<%BANNER%>

Regulation of Prostaglandin F2alpha Biosynthesis by Long Chain Fatty Acids in Cattle

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PAGE 1

REGULATION OF PROSTAGLANDIN F2 BIOSYNTHESIS BY LONG CHAIN FATTY ACIDS IN CATTLE By CARLOS J. RODRGUEZ SALLABERRY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Carlos J. Rodrguez Sallaberry

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Este trabajo se lo quiero dedicar a las dos L ourdes de mi vida: Lourdes Antonia, fuente de inspiracin, lucha y sacrific io; Lourdes Paola, quien con su mera existencia me da fuerzas para seguir adelan te todos los das y repr esenta lo mejor de m.

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iv ACKNOWLEDGMENTS I like to express my gratitude to Dr. Lokenga Badinga, for his guidance throughout the process of completing this project, including reading and editing this dissertation, and for the excellent scientific and intellectual tr aining I received in hi s laboratory during this time. I thank him for being an excellent mentor and advisor, by being there every time I needed advice and setting a great example for the perfect balance between work and family. Acknowledgements are extended to Dr. Charles Staples, Dr. William Buhi, and Dr. Ramon Littell for serving as members of my supervisory committee and for their suggestions and insights that helped towards the completion of this study. I would also like to thank David Arms trong and the whole crew of the DRU, especially Mary, Eric, and Jerry; without th eir assistance and coope ration the trial would not have been a success. I want also to thank Werner Collante for his help in the laboratory and throughout the project. I wish to give special thanks to Dr. Andrs Kowalski, Jeremy Block, Dervin Dean, Mar a B. Padua, and Elizabeth Johnson-Greene; their help and friendship is going be eternally appreciated. Gratitude and love are extended to my da ughter, Lourdes, and my family for their neverending support in all my endeavors. Lastly, I want to thank my best friend a nd love, Cristina, for being there for me every time I needed a push and words of encouragement.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix ABBREVIATION KEY...................................................................................................xii ABSTRACT.......................................................................................................................xv CHAPTER 1 INTRODUCTION........................................................................................................1 2 LITERATURE REVIEW.............................................................................................5 Estrous Cycle................................................................................................................5 Neuro-Endocrine System.......................................................................................6 Stages of the Estrous Cycle...................................................................................7 Follicular Development during the Estrous Cycle................................................8 Ovulation and Development of the Corpus Luteum............................................11 Prostaglandin Biosynthesis a nd Luteolysis in Cattle..................................................15 Prostaglandin Biosynthesis..................................................................................16 Luteolysis in Cattle..............................................................................................17 Pregnancy Establishment in Domestic Ruminants.....................................................19 Energy Balance and Fertility Responses in Postpartum Dairy Cows.........................21 Energy Balance in Tr ansition Dairy Cows..........................................................21 Fertility Responses in Postpartum Dairy Cows...................................................24 Effects of Dietary Fats on Re productive Response in Cattle.....................................29 Regulation of Prostaglandin F2 Synthesis by Polyunsaturated Fatty Acids......39 Conjugated Linoleic Acid and Reproduction......................................................43 3 EFFECTS OF POLYUNSATURATED FATTY ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS........................................................................................................................46 Introduction.................................................................................................................46 Materials and Methods...............................................................................................49

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vi Materials..............................................................................................................49 Cell Culture and Treatment.................................................................................50 PGF2 Radioimmunoassay..................................................................................51 RNA Isolation and Analysis................................................................................51 Western Blot Analysis of PGHS-2, PGES and PPAR ......................................52 Statistical Analyses..............................................................................................53 Results........................................................................................................................ .54 Discussion...................................................................................................................55 Summary.....................................................................................................................56 4 EFFECTS OF CONJUGATED LINO LEIC ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS.........................................70 Introduction.................................................................................................................70 Materials and Methods...............................................................................................72 Materials..............................................................................................................72 Cell Culture and Treatment.................................................................................73 Statistical Analyses..............................................................................................73 Results........................................................................................................................ .74 Discussion...................................................................................................................75 Summary.....................................................................................................................76 5 EFFECTS OF CIS AND TRANS -OCTADECENOIC ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS........................................................................................................................85 Introduction.................................................................................................................85 Materials and Methods...............................................................................................87 Materials..............................................................................................................87 Cell Culture and Treatment.................................................................................88 Statistical Analyses..............................................................................................88 Results........................................................................................................................ .89 Discussion...................................................................................................................89 Summary.....................................................................................................................91 6 EFFECTS OF DIETARY TRANS FATTY ACIDS ON PROSTAGLANDIN F2 CONCENTRATIONS IN POSTPARTUM HOLSTEIN COWS............................100 Introduction...............................................................................................................100 Materials and Methods.............................................................................................105 Materials............................................................................................................105 Cows and Diets..................................................................................................105 Collection of Blood Samples.............................................................................108 Metabolite and PGFM Assays...........................................................................108 Ultrasonography................................................................................................110 Statistical Analyses............................................................................................110 Results.......................................................................................................................110

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vii Production Responses........................................................................................110 Metabolic Responses.........................................................................................112 Reproductive Responses....................................................................................113 Discussion.................................................................................................................114 Summary...................................................................................................................118 7 GENERAL DISCUSSION.......................................................................................135 LIST OF REFERENCES.................................................................................................141 BIOGRAPHICAL SKETCH...........................................................................................171

