
other was born dead as a result of complications with calving.








71





Experiment 1- Bolivia


30



E 20-

S15-

10
Nov 1 Nov 15 Dec1 Decl5 Dec 30


100
90
80
[Y 70
60
Experiment 2 Florida s
35-





iEi



Q 10-


June 1 July1 Aug 1 Sept1 Oct1 Nov1


Figure 3-1. Maximum (open circles) and minimum (closed circles) daily air temperatures
and relative humidities (RH) during the experiments.















CHAPTER 4
EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B
TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED
BOVINE EMBRYOS PRODUCED IN VITRO

Introduction

In vitro production of embryos is an important tool for improving genetic merit and

fertility of cattle and is an indispensable component of other technologies such as somatic

cell cloning and transgenesis (Hansen and Block, 2004). One limitation to the

widespread use of in vitro produced embryos in the cattle industry is the poor

survivability of in vitro produced embryos to cryopreservation. In vitro survival rates

following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann,

2000a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates following

thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-

Katanani et al., 2002a) are consistently lower for embryos produced in vitro when

compared to embryos produced in vivo by superovulation.

The poor survival of the in vitro produced embryo is associated with culture-

induced changes in ultrastructure (Rizos et al., 2002), gene expression (Bertolini et al.,

2002a; Lazzari et al., 2002; Lonergan et al., 2003), and metabolism (Krisher et al., 1999;

Khurana and Niemann, 2000b) that make it distinct from the embryo produced in vivo.

Among the metabolic changes are an increase in lipid content (Abe et al., 1999; Rizos et

al., 2002) and this condition has been linked to poor freezability. Mechanical delipidation

(Tominaga et al., 2000; Diez et al., 2001) and addition of inhibitors of fatty acid synthesis

(De la Torre-Sanchez et al., 2005) can improve survival following cryopreservation.









In the current study, two approaches for enhancing survival of bovine embryos

following cryopreservation were evaluated. The first was to culture embryos in the

presence of hyaluronic acid. This unsulphated glycosaminoglycan is present in follicular,

oviductal and uterine fluids in several species including cattle (Lee and Ax, 1984).

Receptors for hyaluronic acid (CD44) have been reported on the bovine oocyte, cumulus

cell, and preimplantation stage embryo (Valcarcel et al., 1999). Addition of hyaluronic

acid to culture medium has been reported to increase blastocyst re-expansion and

hatching after freezing (Stojkovic et al., 2002; Lane et al., 2003). The second approach

was to determine whether altering the cytoskeleton before cryopreservation would

enhance embryo survival. The rationale for this treatment is that cryoinjuries such as

intracellular ice formation and osmotic shock induce irreversible disruption in

microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) and that

temporary depolymerization of actin microfilaments before cryopreservation could

reduce cytoskeletal damage and plasma membrane fracture caused by alterations in

cytoskeletal architecture (Dobrinsky, 1996). Addition of cytochalasin B to cause actin

depolymerization had no effect on survival of eight-cell embryos in the mouse (Prather

and First, 1986) but enhanced survival of expanded and hatched blastocysts without

effecting survival of morula and early blastocysts in the pig (Dobrinsky et al., 2000).

Materials and Methods

Embryo Production

Procedures, reagents, and media formulation for oocyte maturation, fertilization,

and embryo culture were as previously described (Roth and Hansen, 2005) with some

modifications. Briefly, cumulus oocyte complexes (COCs) were harvested from ovaries

of a variety of breeds collected at a local abattoir located at a travel distance of









approximately 1.5 h from the laboratory. The COCs were matured in Tissue Culture

Medium-199 with Earle's salts supplemented with 10% (v/v) steer serum, 2 Cpg/mL

estradiol 17-P, 20 Cpg/ml follicle stimulating hormone, 22 Cpg/ml sodium pyruvate, 50

Cpg/ml gentamicin and an additional 1 mM glutamine for approximately 22 h at 38.50C in

an atmosphere of 5% (v/v) CO2 in humidified air. Insemination with a cocktail of

Percoll-purified spermatozoa from three different bulls was performed in In Vitro

Fertilization Tyrode's Albumin Lactate solution. At 8 12 h post-insemination (hpi),

putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium

containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and

vortexing in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in

groups of ~30 in 50 Cll microdrops of a modified Synthetic Oviductal Fluid (SOF)

prepared as described by Fisher-Brown et al. (2002). Embryos were cultured at 38.50C in

a humidified atmosphere of 5% (v/v) CO2, 5% 02, and with the balance N2. Blastocysts

were collected for cryopreservation on day 7 after insemination.

Experimental Design and Embryo Manipulation

The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid

during culture (+ or -) and cytochalasin B before cryopreservation (+ or -). Data on

development were obtained from 18 replicates using 5022 oocytes while data on

cryopreservation were obtained from 7 replicates using a total of 197 blastocysts.

Following insemination and transfer to fresh microdrops, embryos cultured without

hyaluronic acid were cultured in SOF for 7 days beginning after insemination. Embryos

treated with hyaluronic acid were cultured in SOF until day 5 when all embryos were

transferred to a fresh microdrop of SOF containing 6 mg/ml hyaluronic acid from

Streptococcus zooepidemicus (Sigma).









Blastocysts and expanded blastocysts were harvested on the morning of day 7 after

insemination and washed twice in holding medium consisting of Hepes-TALP (Parrish et

al., 1989) containing 10% (v/v) fetal calf serum (FCS). Embryos treated with

cytochalasin B were incubated for 10 min at 38.50C in air while in Hepes-TALP

containing 10% (v/v) FBS and 7.5 Cpg/ml cytochalasin B (Sigma) in a 1.5 ml

microcentrifuge tube (Tominaga et al., 2000). Cytochalasin B was initially dissolved in

DMSO at a concentration of 5 mg/ml and was then added to HEPES-TALP to achieve a

final concentration of 7.5 Cpg/ml. Control embryos were incubated similarly in HEPES-

TALP containing 10% (v/v) FBS.

Cryopreservation

Procedures for freezing were modified from those reported elsewhere (Hasler et al.,

1995; Enright et al., 2000). In brief, blastocysts were transferred in groups of 10 to a fresh

100 Cl~ microdrop of Hepes-TALP containing 10% FCS at 3 8.5oC for the time it took to

harvest all embryos (~ 10 min). Next, embryos in groups of 5 8 per treatment

hyaluronicc acid or control) were randomly selected to receive cytochalasin B treatment

before freezing or not as described above. Afterwards, each group of 5 8 embryos was

placed in a 50 Cl~ microdrop of 10% (v/v) glycerol in Dulbecco's phosphate-buffered

saline (DPBS) containing 0.4% (w/v) bovine serum albumin (freezing medium) in a grid

plate over a slide warmer at 30oC. Within 10 min, embryos were loaded in a 50 Cl1 volume

into 0.25 ml plastic straws (Agtech, Manhattan, KS). Up to 8 embryos were loaded in

each straw. Two columns of 50 Cl1 freezing medium separated by air bubbles were always

placed above and below the column of embryos. Straws were transferred to a freezing

chamber (Cryologic Model CL5500 (Mulgrave, Victoria, Australia) for 2 min at -5oC and

then ice crystals were induced by touching the straw where the top column of medium









resided with a cotton plug that had been immersed in liquid nitrogen. After an additional

3 min at -5oC, embryos were cooled to -32oC at a rate of -0.6oC/min. After 2 min at -

32oC, straws were directly immersed in liquid N2 and stored until thawing (4 days 1

week later).

Thawing and Determination of Survival

Straws containing embryos were thawed by warming for 10 sec in air at room

temperature and 20 sec in a 32oC water bath. All subsequent steps before culture were

performed with media prewarmed to ~30oC and with dishes placed on a slide warmer set

at 30oC. Embryos were then expelled into an empty petri dish and immediately

transferred to a fresh 60 Cl1 drop of DPB S containing 6.6% (v/v) glycerol and 0.3 M

sucrose in an grid dish. After 5 min, embryos were sequentially transferred to DPBS

containing 3.3% (v/v) glycerol and 0.3 M sucrose for 5 min and DPBS + 0.3 M sucrose

for 5 min. Embryos were then washed three times in HEPES-TALP + 10 % (v/v) FCS

and placed into culture in groups of 5- 8 in 25 Cl1 microdrops of SOF containing 10%

(v/v) FCS. Culture was at 38.50C in a humidified atmosphere of 5% (v/v) CO2, 5% 02,

and 90% N2. Re-expansion was determined at 48 h after thawing and hatching at 72 h.

Statistical Analysis

The proportion of oocytes that cleaved and the proportion of embryos that

developed to the blastocyst stage on day 7 and day 8 were determined for each replicate.

Treatment effects were determined by least-squares analysis of variance using the proc

GLM procedure of SAS (SAS for Windows 90, Cary, NC). The model included the main

effects of replicate and treatment. Data for the proportion of frozen/thawed embryos that

re-expanded and on the proportion that hatched by 72 h of culture were analyzed using

the CATMOD procedure of SAS. The initial model included all main effects and two-









way interactions. After removing nonsignificant effects, the final model included

replicate, hyaluronic acid, preparation prior to freezing (none, cytochalasin B), and the

interaction of hyaluronic acid and preparation before freezing.

Results

Effect of Hyaluronic Acid on Embryonic Development

As shown in Table 1, addition of hyaluronic acid at day 5 after insemination caused

a slight reduction in the yield of blastocysts on day 7 and day 8 after insemination

regardless of whether data were expressed as the proportion of oocytes developing to the

blastocyst stage (P < 0.05) or the proportion of cleaved embryos developing to the

blastocyst stage (P < 0.01). Of the blastocysts that were recovered, 62-68% were

recovered at day 7 and the balance at day 8. There was no effect of hyaluronic acid on

the proportion of blastocysts collected at day 7 (Table 4-1).

Survival after Cryopreservation

Overall, cytochalasin B increased the percent of embryos that re-expanded

following thawing (P < 0.0001) and that hatched following thawing (P < 0.05) (Table 4-

2). Re-expansion rates were 51.2% (22/43) for embryos treated with cytochalasin B and

18.2% (8/44) for embryos not subjected to cytochalasin B. Hatching rates were 39.5%

(17/43) for embryos treated with cytochalasin B and 4.5% (2/44) for embryos not

subj ected to cytochalasin B.

While there was no significant effect of hyaluronic acid on cryosurvival, there was

a tendency (P=0.09) for a hyaluronic acid x cytochalasin B interaction affecting percent

of blastocysts that hatched following thawing. This interaction reflects the fact that

hyaluronic acid increased the percent hatching for embryos not subj ected to cytochalasin

B treatment and decreased percent hatched for embryos subj ected to cytochalasin B.









Discussion

Of the two treatments evaluated for enhancing cryosurvival of in vitro produced

bovine embryos, cytochalasin B treatment was the most effective as determined by an

improvement in both embryo re-expansion and hatching. The rationale for this treatment

is to reduce cellular injury caused by disruption in microtubules and microfilaments

(Kuwayama et al., 1994; Fair et al., 2001) and to increase flexibility of the plasma

membrane to allow it to tolerate forces associated with freezing that lead to membrane

damage. In other studies, addition of cytochalasin B had no effect on survival of eight-

cell embryos in the mouse (Prather and First, 1986), enhanced survival of expanded and

hatched pig blastocysts without effecting survival of morula and early blastocysts

(Dobrinsky et al., 2000), and improved survival of in vivo derived bovine blastocysts

subj ected to vitrification (Dobrinsky et al., 1995).

For embryos not exposed to cytochalasin B, there was a tendency for those cultured

in hyaluronic acid to have a higher re-expansion rate and hatching rate than embryos

cultured without hyaluronic acid. Both Stojkovic et al. (2002) and Lane et al. (2003)

reported improved survival rates to freezing when embryos were cultured in hyaluronic

acid; such a beneficial effect has not always been observed (Furnus et al., 1998).

Surprisingly, embryos cultured in hyaluronic acid were less likely to survive freezing

than control embryos when the cytochalasin B treatment was applied. Perhaps

physiological changes induced by hyaluronic acid cause the embryo to be less able to

adjust to the cellular actions of cytochalasin B. Those changes are potentially numerous

because hyaluronic acid acts to affect cell function through several means including

signaling through cell surface receptors, modifying the biophysical properties of

extracellular and pericellular matrices by attracting water, and by interacting physically









with a variety of ions and other molecules (Laurent, 1987; Ruoslahti and Yamaguchi,

1991; Hardingham and Fosang, 1992; Yasuda et al., 2002; Toole et al., 2005). One

possible mechanism by which hyaluronic acid could increase embryo survival to freezing

is by increasing the total number of cells in the embryo (Stojkovic et al., 2002; Jang et al.,

2003; Kim et al., 2005)

One unexpected finding was the reduction in the percentage of embryos that

became blastocysts caused by hyaluronic acid. In other studies, hyaluronic acid either had

no effect (Stojkovic et al., 2002; Lane et al., 2003) or caused an increase in blastocyst

yield (Furnus et al., 1998; Jang et al., 2003). Differences in origin and concentration of

hyaluronic acid could explain some of this difference between studies. Hyaluronic acid

can be isolated from different sources (ex., bacteria, rooster comb, and umbilical cord)

and preparations can differ in protein, endotoxin, and nucleotide content (Shiedlin et al.,

2004). Stojkovic et al. (2002) reported that preliminary results indicated that embryo

development in vitro was dependent upon the origin of the commercially-avail able

hyaluronic acid. However, embryos cultured with hyaluronic acid experienced a change

in culture medium at day 5 whereas control embryos did not. Such a difference could

have obscured beneficial effects of hyaluronic acid although another paper indicates no

effect of changing culture medium at 72 hpi on blastocyst yield in cattle (Ikeda et al.,

2000).

The percent of embryos that underwent hatching after freezing in glycerol and

thawing has varied from 0% (Enright et al., 2000) 22% (Diez et al., 2001; Nedambale et

al., 2004a), 32% (Guyader-Joly et al., 1999) and 69% (Hasler et al., 1997). The best

survival achieved in this study was for embryos cultured without hyaluronic acid and









treated with cytochalasin B. In this group, 51.2% of cryopreserved embryos were

capable of re-expansion and 39.5% hatched. It is likely that the percent hatching can be

further improved by modifying post-thaw culture-conditions. Massip et al. (1993) found

hatching rates for frozen/thawed, in vitro produced embryos were 41% when culture was

performed in the presence of bovine oviductal epithelial cells while hatching rate using

other culture conditions not involving co-culture was 0-6%. Nonetheless, one would not

expect optimal pregnancy rates to be achieved following direct transfer of embryos

frozen in glycerol even with the inclusion of cytochalasin B treatment. Rather, it is

suggested that pregnancy rates following transfer of embryos cryopreserved using slow-

freezing procedures can be optimized by selecting embryos for transfer based on

development in culture shortly after thawing.

In contrast to the poor survival of in vitro-produced embryos frozen using

conventional slow-freezing techniques, several experiments indicate that cryosurvival can

be enhanced by using vitrifieation (Vajta, 2000). It remains to be tested whether survival

of embryos produced in vitro after vitrifieation can be improved by cytochalasin B

treatment. There was a beneficial effect of cytochalasin B treatment on cryosurvival of

embryos derived in vivo following vitrifieation (Dobrinsky et al., 1995).

In conclusion, cytochalasin B treatment before freezing improved cryosurvival of

bovine embryos produced in vitro and subj ected to slow-freezing in glycerol. Such a

treatment could be incorporated into methods for cryopreservation of bovine embryos

provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was

of minimal benefit the increased cryosurvival in the absence of cytochalasin B was not

sufficient to allow an adequate number of embryos to survive.









Table 4-1. Effect of hyaluronic acid added at day 5 after insemination on production of
blastocysts at day 7 and 8 after inseminationab.
Culture Number Percent Blastocysts/oocyte Blastocysts/cleaved Percent of
medium of cleaved (%) c embryo (%) c total
oocytes blastocysts
that were
collected at
day 7
Control 1935 76.0 + 36.0 + 1.2* 47.2 + 1.3** 68.8 + 2.4
0.9
Haluronic 3087 77.7 + 31.5 + 1.2 40.7 + 1.3 62.2 + 2.4
acid 0.9

a n=18 replicates
b Means within a column that differ significantly are indicated by (P < 0.05) and ** (P < 0.01)
Includes blastocvsts collected at day 7 and those collected at day 8.


Table 4-2. Effect of culture in hyaluronic acid and treatment with cytochalasin B on
survival after cryopreservation. a
Cytochalasin
Culture medium treatment Re-expansion by 72 hb Hatching by 72 he

Control Control 8/44 (18.2%) 2/44 (4.5%)
Control Cytochalasin B 22/43 (51.2%) 17/43 (39.5%)
Hyaluronic acid Control 16/55 (29.0%) 7/55 (12.7%)
Hyaluronic acid Cytochalasin B 26/55 (47.3%) 12/55 (21.8%)
a Data are the fraction of embryos, and in parentheses, percent. Number of replicates was 7.
b Effect of cvtochalasin B (P < .0001).
Effect of cvtochalasin B (P < 0.05), hyaluronic acid (P < 0.10), and the cytochalasin B x
hyaluronic acid interaction (P = 0.09).















CHAPTER 5
GENERAL DISCUSSION

As alluded to at the beginning of this thesis, there has been a precipitous decline in

fertility of dairy cows over the last 10-40 years in North America (Butler, 1998), Ireland

(Roche, 2000), Spain (L6pez-Gatius et al., 2003), and the United Kingdom (Royal et al.,

2000). In addition, heat stress can compromise fertility in lactating dairy cows (Putney et

al., 1989b; Al-Katanani et al., 1999). The purpose of the present series of experiments

described in the thesis was to 1) evaluate strategies for enhancing fertility after AI using

GnRH treatment (Chapter 2) and 2) further develop ET using in vitro produced embryos

as a tool for increasing fertility by testing whether pregnancy rate could be improved by

transfer of twin embryos (Chapter 3) and whether the developmental competence of

embryos after cryopreservation could be improved by hyaluronan or cytochalasin B

treatment (Chapter 4). Results indicated no consistent benefit of injection of GnRH at

Day 11-15 after anticipated ovulation or insemination on pregnancy rates in heifers or

lactating cows. While unilateral transfer of two embryos was not shown to be an effective

treatment for increasing pregnancy rate in recipients, the high pregnancy rates achieved

in this study point to the potential usefulness of ET as a tool for enhancing fertility.

Large-scale use of embryo transfer will require the ability to freeze embryos successfully.

Results suggest that treatment of embryos with cytochalasin B before freezing is a

promising tool for enhancing survival of embryos following cryopreservation. A large

number of studies have been performed to test the effect of GnRH administration after

expected ovulation on fertility of cattle. Previous results indicated that GnRH was









sometimes effective at increasing pregnancy rate, but this beneficial effect was often not

observed (Peters et al., 2000). Despite this knowledge, we chose to reevaluate the

effectiveness of GnRH treatment because of a report that GnRH treatment at Day 11 after

estrus increases pregnancy rates in lactating cows exposed to heat stress (Willard et al.,

2003). Accordingly, it was hypothesized in Chapter 2 that the beneficial effect of GnRH

treatment would be greater during the summer than winter. This may be so because the

antiluteolytic process may be compromised by heat stress because of decreased growth of

the filamentous stage concepts (Biggers et al., 1987) and increased uterine PGF2a

secretion from the uterus (Wolfenson et al., 1993).

Overall, the results of GnRH treatment were generally negative. For treatment at

Day 11i, a positive effect of GnRH on fertility was never seen. This was the case for

heifers and lactating cows subj ected to AI or whether animals were exposed to heat stress

or not (Chapter 2; experiment 1 and 2). Treatment of lactating recipients with GnRH at

Day 11 also failed to increase pregnancy rate during heat stress in ET recipients (Chapter

3, experiment 2). Effectiveness of treatment with GnRH or its analogues at 11 to 12 d

after estrus for inseminated, heat-stressed lactating cows has yielded variable results, as

some reports indicated a positive effect (Sheldon et al., 1993; Willard et al., 2003), while

others indicated no effect (Jubb et al., 1990). Also, administration of GnRH at Day 1 1

after anticipated ovulation tended to increase pregnancy and calving rates in lactating

Holstein embryo transfer recipients exposed to heat stress (Block et al., 2003).

One factor that could influence the effectiveness of GnRH treatment at Day 11 is

the number of follicular waves that a female experiences during an estrous cycle. Females

with estrous cycles characterized by three follicular waves have larger second-wave









dominant follicles at Day 11 than females with two-wave cycles (Ginther et al., 1989;

Savio et al., 1990; Ko et al., 1991). Given that a follicle must reach 10 mm in diameter to

ovulate in response to LH (Sartori et al., 2001), the preponderance of cycle type (two-

wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at

Day 11.

In one experiment (Chapter 2; experiment 3), administration of GnRH at Day 14

after anticipated ovulation in cows subj ected to TAI increased pregnancy rates of

lactating cows in the summer and winter at two locations. In the following year, though,

GnRH failed to improve fertility when treatment was administered either at day 14 in

cows subj ected to TAI (experiment 4) or at day 14 or 15 in cows previously diagnosed

coming in estrus (experiment 5). It is important to recognize that GnRH treatment should

improve fertility only when triggering luteinization or ovulation of developing

estrogenicc) follicles. Thus, there are at least two possible reasons for a lack in response

upon GnRH treatment at day 14 or 15. One possibility relates to the timing of ovulation

relative to the GnRH treatment and whether these animals failed to ovulate after being

diagnosed as coming into estrus. Although after observing estrus one does not expect

ovulation to fail, this expression does not necessarily mean that subsequent ovulation

occurred (L6pez-Gatius et al., 2005b) and insemination after a false identified estrus

often occurs (Heersche and Nebel 1994). According to L6pez-Gatius et al. (2005b), the

risk of cows failing to ovulate (12%) during the summer was greater than in the cool

period (3%).

During experiment 3 all cows received a GnRH inj section at 72 h following

PGF2a to insure an ovulation of the synchronized dominant follicle. Perhaps, the positive









GnRH effect observed during experiment 3 was masked in the following experiment

because cows did not receive an additional GnRH dose at estrus to ensure subsequent

ovulation. According to Lopez-Gatius et al. (2005a), there is evidence demonstrating the

benefits upon GnRH treatment when given on the day of insemination compared to

controls (30.8% vs. 20.6%), but conception rates were greater if cows received an

additional dose at day 12 post-insemination (35.4%). On the other hand, when GnRH

treatment took place on day 15 to ensure a responsive estrogenicc) dominant follicle

would ovulate at the time of GnRH treatment, it failed to improve fertility as well.

Similarly, in a recent study (Bartolome et al., 2005) there was no effect of GnRH

treatment on pregnancy rates of lactating cows when administered either on day 15 or day

5 and 15 after TAI.

It remains possible that inconsistency in effects of GnRH treatment is caused in

part by the low number of animals per treatment group. The pitfalls associated with

interpretation of experiments with low numbers has been discussed (Dransfield et al.,

1998) and could be responsible for the variation in results for trials to test effects of

GnRH on pregnancy rates in embryo transfer recipients and for inseminated cows.

