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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 |