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viii LIST OF TABLES Table page 6-1 Incidence of health disorders of heif ers and cows fed diet s containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA)...........................119 6-2 Fatty acid profile according to the ma nufacturers of a highly saturated fat (RBF; Cargill, Minneapolis, MN) and a Ca salt lipid enriched in trans C18:1 ( t FA).........120 6-3 Ingredient composition of pr epartum and postpartum diets...................................121 6-4 Chemical composition of pr epartum and postpartum diets....................................122 6-5 Performance of lactating heifers a nd cows fed a diet containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA) at wk 3 postpartum..............................................................................................................123 6-6 Distribution of follicles* in lactating heifers and cows fed a diet containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA)................124

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ix LIST OF FIGURES Figure page 3-1 Effect of phorbol 12, 13 dibut yrate (PDBu) on prostaglandin F2 (PGF2 ) secretion in bovine endo metrial (BEND) cells........................................................58 3-2 Effect of phorbol 12, 13 dibutyrate (PDBu) on pr ostaglandin endoperoxide synthase (PGHS-2) mRNA abundance in bovine endometrial (BEND) cells.........59 3-3 Effect of phorbol 12, 13 dibutyrate (PDBu) on pr ostaglandin endoperoxide synthase (PGHS-2) protein levels in bovine endometrial (BEND) cells.................60 3-4 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostagl andin E synthase (PGES) mRNA abundance in bovine endometrial (BEND) cells.........................................61 3-5 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostagl andin E synthase (PGES) protein levels in bovine e ndometrial (BEND) cells.................................................62 3-6 Effect of fatty aci ds on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells.........................................63 3-7 Effect of fatty acids on prostagla ndin endoperoxide synt hase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDB u) in bovine endometrial (BEND) cells.......................................................................................................................... .64 3-8 Effect of fatty acids on prostaglandi n endoperoxide syntha se (PGHS-2) protein response to phorbol 12, 13 dibutyrate (PDB u) in bovine endometrial (BEND) cells.......................................................................................................................... .65 3-9 Effect of fatty acids on prostagla ndin E synthase (PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovi ne endometrial (BEND) cells.................66 3-10 Effect of fatty acids on prostaglandin E synt hase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovi ne endometrial (BEND) cells.................67 3-11 Effect of fatty acids on peroxiso me proliferator-ac tivated receptor (PPAR ) mRNA response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells............................................................................................................68

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x 3-12 Effect of fatty acids on peroxiso me proliferator-ac tivated receptor (PPAR ) protein response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells............................................................................................................69 4-1 Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrate (PDB u) in bovine endometrial (BEND) cells.......................................................................................................................... .78 4-2 Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin endoperoxide synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................79 4-3 Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin E synthase (PGES) mRNA response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells............................................................................................................80 4-4 Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin endoperoxide synthase (PGHS-2) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................81 4-5 Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin E synthase (PGES) protein response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells............................................................................................................82 4-6 Effect of c 9, t 11 and t 10, c 12 CLA isomers on peroxisome proliferator-activated receptor (PPAR ) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................83 4-7 Effect of c 9, t 11 and t 10, c 12 CLA isomers on peroxisome proliferator-activated receptor (PPAR ) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................84 5-1 Effect of cis and trans isomers of octadecenoic acid on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrat e (PDBu) in bovine endometrial (BEND) cells............................................................................................................93 5-2 Effects of cis and trans isomers of octadecenoic acid on prostaglandin endoperoxide synthase (P GHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells..........................................................94 5-3 Effects of cis and trans isomers of octadecenoic acid on prostaglandin endoperoxide synthase (PGHS -2) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells..........................................................95 5-4 Effects of cis and trans isomers of octadecenoic acid on prostaglandin E synthase (PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................96

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xi 5-5 Effects of cis and trans isomers of octadecenoic acid on prostaglandin E synthase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells............................................................................97 5-6 Effects of cis and trans isomers of octadecenoic acid on peroxisome proliferator-activated receptor (PPAR ) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells.........................................98 5-7 Effects of cis and trans isomers of octadecenoic acid on peroxisome proliferator-activated receptor (PPAR ) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells.........................................99 6-1 Average dry matter intake (DMI) as a percentage of body weight (BW) of periparturient Holstein heifers (A ) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet........................................................................................125 6-2 Average body weight (BW) of peripartur ient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet.............................................126 6-3 Calculated energy balance by week relati ve to parturition for Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet................127 6-4 Average body condition score (BCS) of periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet.............................128 6-5 Temporal patterns of milk yield by peri parturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet.......................................129 6-6 Average feed efficiency as a function of milk yield over intake of periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet...................................................................................................130 6-7 Plasma NEFA concentrations by week relative to calving in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet...................................................................................................131 6-8 Plasma BHBA concentrations by week relative to calving in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet...................................................................................................132 6-9 Plasma glucose concentrations by week relative to calving in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet...................................................................................................133 6-10 Plasma PGFM concentrations by week relative to calving in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet...................................................................................................134