With an existent variation among trials regarding the use of GnRH at day 1 1-15

post-insemination, one could speculate that such inconsistency regarding treatment is due

to the fact that herds of cattle determine the result that an experiment achieves. However,

our results indicate that such a hypothesis is not likely because when an experiment was

replicated the next year using the same herd, GnRH treatment once again proved to be

inconsistent in improving pregnancy rates.









According to Thatcher et al. (2005), hCG results in a more prolonged rise in LH

activity than is achieved following GnRH treatment. Perhaps the likelihood of ovulating

or luteinizing the dominant follicles present at the time of treatment would be higher

using hCG. Although low numbers of inseminated animals were used (n=8; n=49) hCG

treatment on d 14 after estrus improved pregnancy rates (Raj amahendran and

Sianangama, 1992; Sianangama and Rajamahendran, 1992). Use of hCG warrants further

investigation for any additional effect or response during the summer to enhance

pregnancy rates of lactating cows.

Recent work has focused on use of ET to bypass early embryonic death (Putney et

al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a). Given that ET can be more

effective at increasing pregnancy rates than AI for lactating cows during periods of heat

stress (Putney et al., 1989b; Ambrose et al., 1999; Drost et al., 1999; Al-Katanani et al.,

2002a), the potential benefit of ET can be realized. For ET to become an economical

alternative to AI on a wide scale basis in commercial herds, embryos must be inexpensive

to produce (Hansen and Block et al., 2004). Although embryos produced using IVP

systems are relatively inexpensive as compared to embryos produced by superovulation,

pregnancy rates achieved following transfer of an IVP embryo are often less than what is

obtained following transfer of an embryo produced by superovulation (Hasler et al.,

1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a). In addition,

IVP embryos are less likely to survive freezing than superovulated embryos (Hasler et al.,

2003), likely due to their increased lipid content (Abe et al., 1999; Rizos et al., 2002).

Accordingly, the second approach for the thesis focused on improvements in ET by










comparing pregnancy rates following the transfer of two embryos compared to one and

by increasing the viability of embryos that were cryopreserved.

The first effort was to determine whether transfer of two IVP embryos into the

uterine horn ipsilateral to the CL could increase pregnancy rates during periods of heat

stress. It was hypothesized that such a treatment might increase pregnancy rates because

the likelihood is increased that the cow receives at least one embryo competent for

sustained development. In addition, the transfer of two embryos into the ipsilateral

uterine horn is likely to increase the amounts of interferon-z and other embryonic

signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis.

Transferring two embryos into the uterine horn ipsilateral to the CL failed to

increase pregnancy rates. Instead, the transfer of two embryos into recipients led to

pregnancy loss, which occurred earlier for heifers than for cows. The most likely

explanation for the increased frequency of pregnancy loss in recipients receiving two

embryos is uterine crowding, with the effects of crowding occurring sooner in gestation

for nulliparous animals than for multiparous animals. Regardless of whether twin

transfers were performed via bilateral or unilateral placement, similar results were

obtained in another study (Anderson et al., 1979). In contrast, calving rate and twinning

rate in heifers was lower for unilateral twin transfers than for bilateral transfers.

Similarly, Rowson et al. (1971) also found lower embryonic survival rates and twinning

rates for recipients of unilateral twin transfers than for recipients of bilateral transfers in

heifers.

It is evident that uterine capacity can vary between herds of cattle. Thus, there were

no differences in pregnancy success between recipients of twin embryos placed









unilaterally or bilaterally for heifers (Sreenan et al., 1989, Reichenbach et al., 1992) or

cows (Sreenan et al., 1989). Similarly, embryonic survival rate for beef cows selected for

twinning was similar for those having unilateral or bilateral multiple ovulations

(Echternkamp et al., 1990). In lactating dairy cows, in contrast, the likelihood of a twin

pregnancy resulting from multiple ovulations going to term was higher if ovulations

occurred bilaterally than if unilateral ovulations occurred (L6pez-Gatius and Hunter,

2005). Perhaps, identification of the biological processes controlling uterine capacity

will lead to new approaches for increasing the efficacy of producing twins in cattle.

An additional limitation to the widespread use of IVP embryos in cattle is their

poor survival following cryopreservation. In vitro survival rates following thawing

(Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al.,

2001; Guyader-Joly et al., 1999) and pregnancy rates following thawing and transfer

(Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a)

are consistently lower for IVP embryos when compared to embryos produced in vivo by

superovulation.

The percent of embryos that underwent hatching after freezing in glycerol and

thawing has varied from 0% (Enright et al., 2000), 22% (Diez et al., 2001; Nedambale et

al., 2004a), 32% (Guyader-Joly et al., 1999), and 69% (Hasler et al., 1997). Of the two

treatments evaluated for enhancing cryosurvival of IVP bovine embryos, cytochalasin B

treatment was the most effective as determined by an improvement in embryo re-

expansion and hatching rates. In this treatment, 51.2% of cryopreserved embryos were

capable of re-expansion and 39.5% hatched. Nonetheless, one would not expect optimal




Full Text

PAGE 1

STRATEGIES TO ENHANCE FERTILIT Y IN DAIRY CATTLE DURING SUMMER INCLUDING USE OF CRYOPRESERVAT ION OF IN VITRO PRODUCED EMBRYOS By C. MOIS S FRANCO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by C. Moiss Franco

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This professional achievement reflects the sacr ifice and guidance of my family especially that of my mother, Mercedes Y. Vaca El-H age, who laid the foundations with strong pillars in my life. This dissertation is dedicated to my be loved son Talyn Izaak Franco Benton and astonishing father Antonio Vicente Franco M onasterio (†) for their endless love, support and most important, inspiration. “EL HOMBRE SE AUTORREALIZA EN LA MISMA MEDIDA EN QUE SE COMPROMETE AL CUMPLIMIENTO DEL SENTIDO DE SU VIDA” Victor Frankl (1905-1997)

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iv ACKNOWLEDGMENTS This thesis would not have been po ssible without the enthusiasm, knowledge, guidance, tenacity, and, perhaps most importantly faith that I received from my academic advisor, Peter J. Hansen. From the very fi rst interview to the last queries on research accomplishments and career plans, he was always eager to entertain my ideas in hope that I fulfilled my dream(s) and become successful. I was not sure I could handle an undertaking of such a magnitude, but was able to thanks to hi s consistent effort and true desire to keep me on track. I would like to extend my sincere appreci ation to my committee member Dr. Karen Moore, for her insight and willingness to help me academically without fail and regards to time. Despite having other major responsibil ities, Dr. Carlos Risco was willing to help whenever asked. I thank him for his assistance and especially for the desire to help me learn to palpate. Thanks are also extende d to Dr. Alvin Warnick for his advice and suggestions for improving my research projec ts and academic training. I would also like to thank Dr. Joel Yelich for his teaching, support, and enthusiasm while providing me with ideas that can help me achieve my goals. Special thanks are extended to my family for encouraging me to seek for myself a demanding and meaningful education. This thes is could not have taken place without that precious gift. Most sincere appreciation is also due to to my colleague and friend Dr. Roco M. Rivera, whose willingness to assist me in my ea rly stages as a master's student helped to

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v kindle my interest in this expl oration. I would not have gotte n this far if it was not for her unique and excellent training doing IVF. Dr. Zv i Roth was an inspirational friend whose passion for science was transmitted to me. He also expressed his kindness and love towards my son. I also thank Dr. Joel Hernandez for his support, friendship, and guidance. Thanks are given to Maria B. Padua for he r assistance with the completion of this manuscript and Luis Augusto Castro e Paula. Their unconditional fr iendship and help at any given time is sincerely appreciated. I am grateful to Dean Jousan for making the time to proofread my writings throughout th e years and for his assistance in various research experiments. Special thanks go to Amber Brad for her personality and joy that helped the lab be united. Best of all has b een my colleague and friend Jeremy Block for his patience, expertise and engaging convers ations that helped develop in me new dreams. In addition, he always remained motiv ated throughout my transfer experiments. I also would like to thank Central Pa cking Co. management and personnel at Center Hill, FL, for providing the ovaries used for various experiments and William Rembert for his assistance in collecting these ovaries. Special thanks go to Mary Russell and Elise Griffin, for their assistance at the Un iversity of Florida Dairy Research Unit. I thank Luther White and Mark Saulter of H illtop Dairy, R.D. Skelton and Mathew Steed of Levy County Dairy, and Mauricio Franco and Faby Grisel of Sausalito Dairy for cooperation and assistance with th e projects. And last but not least, I would like to thank Todd Bilby, Osiloam Gomez, Reinaldo Cooke, Patrick Thompson, Saban Tekin, and Paolette Soto.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xi CHAPTER 1 REVIEW OF LITERATURE.......................................................................................1 Infertility in Modern Dairy Cattle.................................................................................1 Causes for the Decline in Fertility in Dairy Cattle.......................................................2 Milk Yield.............................................................................................................2 Milk yield and energy balance.......................................................................3 Milk yield and endocrine milieu....................................................................5 Milk yield and heat stress...............................................................................6 Milk yield and diseases..................................................................................9 Milk yield, estrus detection, and fertility.....................................................10 Changes in Herd Size as a F actor in Reduced Fertility.......................................11 Inbreeding............................................................................................................12 Strategies to Improve Fertil ity in Lactating Dairy Cattle...........................................12 Treatment with Bovine Somatotropin (bST) to Enhance Fertility......................13 Treatment with GnRH to Delay Luteolysis.........................................................14 Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus Luteum.............................................................................................................17 Induction of an Accessory Corpus Luteum.........................................................19 Progesterone Supplementation............................................................................20 Inhibition of Luteolysis.......................................................................................21 Nutritional Strategies...........................................................................................22 Fat feeding to improve energy balance........................................................22 Administration of antioxidants.....................................................................25 Crossbreeding......................................................................................................26 Embryo Transfer..................................................................................................27 Limitations to Optimal Pregnancy Rates Using IVP TET................................28 Cryopreservation of IVP Embryos......................................................................30 Summary and Objectives of the Thesis......................................................................31

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vii 2 EFFECTIVENESS OF ADMINIS TRATION OF GONADOTROPIN RELEASING HORMONE AT DAY 11, 14 OR 15 AFTER ANTICIPATED OVULATION FOR INCREASING FERTILITY OF LACTATING DAIRY COWS AND NON-LACTATING HEIFERS............................................................34 Introduction.................................................................................................................34 Materials and Methods...............................................................................................35 Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artific ial Insemination during Heat Stress.....35 Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................37 Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................38 Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Time d Artificial Insemination During Heat Stress................................................................................................................39 Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus................................................................................................................40 Statistical Analysis..............................................................................................40 Results........................................................................................................................ .42 Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artific ial Insemination During Heat Stress....42 Experiment 2 GnRH administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................42 Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination......................43 Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Time d Artificial Insemination During Heat Stress................................................................................................................43 Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus................................................................................................................44 Overall Effectiveness of GnRH Treatme nt as Determined by Meta-Analysis....44 Discussion...................................................................................................................44 3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED EMBRYOS AND POST-TRANSFER ADMINISTRATION OF GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE.......................................................................52 Introduction.................................................................................................................52 Materials and Methods...............................................................................................54 Experiment 1 Single or Twin Transf er of IVP Embryos into Crossbred Dairy Recipients...............................................................................................54 Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in Lactating Recipients that Received an IVP Embryo..................57 Statistical Analysis..............................................................................................59 Results........................................................................................................................ .60

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viii Experiment 1 Single or twin transfer of IVP embryos......................................60 Pregnancy and calving rates.........................................................................60 Characteristics of gestati on, parturition, and calves.....................................61 Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation..........................................................................................................62 Discussion...................................................................................................................62 4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED BOVINE EMBRYOS PROD UCED IN VITRO........................................................72 Introduction.................................................................................................................72 Materials and Methods...............................................................................................73 Embryo Production..............................................................................................73 Experimental Design and Embryo Manipulation................................................74 Cryopreservation.................................................................................................75 Thawing and Determination of Survival.............................................................76 Statistical Analysis..............................................................................................76 Results........................................................................................................................ .77 Effect of Hyaluronic Acid on Embryonic Development.....................................77 Survival after Cryopreservation..........................................................................77 Discussion...................................................................................................................78 5 GENERAL DISCUSSION.........................................................................................82 LIST OF REFERENCES...................................................................................................91 BIOGRAPHICAL SKETCH...........................................................................................123

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ix LIST OF TABLES Table page 2-1 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and ovul ation synchronization prot ocol on pregnancy rates of heifers during heat stress......................................................................................49 2-2 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and s eason of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination.......................................................50 2-3 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and s eason of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination.......................................................50 2-4 Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and Da ys in milk (<150 d vs > 150) at insemination on pregnancy rates of lactating cows subj ected to timed artificial insemination during heat stress......................................................................................................51 3-1 Effect of recipient type and number of embryos transferred per recipient on pregnancy rates and losses.......................................................................................68 3-2 Effect of recipient type and number of embryos transferred per recipient on characteristics of pregnancy and parturition............................................................69 3-3 Effect of recipient type and number of embryos transferred per recipient on characteristics of calves born...................................................................................70 4-1 Effect of hyaluronic acid added at day 5 after insemination on production of blastocysts at day 7 a nd 8 after insemination. .........................................................81 4-2 Effect of culture in hyaluronic acid and treatment with cytochalasin B on survival after cryopreservation. ...............................................................................81

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x LIST OF FIGURES Figure page 1-1 Rolling herd average (RHA, kg milk pe r lactation), calving interval (CI), and services per conception (SPC) for 143 da iry herds continuously enrolled in the Raleigh DHIA record system from 1970 to 1999. ..................................................32 1-2 Temporal changes in first service pregnancy rate and annual average milk production from high-producing Holstein-Fri esian dairy herds in north-eastern Spain. Data for pregnancy rate were r ecorded in the cool (October April months) and warm season (May-September months). ...........................................33 3-1 Maximum (open circles) and minimum (c losed circles) daily air temperatures and relative humidities (RH) during the experiments..............................................71

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xi Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science STRATEGIES TO ENHANCE FERTILIT Y IN DAIRY CATTLE DURING SUMMER INCLUDING USE OF CRYOPRESERVAT ION OF IN VITRO PRODUCED EMBRYOS By C. Moiss Franco Vaca May 2006 Chair: Peter J. Hansen Major Department: Animal Sciences There has been a precipitous decline in fer tility of lactating dair y cows. In addition, heat stress can further compromise fertility. The goals of this thesis were to 1) evaluate strategies for enhancing fer tility after artificial insemi nation using mid-cycle GnRH treatment and 2) further develop embryo tran sfer using in vitro produced embryos as a tool for increasing fertility. For the sec ond objective, experime nts tested whether pregnancy rate could be improved by tran sfer of twin embryos and whether the developmental competence of embryos afte r cryopreservation could be improved by hyaluronic acid or cytochalasin B treatment. A series of six experiments were conduc ted to test the efficacy of GnRH for increasing fertility. Except for one experime nt, in which GnRH administration at day 14 after insemination increased pregnancy rate, GnRH was without effect whether given at

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xii day 11, 14 or 15 after insemination or at da y 11 after anticipated ovulation in embryo transfer recipients.. Neither unilateral transfer of two embr yos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnanc y rates of dairy cattle exposed to heat stress. Cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro. In contrast, cu lture with hyaluronic acid was of minimal benefit. Taken together, GnRH treatment did not consistently increa se pregnancy rates when administered at Day 11-15 after insemina tion and is not recommended as a fertilityenhancing treatment. Similarly, tr ansfer of two embryos to th e uterine horn ipsilateral to the CL was not an effective method for increas ing pregnancy rates in recipients. Transfer of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B since this molecule increased in vitro surv ival, and it remains to be tested whether survival of IVP embryos afte r vitrification can be improve d by cytochalasin B treatment.

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1 CHAPTER 1 REVIEW OF LITERATURE Infertility in Modern Dairy Cattle Fertility is defined as the ability of a cycl ic animal to establish pregnancy and is an important economic trait that a ffects herd productivity in da iry cattle (Pecsok et al., 1994; Plaizier et al., 1998). Unfortuna tely, there has been a decline in fertility in dairy cows over the last 10-40 years. Fertility, whethe r traditionally measured as conception rate (number of pregnant animals divided by the number of inseminated animals) or herd pregnancy rate (number of pregnant animals di vided by the number of animals eligible to be bred), has declined in North Ameri ca (Butler, 1998), Ireland (Roche, 2000), Spain (Lpez-Gatius, 2003), and the United Kingdom (Royal et al., 2000). Other important reproductive measurements have changed during this time as well, including increases in days to first service, days to conception, and calving interv al (de Vries and Risco, 2005). The magnitude of these changes in reproducti ve function over time is illustrated for data from herds in the United States (Figure 11) and northeastern Sp ain (Figure 1-2). The incidence of infertility of dairy cows has been correlated with changes in dairy cattle physiology and improvements in gene tic progress, nutrition, and management practices. This litera ture review will seek to identi fy physiological causes for this decrease in fertility and describe efforts to improve fertility.

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2 Causes for the Decline in Fertility in Dairy Cattle Milk Yield The Animal Improvement Programs Laborator y of the United States Department of Agriculture (USDA) has estimated the genetic trend for milk yield with an average of 37 kg/yr during the 1960s, 79 kg/yr during th e 1970s, 102 kg/yr during the 1980s, and 116 kg/yr for the period from 1990 to 1996 ( http://aipl.arsusda.gov ; Hansen, 2000). It has long been known that fertility is reduced in lactating cows as compared to non-lactating heifers (Ron et al., 1984; Ne bel and McGilliard, 1993). Gi ven that milk yield has increased over time as fertility has declined, the possibility must be considered that the increase in milk yield is one reason that has contributed to the decreased fertility in dairy cattle. There are indications that the genetic correlation between female fertility and milk production is antagonistic (Kadarmideen et al ., 2000; Royal et al., 2002). In contrast, Mahanna et al. (1979) suggested that there was no negative genetic correl ation between milk yield and reproduction because there wa s no difference in fertility among heifers with different genetic abilities for milk yield. There may be an environmental effect of milk yield on fertility, however. As describe d by Lucy (2001), the increase in milk yield over the period from 1970 has been associated with a corresponding decrease in fertility as measured by increased services per con ception and calving interval (Figure 1-1). According to Nebel and McGilliard (1993) ther e was little or no asso ciation of increased milk yield compromising fertility prior to the 1970s (Gaines, 1927; Boyd et al., 1954; Currie, 1956; Smith and Legates, 1962) but a dverse effects of milk yield have been correlated with reduced fertility in studie s conducted since 1975 (S palding et al., 1975;

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3 Laben et al., 1982; Fonseca et al., 1983; St evenson et al., 1983; Hillers et al., 1984; Wiggans et al., 1987; Faust et al., 1988). Using a data set of Holstein, Jersey, and Guernsey cows, it was found that 0.014 more services per conception were required for each additional 100 kg of 120-d milk for Holsteins and 0.028 services per conception for Jersey and Guernsey cows (Olds et al., 1979). Similarly, cows with the highest milk yield had the lowest first service conception rate (Faust et al., 1988) or 90-d non-return rate (Al-Katana ni et al., 1999) and highest number of services (Faust et al., 1988). Days to first in semination and days open also increased linearly as milk yield increased in Jersey dairy cattle (Fonseca et al., 1983). Expression of estrus at first postpartum ovulation is less li kely in cows with higher milk production (Westwood et al., 2002). Some st udies (Nielen et al., 1989; Kinsel et al., 1998), but not others (Deluyker et al., 1991), co rrelate the incidence of twins to milk yield. Amount of milk yiel d, however, was not correlated to increased incidence of multiple ovulations (Lpez-Gatius et al., 2005b), yet the incidence of double ovulations and twinning rate has increased in modern dairy cattle (Wiltbank et al., 2000). Taken together, the associations of milk yield with reduced duration of estr us, increased days to first insemination, increased number of in seminations per conception, reduced first service conception rate s, and reduced progesterone le vels post-ovulation compromise herd fertility. Milk yield and energy balance One way in which milk yield could aff ect fertility is through effects on energy balance. A critical phase exists in the period following calving when dry matter intake does not meet the increased metabolic demands of lactation, and as a result, the animal

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4 enters a state classified as “negative ener gy balance” (NEB). Duri ng the period of NEB, body reserves of fat and protein are mobili zed (Bauman and Currie, 1980; Butler and Smith, 1989). An animal under NEB tends to have low body condition score (BCS), and both NEB and low BCS are associated with low fertility (O’Ca llaghan, 1999; Butler, 2000; Pryce et al., 2001; Pus hpakumara et al., 2003). Energy deficiency reduces or impairs gona dotropin secretion, a nd as an animal reaches this state around parturition, gonadot ropin secretion to support follicular development and ovulation is compromise d and reproductive problems (i.e., cystic ovaries) associated with onset of ovarian activ ity become prevalent (Zulu et al., 2002ab). Growth hormone stimulates insulin-like grow th factor 1 (IGF-1) production by the liver (Jones and Clemmons, 1995), but during NEB growth hormone receptors are downregulated in a process referred to as “Growth Hormone Resistance” (Donaghy and Baxter, 1996). As milk production increases during early lactation and the cow is under NEB, the liver becomes refractory to growth hormone because growth hormone receptors are decreased (Vicini et al., 1991), and this result in reduced plasma concentration of IGF-1 (Pell et al., 1993). Follicular growth is stimulated by IGF1 (Webb et al., 2004) and reduced plasma concentrations of this growth factor are observed in cows wi th high milk yield (Rose et al., 2004) and together are highly correlated to delayed return to ovarian cyclicity (Taylor et al., 2004). After calving, cows with IGF-I concentrations gr eater than 50 ng/ml at first service were 5 times more likely to concei ve than those with lower concentrations (Taylor et al., 2004).

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5 The fact that high-producing cows have greater energetic demands for lactation does not necessarily mean that these cows ha ve greater NEB or low BCS. Staples et al. (1990) found that low-producing cows had lower dry matter intake and were at a greater risk for failure to conceive due to anestrus and infertility than high-producing cows. It was observed that the low-producing group, cla ssified as non-responders, sustained milk production from 28% of body tissue reserve vs 15.9 and 16.7% in the early responder and late responder groups. This interaction was confirmed when low-producing cows had lost the most body weight during the first 2 w eeks of lactation and we re in the greatest energy deficit (Staples et al., 1990). Milk yield and endocrine milieu Cows displaying greater milk producti on often have higher dry matter intakes (Staples et al., 1990; Hommeida et al., 2004), which has been demonstrated to decrease circulating progesterone concentrations in lactating (Hommeida et al., 2004) and nonlactating cows (Rabiee et al ., 2001). Acute feeding reduced circulating progesterone by 25% in pregnant cows (Vasconcelos et al., 2003). Lucy and co-workers (1998) found that circulating progesterone was lower in cattle genetically se lected for high milk production. Sangsritavong et al. (2002) demonstrated that lactating cows have a much greater steroid metabolism than non-lactating cows. As a result, lactating cows may have larger luteal tissue volume on the ovary (Sartori et al., 2002; Sartori et al., 2004) yet experience lower circulating progesterone and estradiol co ncentrations than heifers and dry cows (De la Sota et al., 1993; Wolfenson et al., 2004). There is evidence that low progesterone

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6 secretion can compromise fertility in da iry cattle (Mann and Lamming, 1999) and an increase in progesterone secretion ma y facilitate embryonic development. Progesterone provides nourishment for the co nceptus via induction of secretion of proteins and other molecules from the e ndometrium (Garrett et al., 1988a). Low peripheral concentrations of pr ogesterone are also associated with increased luteinizing hormone (LH) pulses (Ireland and Roche, 1982) that can stimulate lu teolytic signals in favor of pregnancy failure. Skarzynski and Okuda (1999) reporte d that blocking the progesterone receptor with a progesterone antagonist (onapristone) increased prostaglandin F2 (PGF2 ) production by bovine luteal cells harvested from mid-cycle corpora lutea (CL) (Days 8–12). In addition, it was revealed that the bovine corpus luteum (CL) does not undergo apoptosis until progesterone production has declined (Juengel et al., 1993; Rueda et al., 1995). Milk yield and heat stress One reason why milk yield might decrease fe rtility of lactating cows is because it increases their susceptibility to heat stress. Infertility is a particul ar problem during heat stress (Ingraham et al., 1974; Putney et al., 1989b; Al-Katanani et al., 1999) and air temperatures as low as 27oC can induce hyperthemia in lact ating dairy cows (Berman et al., 1985). Cows exposed to elevated temper atures to induce heat stress experienced reduced pregnancy rates (Dunlap and Vincen t, 1971) and increased embryonic mortality (Putney et al., 1988ab; Ealy et al., 1993). On the other hand, provisi on of cooling in the summer increased pregnancy rates as compared to non-cooled cows (Stott et al., 1972; Roman-Ponce et al., 1981; Ealy et al., 1994).

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7 The ability to regulate body temperatur e during heat stress is exacerbated by lactation because of the excess heat produc tion. The increase in body temperature in response to heat stress is great er for lactating cows than heifers (Cole and Hansen, 1993) and greater for high-producing cows than low-producing cows (Berman et al., 1985). Data collected on fertility at first service from 8124 Holstein cows located in South Georgia as well as North and South Florida support the idea that a high level of milk production reduces fertility of lactating cows When cows were grouped according to mature equivalent milk yield, there was a milk yield class x month of breeding interaction that resulted from the fact that the duration and magnitude of summer infertility increased as milk yield increased (Al-Katanani et al., 1999). Heat stress before, shortly after, and on the day of breeding is associated with reduced fertility. Heat stress can compromi se fertility throughout various reproductive processes such as oocyte developmental comp etence (Picton et al., 1998; McNatty et al., 1999) since the oocyte becomes sensitive to da mage throughout the various stages of follicular growth (Badinga et al., 1993). Indeed, follicular steroidogenesis, follicular dynamics and altered concentra tions of FSH and inhibin beco me altered in response to heat stress (Badinga et al., 1994; Wolfenson et al., 1997; Roth et al., 2000). During heat stress sperm can be damaged after insemina tion due to the generation of reactive oxygen species (Ishii et al., 2005) and embryonic de velopment can be co mpromised directly (Monty et al., 1987). Not surprisingly the heat stress problem is mu ltifactorial (Hansen et al., 2001). Heat stress of superovulated cows at day 1 after breedi ng reduced the proportion of embryos that were blastocysts at day 8 afte r breeding, but heat stress on day 3, 5 or 7

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8 after breeding did not affect subsequent embryonic development (Ealy et al., 1993). Superovulated heifers experien ced a high percentage of reta rded embryos recovered on day 7 after insemination after exposure to hi gh temperature and humidity at the onset of estrus for 10 h (Putney et al., 1989a). In another study heat stress was induced in Holstein heifers by submitting them from day 1 to day 7 after estrus to 42oC for 7 h (treatment) or 30oC for 16 h (control) and results obtained revealed more retarded embryos with degenerate blastomeres on the day of recovery (20.7% vs. 51.5%, respectively; Putney et al., 1988a). One cause for the observed reduction in re productive performance under heat stress conditions is steroidogenic capacity and its e ffects on oocyte function (Roth et al., 2001; Al-Katanani et al, 2002b; Roth and Hansen, 2004). Under heat st ress, low estradiol concentration in the follicular fluid of do minant follicles involves reduced aromatase activity in the granulosa cells (Badinga et al., 1993) and reduced androstenedione production by theca cells (Wolfenson et al ., 1997). Although earlier studies were inconsistent in demonstrating that plasma c oncentrations of estrad iol are reduced under heat stress (no change– Gwazdauskas et al., 1981; increase – Rosenberg et al., 1982; decrease – Gwazdauskas et al., 1981), recent work points toward heat stress resulting in lower estradiol concentrations in the follicular fluid (Badinga et al., 1993; Wolfenson et al., 1995; Roth, 1998; Wilson et al., 1998ab). Heat stress also has been reported to d ecrease (Rosenberg et al., 1982, Younas et al., 1993; Howell et al., 1994), in crease (Abilay et al., 1975; Roman-Ponce et al., 1981; Trout et al., 1998), or have no effect (Wise et al., 1988; Wolfenson et al., 1995) on peripheral concentrations of progesterone. Elev ated temperatures in culture can directly

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9 influence endometrium explants by increasing PGF2 secretion (Putney et al., 1988c; Malayer and Hansen, 1990) and from days 816 of pregnancy can reduce the size of the embryo at day 17 (Biggers et al., 1987). A retrospective survey involving 12,711 lact ations from high-yielding dairy herds in northeast Spain demonstrat ed that milk yield per cow increased from 1991-2000 (Lpez-Gatius, 2003; see Figure 2). For each 1000 kg increase in average milk yield in the warm period, there was a decrease of 6% in pregnancy rate, and 7.6% in cyclicity, and an increase of 8% in the incidence of inactive ovaries. During the cool period, however, there was no change in fertility over time. Thus, the continual increase in milk yield might have reduced fertility in Spain, at least, by exacerba ting effects of heat stress. Milk yield and diseases Increased incidence of certain diseases has been associated with elevated milk yield. High somatic cell score and clinical mastitis (Schukken et al., 1990; Barkema et al., 1998; Chassagne et al., 1998; Fleischer et al., 2001); lameness (Green et al., 2002); cystic ovarian disease (Fleis cher et al., 2001; Lpez-Ga tius et al., 2002); milk fever (Fleischer et al., 2001); and acu te metritis (Kelton et al., 1998) are all co rrelated with milk yield. Compared to non-mastitic herd-mates, high producing cows were at a greater risk of developing clinical mastitis (Gr hn et al., 2004). Number of days to conception, artificial inseminations per conception and num ber of days to first artificial insemination (AI) were significantly greater for cows with clinical mastitis (B arker et al., 1998), and may affect embryonic survival when occurr ing after insemination (Soto et al., 2003). According to Jousan et al., (2005) an elev ated somatic cell count score among lactating

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10 females influenced mid-to-late fetal loss (re presented as occurring after day 70 to 90 of gestation) and mastitis has been reported to affect pregnancy lo ss during the period of embryonic (Chebel et al., 2004) and fetal develo pment (Risco et al., 1999; Santos et al., 2004a). High yielding cows had an increased likelihood of becoming lame (Green et al., 2002) and cows that had been treated for la meness had a negative influence on pregnancy to first insemination and numbers of insemi nations per service period (Petersson et al., 2005). Similarly, non-lame cows were more likel y to conceive at first service than lame cows and lameness within the first 30 days after calving was associated with reduced pregnancy rates at first AI a nd a higher number of services per conception (Hernandez et al., 2001; Melendez et al., 2003). In a meta-a nalysis of several published papers, leg problems were associated with an average increase of 12 days to conception (Fourichon et al., 2000). Cows that develop cysts remain infertile as long as this conditi on persists and early spontaneous cyst recovery was negatively corr elated with milk yiel d (Lpez-Gatius et al., 2002). Similarly, elevated milk yield increase d the risk of cows developing cysts (LpezGatius et al., 2002) and days from metritis o ccurrence to first AI is also correlated to infertility (Loeffler et al., 1999) Milk yield in the current lact ation is also correlated with incidence of milk fever (Fleis cher et al., 2001) and this dis ease reduces fertility (Chebel et al., 2004). Milk yield, estrus de tection, and fertility Milk yield may affect fertility indirectly by reducing the ability to accurately detect estrus. An antagonistic rela tionship between increased milk production and days to first

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11 visual estrus has already b een reported. According to Lpez et al. (2004), duration, standing events, intensity (determined by th e number of standing events per hour), and standing time were reduced for high-producing cows as compared to low producers. Similarly, Harrison et al. (1990) reported that elevated milk yield was correlated to a longer period of estrus suppr ession. Westwood et al., (2002) indicated that high genetic merit for milk yield influenced significan tly the chance a cow showed weak signs of estrus as compared to lo w milk producing cows. Cows with elevated milk yield also had re duced circulating estr adiol concentrations on the day of estrus expression and shorter duration of estrus de spite having larger preovulatory follicle diameters (Lpez et al., 2004). Changes in Herd Size as a Factor in Reduced Fertility Increased milk yield is not the only change in dairy farming over the last 50 years and some of these other changes could also contribute to decreased fertility. One major change has been the trend towards large farms. In a review, Lucy et al. (2001) cited data from the USDA National Agricultural Statistics Services that nearly 30% of all dairy farms in the United States have more than 500 cows. In additi on, Stahl et al. (1999) reported that the expansion of dairy herds comes in large part through the purchase of first-lactation cows. Thus, as Lucy et al (2001) pointed out, these more infertile primiparous cows (Stahl et al., 1999) may ha ve represented an increasingly larger percentage of the herd as dairy herds have expanded ove r the last 10-40 years. The importance of changes in herd size as a cause for infertility have been questioned by de Vries and Risco (2005) who found no clear association with reproductive function. Nevertheless, as the herd size is increased one would expect th at the likelihood that it becomes harder for accurately detecting estrus becomes a challenge because factors

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12 associated with herd size such as the su rface (concrete floor) on which the cow stands will reduce the preponderance of cows displa ying estrus activity (Britt et al., 1986; OÂ’Connor and Senger, 1997). Inbreeding Inbreeding represents increased frequency of identical al leles at a gene locus and the inbreeding percent is a measure for the ge nes of an individual that are identical by descent (Wright, 1922; Falconer, 1981). It is generally considered that reproductive function declines when inbreed ing levels in a population ri se above 6.25% (Hansen et al., 2005). Increased degree of inbreeding as the result of use of AI could explain some of the declines in fertility experienced by dairy cattle because inbreeding coefficients have increased in all the major U.S. dairy breeds. Estimates of inbreedi ng in the U.S. dairy population are near 5% currently (Short et al., 1992; Wiggans et al., 1995; Young et al., 1996; Hansen, 2000; Wall et al., 2005) and increa sing at a constant ra te of about 0.1% per year for U.S. Holsteins (Hansen et al., 2005). At an average of 5%, it is likely that many dairy cows have inbreeding coefficients above 6.25% (Hansen et al., 2005). Thompson et al. (2000ab) found calving inte rvals to increase by 12 and 17 d for Jersey and Holsteins cows, respectively, with levels of inbreeding >10%. Similarly, inbreeding had pronounced negati ve effects on fertility at higher levels (10%) of inbreeding (Wall et al., 2005). In another study, animals with an inbreeding coefficient >9% had fewer transferable embryos following superovulation than animals with a lower inbreeding coefficient (Alvarez et al., 2005). Strategies to Improve Ferti lity in Lactating Dairy Cattle Four general approaches to improve repr oductive function in dairy cattle have been developed. The first is to regula te the timing of ovulation using gonadotropin

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13 releasing hormone (GnRH) and PGF2 utiliz ed in timed AI (TAI) programs. The advantage of this approach is that this program maximizes the number of animals inseminated and allows inseminations to be made at some pre-planned time to eliminate the need for estrus detection. Pioneering st udies (Thatcher et al ., 1989; Twagiramungu et al., 1992; Wolfenson et al., 1994) were able to synchronize estrus effectively, however, subsequent studies at the University of Fl orida (Schmitt et al., 1996a) and University of Wisconsin (Pursley et al., 1995) led to the development of the Ov synch TAI program and the demonstration that good pregnancy rate s can be achieved (Thatcher et al., 2001; Thatcher et al., 2002). Although th is approach is an effective one and is widely used in dairy herds, it involves regul ation of events occurring be fore conception and is beyond the scope of the present review The second approach is to use information regarding the hormonal basis for establishment of pregna ncy and signaling between the maternal and embryonic units during early pregnancy as th e basis for pharmacological treatments to improve embryonic survival. Failure of essential bioche mical dialogue between the conceptus and the maternal unit undoubtedly contributes to embryonic mortality and termination of pregnancy (Spencer et al ., 1996; Spencer and Bazer, 2002). The third approach has been to regulate the nutrition of the dairy cow to improve energy balance or to provide specific nutrients that favor esta blishment and maintena nce of pregnancy. Finally, recent work has focused on use of embryo transfer to bypass early embryonic death and perhaps coupled with crossbreeding may become an important alternative since Holsteins have become more inbred (Hansen et al., 2005). Treatment with Bovine Somatotrop in (bST) to Enhance Fertility Circulating concentrations of IGF-I, glucose, and c holesterol are reduced in lactating animals (de la Sota et al., 1 993; Beam and Butler 1997). Circulating

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14 concentrations of IGF-I is influenced by nut rition (Adam et al., 1997) and closely related to energy balance of the cow (Ginger et al ., 1997; Beam and Butler, 1998; 1999). Present in serum and in various tissues, IGF-I is produ ced mainly by the liver but other organs as well (Murphy et al., 1987; Thi ssen et al., 1994). IGF-I regula tes ovarian function in dairy cattle (Breukink et al., 1998; Chase et al ., 1998), is necessary for proper follicular development in which a fully competent ooc yte capable of inducing ovulation develops (Lucy et al., 1992a), and is required for nor mal CL formation and function (Leeuwenberg et al., 1996; Chase et al., 1998). Dairy cows that initiated estrous cyclicity during the postpartum period had higher plasma IGF-I than anestrous cows (Thatcher et al., 1996), cystic and inactive ovary or persistent CL cows (Zulu et al., 2002a). Bovine somatotropin (bST) increases plasma concentrations of insulin, IGF-I, and growth hormone (Bilby et al., 2004), perhaps by stimulating ovarian function especially after IGF-1 plasma levels are reduced in lact ating animals (de la Sota et al., 1993). In addition, injection of bST stimulates concep tus growth by day 17 of pregnancy (Bilby et al., 2004). Additional studies provided evidence that bST can improve pregnancy rates in lactating cows (Moreira et al ., 2000b; Morales-Roura et al., 2001; Santos et al., 2004b). Superovulated donor cows that received bST treatment experienced reduced number of unfertilized oocytes, increased number of embryos that developed to the blastocyst stage, and increased number of transferable embryos (Mor eira et al., 2002). Collectively, these studies indicate that critical thresholds of GH and IGF-I concentr ations are needed to stimulate reproductive performance (Bilby et al., 2004). Treatment with GnRH to Delay Luteolysis The estrous cycle is characterized by 2, 3, and sometimes 4 waves of follicular growth (Sirois and Fortune, 1988; Ginther et al., 1996). During the second half of the

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15 luteal phase, development of an estrogenic foll icle facilitates the luteolytic process via secretion of estradiol. Nonpregnant cows have higher pe ripheral concentrations of estradiol on days 16 and 18 after breeding comp ared to pregnant animals (Ahmad et al., 1997). Thatcher et al. (1991) examined the la rgest and second larges t follicles present on day 17 after estrus in pregnant and cyclic dairy cows. In the cyclic cows, the largest follicle had greater aromatase activity and c ontained more estradiol and less progesterone in the follicular fluid than the second larges t follicle. These relati onships were reversed in pregnant animals, which indicated an earlier recruitment of the third wave of follicular development in the pregnant animal associ ated with delayed luteolysis and higher pregnancy rates. That these follicles play an important role in luteolysis was shown by Villa-Godey et al. (1985), who re ported that electrocautery to destroy large follicles was associated with an extension of the estrous cycle. Estradiol is now known to be one of three hormones that contro l uterine secretion of PGF2 with progesterone and oxytocin also bei ng involved. Pulsatile release of PGF2 from the luminal epithelium of the endometr ium is stimulated via oxytocin (Roberts and McCracken, 1976; Silvia and Taylor, 1989; Mi lvae and Hansel, 1980). Progesterone and estradiol regulate this process because estr adiol induces formation of oxytocin receptors (Silvia and Taylor, 1989; Zi ngg et al., 1995; Robinson et al ., 2001) after progesterone exposure (Ginther, 1970; Garrett et al ., 1988b; Lafrance and Goff, 1988). While progesterone initially suppresses PGF2 secretion by blocking oxytocin receptors during the early and mid-luteal phase of the estrous cycle, the e ndometrium becomes responsive to oxytocin and progesterone receptors become down regulat ed as the estrous cycle progresses (Lafrance and Goff, 198 8; Spencer and Bazer, 1995).

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16 Delaying luteolysis might improve pregnanc y rate by allowing embryos more time to produce sufficient quantities of interferon(IFN). Eliminating or decreasing estradiol production from the dominant follicle during the critical period of early pregnancy could be one strate gy to improve pregnancy establ ishment (Thatcher et al., 2000; Binelli et al., 2001). One approach fo r doing this is to use GnRH to regulate follicular function. Gonadotropin releasing hormone is a decap eptide that plays a central role in regulating reproductive processe s. Release of GnRH from the hypothalamus occurs in a pulsatile fashion and can be regulated by va rious internal and external signals. Hypothalamic GnRH is synthesized in cell bodies of neurosecretory neurons, and is transported to and released from th e median eminence into the hypothalamichypophyseal portal system (Loucopoulos and Feri n, 1984). GnRH has its primary effects at the pituitary gonadotrope a nd stimulates the pulsatile release of the gonadotropins luteinizing hormone (LH) and follicle-stim ulating hormone (FSH) into the peripheral circulation (Chenault et al., 1990). Two potential gonadotropi n responsive tissues within the ovary are the CL and the follicle. LH re lease induces ovulation or luteinization of large ovarian follicles present at the time of treatment (Thatcher and Chenault, 1976). One strategy tested for in creasing pregnancy rate is to inject GnRH or GnRH analogues at day 11-14 after estrus to increa se progesterone secretion (Willard et al., 2003) and delay luteolysis (Macmillan and Thatcher, 1991), thereby increasing the chance for an embryo to initiate its own antiluteolytic mechanism. Injection of GnRH at this time can lead to decreased estrogen secretion (Rettmer et al., 1992a; Mann and

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17 Lamming, 1995a) in an action that likely involves luteinizat ion of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ryan et al., 1994). Improvement of fertility has been seen by administration of GnRH or its analogues at day 11-14 in nulliparous beef heifers (R ettmer et al., 1992b) and lactating dairy cows (Macmillan et al., 1986; Lajili et al., 1991; Sh eldon and Dobson, 1993; Drew and Peters, 1994; Willard et al., 2003; Lpez-Gatius et al., 2005a). In contrast to these positive results, there was no favorable effect of simila r treatments of GnRH or GnRH analogues on pregnancy rates in other studies (Jubb et al ., 1990; Stevenson et al ., 1993; Ryan et al., 1994; Bartolome et al., 2005). In a meta-analysi s of published results, Peters et al. (2000) concluded that the overall effect of GnRH administration between day 11 and 14 after anticipated ovulation was positive but that resu lts were not consistent between studies. Increase in the Size of the Preovulatory Follicle to Generate a Larger Corpus Luteum As mentioned earlier, high-yielding dair y cows are more likely to have lower circulating concentrations of progesterone th roughout the estrous cycle than cows with lower milk yields because of increased rate of progesterone catabolism (Lucy et al., 1998; Vasconcelos et al., 1999). Given the impor tance of progesterone concentration for embryonic survival (Man and Lamming, 2001), efforts have been made to increase progesterone secretion in cows. One possible e ffect of mid-cycle trea tment with GnRH is to increase progesterone secretion (Schmitt et al., 1996b; Willard et al., 2003). Another approach for increasing progester one concentrations has been to regulate the size of the preovulatory follicle to affect subsequent CL function. Optimum differentiation and growth rate of the CL varies according to the duration and amplitude of the ovulatory LH surge such that inhibition of LH release preceding the

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18 preovulatory surge of LH result ed in development of a smaller CL in diameter (QuintalFranco et al., 1999). Induced ovulation of sm all follicles resulted in a smaller CL and reduced secretion of progesterone than when a larger follicle ovulat ed (Vasconcelos et al., 2001). In another study (Perry et al, 2005), regression analys is indicated that pregnancy rate for cows with induced ovulat ion with an ovulating follicle of 14.5 mm was higher than for cows ovulating follicles <10.3 mm in diameter. It was further revealed that 39% of cows that lost their pregnancy had ovulatory follicles < 11 mm in diameter. Among cows that ovulated spontan eously, however, pregnancy rates at day 27 and 68 were independent of ovulatory follicle size (Perry et al., 2005). In contrast to this result, Vasconcelos et al (1999) found that the group of cows ovulating larger follicles had lower pregnancy rates on day 28 and 98 af ter AI and higher pregnancy loss between these times. Administration of GnRH just prior to or at the time of the LH surge causes an amplified preovulatory surge of LH (Lucy a nd Stevenson, 1986; Yoshioka et al., 2001). Injection of GnRH at or near the time of estrus increased the proportion of large luteal cells in the CL on day 10 of the estrous cycl e (Mee et al., 1993), pe ripheral progesterone concentrations during the first 7 days of the estrous cycle (Lucy and Stevenson, 1986), and increased pregnancy rates in repeat breed ing cows (Stevenson et al., 1990; Mee et al., 1993). Ullah et al. (1996) observed th at GnRH treatment at estrus in dairy cows improved pregnancy rates and increased peripheral progesterone con centration. Conversely, GnRH administered to lactating dairy cows at th e time of AI did not a ffect pregnancy rates (Ryan et al., 1994). Similarly, Mee et al. (1990) concluded that GnRH treatment at 1 h or