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xii ABBREVIATION KEY AA Arachidonic acid Ang Angiotensin ANPT Angiopoietin BEND cells Bovine endometrial cells BHBA Beta hydroxybutyric acid CAT-1 Carnitine acyl transferase CL Corpus luteum CLA Conjugated linoleic acid DHA Docosahexaenoic acid E2 Estrogen EC Endothelial cells EPA Eicosapentaenoic acid ER Estrogen receptor ET-1 Endothelin-1 FGF Fibroblast growth factor FSH Follicle-stimulating hormone GH Growth hormone GnRH Gonadotropin-releasing hormone IGF Insulin-like growth factor IFNInterferon gamma

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xiii IFNInterferon tau IL-1 Interleukin JAK/STAT Janus kinase signa l transducer and activator of transcription LA Linoleic acid LCFA Long-chain fatty acid LH Luteinizing hormone LNA Linolenic acid MUFA Monounsaturated fatty acid NEB Negative energy balance NEFA Non-esterified fatty acid OT Oxytocin OTR Oxytocin receptor P4 Progesterone PDBu Phorbol-12,13-dibutyrate PG Prostaglandin PGG Prostaglandin G PGE2 Prostaglandin E2 PGES Prostaglandin E synthase PGF2 Prostaglandin F2 PGFS Prostaglandin F synthase PGFM PGF2 metabolite (13,14-dihydro-15-keto prostaglandin F2 )

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xiv PGH2 Prostaglandin H2 PGHS Prostaglandi n endoperoxide synthase PGI2 Prostaglandin I2 (Prostacyclin) PKC Protein kinase C cPLA2 Cytosolic phospholipase A2 PLC Phospholipase C PPARs Peroxisome proliferat ors-activated receptors PPRE PPAR response element PUFA Polyunsaturated fatty acid SFA Saturated fatty acid TAG Triacylglycerol TNFTumor necrosis factor VEGF Vascular endot helial growth factor

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xv Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy REGULATION OF PROSTAGLANDIN F2 BIOSYNTHESIS BY LONG CHAIN FATTY ACIDS IN CATTLE By Carlos J. Rodrguez Sallaberry May, 2006 Chair: Lokenga Badinga Major Department: Animal Sciences A series of in vitro experiments and an in vivo experiment were conducted to examine the effects of long-chain fatty acids (LCFA) on PGF2 biosynthesis in cattle. Treatment of bovine endometrial (BEND) ce lls with phorbol 13, 14-dibutyrate (PDBu) resulted in induction of PGF2 secretion. The PDBu-induced PGF2 secretion coincided with increased PGHS-2 mRNA and protein expression. There was no evidence for PDBu modulation of PPAR mRNA or protein synthesi s in cultured BEND cells. Priming of BEND cells with ST, LNA, and EPA reduced PGF2 response to PDBu by 17%, 14%, and 66%, respectively. Both sa turated and unsaturated fatty acids had no detectable effects on PGHS-2, PGES or PPAR mRNA response to PDBu. Similarly, supplementation of BEND cells with cis -9, trans -11 or trans -10, cis -12 CLA isomers greatly decreased PGF2 response to PDBu. Co-incubation with both CLA isomers increased PGHS-2 and PPAR mRNA abundance in PDBu-stimulated BEND cells, suggesting that these fatty acids alter PGF2 production through a mechanism that does

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xvi not require repression of PGHS-2 or PPAR gene expression. Priming of BEND cells with cis -9, trans -9, cis -11, and trans -11 isomers of octadecenoic acid further enhanced PGF2 and PGHS-2 mRNA response to PDBu. Pre-incubation of BEND cells with cis and trans monounsaturated fatty acids decreased PGES mRNA response to PDBu. None of the fatty acids studied altered PPAR mRNA or protein levels. Feeding trans fatty acids to primiparous or multiparous Holstein cows did not induce any significant alterations in production or metabolic responses when compared to supplementation with saturated fatty acids. Dietary supplementation of cows with t FA significantly increased plasma PGFM concentra tion within the first week of lactation. Whether this augmentation in PGF2 production results in improved fertility and immune competency warrants further research.

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1 CHAPTER 1 INTRODUCTION In recent years, the effect of nutrition on reproduction ha s generated much attention and has become increasingly important in the da iry industry. In the past decade, genetic selection for high milk production has been associated to a decrease in reproductive efficiency in lactating dairy cows (Butler, 2000). Poor reproductive efficiency includes early embryonic loss (Thatcher et al., 1995), im paired ovarian cyclicity and low fertility rates (Butler, 2000), which collectively result in reduced milk production (Plaizier et al., 1997). Early lactating dairy cows have highe r energy requirements than can be supported by dietary energy intake, which creates a ne gative energy state and leads to impaired reproductive function (Butler, 2000). To discuss the effects of nutrition on repr oduction during early pregnancy, it is very important to understand the changes brought abou t by lactation. The most critical period of lactation is considered to be the trans ition period, which extends from 3 weeks before calving to 3 weeks after part urition. During this period, dramatic metabolic changes (homeorhesis) take place in order to suppor t lactation. Parturition and the onset of lactation cause an abrupt shif t in nutritional requirements. Essentially all of the energy that is consumed by the lactating animal is used by mammary tissue for milk production, leaving an insignificant amount of energy to be distributed for other physiological processes (Bell, 1995). Hence, the lacta ting animal experiences a state of negative energy balance (NEB). This NEB represents a state of undernutri tion, which results in massive mobilization of fat from adipose tissue, increasing plasma levels of non-

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2 esterified fatty acids (NEFA). This massive fat mobilization, in combination with reduced energy intake (dry matter intake), re sults in loss of body c ondition of lactating animals. Extensive data have shown that the aforementioned conditions (NEB and loss of body condition) invariably affect reproducti ve efficiency in lactating dairy cows. The mechanisms by which the periparturie nt metabolic upsets result in reduced reproductive efficiency are not well underst ood. However, available evidence indicates that reproductive efficiency is dependent on the growth and development of a viable oocyte that can then be fertilized. This is directly dependent on a normal estrous cycle characterized by folliculogenesis, ovulation, corpus luteum (CL) formation and regression (~21 days). It has been shown that with inappropri ate nutrition, ovarian cyclicity may cease. The estrous cycle consists of two major phases: the follicular phase (formation of preovulatory follicles) and lute al phase (CL formation and regression). These two phases are controll ed through the action of gona dotropins (FSH and LH), which in turn are regulated by the ac tion of hypothalamic gonadotropin releasing hormone (GnRH) on the pituitary. Therefore, nutrition can affect reproductive efficiency through modulation of the hypothalamic-pituitary ax is and/or direct effects at the ovarian level. The hypothalamic-pituitary axis is essen tial in the modulation of gonadotropins secretion, which in turn plays a pivotal role in control of th e estrous cycle. Even though FSH is critical for follicular growth, LH pulsatility is responsible for normal ovarian activity since it is critical fo r ovulation. However, results from numerous studies have been inconsistent establishing the relationship between nutriti onal status and secretion of gonadotropins.

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3 Metabolic upsets, like NEB, have been shown to change the profile of metabolic hormones such as growth hormone (GH), insuli n, and insulin-like gr owth factor I (IGFI), all of which play an important role in control of follicular development in cattle. Therefore, it is likely that ch anges in any of these factor s due to NEB could alter the pattern of ovarian follicular growth a nd development during early postpartum period resulting in reduced reproductive efficiency. Several studies have shown that animals in a less severe NEB state have increased leve ls of plasma insulin and IGF-I, which coincide with a gonadotropin-independent e nhancement of follicu lar growth. These hormones can also influence steroidogenic activity of growing follicles, which is critical for recruitment, selectio n, dominance and ovulation. Long-chain fatty acids (LCFA) are generally added to dairy rations to increase the energy density of the diet. It is expected that supplementati on of the diet with fatty acids may enhance reproductive efficiency by enhanci ng the energy status of the dairy cow. However, recent studies indi cate that dietary fatty aci ds may affect reproductive efficiency in farm animals through an ener gy-independent mechanism. One way that fatty acids could enhance reproductive e fficiency would be through regulation of prostaglandin biosynthesis. Prostaglandin production can be influenced by nutrition since the precursor for the biologically ac tive prostaglandin of the two series is arachidonic acid (AA), an n-6 fatty acid s ynthesized from elongation/desaturation of linoleic acid (LA). Prostaglandi ns (PG) of the 2 series (PGF2 PGE2) have been implicated in the process of reproduction, including ovulation, follicular development, and corpus luteum functions. Hence, any effects of fatty acids on PGF2 synthesis are likely to affect overall reproductive performance.

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4 In summary, it is well established that the energy state of the cow during early lactation can influence reproduc tive efficiency by modulating the estrous cycle. This could be through the neuroendocrine modula tion of gonadotropin secretion, specifically LH pulsatility (hypothalamic-p ituitary axis), although more consistent evidence is needed. Alternatively, local ovarian eff ects could take place through modulation of follicular growth and development by metabolic hormones (IGF-I and insulin), which could determine oocyte quality and viability and steroidogenic activity of the CL. However, energy-independent effects ma y also be observed after fatty acid supplementation, which could involve prostaglan din biosynthesis. Therefore, nutrition can influence reproductive efficiency in dair y cows not only by altering the energy status of the animal but also by influencing factor s involved in the regul ation of reproductive processes like follicular dynamics, ovulati on, CL function and embryo survival among others. The goal of this research project was to examine the physiological effects of supplemental fatty acids on endometrial PGF2 production in cattle. This dissertation begins with a brief overview of the physio logy of reproduction and how dietary fatty acids affect energy balance and reproductiv e processes in cat tle (Chapter 2). Experiments described in Chapters 3 and 4 were designed to eluc idate endometrial PGF2 responses to omega-6 and omega-3 fatty acids (Chapter 3) and to cis and trans conjugated isomers of linoleic aci d (Chapter 4). Experiments described in Chapters 5 and 6 evaluated the effects of trans fatty acids on bovine endometrial PGF2 production in vitro (Chapter 5) and in vivo (Chapter 6). The dissertati on is concluded with a general discussion of the major findings of th is research project (Chapter 7).