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19 12 to 16 h after first detected es trus did not improve pregnancy rates at first service. Mee et al. (1990) mentioned that 16 studies in th e literature suggest an overall advantage in pregnancy rate of 6 percentage points ( 53 vs. 59%) or an 11% improvement for cows receiving GnRH treatment at the time of AI or up to 6 h preceding AI. Induction of an Accessory Corpus Luteum Progesterone concentrations following ovulatio n have been positively correlated to volume of uterine secretions (Garrett et al., 1988a) conceptus development (Garrett et al., 1988a; Mann et al., 1996), the em bryos ability to secrete IFN(Kerbler et al., 1997; Mann et al., 1998), embryo viability for subse quent survival (Stronge et al., 2005), and perhaps most importantly conception rates (H ansel, 1981; Fonseca et al., 1983; Shilton et al., 1990; Larson et al., 1997). One possibl e approach to incr easing progesterone secretion has been to induce formation of an accessory CL by administering GnRH or hCG, LH or their analogues at a time when the first wave dominant follicle is present after ovulation (metestrus) (Rajamahendran and Sianangama, 1992; Schmitt et al., 1996b; Santos et al., 2001). Santos et al. (2001) reported that hCG treatment on d 5 of a synchronized estrous cycle induced an accessory CL in 86.2% of treated cows, increased plasma progesterone by 5 ng/ml, and increase d conception rates on day 28 from 38.7% to 45.8% and on day 90 of pregnancy from 31.9% to 38.4%. Lactating dairy cows treated with GnRH on d 5 (Willard et al., 2003) and hCG on day 7 (Rajamahendran and Sianangama, 1992) or day 4 in heifers (Breue l et al., 1989) reported successful accessory CL formation and an increase in co nception rates and pregnancy rate. Besides stimulating luteal tissue formati on, treatment of cows to induce ovulation of the first wave dominant follicle with GnRH or GnRH analogues also reprograms follicular growth to increase the proportion of estrous cycles composed of three follicular

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20 waves as compared to two waves (Diaz et al ., 1998). Such an effect could reduce the probability that a large, highly estrogenic foll icle is present during the critical period of pregnancy recognition. Compared to animal s with two-wave cycles, Holstein cows (Townson et al., 2002) and beef cows (Ahmad et al., 1997) with a three-wave cycle had higher conception rates and a longer lu teal phase (Ginther et al., 1989). Progesterone Supplementation The ability of the conceptus to secrete IFNis related to its developmental progress and progesterone concen tration of the pregnant fema le (Mann et al., 1999). Low progesterone concentration in plasma as ea rly as day 6 after insemination has been implicated as a contributing factor for cows failing to conceive (Bulman and Lamming, 1978; Lukaszewska and Hansel, 1980; Kimu ra et al., 1987; Lamming and Darwash, 1995; Inskeep, 1995; Mann and Lamming, 1999; Hommeida et al., 2004). Enhanced luteolytic signals also result from subop timal progesterone concentrations after insemination (Mann and Lamming, 1995b). Anothe r approach to increase fertility of lactating dairy cows has been to directly supplement cows with progesterone. A metaanalysis of 17 studies rev ealed that progesterone suppl ementation after insemination produced an overall improvement in concep tion rate of 5% and that the timing of progesterone supplementation was a critic al factor (Mann an d Lamming, 1999). One study revealed depressed conception rates when controlled in ternal drug releasing (CIDR) devices containing progesterone were inserted in heifers on day 1 or day 2 following estrus (Van Cleef et al., 1989). In contrast, injection of progesterone (100 mg) on day 1, 2, 3, and 4 of pregnancy advanced de velopment of conceptuses to 14 days of gestation in beef cows (Garre tt et al., 1988a). These conc eptuses had incr eased length and secreted a greater array of proteins in to medium following a 24 hour culture. When

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21 progesterone supplementation was initiated be ginning at day 10 of pregnancy, Macmillan et al. (1991) found a slight decrease in pregnancy rate (-2.7%), Sreenan and Diskin, (1983) obtained a small increase (4.3%), a nd Robinson et al. (1989) obtained a large increase (29.3%) in pregnancy rate. Villarro el et al. (2004) found that first and second lactation repeat-breeder Hols tein cows were 3.26 times more likely to become pregnant when cows received progesterone releasing intravaginal device (PRID, 1.55g of progesterone) on day 5 through 19 post-AI. Inhibition of Luteolysis The maintenance of a functional CL de pends directly upon the intensity of embryonic signals that attenuates endometrial secretion of PGF2 Pregnancy fails if an embryo does not produce sufficient amounts of IFNor if production is delayed until after the critical time-period between days 14 and 17 when the luteolysis would otherwise occur. Intrauterine infusions of recombinant bovine IFNfrom days 14 to 24 of the estrous cycle increased lifespan of the CL a nd duration of the estrous cycle (Meyer et al., 1995). Further studies with a la rge number of cows needs to test whether this treatment increases pregnancy rates. Co-transfer of embryonic vesicles to increase trophoblastic signals has been reported to increase pregnancy rates in embryo transfer recipients (Heyman et al., 1987). Administration of IFNby intramuscular injection, which can also block luteolysis, decreased pregnancy ra tes in heifers (Barros et al., 1992) because IFNhas several adverse actions such as cau sing hyperthermia (Newton et al., 1990). Administration of a prostanoid synthesis inhibitor could suppress the luteolytic stimulus in early pregnancy. Injection of flunixin meglumine (a pr ostaglandin synthesis inhibitor) neutralized oxytocin-induced PGF2 release, reduced the frequency of short

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22 cycles, and increased pregnancy rate from 33.3% in oxytocin challenged cows to 80% in oxytocin treated cows that received a flunixin meglumine injection (Lemaster et al., 1999). In another study, effects of flunixin meglumine on pregnancy rate were farm or location dependent (Purcell et al., 2005). T ogether, these results suggest that certain conceptuses are unable to inhibit uterine PGF2 secretion and that reducing prostaglandin synthesis and stimulating IFNsecretion could improve pregnancy rates. Nutritional Strategies Dairy cows reach peak production on averag e within the first 4 to 6 weeks after parturition. Unfortunately, feed and energy intake do not reach ma ximum levels until approximately 10 – 12 weeks postpartum. Th e end result is a lactating cow with insufficient nutritional requirements that enters a NEB status. As mentioned before, energy balance is defined as the difference between energy gain from feed intake minus the energy e xpenditure associated with maintenance of physiological function, growth, and milk pr oduction (Staples et al., 1990). Several studies have reported that negative ener gy status impaired repr oductive performance (Butler and Smith, 1989; Jorritsma et al., 2000 ). Different nutritional strategies to improve energy balance or alter nutrient de livery to improve re productive function are described in this section. Fat feeding to improve energy balance Fats are glyceride esters of fatty acids that can have a direct effect on the transcription of genes that encode proteins that are essential to reproductive events (Mattos et al. 2000). Dietary fats typically increase concentrations of circulating cholesterol, the precursor of progesterone (Grummer and Ca rroll, 1991). Ruminants fed

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23 supplemental fat often have a slight increas e in blood progesterone concentrations [see Staples et al. (1998) for review]. Hawkins et al. (1995) suggested th at the increase seen in circulating progesterone when cows are fe d supplemental fat was from a reduced rate of clearance of progesterone ra ther than an increase in progesterone synthesis. Fat supplementation has also been shown to stim ulate programmed growth of a preovulatory follicle (Lucy et al., 1993), total number of follicles (Lucy et al., 1991ab; Wehrman et al., 1991; Thomas and Williams, 1996; Beam a nd Butler, 1997; Lammoglia, 1997), and size of preovulatory follicles (Lucy et al., 1990, 1991a, 1993; Beam and Butler, 1997; Oldick et al., 1997). Garcia-Bojalil et al. (1998) reported that accumulated plasma progesterone from 0 to 50 days in milk (DIM) wa s greater, pregnancy rates im proved, and energy status did not change when cows were fed diets of 2.2% calcium salts of fatty acids (CSFA) compared to non fat-supplemented cows. Sim ilarly, Scott et al. (1995) fed CSFA at 0 or 450 g/d from 1 to 180 or 200 DIM and reporte d a tendency for CSFA to increase the proportion of cows exhibiting standing estr us (71.4% vs. 65.6) and a reduction in the proportion of cows with inactive ovaries. Other studies have also found a beneficial effect of feeding supplemental fats on fertility of lactating cows (E rickson et al., 1992; Sklan et al., 1994) while some studies have found no beneficial effect. Although fer tility results are inconsistent when cows were evaluated after being fed supplemental fat, Staples et al. (1998) suggested that positive effects (17 percentage unit improvement) are more often reported. When first AI service and conception or pregnancy rate data was examined, ten studies (Schneider et al., 1988; Bruckental et al., 1989; Sklan et al., 1989; Armstrong et al., 1990; Ferguson et

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24 al., 1990; Sklan et al., 1991; Ga rcia-Bojalil, 1993; Scott et al., 1995; Burke et al., 1996; Son et al., 1996) report an improvement (P < 0.10) while two studies (Erickson et al., 1992; Sklan et al., 1994) revealed a strong ne gative influence accompanied by a large increase in milk production. Among studies that reported an improvement (Armstrong et al., 1990; Ferguson et al., 1990; Sklan et al., 1991), a reduced number of services per conception by feeding a fat supplemen ted diet occurred as well. Dietary fats could favor reproductive processes through actions related to energy balance or through specific actions of indivi dual fatty acids on tissue function. Mattos et al (2000) has suggested that altered uterine and ovarian function can be mediated through specific fatty acid precursors in the diet to allow increased steroid and/or eicosanoid secretion. There are many examples of effect s of feeding diets high in specific fatty acids. Linoleic acid supplemented in the diet prepartum can stimulate arachidonic acid synthesis and lead to higher concentrations of the series 2 prostagla ndins (Thatcher et al., 1994). It is speculated that linolenic acid may compete with arachidonic acid for binding sites of a key enzyme, cyclooxygenase 2 (PGHS-2 ), which is necessary for the synthesis of PGF2 (Mattos et al., 2000; 2004). Supplementation of the diet with fish m eal has been reported to reduce uterine PGF2 secretion of lactating dairy cows (Thatc her et al., 1997). Fish meal contains relatively high concentrations of two polyunsat urated fatty acids of the n-3 family, EPA (eicosapentaenoic acid) and DHA (docosahexaen oic acid). Concentrations of EPA and DHA in fish oil have been reported to be 10.8 and 11.1% of total fatty acids (Donovan et al., 2000). EPA and DHA can inhibit secretion of PGF2 in different cell culture systems (Levine and Worth, 1984; Achard et al., 1997) including bovine endometrial cells

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25 (Mattos et al., 2001). Using fish meal to re place soybean meal as a source of protein, Bruckental et al. (1989) a nd Armstrong et al. (1990) re ported higher pregnancy and conception rates. These results suggest that high concentrations of EPA and DHA in the diet can reduce PGF2 endometrial secretion and aid in es tablishment of pregnancy rates. Administration of antioxidants Reactive oxygen species are a possible s ource of infertility because ovarian steroidogenic tissue (Carlson et al., 1993; Margolin et al., 199 2), spermatozoa (Rivlin et al., 2004), and preimplantation embryos (Fujit ani et al., 1997) become compromised as a consequence of free radical damage. Vitamin E (i.e., -tocopherol) and -carotene are major antioxidants present in plasma membranes of cells (Wang and Quinn, 1999; 2000). Treatment of cows with vitamin E and selenium can increase the rate of uterine involution in cows with metritis (Harrison et al., 1986) and improve fe rtilization rates in ewes (Segerson and Ganapathy, 1980) and co ws (Segerson et al., 1977). In general, however, treatment of lactating cows with vitamin E alone, through feeding or injection, had little or no benefits on postpartum cows (Kappel et al., 1984; Stowe et al., 1988; Archiga et al., 1998a; Paul a-Lopes et al., 2003). -carotene is another cellular antioxidant and is thought to be present at the interior of membranes or lipoproteins (Niki et al ., 1995). Cows fed diets deficient in -carotene had lower amounts of progesterone in the CL (Ahlswede and Lotthammer, 1978). In spite of this, its effect on fertility is cont roversial. Some author s report benefits of feeding supplemental -carotene (Ahlswede and Lotthammer, 1978; Rakes et al., 1985; Archiga et al., 1998b) whereas others do not (Wang et al., 1982; Akordor et al., 1986). There was no strong relationship be tween serum concentrations of -carotene and fertility

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26 in dairy cattle (Gossen et al., 2004; Gossen a nd Hoedemaker, 2005). Injection of vitamin A, a metabolite of -carotene, resulted in an increase in the number of recovered blastocysts from superovulated cows (Shaw et al., 1995). Crossbreeding Two bulls (Chief and Elevation) make up about 30% of the gene pool of U.S. Holsteins (Hansen et al., 2005). As mentione d previously, inbreedi ng coefficients are rising in American dairy cat tle (Short et al., 1992; Wigga ns et al., 1995; Young et al., 1996; Hansen, 2000; Wall et al., 2005) and th ere is some evidence that this has contributed to the decline in fertility seen in dairy cat tle (Thompson et al., 2000ab; Alvarez et al., 2005; Wall et al., 2005). Crossbreeding represents a strategy for preventing effects of inbreeding especially if the milk yield of crossbreds can approach that of Holstein cattle. A study in Canada revealed that some groups of crossbred cattle were equivalent to Holstein controls in lifetime net profit (McA llister et al., 1994). Hansen et al. (2005) conducted a study using seven large dairies in California to compar e characteristics of several crossbred animals (Normande-H olstein, Montebeliarde-Holstein, and Scandinavian Red-Holstein) versus Holsteins. Milk production as we ll as fat and protein production during the first 150 DIM among firs t lactation cows was not significantly different among breed types. Holsteins produced an average of 29.9 kg, followed by Scandinavian Red-Holstein with 29.7 kg, Montebeliarde-Holstein with 28.8 kg, and Normande-Holstein with 26.5 kg. Calving diffi culty and stillbirths were reduced in crossbred animals. Survival rates indicate that purebred animals left these dairies sooner. The first service conception rate was 22% for Holsteins compared to 30 35% for crossbreds. There were also significantly fewer days open for crossbred cows. Thus,

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27 crossbreeding offers some promise for enha ncing fertility. One unanswered question is the optimal type of mating scheme for the cr ossbred animals themselves and whether the resultant loss of heterosis in the F2 animals will reduce any advantage over purebred cows. Embryo Transfer The concept of using embryo transfer (ET) as a tool to increase pregnancy rates is based on the observation that disruptive even ts such as anovulation, ovulation of oocytes with low developmental competence, compromised oviductal transport or uterine environment, and insemination errors or dama ged spermatozoa all occur before the time when embryos are ordinarily transferred (d ay 6 8 after estrus) (Hansen and Block, 2004). Selection of morula and blastocyst stag e embryos for transfer offers the chance to avoid pregnancy failure associated with the early stages of embryonic development (day 0 8 after estrus). It has been proposed that during absence of heat stress, pregnancy rates following embryo transfer as compared to AI in lact ating cows are not optimal (Putney et al., 1989b; Drost et al., 1994; Ambrose et al., 1997 ). However, ET may become a more effective strategy to increase pr egnancy rates as compared to AI in lactating cows during periods of heat stress, and the magnitude of the increased temperature does not seem to influence overall success following transfer (Hansen and Archiga, 1999). As embryos advance in their development, the effect s of elevated temperatures become less significant because embryos become more resist ant to the deleterious effects of elevated temperatures (Ealy et al., 1992; Ealy and Hansen, 1994; Ea ly et al., 1995; Edwards and Hansen, 1997; Rivera and Hans en, 2001). As a result, pregnancy rates following ET

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28 during heat stress are higher th an pregnancy rates to AI (Put ney et al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a) although no t in the absence of heat stress. One potential constraint for embryo transfer in lactating cows is the short duration of estrus and lack of intense mounting activ ity seen in dairy cows (Dransfield et al., 1998). This phenomenon is exacerbated by heat stress (Nebel et al., 1997) and will limit the number of embryos transferre d in lactating cows in a pr ogram that is dependent upon estrus detection. The first report of a timed embryo transfer (TET) protocol, where ovulation was synchronized using an Ovsync h protocol, was by Ambrose et al. (1999) who evaluated the efficiency of TET using either fresh or frozen-thawed in vitro produced (IVP) embryos and TAI under heat st ress conditions. Pregnancy rates in cows that received a fresh IVP embryo were high er compared to cows in the TAI group. Limitations to Optimal Pregnancy Rates Using IVP TET For ET to replace AI on a wide scale in commercial herds ET must become an economical breeding alternative and embryos must be inexpensive to produce (Hansen and Block et al., 2004). Supe rovulation provides the best source of embryos while the most likely inexpensive source of embryos will be produced from slaughterhouse oocytes by IVP since superovulation is costly and requires intens ive management and careful synchronization of the donor cows. Although embryos produced using IVP sy stems are relativel y inexpensive as compared to embryos produced by superovula tion, pregnancy rates achieved following transfer of an IVP embryo are often less than what is obtain ed following transfer of an embryo produced by superovulation. For example, Hasler (2003) reported a 36.7% pregnancy rate for in vitro derived embryos vs. 54.8% for in vivo embryos. The reason for the poor survival of IVP embryos is not known. However, IVP embryos are different

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29 from in vivo embryo in terms of morphology (Massip et al., 1995; Cr osier et al., 2001; Rizos et al., 2002), gene e xpression (Bertolini et al ., 2002a; Lazzari et al., 2002; Lonergan et al., 2003), metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b) and chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). One or more of these alterations likely contribu tes to the poor embryo survival after transfer. Calves born as the result of in vitro production are also more likely to experience developmental defects (Hasler et al., 2003; Farin et al., 2006). One possible strategy for increas ing pregnancy rates is to transfer two embryos into the uterine horn ipsilateral to the CL. Th is approach is based on the idea that the likelihood is increased that th e cow receives at least one em bryo competent for sustained development. In addition, the transfer of tw o embryos into the ipsilateral uterine horn to the CL is likely to increase the amounts of IFNand other embryo-derived signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of embryonic vesicles to incr ease trophoblastic signals has been reported to increase pregnancy rates in ET recipi ents (Heyman et al., 1987). In a recent study, there was a tendency for higher calving rates fo r recipients that received two embryos in the uterine horn ipsila teral to the CL as compared to recipients that received one embryo (Ber tolini et al., 2002a). The requir ement for the antiluteolytic signal in cattle to be locally administered (del Campo et al., 1977, 1983) means that one should expect pregnancy rates to be higher in cows that receive d two embryos in the same uterine horn (unilateral transfer) th an for cows that received two embryos distributed in both uterine hor ns (bilateral transfer). Th e opposite was true for heifers (Anderson et al., 1979). In other studies, transfer of embryos to create two pregnancies in

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30 the uterine horn ipsilateral to the CL has pr oduced a similar pregnancy rate as bilateral twins and single pregnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to bilateral transfer (Rowson et al., 1971). Cryopreservation of IVP Embryos An additional limitation to the widespread use of IVP embryos in cattle is their poor survival following cryopreservation. Hasler et al. (1995), Ambros e et al., (1999) and Al-Katanani et al. (2002a) indi cated that IVP embryos do not survive freezing as well as embryos produced in vivo based on pregnancy rates following transfer as compared to non-frozen embryos. In vitro survival rate s following thawing (Po llard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000 a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a) are consistently lower for IVP embryos as compared to embryos produced in vivo by superovulation. Among the metabolic changes associated with IVP embryos linked to poor freezability is an increase in lipid conten t (Abe et al., 1999; Rizos et al., 2002). Mechanical delipidation (Tominaga et al ., 2000; Diez et al., 2001) and addition of inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can improve embryo survival following cryopreserv ation. Hatching rates were hi gher for delipidated embryos compared to controls when day 7 blastocy sts were frozen (Murakami et al., 1998), but pregnancy rates after the tran sfer of delipidated embryos was 10.5% compared to 22% for control embryos (Diez et al., 2001). Although delipidated em bryos can survive freezing conditions when tested in vitr o, special consideration must be taken since these embryos do not reflect higher pregnanc ies and remain less viable than control embryos.

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31 Manipulating the cryopreservation process to minimize damage to the embryo has also been considered. Of most promise ar e procedures based on vitrification, which is defined as “the solidification of a solu tion (glass formation) brought about not by crystallization but by extreme elevation in vi scosity during cooling” (Fahy et al., 1984). Vitrification depends on ra pid cooling and thawing of embryos while using high concentrations of cryoprotectants associ ated with elevated cooling rates (~2500oC/min, Palasz and Mapletoft, 1996). Although vitrif ication does not elimin ate toxic effects of cryoprotectants and osmotic damage, the rapi d cooling has been reported to decrease chilling injury and prevent damage associat ed with high lipid content (Dobrinsky, 1996; Martino et al., 1996ab). In vi tro survival rates following th e thawing of vitrified IVP embryos was either equal (Van-Wagtendonk et al., 1995) or superior to embryos frozen conventionally (Dinnys et al., 1995; Agca et al., 1998; O’Kearney-Fl ynn et al., 1998). Sensitivity of in vivo derived embryos to cryopreservation is much less and the complex environment where the embryo develops is key. It has been reported that embryos cultured in the sheep oviduct (26%) compared to synthetic oviductal fluid in culture systems (7%) were better able to to lerate freezing conditions. Embryos cultured in Buffalo rat liver cells or oviductal cells were more resistant to freezing as well as compared to embryos not subjected to co -culture (Massip et al., 1993; Leibo and Loskutoff, 1993; Te rvit et al., 1994). Summary and Objectives of the Thesis There has been a precipitous decline in fe rtility of dairy cows over the last 10-40 years and heat stress is associated with infert ility in lactating dairy cows. To characterize events associated with infertility is importa nt and the purpose of th e present series of experiments described in this thesis was to evaluate strategies that help overcome

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32 reproductive failure. Improving reproductive functi on in dairy cattle is of major interest and experiments were designed to 1) evaluate strategies for enhancing fertility after AI using GnRH treatment and 2) further deve lop ET using IVP embryos as a tool for increasing fertility by testing whether pregna ncy rate could be im proved by transfer of twin embryos and whether the developmental competence of embryos after cryopreservation could be improved. Figure 1-1. Rolling herd average (RHA, kg milk per lactation) calving interval (CI), and services per conception (SPC) for 143 da iry herds continuously enrolled in the Raleigh DHIA record system from 1970 to 1999 (Lucy, 2001).