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5 CHAPTER 2 LITERATURE REVIEW Estrous Cycle The onset of puberty is regarded as the st art of the reproductiv e life of a female, when she enters a period of reproductive cycl icity that continues th roughout most of her productive life. This period of reproductive cyclicity is known as the estrous cycle and its importance is stressed by the fact that it provides the female with several opportunities throughout life to be bred and b ecome pregnant in order to pe rpetuate the existence of her species. The estrous cycle consists of a series of predictable events, which in general terms are estrus, ovulation, and the beginning of a new cycle with the onset of a subsequent estrus. At the beginning of the estrous cycle, the female enters a state of sexual receptivity, known as estrus, which is follo wed by mating. Usually, mating takes place prior to ovulation to increase th e chances of the newly released oocyte to be fertilized. However, if conception does not occur, anot her cycle begins, provi ding the female with another opportunity to reproduce. On the other hand, if conception does occur, the female enters a period in which she will not exhibit regular estrous cycles during pregnancy. This is known as a period of ge stational anestrus, which allows for fetal growth and development, and ends after pa rturition and uterine involution. This and other examples of anestrus or the absence of a regular estrous cy cle will be discussed later in this chapter.

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6 The estrous cycle and the events gover ning reproductive cycl icity are tightly regulated by a series of hormones and factors that will eventually determine normal reproductive efficiency. Neuro-Endocrine System All reproductive processes, including the es trous cycle, are under the control of two systems: the central nervous system and the endocrine system. These two systems interact with each other through the hypotha lamus, resulting in a neuro-endocrine network responsible for the initi ation, coordination and regula tion of the functions of the reproductive system (Senger, 1997; Hafez and Hafez, 2000). The hypothalamus consists of clusters of nerve cell bodies, which are responsible for production of gonadotropin releasing hor mone (GnRH), and the paraventricular nucleus, which produces oxytocin (Senger, 1997) Each set of hypothalamic nuclei has different functions and is stimulated under different conditions. However, the communication between the hypothalamus and the pituitary is necessary for these hormones to exert their action. This co mmunication between the hypothalamus and the pituitary is referred to as the hypothalamic-pituitary axis. The pituitary gland is comprised of the posterior and the anterior lobe. The hypothalamus communicates with the posterior lobe through neural connections, and therefore the posterior pituitary is referre d to as neurohypophysis. For example, oxytocin is synthesized in neurons in the paraventri cular nucleus and it is transported down the axon to the posterior pituitary where it is re leased into the blood (Senger, 1997). On the other hand, the hypothalamus communicates wi th the anterior lobe (adenohypophysis) through the hypothalamo-hypophyseal portal system, which prevents hormones synthesized in the hypothalamus from en tering and being diluted by the systemic

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7 circulation. Gonadotropin-releas ing hormone is synthesized in either the surge or tonic center of the hypothalamus and is released into the primary capillary pl exus at the stalk of the pituitary. Blood enters this capillary system from the superior hypophyseal artery and transports GnRH to a secondary capillary plexus in the anterior pitu itary where it acts on target cells to release either follicle stim ulating hormone (FSH) or luteinizing hormone (LH) (Senger, 1997; Hafez and Hafez, 2000). Hormones that are secreted by the pituitar y are then transported through systemic circulation to the target tissu e to elicit the physiological re sponse. This is where the neural and the endocrine systems interact to modulate reproductive f unctions such as the estrous cycle. Stages of the Estrous Cycle The estrous cycle is divided into four stages: proestrus, estrus, metestrus and diestrus. Proestrus begins when circ ulating concentrati on of progesterone (P4) declines as a result of the regression of the corpus luteum from the previous cycle. It lasts from 2 to 5 days, depending on species, and terminates with the onset of estrus. Proestrus is characterized by an endocrine transition from a period of P4 dominance to a period of estradiol (E2) dominance under the control of FSH and LH. During this period, recruitment of follicles takes place and the reproductive tract prepares for the next stage, estrus (Senger, 1997). Estrus is the stage of the estrous cycle during which the female undergoes behavioral as well as endocrine changes characterized by sexual receptivity. These behavioral and physiological change s in the female are induced by E2, which is the dominant hormone during this stage. Sexual receptivity during estrus increases gradually

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8 until the animal reaches the typical standing es trus or lordosis. In cattle, estrus can extend from 6 to 24 h, with an average of 15 h (Senger, 1997). Metestrus is characterized by the transition from E2 dominance to P4 secretion as a result of the formation of a functional CL. At the beginning of this stage, concentration of both E2 and P4 is relatively low. However, the newly ovulated follicle undergoes cellular and structural changes that result in luteinization of the follicle and formation of the CL, which starts producing P4 soon after ovulati on (Senger, 1997). Diestrus is the longest stage of the estrous cycle, encompassing maximal CL function and sustained P4 secretion, and ending with the regression of the CL. High P4 levels prepare the uterus and its microe nvironment for early embryo development and implantation. The duration of diestrus (10 to 14 days) is directly related to CL function and P4 secretion. Under certain physiological conditions the female does not exhibit regular estrous cycles and is said to be in anestrus. Anestrus or lack of estrous cy clicity, is the result of insufficient GnRH release from the hypothala mus to stimulate and maintain gonadotropin secretion. This can be caused by pregnancy, lactation, season or stress (Senger, 1997). Follicular Development during the Estrous Cycle The follicular phase is the stage of the estrous cycle when preovulatory follicle development is stimulated by increasing concen trations of FSH and LH in plasma. This phase coincides with a marked reduction in plasma P4, which leads to significant increase in tonic GnRH secretion. Increased basal co ncentration of GnRH stimulates FSH and LH secretion from the pituitary and prom otes follicular development and E2 production. In cattle, growth of early antral follicles (~3 mm) is considered to be gonadotropinindependent (Scaramuzzi et al., 1993). The tr ansient rise in FSH at the time of luteal

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9 regression initiates the process of recruitment, which consists of stimulation of a cohort of small antral follicles to grow beyond 4 mm in diameter around da ys 1-2 of the cycle (Adams et al., 1992; Sunderland et al., 1994; Gi nther et al., 1997). Follicle-stimulating hormone stimulates ovarian follicles to acquire key properties associated with E2 synthesis such as increased cytochrome P450 side-chain cleavage and cytochrome P450 aromatase enzyme activities (Bao and Garverick, 1998; Garv erick et al., 2002). The process of recruitment is then followed by a decline in FSH due to the negative feedback by E2 and inhibin from the domi nant follicle (Ginther et al., 1998; Hunter et al., 2004). This decline in FSH has been identified as an important component of the selection process (Mihm et al., 1997). Follicle selection then results in a decrease in the number of growing follicles and is thought to end with growth diverg ence or deviation of a dominant follicle from the subordinate foll icles (Evans, 2003). During this time, the dominant follicle can use LH for its continue d growth, and this is supported by the fact that there is a switch from FSHto LH-dependency as the follicle matures and FSH concentration declines in cattle (Gong et al., 1996; Webb et al., 2003). Additionally, there are reports of a transi ent increase in circ ulating LH (Kulick et al., 1999, 2001) as well as increased expression of LH receptors in granulosa cells (Xu et al., 1995; Goudet et al., 1999; Beg et al, 2001) surrounding devi ation of the dominant follicle in cattle. Acquisition of LH receptors in granulosa cells might allow the transient increase in LH to have a functional effect in follicle se lection, since LH is known to increase E2 concentration in follicular fluid, thus facili tating the establishment of dominance (Ginther et al., 2000b). The freshly selected dominant follicle grows to a much larger size (12-20 mm) than all other subordinate fol licles, resulting in enhanced E2 secretion (Ginther et al.,

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10 1997). This enhanced E2 secretion is associated with increased expression of the genes encoding aromatase, 3 -hydroxy-steroid dehydrogenase and FSH-receptor, as well as the acquisition of LH-receptors in granulosa cells (Ireland and Roche, 1983; Xu et al., 1995; Bao et al., 1997a, b; Evans and Fortune, 1997). High E2 secretion is also responsible for maintaining low FSH concentrations to prevent growth of another cohort of follicles (Ireland et al., 1984, Ginther et al., 1999, 2000a, b). The growth of the first wave dominant fo llicle does not take place for more than 34 days, as P4 secreted from the developing CL inhi bits LH pulse frequency and the LHdependent dominant follicle becomes atretic (Sunderland et al., 1994; Evans et al., 1997). As a result, the first wave dominant follicle loses its ab ility to produce E2 between days 7 and 9 of the estrous cycle. The loss of domi nance is followed by another transient rise in FSH, resulting in the emergence of a new follic ular wave (Sunderland et al., 1994). If the CL fails to regress, the second dominant folli cle will undergo atresia and a third wave of follicles emerges. However, if luteolysis occurs during this second wave of follicular development, then the dominant foll icle will ovulate (C ooke et al., 1997). In cattle, most estrous cycles consist of two to three waves of follicular development (Fortune et al., 1988; Savio et al ., 1988; Ginther et al., 1989a) that emerge on about days 2 and 11, or days 2, 9 and 11 for animals with two or three follicular waves, respectively (Sirois and Fortune, 1988). Lactating Holstein cows tend to have two waves per cycle (Taylor and Rajamahendran, 1991, Townson et al., 2002), whereas beef and dairy heifers tend to have two or three waves per cycle (S avio et al., 1988; Sirois and Fortune, 1988; Ginther et al., 1989c). Cattle with two follicular waves tend to have

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11 shorter estrous cycles, ovulate larger and older follicles and to be less fertile than those with three waves (Townson et al., 2002). Ovulation and Development of the Corpus Luteum The luteal phase encompasses about 80% of the estrous cycle and covers the period of time that includes ovulation of the dominant preovulatory follicle, CL formation, and regression of the CL. A landmark event that precedes ovulation is the preovulatory LH surge, which is stimulated by increased concentrations of E2 in plasma (Senger, 1997). The LH surge triggers a cascade of events that cause biochemical and structural changes in the preovulatory follicle that lead to the rupture of the follicle wall resulting in the release of the oocyte and subsequent development of the CL (Berisha and Schams, 2005; Senger, 1997). Ovarian blood flow increases at th e time of ovulation and subsequent CL formation (Senger, 1997), which emphasizes the importance of angiogenesis during metestrus and diestrus. Angiogenesis is defined as the generati on of new blood vessels through sprouting from already existing ones. This process i nvolves degradation of the capillary vessel membrane, through which endothelial cells (E C) from pre-existing vessels migrate and proliferate to create a new lumen and furt her vessel maturation (Abulafia and Sherer, 2000). Angiogenesis is critical for fina l development and differentiation of the preovulatory follicle as an increase in both vascular area and blood flow has been demonstrated during this stage (Acosta et al ., 2003). Acosta et al. (2003) showed that there was a marked increase in blood flow and volume in the subsequent CL, which is closely associated with increased plasma P4 concentrations. Moreover, it has been reported that follicular diameter, E2 concentration in the follicul ar fluid, and vascular area