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33 Figure 1-2. Temporal changes in first servi ce pregnancy rate and annual average milk production from high-producing Holstein-Fri esian dairy herds in north-eastern Spain. Data for pregnancy rate were r ecorded in the cool (October April months) and warm season (May-September months). Data were drawn by P.J. Hansen (unpublished) based on da ta of Lopez Gatius (2003). Milk yield (kg) 7500 8000 8500 9000 9500 10000 10500 1 9 9 1 1 9 9 2 1 9 9 3 1 9 9 4 1 9 9 5 1 9 9 6 1 9 9 7 1 9 9 8 1 9 9 9 2 0 0 0 First service pregnancy rate, (%) 15 20 25 30 35 40 45 50 Warm seaso n Milk y iel d Cool season Warm Season Milk Yield

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34 CHAPTER 2 EFFECTIVENESS OF ADMINISTRATI ON OF GONADOTRO PIN RELEASING HORMONE AT DAY 11, 14 OR 15 AF TER ANTICIPATED OVULATION FOR INCREASING FERTILITY OF LACT ATING DAIRY CO WS AND NONLACTATING HEIFERS Introduction One of the approaches proposed to improve fertility in cattle is administration of GnRH or GnRH analogues at day 11-15 after estr us. Injection of GnRH at this time can lead to decreased estrogen secretion (R ettmer et al., 1992a; Mann and Lamming, 1995a) in an action that likely involves luteinizati on of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992a; Ry an et al., 1994). In some cases, extended estrous cycle length (Lynch et al., 1999) and in creased progesterone secreti on also results (Rettmer et al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). Improvement of fertility has been seen by administrati on of GnRH or its analogues at day 11-14 in nulliparous beef heifers (Rettmer et al., 1992b) and lactating dairy cows (Macmillan et al., 1986; Lajili et al., 1991; Sheldon et al., 1993; Drew and Peters, 1994; Willard et al., 2003; Lpez-Gatius et al., 2005a). In contrast to these positive results, there was no favorable effect of similar treatments of Gn RH or GnRH analogues on pregnancy rates in other studies (Jubb et al., 1990; Stevenson et al., 1993; Ryan et al., 1994; Bartolome et al., 2005). In a meta-analysis of published result s, Peters et al. (2000) concluded that the overall effect of GnRH administration betw eendDay 11 and 14 after anticipated ovulation was positive, but that results were not consistent between studies.

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35 It is possible that GnRH treatment is mo re effective at increasing pregnancy rate per insemination during periods of heat stress than in cool weather because circulating concentrations of progesterone can be re duced in cows subjected to heat stress (Wolfenson et al., 2000). In addition, the anti-luteolytic process may be compromised because heat stress can decrease growth of the filamentous stage conceptus (Biggers et al., 1987) and increase uterine prostaglandin-F2 secretion from the uterus (Wolfenson et al., 1993). Beneficial effect s of GnRH treatment at da y 11-12 after insemination on fertility have been observed in lactating dairy cows during heat stress (Willard et al., 2003; Lpez-Gatius et al., 2005a). The purpose of the present series of experiments was to evaluate the effectiveness of GnRH treatment at either day 11, 14 or 15 after anticipated ovulation for improving fertility of lactating cows and heifers and determine whether the beneficial effect of GnRH was greater during summer than winter. Materials and Methods Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artificial Insemination during Heat Stress The experiment was conducted at a commer cial dairy located in Trenton, Florida (29o37Â’ N 82o49Â’ W) from July to September, 2003 using 149 Holstein heifers. The heifers ranged in age from 13-23 mo (mean =539 d, SD=76) and ranged in weight from 316 to 448 kg (mean=360 kg, SD=32). Heifers we re maintained on grass pasture with supplemental grass hay. Heifers were randomly allocated to one of f our treatments in a 2 x 2 factorial design with main effects of timing of insemi nation (protocol A vs B) and treatment (vehicle vs GnRH). The experime nt was replicated twi ce with between 70 and 79 heifers per replicate. Heifers were subjected to timed artificial insemination (TAI) based on a protocol published previously (Mar tinez et al., 2002ab). On Day -10 of the

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36 protocol (Day 0 equals the day of anticipated ovulation), heifer s received 100 g (i.m.) of GnRH (Fertagyl, equivalent to 50 g /ml gonadorelin diaecetate tetrahydrate; (Intervet Inc. Millsboro, DE) and an unused intravag inal progesterone-rel easing device insert (EAZI-BREED CIDR insert, 1.38 g of progesterone, Pf izer Animal Health, New York, NY, USA). At Day -3, CIDR devices were re moved and 25 mg (i.m.) of prostaglandin F2 (PGF2 ; 5 ml Lutalyse, Pfizer Animal Health, New York, NY, USA) was administered. A second 100 g GnRH injec tion was given 48 h afte r CIDR withdrawal (Day -1). Regardless of estrus behavior, heifers in protocol A were inseminated 24 h after the second GnRH injection (d 0) and heif ers in protocol B were inseminated at the same time as the second GnRH injecti on (d -1). Two individuals conducted all inseminations and semen from one sire was used for all heifers. Heifers from each synchronization treatment protocol were random ly allocated to receive either 100 g of GnRH, (i.m.) or an equivalent volume (2 ml ) of vehicle (9 mg/ml of benzyl alcohol and 7.47 mg/ml of sodium chloride in water) at Day 11 after anticipated ovulation. On the day of insemination and on Day 11 after anticipated ovulation, a 10-ml blood sample was collected via coccygeal or jugular venipuncture into heparinized tubes (Becton Dickinson, Franklin Lakes, NJ) to measure the proportion of heifers successfully synchronized. An animal was considered s ynchronized if progester one concentrations were lower than 1 ng/ml on the day of insemination and greater than 1 ng/ml on Day 11 after anticipated ovulation. A third blood sample was collected in a subset of 76 heifers at Day 15 after anticipated ovulation (i.e., 4 d af ter the injection of Gn RH or vehicle) to determine the effect of GnRH treatment on serum concentrations of progesterone. Pregnancy was diagnosed by palpation per rectum at Day 44-51 after insemination.

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37 Blood samples were stored on ice (~2-4 h) until centrifugatio n at 2,000 x g for 20 min at 4 oC to obtain plasma. Plas ma was stored at -20 oC until assayed for progesterone concentrations using a progesterone radioi mmunoassay kit (Coat-a-Count; Diagnostic Products Corp., Los Angeles, CA). The sens itivity of the assay was 0.1 ng/ml and the intrassay and interassay CV were each 6%. Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination This study took place at the University of Florida Dairy Research Unit (Hague, Florida; 29o46’ N 82o25’ W). A total of 244 primiparous and multiparous lactating Holstein cows housed in freestall barns equi pped with a fan–and-sprinkler system were used. Cows were fed a total mixed ration (TMR) to meet or exceed requirements recommended for lactating dairy cows, were milked three times a day, and received bovine somatotropin (Posilac, Monsanto Corp., St. Louis, MO) according to manufacturer’s recommendation. Cows were subjected to the OvSynch TAI program (Schmitt et al., 1996a; Pursley et al., 1998); 100 g (i.m.) GnRH (Fertagyl equivalent to 50 g /ml gonadorelin diaecetate tetrahydrate, Intervet, Millsboro, DE) was injected at Day 0 of the protocol, 25 mg (i.m.) PGF2 (5 ml of Lutalyse, Pfizer Animal Health, New York, NY, USA) was given at Day 7, 100 g (i .m.), GnRH was again injected, i.m., at Day 9, and cows were inseminated 16 h later (t he day of anticipated ovulation). At the time of insemination (from January September, 2004), 244 cows were between 76 and 594 days in milk (DIM; mean= 176, SD= 114). Multiple individuals conducted inseminations (n=7) and multiple AI sires were used (n=45). Cows were randomly assigned within pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

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38 chloride in water) at Day 11 after anticipat ed ovulation (i.e., 11 d after insemination). Rectal temperature was record ed in a subset of cows (n =134) on the afternoon of Day 11 after TAI at 1500 – 1600 h. Pregnancy was diag nosed by rectal palpation at ~Day 46 after insemination. Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination This study was conducted at two different locations using lactating Holsteins. Farm 1 was the University of Florida Dairy Research Unit at Hague, Florida while farm 2 was a commercial dairy in Chiefland, Florida (29o30’ N 82o52’ W). Cows from farm 1 (n=307) were inseminated from February November 2004 and cows in farm 2 (n=170) were inseminated from June October 2004. At both farms, primiparous and multiparous cows were used. At farm 1, 307 cows were TAI between 76 – 590 DIM (mean= 187, SD= 102). Multiple individuals conducted in seminations (n=7) and multiple AI sires were used (n=42). At farm 2, 170 cows were used for first service after calving using seven different sires and one inseminator. The TAI protocol was designed to achieve insemination at 60 + 3 d in milk. Cows in both farms were housed in freestall barns equipped with fans and sprinklers, were fed a TMR, were milked three times a day, and received Posilac (Monstanto, St. Louis, MO) according to manufacturer’s directions. Cows in farm 1 were subjected to an OvSynch protocol as described for Experiment 2. Cows for farm 2 were subjecte d to a TAI protocol that incorporated a presynchronization with PGF2 (Moreira et al., 2001) a nd the CIDR-Synch ovulation synchronization protocol (Portaluppi a nd Stevenson, 2005). Cows received two injections of 25 mg PGF2 (i.m.) (Lutalyse) 14 d ap art starting on Day 21-27 DIM. Twelve days after the second PGF2 injection, a timed ovulation synchronization protocol

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39 was initiated. Cows received 100 g (i.m.) GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ, USA) and an unused EAZI-BREED CIDR intravaginal progesteronereleasing device insert. Seven days later, CIDR devices were removed and 25 mg (i.m.) PGF2 was given. Cows received a second 100 g (i.m.) injection of GnRH at 72 h after CIDR withdrawal. Estrus was detected us ing tail chalk or KaMar estrus detection patches (KAMAR Inc., Steamboat Springs, CO, US A). Cows observed in estrus at 24 or 48 h after CIDR removal were inseminated at estrus. Cows not observed in estrus were inseminated at 72 h after CIDR withdrawal. Ovulation was anticip ated to occur 72 h after CIDR withdrawal. All an imals received the GnRH inj ection at 72 h regardless of estrus behavior. Cows were also randomly a ssigned within pair to receive either 100 g (i.m.) GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ USA), or vehicle (as for experiment 2) at 14 d after anticipated ovul ation. Pregnancy was diagnosed by rectal palpation at ~Day 45 after insemination. Rectal temperature was recorded in a subset of 100 cows in Farm 1 and 39 cows in Farm 2 at 1500 h of Day 14 after anticipated ovulation. Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artifi cial Insemination Du ring Heat Stress This study took place at the University of Florida Dairy Research Unit with inseminations in April to June, 2005. A total of 137 primiparous and multiparous lactating Holstein cows ranging in DIM from 78 to 566 d (mean= 185, SD= 110) were subjected to an OvSynch protoc ol as described for Experime nt 2. Multiple individuals conducted inseminations (n=4) and multip le AI sires were used (n=22). Cows were randomly assigned within pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium

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40 chloride in water) at Day 14 after anticipat ed ovulation (i.e., 14 d after insemination). Pregnancy was diagnosed by rectal palp ation at ~Day 46 after insemination. Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus This study took place at a commercial dair y in Chiefland, Florida. A total of 296 primiparous and multiparous lactating Holstein cows inseminated at detected estrus were used. Cows were inseminated from April – August, 2005. At the time of insemination, cows were between 51 and 235 DIM (mean= 122, SD= 40). Estrus was detected using tail chalk or KaMar estrus detec tion patches (KAMAR Inc., Steamboat Springs, CO, USA). Estrus de tection patches were visually monitored twice (morning and afternoon) daily by the insemi nator. When cows were first diagnosed in estrus in the afternoon, insemination was performed the next morning. When estrus was first detected in the morning, cows were inseminated at that time. Cows were bred by one inseminator and 31 different sires use d. Every other day of the experiment, cows were selected to receive inje ctions at Day 14 or 15 after in semination. Within each day, cows were randomly assigned within a pair to receive 100 g (i.m.) GnRH or an equivalent volume (2 ml) of vehicle (9 mg/ml benzyl alcohol and 7.47 mg/ml sodium chloride in water). Pregnancy was diagnos ed by rectal palpation at ~Day 45 after insemination. Statistical Analysis Data on pregnancy rate were analyzed by logistic regression with the LOGISTIC and GENMOD procedures of SAS (SAS fo r Windows, Release 8.02; SAS Inst., Inc., Cary, NC). For the LOGISTIC procedure, a ba ckward stepwise logistic model was used. Variables were continuously removed from th e model by the Wald statistic criterion if the significance was greater than 0.20. The Wald 2 statistic was used to determine the

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41 significance of each main effect that remained in the reduced model. The adjusted odds ratio (AOR) estimates and the 95% Wald conf idence intervals from logistic regression were obtained for each variable that remained in the final statistical model following the backward elimination. Data were also analyzed by PROC GENMOD and P values for significant treatment effects are reported from this analysis. The full mathematical model for experiment 1 included main effects of in seminator, treatment, protocol, replicate, replicate x protocol, replicate x treatment, re plicate x inseminator, protocol x treatment, protocol x inseminator, treatment x inseminator. The full mathematical model for experiment 2 included the effects of season of insemination (January to March vs April to September), treatment, and season x treatment. For experiment 3, the full mathematical model included the effects of farm, treatme nt, season of insemination (warm vs cool season; farm 1 = October to March vs April to September; farm 2 = June to September vs October to November), and season x treatment, season x farm, and treatment x farm. In addition, a subset of data composed of cows from farm 2 only was analyzed where the additional factor of estrus detection (yes or no) was included in the model. For experiment 4, the full mathematical model incl uded the effects of treatment, month of insemination, parity (1 vs ot hers), sire, DIM at insemina tion class (<150 d vs > 150 d), parity x treatment, DIM class x treatment a nd month x treatment. For experiment 5, the full mathematical model included the effects of treatment, season of insemination (April and May vs June to August), parity (1 vs > 1) number of services (1, 2 and >2), DIM at insemination class (<150 d vs > 150 d) and interactions of main effects with treatment. Since interactions were not si gnificant, data were reanaly zed with main effects only.

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42 Data on rectal temperatures were analyzed by least-squares analysis of variance using the GLM procedure of SAS. The m odel included effects of season (Exp.2) or season, farm and farm x season (Exp. 3). A meta-analysis was performed using Mant el-Haenszel procedures available using software downloaded from http://www.pitt.edu/~super1 /lecture/lec1171/index.htm Three analyses were performed – using all experiments, experiments with GnRH treatment at Day 11, and experiments with GnRH treatment at Day 14 or 15. Results Experiment 1 GnRH Administration at Day 11 after Anticipated Ovulation in Heifers Subjected to Timed Artificial Insemination During Heat Stress Based on progesterone concentrations measur ed at insemination and at Day 11 after anticipated ovulation, estrous cycles of 137/149 (92%) of th e heifers were successfully synchronized. Pregnancy rate was not signi ficantly affected by GnRH treatment or insemination protocol. This is true whether all heifers were considered (Table 1) or only those successfully synchronized (results not shown). There was also no effect (P > 0.10) of GnRH treatment at Day 11 on concentra tions of plasma progesterone on Day 15. Values were 3.5 0.19 ng/ml for heifers receiving vehicle and 3.6 0.19 ng/ml for heifers receiving GnRH. Experiment 2 GnRH Administration at Day 11 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination Treatment with GnRH did not significantly (P > 0.10) affect pregnancy rate per insemination (Table 2). This was true for inse minations in both cool seasons (January to March) and warm season (April to September) (results not shown). There was also no significant difference in pregna ncy rate between seasons.

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43 Rectal temperatures were higher (P < 0.001) for cows in the warm season (leastsquares means + SEM; 39.3 + 0.07 oC) than for cows in the cool season (38.9 + 0.07 oC). Experiment 3 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artificial Insemination Injection of GnRH increased pregnancy rates at both farms (treatment, P < 0.02; treatment x farm, non-significant) (Table 3) While pregnancy rates were lower in summer than winter (P < 0.05), the effect of GnRH was ap parent in both seasons and the season x treatment interaction was not significant. Cows in farm 2 were monitored for estrus. No cows were seen in estrus at 24 h after PGF2 4.7% (8/171) were detected in estr us at 48 h, 32.2% (55/171) at 72 h, and 63.1% (108/171) were not detected in estrus. Co ws in estrus at 48 h were inseminated at that time while other cows (those seen in estrus at 72 h and those not seen in estrus) were inseminated at 72 h. There was an estrus de tection class (detecte d in estrus vs not detected) x treatment interaction (P < 0.03) on pregnancy rate per insemination that reflected the fact that GnRH was effective at increasing pregnancy rate for those cows displaying estrus [3/29 (10%) for control and 12/34 (35%) for GnRH[ but had no effect for those cows not displaying estrus [7/54 ( 13%) for control and 4/54 (8%) for GnRH]. Rectal temperatures were higher (P < 0.01) for cows in the warm season (leastsquares means + SEM: 39.4 + 0.06 oC) than for cows in the cool season (39.1 + 0.11 oC) and higher (P < 0.001) for farm 2 (39.5 + 0.10 oC) than for farm 1 (39.1 + 0.07 oC), but there was no farm x season interaction. Experiment 4 GnRH Administration at Day 14 after Anticipated Ovulation in Lactating Cows Subjected to Timed Artifi cial Insemination Du ring Heat Stress Treatment with GnRH did not significantly affect pregnancy rate (Table 4). Pregnancy rate was higher (P<0.02) for cows inseminated at or before 150 DIM (30.3%,

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44 20/66) than for cows inseminated after 150 DIM (12.7%, 9/71). There were no other significant main effects or in teractions of GnRH treatmen t with other effects. Experiment 5 GnRH Administration at Day 14 or Day 15 after Detected Estrus Overall, pregnancy rate was higher (P< 0.0001) for cows inseminated in April and May (55/171, 32.2%) than for animals inseminated in June, July or August (12/125, 9.6%). There were, however, no other significan t main effects or interactions of GnRH treatment with other effects. Pregnancy rates were 25.6% (32/125) for cows receiving vehicle at day 14 or 15, 20.7% (19/92) for cows receiving GnRH at Day 14, and 20.3% (16/79) for cows receiving GnRH at Day 15. Overall Effectiveness of GnRH Treatme nt as Determined by Meta-Analysis When data from multiple experiments were considered together by meta-analysis, there was no significant effect of GnRH on pregnancy rate. This was the case when all experiments were consider ed (odds ratio=0.97; 95% CI=0.63, 1.50), or whether experiments with GnRH treatment on Day 11 (odds ratio=0.87; 95% CI=0.50, 1.50) or Day 14 or 15 (odds ratio=1.06; 95% CI=0.68, 1.65) were considered separately. Discussion Overall, there was no significant effect of GnRH treatment on pregnancy rate. In particular, GnRH treatment at Day 11 af ter anticipated ovulation did not improve pregnancy rate of heifers or lactating cows in any expe riment, whether animals were exposed to heat stress or not. Moreover, Gn RH did not consistently improve fertility when given at Day 14 after anticipated ovulatio n or at Days 14 or 15 after insemination. In one experiment (experiment 3), administ ration of GnRH at Day 14 after anticipated ovulation in cows subjected to TAI increased pregnancy rate of lactating cows in

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45 summer and winter at two locations. However, this positive effect c ould not be replicated either in lactating cows subjected to TAI or for cows inseminated at standing estrus. The variability in response to GnRH is reminiscent of the results of the metaanalysis of published studies performed by Pe ters et al. (2000) in which inconsistency between studies was noted. Variability in results could reflect either error in estimates of treatment effects because of small numbers of experimental units or variability in biological responses to GnRH. The number of animals used for the present studies varied and could have been too small in some studies to detect significan t differences or have lead to sampling errors that obscured the magnitude or direction of the treatment differences. However, meta-analysis of the entire data set, involving 1303 cows, indicated that there was no overall effect of GnRH. It is also possible that herds differ between each other or over time in the predominant biological response to GnRH treat ment. Presumably, beneficial effects of GnRH post-insemination on fertility are relate d to its actions to cause LH release. Treatment with GnRH at Day 11-15 of the es trous cycle can decrea se function of the dominant follicle (Thatcher et al., 1989; Rettmer et al., 1992 a; Ryan et al., 1994; Mann and Lamming, 1995a) and increase progester one secretion (Re ttmer et al., 1992a; Stevenson et al., 1993; Ryan et al., 1994; Willard et al., 2003). The reduction in estradiol-17 secretion caused by GnRH should dela y luteolysis and conceivably allow a slowly-developing conceptus additional time to initiate secretion of interferon. Low progesterone secretion may also compromise fertility in dairy cattle (Mann and Lamming, 1999; Lucy, 2001) and an increase in progesterone secretion caused by GnRH may facilitate embryonic development. Whether a herd responds to GnRH by undergoing

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46 follicular changes may depend upon the character istics of follicular growth because a follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001). Perhaps, herds that do not respond to GnRH w ith an increase in fert ility are herds where many cows have lower follicular growth or follicular wave characteristics that do not result in sufficient follicular development at the time of injection. One example of the potential importance of follicular dynamics in determining responses to GnRH is the expected res ponse to GnRH treatment at Day 11 after anticipated ovulation. In the current studi es, injection of GnRH at Day 11 after anticipated ovulation did not incr ease pregnancy rates in either lactating Holstein cows or nulliparous heifers. For lactating cows, the absence of an effect of GnRH at Day 11 was seen in both summer and winter. This result which agrees with other studies in which injection of GnRH at Day 11 does not affect fertility (Stevenson et al., 1993; Jubb et al., 1990), is in contrast to othe r studies indicating that GnRH treatment at Day 11 can increase fertility of heifers (Rettmer et al., 1992b) and lactating cows (Sheldon and Dobson, 1993; Willard et al., 2003). One factor that could influence the effectiveness of GnRH treatment at Day 11 is the number of follicular waves that an individual animal expresses. Animals with estrous cycles characterized by three follicular waves have larger second-wave dominant follicles at Day 11 of the estrous cycl e than animals with two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) and thus the preponderance of cycle type (two-wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at Day 11. There is variation from study to study in the relative frequency of three-wa ve vs two-wave cycles, at least among Holstein heifers (Ginther et al., 1989; Knopf et al., 1989; Ra jamahendran et al., 1991; Gong et al., 1993),

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47 and this variation is evidence for herd-to-herd variation in frequenc y of follicular wave patterns. Even in animals with three-wave follicul ar cycles, Day 11 woul d appear to not be an optimal time of the estrous cycle for us ing GnRH to cause luteinization because the second-wave dominant follicle is smaller at Da y 11 than at 14-15 in heifers (Ginther et al., 1989; Ko et al., 1991) and lactating cows (Ko et al., 1991). Results from a limited number of cows in Experiment 3 suggested that the effectiveness of GnRH at Day 14 after anticipated ovulation depends upon wh ether cows are detected in estrus. Presumably, ovulation occurred on average sooner for cows in estrus at 48 and 72 h after prostaglandin than for cows not detected in estrus (which contains cows that had not initiated estrus by 72 h as well as some cows in which estrus occurred by 72 h but was not detected). Among those detected in estr us, GnRH injection improved fertility from 10.3% to 35.3%. Among animals not dete cted in estrus, however, there was no difference in pregnancy rate between anim als treated with vehicle (13.0%) or GnRH (7.6%). It is likely that GnRH did not affect pregnancy rate in the cows not detected in estrus because this group included cows that were anovulatory at insemination or that were not successfully synchronized; GnRH w ould be unlikely to increase pregnancy rate in these animals. It was hypothesized that bene ficial effects of GnRH would be greater during heat stress because this condition can decrease gr owth of the filamentous stage conceptus (Biggers et al., 1987), increas e uterine prostaglandin F2 secretion from the uterus (Wolfenson et al., 1993) and reduce circul ating concentrations of progesterone (Wolfenson et al., 2000). Bene ficial effects of GnRH tr eatment at Day 11-12 after