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12 are highly correlated (Mattioli et al., 2001). Thus, ovulation is the result of an interaction between LH surge and local factors that incl ude steroids, prostagla ndins and vasoactive peptides (Acosta and Miyamoto, 2004). Lutein izing hormone has been shown to increase ovarian blood flow in rats (Varga et al., 1985), rabbits (Janson, 1975), and sheep (Niswender et al., 1976). Thus, increased va scular dilatation and permeability, together with degradation of collagen layers that pr ovide strength to the follicular wall, are necessary for facilitating follic ular rupture (Murdoch et al., 1986; Abisogun et al., 1988) and oocyte release. Increased ovarian blood flow may increas e the availability of gonadotropins, nutrients, hormonal substrates and other blood components that are necessary for ovulation (Acosta and Miyamoto, 2004). Hence, vasoactive peptides re leased within the follicular wall may modulate local changes in blood flow observed in the ovulatory follicle (Brannstrom et al., 1998; Acosta et al ., 2003). This cascade could then mediate LH action to increase prostaglandin produc tion during ovulation. Vasoactive peptides have been shown to play important roles in the ovulation process as well as early luteal development, through modulation of local s ecretion of prostagl andins and steroid hormones (Acosta et al., 1999, 2000; Koba yashi et al., 2002). Although several promoters of angiogenesis have been identi fied, vascular endothelial growth factors (VEGF), fibroblast growth f actors (FGF), insulin-like growth factors (IGF) and angiopoietin (ANPT) are thought to be the most important factors modulating follicle maturation and CL formation (Berisha and Schams, 2005). Normal development of the CL and its capacity to produce P4, growth factors, angiogenic factors, and vasoactive substances is dependent on its va scularization (Acosta

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13 and Miyamoto, 2004). Ovarian blood flow decreases shortly after ovulation, but gradually increases from day 2 to day 5 of th e estrous cycle. This period also coincides with marked increases in CL volume and peripheral P4 concentration (Acosta et al., 2003). Angiogenesis within the CL reaches a peak 2-3 days after ovulation (Reynolds et al., 2000) and appears to be locally modul ated by angiotensin (Ang II) and growth factors, which also support P4 synthesis in luteal cells (Kobayashi et al., 2001b). Immediately after ovulation, the follicle wa lls collapse, allowing theca interna and granulosa cells to mix with one another. At the same time, these cells undergo a dramatic transformation during which they become lute inized after stimulati on by preovulatory LH surge (Senger, 1997). Theca interna cells beco me the small luteal cells, which contain numerous lipid droplets, while granulosa ce lls become the large luteal cells with secretory granules containing oxytocin (Senger, 1997). Within the developing CL, large luteal cells increase in size, while small luteal cells incr ease in number (Senger, 1997). The process of luteinization is characterized by increased synthesis and activity of enzymes responsible of switching steroid production from E2 to P4 (Juengel and Niswender, 1999). Progesterone exerts a strong negative feedback on the hypothalamus, reducing GnRH pulses, thus preventing de velopment of the dominant follicle, E2 synthesis and ovulation (Senger, 1997). It also promotes endometrial secretions and quiescence, which ultimately favors embryo implantation. In cattle, LH and growth hormone (GH) are the primary hormones regulating development and function of the CL, and th is is supported by e xpression of mRNA for both LH and GH receptors in the CL during the estrous cycle (Schams and Berisha, 2004). Luteinizing hormone is the principal hormone stimulating P4 synthesis by the

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14 small luteal cells (Niswender and Nett, 1988) an d this is further supported by the fact that most of LH receptors are found in these cells (Schams and Berisha, 2004). On the other hand, receptors for growth hormone are mainly located on large luteal cells (Lucy et al., 1993; Kirby et al., 1996; Koelle et al., 1998) which are res ponsible for 80% of total P4 production by the CL (Niswender et al., 1985) Growth hormone has been shown to stimulate P4 and oxytocin secretion by the bovine CL in vitro (Liebermann and Schams, 1994) and to support CL development in vivo (Lucy et al., 1994; Ju engel et al., 1997). Furthermore, GH is a more powerfu l stimulator of prostaglandin F2 and P4 production than LH in vitro (Kobayashi et al., 2001a). There are many other local autocrine and paracrine regulators that play an important modulatory role during the lifespan of the CL. Growth factors involved in angiogenesis, such as IGFs and FGFs, indu ce CL function by stimul ating secretion of P4 and oxytocin (Einspanier et al., 1990; Sauerewe in et al., 1992). Insulin-like growth factor-1 has been lo calized in the cytopl asm of both small and large luteal cells (Amselgruber et al., 1994). Fi broblast growth factor-2 gene and protein e xpression also have been demonstrated in large luteal ce lls of the bovine CL (Schams et al., 1994). Ovarian peptides such as oxyt ocin (OT), angiotensin II (A ng II), endothelin-1 (ET-1), progesterone and prostaglandin al so are important in CL function. It is well established that oxytocin is highly expr essed in the ruminant CL (Wathes et al., 1983; Schams, 1992), and it has been localized in both small and large luteal cells in cattle (Kruip et al., 1985). The LH surge appears to stimulate ovarian production a nd secretion of OT together with P4 (Schams and Berisha, 2004). There are reports that support a potent luteotropic role of OT during the developi ng phase of the bovine CL (Schams, 1989;