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48 insemination on fertility have been observed in lactating dairy cows during heat stress (Willard et al., 2003; Lpez-Gatius et al., 2005a ). There was no evidence, however, that GnRH was more effective during the summer. In particular, the in crease in pregnancy rate caused by injection of GnRH at Day 14 during experiment 3 was similar for cows inseminated in summer and winter. In ot her experiments conducted during the summer, GnRH was without be neficial effect. In experiment 1, there were no differences in pregnancy rates for Holstein heifers inseminated either at second GnRH injection (24.4%) or 24 after GnRH (19.8%). This result is similar to results of Pursley et al. (1998) who reported little difference in pregnancy rates and no differen ces in calving rates between l actating cows inseminated at 0, 8, 16, or 24 h after the second GnRH injection of the OvSynch regimen. The pregnancy rates achieved with heifers in experiment 1 were lo w compared to other studies in which heifers rece ived a similar ovulation synchronization program (Martinez et al., 2002ab). The low fertility was not a result of delayed pubert y or unresponsiveness to the synchronization protocol because 92% of the heifers had both low progesterone concentrations during the expected pe riovulatory period a nd high progesterone concentrations at the predicted lu teal phase of the cycle. It is possible that some of these heifers classified as synchronized experienced short estrous cycles (Schmitt et al., 1996b; Moreira et al., 2000a). The experiment was co nducted during the summer and it is also possible that heat stress reduced fertility. A lthough fertility in Hols tein heifers does not always decline during the summer (Ron et al ., 1984; Badinga et al., 1985), there is one report (Donovan et al., 2003) that heifers from a dairy farm in north central Florida inseminated in summer were more than four times less likely to become pregnant to first

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49 insemination than heifers insemina ted during the rest of the year. It is also possible that the one sire used to inseminate all heifers was not a fertile bull. In conclusion, injection of GnRH at Day 11-15 after anticipated ovulation or insemination did not consisten tly increase pregnancy rates in heifers or lactating cows. The fact that GnRH administration was eff ective in one study indicates that such a treatment may be useful for increasing pregna ncy rate in some herd s or situations. More work will be required to describe factors that could identify which groups of cows would be most likely to benefit from GnRH treatment. Table 2-1. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and ovulation synchronization protocol on pregnancy rates of heifers during heat stress. 1 Data represent the number of females pregnant at Day 44-51 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =0.54 (N.S). 4 Wald chi-square statistic = 0.40 (N.S.) Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 20/78 25.61.29 0.59 – 2.83 0.41 Vehicle 14/71 19.7 Protocol 4 B 20/79 25.31.34 0.61 – 2.95 0.41 A 14/70 20.0

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50 Table 2-2. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 11 after anticipated ovulation and season of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination. 1Data represent the number of females pregnant at ~d 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 2 Wald chi-square statistic =1.50 (N.S). 4 Wald chi-square statistic = 1.38 (N.S.) Table 2-3. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and season of insemination on pregnancy rates of lactating cows subjected to timed artificial insemination. 1 Data represent the number of females pregnant at ~Day 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =4.94 (P=0.026). 4 Wald chi-square statistic = 5.12 (P=0.024) Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 26/121 21.50.66 0.37 – 1.18 0.16 Vehicle 36/123 29.3 Season 4 January – March 30/103 29.11.38 0.77 – 2.48 0.27 April September 32/141 22.7 Pregnancy rate Proportion 1 % AOR95% Wald CI P -value 2 GnRH Treatment 3 GnRH 49/241 20.31.76 1.07 – 2.89 0.02 Vehicle 30/236 12.7 Season 4 Oct, Nov, Feb, March 40/187 21.41.76 1.08 – 2.87 0.02 May September 39/290 13.5

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51 Table 2-4. Descriptive statistics, adjusted odds ratio (AOR) estimates, and 95% Wald confidence intervals (CI) for effect of GnRH administrati on at Day 14 after anticipated ovulation and Da ys in milk (<150 d vs > 150) at insemination on pregnancy rates of lactating cows subj ected to timed artificial insemination during heat stress. 1 Data represent the number of females pregna nt at ~Day 45 after insemination / total number of females inseminated. 2 Derived from PROC GENMOD. 3 Wald chi-square statistic =3.55 (P=0.060). 4 Wald chi-square statistic = 6.12 (P=0.013) Pregnancy rate Proportion 1 % AOR 95% Wald CI P -value 2 GnRH Treatment 3 GnRH 11/73 15.1 0.43 0.18 – 1.04 0.05 Vehicle 18/64 28.1 Days in milk at insemination 4 < 150 d 20/66 30.3 3.11 1.27 – 7.62 0.02 > 150 d 9/71 12.7

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52 CHAPTER 3 EFFECT OF TRANSFER OF ONE OR TWO IN VITRO-PRODUCED EMBRYOS AND POST-TRANSFER ADMINISTRATI ON OF GONADOTROPIN RELEASING HORMONE ON PREGNANCY RATES OF HEAT-STRESSED DAIRY CATTLE Introduction The in vitro produced (IVP) embryo is di fferent from the embryo produced in vivo in terms of morphology (Iwasak i et al., 1992; Massip et al., 1995; Crosier et al., 2001), gene expression (Bertolini et al., 2002a; L azzari et al., 2002; Lonergan et al., 2003), metabolism (Khurana et al., 2000b), and in cidence of chromosomal abnormalities (Iwasaki et al., 1992; Viuff et al., 2000). No t surprisingly, pre gnancy rates achieved following transfer of an IVP embryo are of ten less than what is obtained following transfer of an embryo produced by superovulatio n and calves born as the result of in vitro production are more likely to experience deve lopmental defects (Hasler et al., 2003). Problems associated with the transfer of IVP embryos have limited the realization of the potential of these embryos for enhanci ng genetic improvement and reproductive performance of lactating dairy cattle (Ru tledge, 2001; Hansen and Block et al., 2004). One method that might be useful for in creasing pregnancy ra tes in dairy cattle recipients that receive an IVP embryo is to transfer two embryos into the uterine horn ipsilateral to the CL. Such a treatment might increase pregnancy rate because the likelihood is increased that th e cow receives at least one em bryo competent for sustained development. In addition, the transfer of tw o embryos into the ipsilateral uterine horn is likely to increase the amounts of interferonand other embryonic signaling molecules in the uterus needed to maintain pregnancy and prevent luteolysis. Co-transfer of

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53 embryonic vesicles to increase trophoblasti c signals has been reported to increase pregnancy rates in embryo transfer recipien ts (Heyman et al., 1987). For the current experiment, both embryos were transferred in to the uterine horn ipsilateral to the CL because of the requirement for the antiluteolytic signal in cattle to be locally administered (Del Campo et al., 1977; 1983). In a recent st udy with a small number of transfers (n=10 to 28 recipients), there was a tendency for hi gher calving rate for recipients that received two embryos in the uterine horn ipsilateral to the CL as compared to recipients that received one embryo (Bertolini et al., 2002b). Anderson et al. (1979) found a tendency for pregnancy rates to be highe r in cows that received two embryos in the same uterine horn (unilateral transfer) than for cows that received two embryos distributed in both uterine horns (bilateral tran sfer); the opposite was true fo r heifers. In other studies, transfer of embryos to create tw o pregnancies in the uterine horn ipsilateral to the CL has produced a similar pregnancy rate as bilatera l twins and single pre gnancies (Sreenan and Diskin, 1989; Reichenbach et al., 1992) or reduced pregnancy rate as compared to bilateral transfer (R owson et al., 1971). Another treatment that has potential for increasing pregnancy rates in embryo transfer recipients is injec tion of GnRH at Day 11 after th e anticipated day of ovulation. Such a treatment was shown to increase pregna ncy rates in heat-stre ssed, lactating cows following insemination (Sheldon and Dobs on, 1993; Willard et al., 2003) and embryo transfer (Block et al., 2003) Treatment with GnRH or its analogues at Day 11 to12 of the estrous cycle has been reported to increase progesterone secretion (Ryan et al., 1994; Willard et al., 2003) and inhibit function of the dominant follicle (Savio et al., 1990; Ryan et al., 1994) to possibly delay luteolysis.

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54 The purpose of the current pair of experiments was to examine the effectiveness of unilateral transfer of twin embryos and treatment with GnRH at Day 11 after the anticipated day of ovulation for increasing pregna ncy rates in dairy catt le recipients that received IVP embryos. Experiments were performed during periods of heat stress because embryo transfer offers benefits as a method for increasing pregnancy rate as compared to AI in females subjected to heat stress (Rutledge, 2001). Materials and Methods Experiment 1 Single or Twin Transfer of IVP Embryos into Crossbred Dairy Recipients The experiment was conducted at a comm ercial dairy located in Santa Cruz, Bolivia (17o48’ S, 63o10’ W) from November – Decembe r, 2004. Data on minimum and maximum air temperatures during the experi ment collected by Servicio Nacional de Meteorologa e Hidrologa ( http://www.senamhi.gov.bo/meteorologia/ ) for Santa Cruz are presented in Figure 1. Females receivi ng embryos included 32 virgin crossbred heifers sired by Simmental, Gyr, or Brown Swiss bulls and Holstein or Holstein crossbred dams and 26 lactating, crossbred cows with the proportion of Holstein varying from 1/2 to 15/16. The heifers ranged in age from 363 to 2070 d (mean = 850 d and median = 664 d; SD = 421 d) and ranged in weight from 247 to 430 kg (mean = 310 kg and median = 288 kg; SD = 52.3 kg). Animals were maintained on grass pasture until two weeks prior to the start of the synchronization program when they also received a supplement of 6 kg/head/d of spent brewers’ grain. The cows ranged in age from 820 to 4075 d (mean = 2083 d and median = 1670 d; SD = 986 d), were maintained on grass pasture, and received 11 kg of brewers’ grains and 2 kg of a soybean-based concentrate mixture before each milking. Cows were milked two times per day and ranged from 110

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55 to 417 d in milk (mean =190 d and median = 170 d; SD = 75 d). Milk yield per day across all days of lactation ranged from 5.9 to 21.1 kg/d (mean = 12.5 kg/d and median = 12.6 kg/d; SD = 3.8 kg/d). Recipients were synchronized for timed em bryo transfer using a modified OvSynch protocol (Portaluppi and Steven son, 2005) with the inclusion of a controlled intravaginal drug releasing device (EAZI-BREED CIDR insert, 1.38 g of progesterone, Pfizer Animal Health, New York, NY, USA). On Day -10 (Day 0 equals the day of anticipated ovulation), females received 100 g (i.m.) of GnRH (1 ml of Profertil; Tortuga Cia. Zootcnica Agrria, So Paulo, Brazil) and an intravaginal progesterone-releasing device insert that had been used one time previous ly. On Day -3, CIDR devices were removed and females received 150 g (i.m.) of PGF2 (2 ml of Prostaglandina Tortuga, Tortuga Cia. Zootcnica Agrria). On Day 0, 100 g (i.m.) of GnRH was administered. Behavioral symptoms of estr us were monitored about 5 times each day for 3 d following CIDR removal and PGF2 injection. On Day 6 after antic ipated ovulation, all females, including those not seen in estrus, were exam ined per rectum for the presence of a CL using an Aloka 210 ultrasound unit equipped wi th a 5 MHz linear array probe (Aloka, Wallingford, CT, USA). A group of females having a CL (n=32 heifers and n=26 cows) were randomly selected within recipient type (heifers or cows) to receive one (n=31 females) or two (n=27 females) embryos on Day 7 after anticipated ovulation. For embryo transfer, an epidural block of 5 ml of lidocaine hydrochloride (2% w/v; Sparhawk Laboratories Inc., Lenexa, KS, USA) was admini stered to each recipi ent, and one or two IVP embryos were deposited in to the uterine horn ipsilatera l to the ovary containing the CL. One technician conducted all transfers.

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56 A total of 85 blastocysts (72 at Day 7 after insemination and 13 and Day 8 after insemination) were transferred in this expe riment. Of these, six were produced by Transova (Sioux City, IA, USA) using Holste in oocytes and a Holstein sire and were cultured in Synthetic Oviductal Fluid (SOF) medium. Embryos were shipped overnight in a portable incubator to Gainesville, FL, USA on Day 4 after insemination. Embryos were transferred to fresh microdrops of a modified SOF (Fischer-Brown et al., 2002) prepared by Specialty Me dia (Phillipsburg, NJ, USA) and cultured at 38.5oC in a humidified atmosphere of 5% O2 and 5% (v/v) CO2 (balance N2). The remainder were produced using oocytes obtained from ovaries of a variety of breeds collected at a local abattoir located at a travel distance of approximately 1.5 h from the Gainesville laboratory. Procedures, reagents, and media formulation for oocyte maturation, fertilization, and embryo culture were as previously described (Roth and Hansen, 2005) with some modifications. Cumulus-oocyte complexes were matured for approximately 22 h at 38.5C in an atmosphere of 5% (v/v ) CO2 in humidified air and then inseminated with a cocktail of Percoll-purified spermatozoa from three different bulls of various breeds. At 8 – 12 h post-insemination (hpi), putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium (Caisson, Rexburg, ID, USA) containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexed in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in 50 l microdrops of KSOM-BE2 (Soto et al ., 2003) (Caisson, Rexburgh, ID, USA) at 38.5C in an atmosphere 5% (v/v) CO2 in air. Regardless of method of production, embryos greater than 16 cells in appearance were collected at 1300 h on Day 6 or 7. Embryos were placed in groups of 21 to 65 into

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57 2 ml cryogenic vials (Nalge Company, Roches ter, NY, USA) filled to the top with KSOM-BE2 that was pre-warmed and equilibr ated in 5% (v/v) CO2 in air. Embryos produced by Transova were kept separately from those produced using ovaries from the local abattoir. Vials containing embryos were placed in a portable incubator (Minitube of America, Verona, WI, USA) that had been pre-warmed to 39oC for 24 h prior to use. Embryos were shipped by air and arrived at Santa Cruz de la Si erra, Bolivia, at 1100 h the next day (Day 7 or 8 after in vitro insemination) and transported by ground to the farm. Embryos were transferred over a time span from 1300 h and 2000 h. One or two embryos were loaded into 0.25 cc straws in Hepes-TALP (Caisson) containing 10% (v/v) bovine steer serum (Pel-Freez, Rogers, AR, USA) and 100 M 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Embryos were transferred to recipients that were palpated the day before and had a detectable CL. Recipien ts were randomly assigned to receive one or two embryos, and all embryos we re transferred into the ipsilateral horn to the CL. Pregnancy diagnosis was performed by rectal palpation at Day 64 and 127 posttransfer, and the number of fetuses was reco rded on Day 127. Data collected at calving included length of gestation (with the day of transfer being considered Day 7 of gestation), occurrence of dystocia (defined as needing assistance), sex, weight and viability of each calf, and o ccurrence of retained placenta (f ailure of the placenta to be expelled within 12 h after calving). Calf surviv al until Day 7 of age was also recorded. Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation in Lactating Recipients that Received an IVP Embryo This study took place at a commercial dairy located in Bell, FL, USA (29o 45Â’ N 82o 51Â’ W) from June to October, 20 04. Data on minimum and maximum air

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58 temperatures and average relative humidity collected by the Florida Automated Weather Service ( http://fawn.ifas.ufl.edu ) for Alachua, FL, USA are presented in Figure 1. A total of 87 multiparous, lactating Holstein cows in la te lactation were used as recipients. Cows were fed a total mixed ration to meet or exceed requirements recommended for lactating dairy cows, milked three times a day, and received bovine somatotropin (Posilac, 500 mg sometribove zinc, Monsanto, St. Loui s, MO, USA) accordi ng to manufacturer’s directions. Cows were housed in a dry lot with access to a permanent shade structure without fans or sprinklers a nd with access to a cooling pond. Cows were prepared for embryo transfer in groups of 6 to 18; a total of 10 replicates were completed. To synchronize re cipients for timed em bryo transfer, cows received 100 g (i.m.) of GnRH (2 ml of Cystorelin; Merial Limited, Iselin, NJ, USA), on Day –10; 25 mg (i.m.) of PGF2 on Day -3; and 100 g (i.m.) of GnRH, on Day 0 (i.e., the day of anticipated ovulation). On Day 7 after anticipated ovulation, all cows were palpated per rectum for the presence of a CL. Cows that had a palpable CL received an epidural block of 5 ml of lidocaine (2%, w/v), and a si ngle embryo was transferred to the uterine horn ipsilateral to the ovary c ontaining the CL. Recipients were randomly assigned to receive 100 g (i.m) of GnRH or vehicle (9 mg/ml of benzyl alcohol and 7.47 mg/ml of sodium chloride in water) on Day 11 after anticipated ovulation. The embryos used for transfer were produ ced in the Gainesville laboratory using oocytes of various breeds and a pool of seme n from three bulls of various breeds as described for Experiment 1. A different pool of semen was used for each replicate. Presumptive zygotes were cultured in groups of ~30 in 50 l microdrops of modified SOF (Fischer-Brown et al., 2002) containing 100 ng/ml of insulinlike growth factor-1

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59 (Upstate Biotechnology, Lake Placid, NY, USA) Embryos were cultured at 38.5C in a humidified atmosphere of 5% (v/v) O2 and 5% (v/v) CO2 with the balance N2. On Day 7 after insemination, blastocysts were harveste d and transported to the farm in 2 ml cryogenic vials (20 to 25 embr yos/tube) filled to the top wi th pre-warmed Hepes-TALP. Tubes containing embryos were placed in a portable incubator (Minitube of America, Verona, WI, USA) that had been pre-warmed to 39oC for 24 h prior to use. Embryos were transported to the farm and loaded in 0.25 cc straws prior to transfer into recipients. Pregnancy was diagnosed by rect al palpation at Day 45 to 53 after anticipated ovulation. Statistical Analysis Categorical data were analyzed by lo gistic regression using the LOGISTIC procedure of SAS for Windows (Version 9, SAS Institute Inc., Cary, NC, USA) with a backward stepwise logistic model. Variable s were continuously removed from the model by the Wald statistic criterion if the significan ce was greater than 0.2. The full statistical model for Experiment 1 included treatment ( one embryo or two embryos), parity (cows vs heifers), estrus (observe d in estrus vs not observed) and treatment x parity on pregnancy rate, pregnancy loss, calving rate, calf mortalit y and twinning rate. The only variable in the final mathematical model fo r Experiment 2 was GnRH treatment as other effects (replicate and replicate x treatment) were not significant. The adjusted odds ratio estimates and the 95% Wald confidence inte rvals (CI) from logistic regression were obtained for each variable that remained in the final statistical model following the backward elimination. Data were also anal yzed with the GENMOD procedure of SAS to determine the significance of each effect that remained in the reduced model; P values for logistic regression analyses repo rted in the tables are derived from these analyses. Data for gestation length and calf bi rth weight were analyzed by analysis of variance using

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60 Proc GLM. The full statistical model incl uded the effects of treatment, parity and treatment x parity. The 2 test was used to determine whether the sex ratio of calves differed from the expected 1:1 ratio. Results Experiment 1 Single or twin transfer of IVP embryos Pregnancy and calving rates Data are summarized in Tabl e 1. At Day 64 of gestation, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. Wh ile there were no si gnificant effects of number of embryos transferred or parity x number transferred, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% for two embryos vs 41% for one embryo) while there was no difference in pregnancy rate due to number of embryos transferred to cows (50% for two embryos vs 57% for one embryo). Pregnancy losses between Day 64 and 127 occurred in one group only – cows receiving two embryos. In that group, pregna ncy rate was 50% at Day 64 but decreased to 17% at Day 127. There was no difference in pregnancy rates at Day 127 between cows and heifers, but recipients that r eceived two embryos had lower pregnancy rates (17% for cows and 20% for heifers) than re cipients that received one embryo (57% for cows and 41% for heifers, P < 0.03). Pregnancy loss after Day 127 occurred in one female only. In particular, a cow receiving a single embryo gave birth to a st illborn calf at 251 d of gestation. Like for pregnancy rate at Day 127, there was no diffe rence in calving rate between cows and heifers, but recipients that received two embryos had lower calving rates (17% for cows

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61 and. 20% for heifers) than reci pients that received one em bryo (50% for cows and 41% for heifers, P < 0.03). Estrus was detected at 24, 48 or 72 h afte r prostaglandin injection in 21/32 heifers (8 at 24 h after injection and 13 at 48 h) and 19/26 cows (1 at 24 h after injection, 14 at 48 h and 4 at 72 h). While not statistically different (P=0.11), th ere was a tendency for pregnancy rates to be lower for animals not de tected in estrus. For example, pregnancy rates at Day 127 for animals receiving one embr yo was 55% (11/20) for animals in estrus vs 36% (4/11) for animals not observed in es trus. Pregnancy rates at Day 127 for animals receiving two embryos were 25% (5/20) for anim als in estrus vs 0% (0/7) for animals not observed in estrus. Characteristics of gestation, parturition, and calves Gestation length was affected by recipient type x number of embryos transferred (P<0.05; Table 2). For cows, gestation length was slightly longer fo r those receiving one embryo as compared to those receiving two embryos while the opposite was true for heifers. Two of 5 females calving that receiv ed two embryos produced twin calves. There was no significant effect of r ecipient type or number of em bryos transferred on dystocia or incidence of retained placenta (Table 2) Sex ratio (including the one stillborn calf) was in favor of males with 15 males compared to 7 female calves born (68% male; Table 3). This ratio tended to be different from the expected 1:1 ratio (P<0.10). While there were no significant differences there was a tendency for calf mortality at birth to be greater for he ifers receiving two embryos than for other groups (Table 3). None of the cows lost their calf at birth a nd only 1 of 7 heifers receiving a single embryo experienced calf death at birth. In contra st, 2 of 3 heifers receiving two embryos experienced calf loss. One heifer had twin fe tuses and both were born dead as a result of

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62 complications with calving. Anot her heifer gave birth to a si ngle calf that was born dead as a result of complications w ith calving. The calf from the third heifer was born alive. All calves born alive were alive 7 d later. Experiment 2 Administration of GnRH on Day 11 after Anticipated Ovulation Administration of GnRH at Day 11 afte r anticipated ovulation had no effect (P>0.10) on pregnancy rates. Recipients treated with GnRH had a pregnancy rate of 17.8% (8/45) while those recipients that re ceived placebo had a pregnancy rate of 16.7% (7/42). The odds ratio was 1.08 with 95% Wald confidence interval of 0.23 and 3.30. Discussion The purpose of the experiments described here was to examine two strategies for increasing pregnancy rates in heat-stressed da iry recipients that receive an IVP embryo. Neither approach, transferring tw o embryos into the uterine horn ipsilateral to the CL or injection of GnRH at Day 11 after anticipated ov ulation, increased pregnancy rates. Results of Experiment 1 indicated that the transfer of two embr yos into recipients led to pregnancy loss and that such loss occurred earlier for heifers than for cows. There was a distinct difference in pregnancy rate between heifers that received one or two embryos as early as Day 64 of gestation. Among cows, in contrast, there were no differences in pregnancy rate at this stage of gestation betw een recipients that received one or two embryos. By Day 127, however, cows that received two embryos experienced substantial mid-to-late fetal loss and pregna ncy rate and subsequent calving rate was lower for this group than for cows that received a single embryo. The most likely explanation for the incr eased frequency of pregnancy loss in recipients receiving two embryos is uterin e crowding, with the effects of crowding occurring sooner in gestation for nulliparous animals than for multiparous animals.

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63 Similar results were obtained in anothe r study (Anderson et al., 1979). In that study, calving rates and twinning rate s were similar for cow recipients regardless of whether twin transfers were performed via bilateral or unilateral placeme nt. For heifers, in contrast, calving rate and twinning rate was lower for unilateral twin transfers than for bilateral transfers. Using heifers, Rowson et al. (1971) also found lower embryonic survival rates and twinning rates for recipien ts of unilateral twin transfers than for recipients of bilateral transfers. It is evident, however, that uterine capacity can vary between herds of cattle. Thus, there were no differences in pregnancy su ccess between recipients of twin embryos placed unilaterally or bilaterally for heifers (Sreenan and Diskin 1989; Reichenbach et al., 1992) or cows (Sreenan and Diskin 1989). Si milarly, embryonic survival rate for beef cows selected for twinning was similar for those having unilateral or bilateral multiple ovulations (Echternkamp et al., 1990). In lact ating dairy cows, in contrast, the likelihood of a twin pregnancy resulting from multip le ovulation going to term was higher if ovulations occurred bilaterally than if unilateral o vulations occurred (Lpez-Gatius et al., 2005b). Perhaps, identification of the biological processes controlling uterine capacity will lead to new approaches for increasing the efficacy of producing twins in cattle. In an earlier study, administration of GnRH at Day 11 after an ticipated ovulation tended to increase pregnancy and calving rates in lactating Holstein recipients (Block et al., 2003). The management of these cows was similar to those in Experiment 2. In both studies, recipients were exposed to heat stress and received an IVP embryo using a timed embryo transfer protocol. Effectiveness of treatment with GnRH or its analogues at 11 to12 d after estrus for inseminated cows has yielded variable results, as some reports

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64 indicated a positive effect (S heldon and Dobson, 1993; Willard et al., 2003) while others indicated no effect (Ryan et al., 1994). One f actor that could influence the effectiveness of GnRH treatment at Day 11 is the number of follicular waves that a female experiences during an estrous cycle. Females with estr ous cycles characterized by three follicular waves have larger second-wave dominant follic les at Day 11 than females with two-wave cycles (Ginther et al., 1989; Sa vio et al., 1990; Ko et al., 1991) Given that a follicle must reach 10 mm in diameter to ovulate in re sponse to LH (Sartori et al., 2001), the preponderance of cycle type (two-wave vs three-wave) within a herd may determine effectiveness of GnRH treatment at Day 11. Finally, it remains possible that failure to observe an effect of GnRH treatment was because the number of animals per group was low. The pitfalls associated with interp retation of experiments with low numbers has been discussed (Amann, 2005) and could be resp onsible for the variation in results for trials to test effects of GnRH on pregnancy rates in embryo transfer recipients. Estrus is difficult to detect in lactating dairy cows because of the short duration of estrus and the large proporti on of cows that do not display intense mounting activity (Dransfield et al., 1998). This problem, whic h is exacerbated by heat stress (Thatcher et al., 1986), makes embryo transfer in lactating co ws inefficient if recipient selection is based solely on estrus detect ion. The first report of a tim ed embryo transfer protocol, where ovulation was synchronized using an OvSynch protocol, was by Ambrose et al. (1999). The suitability of timed embryo transf er as a method for prep aring recipients was demonstrated in Experiment 1 because calving rates were 50 and 41% for cow and heifer recipients that received a si ngle embryo, respectively. Similarl y, using beef recipients, a pregnancy rate of 49% was achieved using tim ed embryo transfer (Bo et al., 2002). In

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65 contrast, pregnancy rate at Day 45 of ge station in Experiment 2 was only 17%. Low pregnancy rates have been reported in othe r studies with timed embryo transfer using lactating, heat-stressed recipients with pregnancy rates at ~ 45 d of gestation following timed embryo transfer ranging from 11 – 26% (Ambrose et al., 1999; Al-Katanani et al., 2002a; Block et al., 2003). The reason for th e differences in pregnancy rates between Experiment 1 and 2 cannot be deduced because of the large number of variables between studies including nutrition, housing, level of milk yield, stage of lactation, breed, synchronization protocol, and embryo culture protocol. Despite the effectiveness of timed em bryo transfer, there was a tendency for pregnancy rates in Experiment 1 to be higher fo r those recipients detect ed in estrus. Most of the animals not detected in estrus likely ovulated after the last GnRH injection because embryos were only transferred to recipients with a detectable CL. Nonetheless, some cows in this group probably were not synchr onized with respect to predicted ovulation time. Transfer of IVP embryos has been associ ated with large calf syndrome, increased rates of fetal loss, sex ratio skewed towards the male and increased rate of dystocia and calf mortality (see Hasler et al., 2000; Ha nsen and Block, 2004; Farin et al., 2004 for review). There are also reports of prolonge d gestation length (Kruip and den Dass, 1997; Rerat et al., 2005). In Experiment 1, most char acteristics of the fetus and calf that were measured in females receiving one embryo were within normal ranges including gestation length, rates of fetal loss, calf birth weight, and calf survival at birth and within the first 7 d of age. The incidence of dystocia among fema les receiving one calf was 21% and it is difficult to determine whether this value is high because of the particular mating

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66 combinations used (embryos of diverse ge notypes transferred into females of several different genotypes). In a study with Holsteins bred by artificial insemination, the frequency of difficult births ranged fro m 6 to 18% (Djemali et al., 1987). The one abnormality identified was a skewed sex ratio with 68% of the calves being male. While previous work suggests th at the altered sex ra tio among IVP embryos is due to toxic effects of concentrations of glucose in excess of 1 mM on female embryos (Kimura et al., 2005), the concentration of gluc ose in the medium used for culture here (KSOM-BE2) contains only 0.2 mM glucose (Soto et al., 2 003). Others have found a tendency for male embryos to become blastocysts sooner in development when cultured in KSOM than female embryos (Nedambale et al., 2004b). Differences in sex ratio have been seen as early as between the eight-cell and morula stages of development (Block et al., 2003). While it is possible that selection of most embr yos for transport done on Day 6 after insemination exacerbated the skewed se x ratio, Block et al. (2003) reported that 64% of calves born as a result of transfer of IVP embryos cultured in modified KSOM were male even though embryos were harveste d for transfer on Day 8 after insemination. In conclusion, results indicate that unilate ral transfer of two embryos to increase pregnancy rate is unwarranted. The fact that fetal loss occurred sooner for heifers than cows points out the importance of uterine capacity as a lim iting factor for maintenance of fetal development of two conceptuses. Ther e was also no evidence that GnRH treatment at Day 11 after anticipated ovulation improves pr egnancy rate. Finall y, the suitability of timed embryo transfer as a method for prepari ng recipients for transfer was evident by the high pregnancy and calving rates achieved with crossbred females that received a single embryo. Additional research is warranted to reduce incidence of skewed sex ratio.

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67 While sexed semen could be used to contro l sex ratio (Wilson et al., 2005), it is likely that the underlying biological cau ses of altered sex ratio aff ect other aspects of embryo physiology also.

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68Table 3-1. Effect of recipient type a nd number of embryos transferred per re cipient on pregnancy rates and losses. Recipient type Pregnancy rate, d 64 of gestationab Pregnancy rate, d 127 of gestationac Pregnancy loss between Day 64 and 127 of gestationd Calving ratee,f Pregnancy loss between Day 127 and calvingg Lactating cow – single embryo 8/14 (57%) 8/14 (57%) 0/8 (0%) 7/14 (50%) 1/8 (13%)h Lactating cow – two embryos 6/12 (50%) 2/12 (17%) 4/6 (66%) 2/12 (17%) 0/2 (0%) Nulliparous heifer – single embryo 7/17 (41%) 7/17 (41%) 0/7 (0%) 7/17 (41%) 0/7 (0%) Nulliparous heifer – two embryos 3/15 (20%) 3/15 (20%) 0/3 (0%) 3/15 (20%) 0/3 (0%) a Data are the proportion of animals pregna nt of those that received embryos and, in parentheses, the percent pregnant. b Logistic regression indicated e ffect of recipient type (P=0.07). The odds ratio estimate wa s 0.38 (heifer/cow) (95% Wald CI = 0.13, 1.14; Wald Chi-Square statistic = 2.96, P=0.08). c Logistic regression indicated an effect of number of embryos tran sferred (P<0.03). The odds ratio estimate was 4.13 (one embryo/two embryos) with a 95% Wald CI of 1.243, 13.690. Wald Chi-Square statistic = 5.36; P<0.03). d Data are the proportion of pre gnant recipients at Day 64 that lost their pregnancy by Day 127 of gestation and, in parentheses the percent pregnancy loss. e Data are the proportion of animals that calved of those th at received embryos and, in pare ntheses, the percent pregnant. f Logistic regression indicated an effect of number of embryos tran sferred (P<0.03). The odds ratio estimate was 3.62 (one embryo/two embryos) with a 95% Wald CI of 1.090, 12.047. Wald Chi-Square statistic = 4.41; P<0.04). g Data are the proportion of pre gnant recipients at Day 127 that lost their pregnancy before calving and, in parentheses, the pe rcent pregnancy loss. h One cow expelled a stillborn calf at 251 d of gestation.

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69 Table 3-2. Effect of recipien t type and number of embryos transferred per recipient on characteristics of pregnancy and parturition. Recipient type Gestation length, da Twin pregnanciesb Dystociac Retained placentad Lactating cow – single embryo 282 + 3 0/7 (0%) 2/7 (29%) 4/7 (57%) Lactating cow – two embryos 274 + 5 1/2 (50%) 0/2 (0%) 1/2 (50%) Nulliparous heifer – single embryo 276 + 3 0/7 (0%) 1/7 (14%) 5/7 (71%) Nulliparous heifer – two embryos 284 + 4 1/3 (33%) 1/3 (33%) 2/3 (67%) a Data are least-squares means + SEM. Gestation length was a ffected by recipient type x number of embryos transferred (P<0.05). b Data are the proportion of pregnancies in which twin calves were born and, in parentheses, the percent pre gnant. Logistic regression indicate d an effect of number of embryos transferred (P<0.02). c Data are the proportion of pregnancies in wh ich dystocia was recorded at birth and, in parentheses, the percent cows experiencing dystocia. d Data are the proportion of cows calving that experienced retained placenta and, in parentheses, the percent cows experiencing retained placenta.

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70 Table 3-3. Effect of recipien t type and number of embryos transferred per recipient on characteristics of calves born. Recipient type Sex ratio (M:F)a Calf birth weight, kgb Calf mortality at birthc Calf mortality to Day 7 of aged Lactating cow – single embryo 5:3e 34 + 3 0/7 (0%) 0/7 (0%) Lactating cow – two embryos 2:1 25 + 5 0/3 (0%) 0/3 (0%) Nulliparous heifer – single embryo 4:3 26 + 3 1/7 (14%)f 0/6 (0%) Nulliparous heifer – two embryos 4:0 25 + 5 3/4 (75%)g 0/1 (0%) a The overall sex ratio of 15 male and 7 female s tended to be different (P<0.10) than the expected 1:1 ratio. b Data are least-squares means + SEM. c Data are the proportion of calves that were born dead and, in parentheses, the percent born dead. d Data are the proportion of calves born alive that died before d 7 of live and, in parentheses, the percent death before Day 7. e Data includes the stillborn calf at 251 d of gestation f One calf was stillborn from a cow not experiencing dystocia. g One heifer had twin fetuses and both were bor n dead as a result of complications with calving. The other two heifers ga ve birth to a single calf. On e calf was born alive and the other was born dead as a result of complications with calving.

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71 Air temperature ( o C) 10 15 20 25 30 35 Nov 1Nov 15Dec 15 Dec 1 Dec 30Experiment 1 BoliviaRH (%) X Data 0102030405060708090100110120130140150160 60 70 80 90 100 Air temperature ( o C) 5 10 15 20 25 30 35 June 1July 1Aug 1Sept 1Oct 1Nov 1Experiment 2 Florida Figure 3-1. Maximum (open circ les) and minimum (closed circ les) daily air temperatures and relative humidities (RH) during the experiments.

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72 CHAPTER 4 EFFECTS OF HYALURONIC ACID IN CULTURE AND CYTOCHALASIN B TREATMENT BEFORE FREEZING ON SURVIVAL OF CRYOPRESERVED BOVINE EMBRYOS PRODUCED IN VITRO Introduction In vitro production of embryos is an impor tant tool for improving genetic merit and fertility of cattle and is an indispensable co mponent of other technologies such as somatic cell cloning and transgenesis (Hansen and Block, 2004). One limitation to the widespread use of in vitro produced embr yos in the cattle industry is the poor survivability of in vitro produced embryos to cryopreservation. In vitro survival rates following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al ., 1999) and pregnancy rates following thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; AlKatanani et al., 2002a) are c onsistently lower for embryos produced in vitro when compared to embryos produced in vivo by superovulation. The poor survival of the in vitro produced embryo is associated with cultureinduced changes in ultrastructure (Rizos et al., 2002), gene expressi on (Bertolini et al., 2002a; Lazzari et al., 2002; Lonergan et al., 2003), and metabolism (Krisher et al., 1999; Khurana and Niemann, 2000b) that make it dist inct from the embryo produced in vivo. Among the metabolic changes are an increase in lipid content (Abe et al., 1999; Rizos et al., 2002) and this condition has been linked to poor freezability. Mechanical delipidation (Tominaga et al., 2000; Diez et al., 2001) and ad dition of inhibitors of fatty acid synthesis (De la Torre-Sanchez et al., 2005) can im prove survival following cryopreservation.

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73 In the current study, two approaches for enhancing survival of bovine embryos following cryopreservation were evaluated. Th e first was to culture embryos in the presence of hyaluronic acid. This unsulphate d glycosaminoglycan is present in follicular, oviductal and uterine fluids in several species including catt le (Lee and Ax, 1984). Receptors for hyaluronic acid (CD44) have been reported on the bovine oocyte, cumulus cell, and preimplantation stage embryo (Valca rcel et al., 1999). Addition of hyaluronic acid to culture medium has been reported to increase blastocy st re-expansion and hatching after freezing (Stoj kovic et al., 2002; Lane et al ., 2003). The second approach was to determine whether altering the cy toskeleton before cryopreservation would enhance embryo survival. The rationale for this treatment is that cryoinjuries such as intracellular ice formation and osmotic s hock induce irreversible disruption in microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) and that temporary depolymerization of actin micr ofilaments before cryopreservation could reduce cytoskeletal damage and plasma me mbrane fracture caused by alterations in cytoskeletal architecture (Dobrinsky, 1996). A ddition of cytochalasin B to cause actin depolymerization had no effect on survival of eight-cell embryos in the mouse (Prather and First, 1986) but enhanced survival of expanded and hatched blastocysts without effecting survival of morula and early blas tocysts in the pig (Dobrinsky et al., 2000). Materials and Methods Embryo Production Procedures, reagents, and media formulation for oocyte maturation, fertilization, and embryo culture were as previously de scribed (Roth and Hansen, 2005) with some modifications. Briefly, cumulus oocyte comple xes (COCs) were harvested from ovaries of a variety of breeds collected at a local abattoir located at a travel distance of

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74 approximately 1.5 h from the laboratory. The COCs were matured in Tissue Culture Medium-199 with Earle’s salts supplemented with 10% (v/v) steer serum, 2 g/mL estradiol 17, 20 g/ml follicle stimulating horm one, 22 g/ml sodium pyruvate, 50 g/ml gentamicin and an additional 1 mM gl utamine for approximately 22 h at 38.5C in an atmosphere of 5% (v/v) CO2 in humidified air. Inse mination with a cocktail of Percoll-purified spermatozoa from three different bulls was performed in In Vitro Fertilization – Tyrode’s Albumin Lactat e solution. At 8 – 12 h post-insemination (hpi), putative zygotes were denuded of cumulus cells by suspension in Hepes-TALP medium containing 1000 units/ml hyaluronidase type IV (Sigma, St Louis, MO, USA) and vortexing in a microcentrifuge tube for 5 min. Presumptive zygotes were then placed in groups of ~30 in 50 l microdrops of a modified S ynthetic Oviductal Fluid (SOF) prepared as described by Fisher-Brown et al (2002). Embryos were cultured at 38.5C in a humidified atmosphere of 5% (v/v) CO2, 5% O2, and with the balance N2. Blastocysts were collected for cryopreservat ion on day 7 after insemination. Experimental Design and Embryo Manipulation The experiment was a 2 x 2 factorial design to test main effects of hyaluronic acid during culture (+ or -) and cytochalasin B before cryopres ervation (+ or -). Data on development were obtained from 18 repl icates using 5022 oocytes while data on cryopreservation were obtained from 7 rep licates using a total of 197 blastocysts. Following insemination and transfer to fr esh microdrops, embryos cultured without hyaluronic acid were cultured in SOF for 7 days beginning after insemination. Embryos treated with hyaluronic acid were cultured in SOF until day 5 when all embryos were transferred to a fresh microdrop of SOF containing 6 mg/ml hyaluronic acid from Streptococcus zooepidemicus (Sigma).

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75 Blastocysts and expanded blastocysts were harvested on the morning of day 7 after insemination and washed twice in holding medi um consisting of Hepes-TALP (Parrish et al., 1989) containing 10% (v/v) fetal calf serum (FCS). Embryos treated with cytochalasin B were incubated for 10 mi n at 38.5oC in air while in Hepes-TALP containing 10% (v/v) FBS and 7.5 g/ml cytochalasin B (Sigma) in a 1.5 ml microcentrifuge tube (Tominaga et al., 2000). Cytochalasin B was initially dissolved in DMSO at a concentration of 5 mg/ml and wa s then added to HEPES-TALP to achieve a final concentration of 7.5 g/m l. Control embryos were incubated similarly in HEPESTALP containing 10% (v/v) FBS. Cryopreservation Procedures for freezing were modified from those reported elsewhere (Hasler et al., 1995; Enright et al., 2000). In brief, blastocysts were transferred in groups of 10 to a fresh 100 l microdrop of Hepes-TALP containing 10% FCS at 38.5oC for the time it took to harvest all embryos (~ 10 min). Next, em bryos in groups of 5 8 per treatment (hyaluronic acid or control) we re randomly selected to receive cytochalasin B treatment before freezing or not as described above. Af terwards, each group of 5 – 8 embryos was placed in a 50 l microdrop of 10% (v/v) glycerol in Dulbecco’s phosphate-buffered saline (DPBS) containing 0.4% (w/v) bovine serum albumin (freezing medium) in a grid plate over a slide warmer at 30oC. Within 10 min, embryos were loaded in a 50 l volume into 0.25 ml plastic straws (Agtech, Manhattan, KS). Up to 8 embryos were loaded in each straw. Two columns of 50 l freezing medi um separated by air bubbles were always placed above and below the column of embryos Straws were transferred to a freezing chamber (Cryologic Model CL5500 (Mulgrave, Victoria, Australia) for 2 min at -5oC and then ice crystals were induced by touching the straw where the top column of medium

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76 resided with a cotton plug that had been immersed in liquid nitrogen. After an additional 3 min at -5oC, embryos were cooled to -32oC at a rate of -0.6oC/min. After 2 min at 32oC, straws were directly immersed in liquid N2 and stored until thawing (4 days – 1 week later). Thawing and Determination of Survival Straws containing embryos were thawed by warming for 10 sec in air at room temperature and 20 sec in a 32oC water bath. All subsequent steps before culture were performed with media prewarmed to ~30oC and with dishes placed on a slide warmer set at 30oC. Embryos were then expelled into an empty petri dish and immediately transferred to a fresh 60 l drop of DPBS containing 6.6% (v/v) glycerol and 0.3 M sucrose in an grid dish. After 5 min, embr yos were sequentially transferred to DPBS containing 3.3% (v/v) glycer ol and 0.3 M sucrose for 5 min and DPBS + 0.3 M sucrose for 5 min. Embryos were then washed thre e times in HEPES-TALP + 10 % (v/v) FCS and placed into culture in groups of 58 in 25 l microdrops of SOF containing 10% (v/v) FCS. Culture was at 38.5C in a humidified atmosphere of 5% (v/v) CO2, 5% O2, and 90% N2. Re-expansion was determined at 48 h after thawing and hatching at 72 h. Statistical Analysis The proportion of oocytes that cleaved and the proportion of embryos that developed to the blastocyst stage on day 7 and day 8 were determined for each replicate. Treatment effects were determined by least-sq uares analysis of vari ance using the proc GLM procedure of SAS (SAS for Windows 90, Ca ry, NC). The model included the main effects of replicate and treatment. Data fo r the proportion of frozen/thawed embryos that re-expanded and on the proportion that hatche d by 72 h of culture were analyzed using the CATMOD procedure of SAS. The initia l model included all main effects and two-

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77 way interactions. After removing nonsigni ficant effects, the final model included replicate, hyaluronic acid, preparation prior to freezing (none, cytochalasin B), and the interaction of hyaluronic acid a nd preparation before freezing. Results Effect of Hyaluronic Acid on Embryonic Development As shown in Table 1, addition of hyaluroni c acid at day 5 after insemination caused a slight reduction in the yi eld of blastocysts on day 7 and day 8 after insemination regardless of whether data were expressed as the proportion of oocyt es developing to the blastocyst stage (P < 0.05) or the pro portion of cleaved embryos developing to the blastocyst stage (P < 0.01). Of the blas tocysts that were recovered, 62-68% were recovered at day 7 and the balance at day 8. There was no effect of hyaluronic acid on the proportion of blasto cysts collected at da y 7 (Table 4-1). Survival after Cryopreservation Overall, cytochalasin B increased th e percent of embryos that re-expanded following thawing (P < 0.0001) a nd that hatched following th awing (P < 0.05) (Table 42). Re-expansion rates were 51.2% (22/43) for embryos treate d with cytochalasin B and 18.2% (8/44) for embryos not subjected to cy tochalasin B. Hatc hing rates were 39.5% (17/43) for embryos treated with cytochal asin B and 4.5% (2/44) for embryos not subjected to cytochalasin B. While there was no significant effect of hyaluronic acid on cryosurvival, there was a tendency (P=0.09) for a hyaluronic acid x cyto chalasin B interaction affecting percent of blastocysts that hatched following thawing. This interaction re flects the fact that hyaluronic acid increased the percent hatching for embryos not subjected to cytochalasin B treatment and decreased percent hatched fo r embryos subjected to cytochalasin B.

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78 Discussion Of the two treatments evaluated for enhanc ing cryosurvival of in vitro produced bovine embryos, cytochalasin B treatment was the most effective as determined by an improvement in both embryo re-expansion and ha tching. The rationale for this treatment is to reduce cellular injury caused by di sruption in microtubules and microfilaments (Kuwayama et al., 1994; Fair et al., 2001) a nd to increase flexibility of the plasma membrane to allow it to tolerate forces a ssociated with freezing that lead to membrane damage. In other studies, addition of cytochal asin B had no effect on survival of eightcell embryos in the mouse (Pra ther and First, 1986), enhan ced survival of expanded and hatched pig blastocysts without effecting survival of mo rula and early blastocysts (Dobrinsky et al., 2000), and improved surviv al of in vivo derive d bovine blastocysts subjected to vitrification (Dobrinsky et al., 1995). For embryos not exposed to cytochalasin B, there was a tendenc y for those cultured in hyaluronic acid to have a higher re-expansion rate and hatching rate than embryos cultured without hyaluro nic acid. Both Stojkovic et al (2002) and Lane et al. (2003) reported improved survival rates to freezing when embryos were cultured in hyaluronic acid; such a beneficial e ffect has not always been observed (Furnus et al., 1998). Surprisingly, embryos cultured in hyaluronic acid were less likely to survive freezing than control embryos when the cytochalasin B treatment was applied. Perhaps physiological changes induced by hyaluronic ac id cause the embryo to be less able to adjust to the cellular actions of cytochalas in B. Those changes are potentially numerous because hyaluronic acid acts to affect cell function through several means including signaling through cell surface receptors, m odifying the biophysical properties of extracellular and pericellular matrices by at tracting water, and by interacting physically

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79 with a variety of ions and other molecu les (Laurent, 1987; Ruoslahti and Yamaguchi, 1991; Hardingham and Fosang, 1992; Yasuda et al., 2002; Toole et al., 2005). One possible mechanism by which hyaluronic acid could increase embryo survival to freezing is by increasing the total number of cells in the embryo (Sto jkovic et al., 2002; Jang et al., 2003; Kim et al., 2005) One unexpected finding was the reduction in the percentage of embryos that became blastocysts caused by hyaluronic acid. In other studies, hyaluronic acid either had no effect (Stojkovic et al., 2002; Lane et al., 2003) or caused an increase in blastocyst yield (Furnus et al., 1998; Jang et al., 2003). Differences in origin and concentration of hyaluronic acid could explain some of this difference between studi es. Hyaluronic acid can be isolated from different sources (ex., bacteria, rooster comb, and umbilical cord) and preparations can differ in protein, endotoxin, and nucleotide content (Shiedlin et al., 2004). Stojkovic et al. (2002) reported that preliminary re sults indicated that embryo development in vitro was dependent upon th e origin of the commercially-available hyaluronic acid. However, embryos cultured w ith hyaluronic acid experienced a change in culture medium at day 5 whereas contro l embryos did not. Such a difference could have obscured beneficial effects of hyaluro nic acid although anothe r paper indicates no effect of changing culture medium at 72 hpi on blastocyst yield in cattle (Ikeda et al., 2000). The percent of embryos that underwent ha tching after freezing in glycerol and thawing has varied from 0% (Enright et al., 2000) 22% (Diez et al., 2001; Nedambale et al., 2004a), 32% (Guyader-Joly et al., 1999) and 69% (Hasle r et al., 1997). The best survival achieved in this study was for em bryos cultured without hyaluronic acid and

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80 treated with cytochalasin B. In this group, 51.2% of cryopreserved embryos were capable of re-expansion and 39.5% hatched. It is likely that the percent hatching can be further improved by modifying post-thaw cult ure-conditions. Massi p et al. (1993) found hatching rates for frozen/thawed, in vitro pr oduced embryos were 41% when culture was performed in the presence of bovine oviductal epithe lial cells while ha tching rate using other culture conditions not i nvolving co-culture was 0-6%. Nonetheless, one would not expect optimal pregnancy rates to be achie ved following direct transfer of embryos frozen in glycerol even with the inclusion of cytochalasin B treatment. Rather, it is suggested that pregnancy rate s following transfer of embr yos cryopreserved using slowfreezing procedures can be optimized by se lecting embryos for transfer based on development in culture shortly after thawing. In contrast to the poor survival of in vitro-produced embryos frozen using conventional slow-freezing techniques, severa l experiments indicate that cryosurvival can be enhanced by using vitrification (Vajta, 2000). It remains to be tested whether survival of embryos produced in vitro after vitrif ication can be improve d by cytochalasin B treatment. There was a beneficial effect of cytochalasin B treatment on cryosurvival of embryos derived in vivo following vitrif ication (Dobrinsky et al., 1995). In conclusion, cytochalasin B treatment before freezing improved cryosurvival of bovine embryos produced in vitro and subjecte d to slow-freezing in glycerol. Such a treatment could be incorpor ated into methods for cryopr eservation of bovine embryos provided post-transfer survival is adequate. In contrast, culture with hyaluronic acid was of minimal benefit the increa sed cryosurvival in the absence of cytochalasin B was not sufficient to allow an adequate num ber of embryos to survive.

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81 Table 4-1. Effect of hyaluronic acid added at day 5 after insemination on production of blastocysts at day 7 a nd 8 after inseminationa,b. Culture medium Number of oocytes Percent cleaved Blastocysts/oocyte (%) c Blastocysts/cleaved embryo (%) c Percent of total blastocysts that were collected at day 7 Control 1935 76.0 + 0.9 36.0 + 1.2* 47.2 + 1.3** 68.8 + 2.4 Hyaluronic acid 3087 77.7 + 0.9 31.5 + 1.2 40.7 + 1.3 62.2 + 2.4 a n=18 replicates b Means within a column that differ significantly are indicated by (P < 0.05) and ** (P < 0.01) c Includes blastocysts collected at da y 7 and those collected at day 8. Table 4-2. Effect of culture in hyaluronic acid and treatment with cytochalasin B on survival after cryopreservation. a Culture medium Cytochalasin treatment Re-expansion by 72 hb Hatching by 72 hc Control Control 8/44 (18.2%) 2/44 (4.5%) Control Cytochalasin B 22/43 (51.2%) 17/43 (39.5%) Hyaluronic acid Control 16/55 (29.0%) 7/55 (12.7%) Hyaluronic acid Cytochalasin B 26/55 (47.3%) 12/55 (21.8%) a Data are the fraction of embryos, and in parentheses, percent. Number of replicates was 7. b Effect of cytochalasin B (P < .0001). c Effect of cytochalasin B (P < 0.05), hyaluronic acid (P < 0.10), and the cytochalasin B x hyaluronic acid interaction (P = 0.09).

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82 CHAPTER 5 GENERAL DISCUSSION As alluded to at the beginning of this thes is, there has been a pr ecipitous decline in fertility of dairy cows over the last 1040 years in North America (Butler, 1998), Ireland (Roche, 2000), Spain (Lpez-Gatius et al., 2003 ), and the United Kingdom (Royal et al., 2000). In addition, heat stress can compromise fe rtility in lactating dairy cows (Putney et al., 1989b; Al-Katanani et al., 1999). The purpos e of the present series of experiments described in the thesis was to 1) evaluate st rategies for enhancing fe rtility after AI using GnRH treatment (Chapter 2) and 2) further develop ET usin g in vitro produced embryos as a tool for increasing fertility by testing whether pregnancy rate could be improved by transfer of twin embryos (Chapter 3) a nd whether the developmental competence of embryos after cryopreservation could be improved by hyaluronan or cytochalasin B treatment (Chapter 4). Results indicated no c onsistent benefit of in jection of GnRH at Day 11-15 after anticipated ovulation or inse mination on pregnancy rates in heifers or lactating cows. While unilateral transfer of tw o embryos was not shown to be an effective treatment for increasing pregna ncy rate in recipients, the high pregnancy rates achieved in this study point to the pot ential usefulness of ET as a tool for enhancing fertility. Large-scale use of embryo transfer will requi re the ability to freeze embryos successfully. Results suggest that treatment of embryos with cytochalasin B before freezing is a promising tool for enhancing survival of embryos following cryopreservation. A large number of studies have been performed to te st the effect of GnRH administration after expected ovulation on fertility of cattle. Previous results indi cated that GnRH was

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83 sometimes effective at increasi ng pregnancy rate, but this bene ficial effect was often not observed (Peters et al., 2000). Despite th is knowledge, we chose to reevaluate the effectiveness of GnRH treatment because of a report that GnRH treatme nt at Day 11 after estrus increases pregnancy rate s in lactating cows exposed to heat stress (Willard et al., 2003). Accordingly, it was hypothesized in Chapter 2 that the beneficial effect of GnRH treatment would be greater during the summer than winter. This may be so because the antiluteolytic process may be compromised by heat stress because of decreased growth of the filamentous stage conceptus (Biggers et al., 1987) and increased uterine PGF2 secretion from the uterus (Wolfenson et al., 1993). Overall, the results of GnRH treatment we re generally negative. For treatment at Day 11, a positive effect of GnRH on fertil ity was never seen. This was the case for heifers and lactating cows subjected to AI or whether animals were exposed to heat stress or not (Chapter 2; experiment 1 and 2). Treatment of lacta ting recipients with GnRH at Day 11 also failed to increase pregnancy rate dur ing heat stress in ET recipients (Chapter 3, experiment 2). Effectiveness of treatmen t with GnRH or its analogues at 11 to 12 d after estrus for inseminated, heat-stressed lact ating cows has yielded variable results, as some reports indicated a positive effect (She ldon et al., 1993; Willard et al., 2003), while others indicated no effect (Jubb et al., 1990). Also, admini stration of GnRH at Day 11 after anticipated ovulation tended to increase pregnancy and calving rates in lactating Holstein embryo transfer reci pients exposed to heat stre ss (Block et al., 2003). One factor that could influence the effec tiveness of GnRH treatment at Day 11 is the number of follicular waves that a female experiences during an estrous cycle. Females with estrous cycles characterized by three follicular waves have larger second-wave

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84 dominant follicles at Day 11 than females w ith two-wave cycles (Ginther et al., 1989; Savio et al., 1990; Ko et al., 1991) Given that a follicle must reach 10 mm in diameter to ovulate in response to LH (Sartori et al., 2001), the preponderance of cycle type (twowave vs three-wave) within a herd may dete rmine effectiveness of GnRH treatment at Day 11. In one experiment (Chapter 2; experime nt 3), administration of GnRH at Day 14 after anticipated ovulation in cows subject ed to TAI increased pregnancy rates of lactating cows in the summer and winter at two locations. In the following year, though, GnRH failed to improve fertility when treatm ent was administered either at day 14 in cows subjected to TAI (experiment 4) or at day 14 or 15 in cows previously diagnosed coming in estrus (experiment 5). It is impor tant to recognize that GnRH treatment should improve fertility only when triggering luteinization or ovulation of developing (estrogenic) follicles. Thus, there are at leas t two possible reasons for a lack in response upon GnRH treatment at day 14 or 15. One possi bility relates to the timing of ovulation relative to the GnRH treatment and whether these animals failed to ovulate after being diagnosed as coming into estrus. Although af ter observing estrus one does not expect ovulation to fail, this expression does not necessarily mean that subsequent ovulation occurred (Lpez-Gatius et al., 2005b) and inse mination after a false identified estrus often occurs (Heersche and Nebel 1994). According to Lpez-Gatius et al. (2005b), the risk of cows failing to ovulate (12%) duri ng the summer was greater than in the cool period (3%). During experiment 3 all cows received a GnRH injection at 72 h following PGF2 to insure an ovulation of the synchronized dominant follicle. Perhaps, the positive

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85 GnRH effect observed during experiment 3 was masked in the following experiment because cows did not receive an additional GnRH dose at estrus to ensure subsequent ovulation. According to Lopez-Gatius et al. ( 2005a), there is evid ence demonstrating the benefits upon GnRH treatment when given on the day of insemination compared to controls (30.8% vs. 20.6%), but conception rates were grea ter if cows received an additional dose at day 12 post-inseminati on (35.4%). On the other hand, when GnRH treatment took place on day 15 to ensure a re sponsive (estrogenic) dominant follicle would ovulate at the time of GnRH treatment it failed to improve fertility as well. Similarly, in a recent study (Bartolome et al., 2005) there was no effect of GnRH treatment on pregnancy rates of lactating cows when administered either on day 15 or day 5 and 15 after TAI. It remains possible that inconsistency in effects of GnRH treatment is caused in part by the low number of animals per treat ment group. The pitfalls associated with interpretation of experiments with low numb ers has been discussed (Dransfield et al., 1998) and could be responsible for the variation in results for trials to test effects of GnRH on pregnancy rates in embryo transfer recipients and for inseminated cows. With an existent variation among trials regarding the use of GnRH at day 11-15 post-insemination, one could specu late that such inconsistenc y regarding treatment is due to the fact that herds of cattle determine th e result that an experiment achieves. However, our results indicate that such a hypothesis is not likely becau se when an experiment was replicated the next year using the same he rd, GnRH treatment once again proved to be inconsistent in improving pregnancy rates.

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86 According to Thatcher et al. (2005), hCG results in a more prolonged rise in LH activity than is achieved fo llowing GnRH treatment. Perhap s the likelihood of ovulating or luteinizing the dominant follicles presen t at the time of treatment would be higher using hCG. Although low numbers of insemina ted animals were used (n=8; n=49) hCG treatment on d 14 after estrus improved pregnancy rates (Rajamahendran and Sianangama, 1992; Sianangama and Rajamahe ndran, 1992). Use of hCG warrants further investigation for any additional effect or response during the summer to enhance pregnancy rates of lactating cows. Recent work has focused on use of ET to bypass early embryonic death (Putney et al., 1989b; Ambrose et al., 1999; Al-Katanani et al., 2002a). Given that ET can be more effective at increasing pregnanc y rates than AI for lactating cows during periods of heat stress (Putney et al., 1989b; Ambrose et al., 1999; Drost et al., 1999; Al-Katanani et al., 2002a), the potential benefit of ET can be realized. For ET to become an economical alternative to AI on a wide scale basis in co mmercial herds, embryos must be inexpensive to produce (Hansen and Block et al., 2004) Although embryos produced using IVP systems are relatively inexpe nsive as compared to embr yos produced by superovulation, pregnancy rates achieved following transfer of an IVP embryo are often less than what is obtained following transfer of an embryo produced by superovulation (Hasler et al., 1995; Agca et al., 1998; Ambrose et al., 1999; Al-Katanani et al., 2002a). In addition, IVP embryos are less likely to survive freezing than superovulated embryos (Hasler et al., 2003), likely due to their increa sed lipid content (Abe et al ., 1999; Rizos et al., 2002). Accordingly, the second approach for the thesis focused on improvements in ET by

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87 comparing pregnancy rates following the tran sfer of two embryos compared to one and by increasing the viability of embryos that were cryopreserved. The first effort was to determine whether transfer of two IVP embryos into the uterine horn ipsilateral to the CL could increase pregnancy rates during periods of heat stress. It was hypothesized that such a treatment might incr ease pregnancy rates because the likelihood is increased that the cow receives at least one embryo competent for sustained development. In addition, the tran sfer of two embryos into the ipsilateral uterine horn is likely to incr ease the amounts of interferonand other embryonic signaling molecules in the uterus needed to ma intain pregnancy and prevent luteolysis. Transferring two embryos in to the uterine horn ipsilateral to the CL failed to increase pregnancy rates. Instead, the transf er of two embryos into recipients led to pregnancy loss, which occurred earlier fo r heifers than for cows. The most likely explanation for the increased frequency of pregnancy loss in recipients receiving two embryos is uterine crowding, with the effect s of crowding occurring sooner in gestation for nulliparous animals than for multiparous animals. Regardless of whether twin transfers were performed via bilateral or unilateral placement, similar results were obtained in another study (Ande rson et al., 1979). In contra st, calving rate and twinning rate in heifers was lower for unilateral twin transfers than for bilateral transfers. Similarly, Rowson et al. (1971) also found lo wer embryonic survival rates and twinning rates for recipients of unilateral twin transfers than for recipients of bilateral transfers in heifers. It is evident that uterine cap acity can vary between herds of cattle. Thus, there were no differences in pregnancy success between recipients of twin embryos placed

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88 unilaterally or bilaterally for heifers (Sr eenan et al., 1989, Reiche nbach et al., 1992) or cows (Sreenan et al., 1989). Similarly, embryoni c survival rate for beef cows selected for twinning was similar for those having un ilateral or bilatera l multiple ovulations (Echternkamp et al., 1990). In lactating dairy cows, in cont rast, the likelihood of a twin pregnancy resulting from multiple ovulations going to term was higher if ovulations occurred bilaterally than if unilateral ovul ations occurred (Lpez-Gatius and Hunter, 2005). Perhaps, identification of the biologi cal processes controlling uterine capacity will lead to new approaches for increasing th e efficacy of producing twins in cattle. An additional limitation to the widespread use of IVP embryos in cattle is their poor survival following cryopreservation. In vitro survival rates following thawing (Pollard and Leibo, 1993; Enright et al., 2000; Khurana and Niemann, 2000a; Diez et al., 2001; Guyader-Joly et al., 1999) and pregnancy rates follo wing thawing and transfer (Hasler et al., 1995; Agca et al., 1998; Ambr ose et al., 1999; Al-Kat anani et al., 2002a) are consistently lower for IVP embryos when compared to embryos produced in vivo by superovulation. The percent of embryos that underwent ha tching after freezing in glycerol and thawing has varied from 0% (Enright et al ., 2000), 22% (Diez et al., 2001; Nedambale et al., 2004a), 32% (Guyader-Joly et al., 1999), and 69% (Hasler et al., 1997). Of the two treatments evaluated for enhancing cryosurvi val of IVP bovine embryos, cytochalasin B treatment was the most effective as dete rmined by an improvement in embryo reexpansion and hatching rates. In this tr eatment, 51.2% of cryopreserved embryos were capable of re-expansion and 39.5% hatched. No netheless, one would not expect optimal

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89 pregnancy rates to be achieved following direct transfer of embryos frozen in glycerol even with the inclusion of cytochalasin B treatment. In contrast to the poor survival of IV P embryos frozen using conventional slowfreezing techniques, several experiments indica ted that embryo survival can be enhanced following vitrification (Vajta, 2000) It remains to be tested whether survival of embryos produced in vitro after vitrific ation can be improved by cytoch alasin B treatment. Rather, it is suggested that pregnancy rates followi ng transfer of embryos cryopreserved using slow-freezing procedures can be optimized by selecting embryos for transfer based on development in culture shortly after thawing. Indeed, fertility issues will continue to dr ive new ideas for developing strategies to improve or at least reduce undesirable con ception and pregnancy rates in any cattle operation. Efficiency among cattle operations is of major interest and ET has the potential to be the vehicle th at can help overcome some fer tility issues associated with oocyte developmental competence, fertiliz ation, and early embryonic development. However, the potential this reproductive technology has is underestimated when pregnancy rates continue to be less than AI during the absence of heat stress. Further research that identifies embryos that are more likely to survive following transfer and establish a pregnancy is warranted. In conclusion, GnRH treatment did not c onsistently increase pregnancy rates when administered at Day 11-15 after insemination and is not recommended as a fertilityenhancing treatment. Similarly, tr ansfer of two embryos to th e uterine horn ipsilateral to the CL was not an effective method for increas ing pregnancy rates in recipients. Transfer of cryopreserved embryos may be enhanced by treatment of embryos with cytochalasin B

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90 since this molecule increased in vitro surviv al. Since several experi ments indicate that cryosurvival can be enhanced using vitrifi cation (Vajta, 2000), it remains to be tested whether survival of IVP embryos after vitr ification can be improved by cytochalasin B treatment.

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91 LIST OF REFERENCES Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. Ultrastructure of bovine embryos developed from in vitro-matured and fertil ized oocytes: comparative morphological evaluation of embryos cultured either in serum-free medium or in serum supplemented medium. Mo l Reprod Dev 1999; 53:325-35. Abilay TA, Johnson HD, Madan M. Influen ce of environmental heat on peripheral plasma progesterone and cortisol during th e bovine estrous cycle. J Dairy Sci 1975; 58:1836-40. Achard D, Gilbert M, Bnistant C, Slam a SB, DeWitt DL, Smith WL and Lagarde M. Eicosapentaenoic and docosahexaenoic acid s reduce PGH synthase 1 expression in bovine aortic endothelial cells. Bi och Biophy Res Comm 1997; 241:513–18. Adam CL, Findlay PA, Moore HA. Eff ects of insulin-like growth factors-I on luteinizing hormone secretion in sh eep. Anim Reprod Sci 1997; 50:45-56. Agca Y, Monson RL, Northey DL, Abas Mazni O, Schaeffer DM, Rutledge JJ. Transfer of fresh and cryopreserved IVP bovine embryos: normal calving, birth weight and gestation lengths. Theriogenology 1998; 50:147-62. Ahlswede L, Lotthammer KH. Studies on a spec ific vitamin A-unrelated effect of beta carotene on the fertility of ca ttle. 5. Studies of organs (ovaries, corpora lutea, liver, fatty tissues, uterine secretion, adrena l glands--determination of weight and content] Dtsch Tierarztl Wochenschr 1978; 85:7-12. Ahmad N, Townsend EC, Dailey RA, Inskeep EK Relationships of hormonal patterns and fertility to occurrence of two or th ree waves of ovarian follicles, before and after breeding, in beef cows and he ifers. Anim Reprod Sci 1997; 49:13-28. Akordor FY, Stone JB, Walton JS, Leslie KE, Buchanan-Smith JG. Reproductive performance of lactating Hols tein cows fed supplemental -carotene. J Dairy Sci 1986; 69:2173-8. Al-Katanani YM, Webb DW, Hansen PJ. Fact ors affecting seasonal variation in 90 day non-return rate to first service in lactating Holstein cows in a hot climate.J Dairy Sci 1999; 82:2611-5. Al-Katanani YM, Drost M, Monson RL, Rutledge JJ, Krininger-III CE, Block J, Thatcher WW, Hansen PJ. Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions. Theriogenology 2002a; 58:171-82.

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123 BIOGRAPHICAL SKETCH C. Moiss Franco was born in 1979 in Sa nta Cruz, Bolivia. He is the youngest of three brothers (Inj. Oscar Antonio Franco; In j. Jorge Mauricio Franco) and three sisters (Dra. Rosario Franco; Mara Isabel Franco; Arq. Erika Lorena Franco). He is the son of Antonio V. Franco Monasterio (may god ble ss his soul) and Mercedes Yolanda Vaca ElHage. He graduated from La Salle High School in the same city in 1997 and enrolled the next year in the Department of Animal Science at the University of Arkansas in Fayetteville, USA, where he received his Bach elor of Science degree in animal science in 2001. During 2002 he did an internship in the Scottish Agricultural College with Dr Tom McEvoy. He enrolled in the graduate program of the Department of Animal Sciences at the University of Florida under supervision of Dr. Peter J. Hansen in January, 2003. He is currently a Master of Science candidate. Upon completion of his degree, he will open an embryo transfer company. In the near futu re he plans to resume his studies through pursuit of the Doctor of Philosophy degree at the University of Florida under Dr P.J. Hansen.