PAGE 31

15 Schams, 1996). Moreover, OT potentiates the luteotrophic e ffects of LH on P4 secretion in vitro (Schams et al., 1995). On the other hand the vasoactive peptides Ang II and ET1 have been shown to directly inhibit P4 production in bovine lut eal cells (Girsh et al., 1996; Miyamoto et al., 1997; Hayashi and Miyamoto, 1999). Prostaglandin Biosynthesis and Luteolysis in Cattle Prostaglandins are members of the eicosa noid family of fatty acids. They are derived from 20 carbon polyunsaturated fatty acids, such as arachidonic acid (AA). Prostaglandins of the 2-series, namely prostaglandin F2 (PGF2 ) and prostaglandin E2 (PGE2), are the most biologically active eicosa noids and are involved in key reproductive processes such as follicular development (W allach et al., 1975), ovulation (Espey, 1980), luteolysis (Wathes and Lammi ng, 1995), and parturition (Cha llis, 1980). Prostaglandin F2 is an important factor modulating luteal function in cattle. There are two different sources of PGF2 within the reproductive tract. Temporal expression and site of synthesis then determine its m odulatory role during luteal fu nction. In contrast to the luteolytic effects of endometrium-derived PGF2 luteal PGF2 seems to be luteotrophic during early and mid-luteal phases (Miyamoto et al., 1993). Bovine CL produces high amounts of PGF2 during early luteal phase, but PGF2 concentrations decrease as the luteal cycle progresses (Milvae and Hansel, 1983; Schams et al., 1995; Skarzynski and Okuda, 1999). Furthermore, in an in vitro study, infusion of Ang II stimulated P4 and PGF2 release from the developing CL (Kobayash i et al., 2001b). Moreover, Kobayashi et al. (2001a) showed that Ang II together with PGF2 highly stimulated P4 secretion from developing bovine CL. In mid-cycle luteal cells, P4 inhibits luteal PGF2 secretion (Pate, 1988; Skarzynski and Okuda, 1999). The other source of PGF2 biosynthesis and

PAGE 32

16 secretion is the endometrial ti ssue of the bovine uterus and th is is associated with the luteolytic cascade. Prostaglandin Biosynthesis Prostaglandins of the 2-seri es are synthesized from arachidonic acid (AA), which uses linoleic acid as the primary precursor. Arachidonic acid is store in an esterified form at the sn 2 position of the membrane phospholipids bilayer (Croffo rd, 2001). The first and rate limiting step of prostaglandin biosynthe sis is the hydrolytic release of AA by the action of cytosolic phospholipase A2 (cPLA2) enzyme (Lapetina, 1982). This enzyme has a preference for phospholipids containing AA at the sn 2 position. Upon cell activation, release of intracellular Ca2+ stimulate cPLA translocati on and binding to the membrane, which is a prerequisite for its enzymatic activity (Murakami et al., 1997; Leslie, 1997). Following its release, AA is converted to prostaglandin H2 (PGH2) by the action of prostaglandin H synthase (PGHS), also know n as cyclooxygenase (COX). Prostaglandin H synthase has cyclooxygenase and peroxidase activities th at convert prostaglandin G (PGG) to PGH2 (Goff, 2004). This enzyme consists of two isomers (PGHS-1 and PGHS2) (Goff, 2004), which are primarily locat ed on the luminal surface of the endoplasmic reticulum and the inner and outer membranes of the nuclear envelope (Spencer et al., 1998). The constitutively expressed PGHS-1 is considered to play a housekeeping role, whereas PGHS-2 is the inducible form, hen ce stimulated by hormones, growth factors, etc. in a variety of tissues (Goff, 2004). After synthesis of PGH2, this endoperoxide is converted to one of several possible prostanoids by the action of specific terminal enzymes. Biosynthesis of prostaglandin E2

PAGE 33

17 (PGE2) and prostaglandin F2 (PGF2 ) are catalyzed by the acti on of prostaglandin E and prostaglandin F synthases, respectively (Goff, 2004). Luteolysis in Cattle In ruminants, ovarian E2, oxytocin (OT), and P4 seem to be the physiological regulators of synthesis and secretion of uterine PGF during the estrous cycle. Prostaglandin F2 released from the endometrium is the principal luteolytic agent in ruminants (Lukaszewska and Hansel, 1970; Mc Cracken, 1971; McCracken et al., 1972). There is clear evidence that luteal regression at the end of the estrous cycle is caused by episodic release of endometrial PGF2 that reaches the CL through a counter current mechanism between the uterine vein and ova rian artery (Senger, 1997, Krzymowski and Stefa czyk-Krzymowska, 2004; Schams and Berisha, 2004). It has been demonstrated that P4 regulates the lifespan of the CL (Ottobre et al., 1980; Schams et al., 1998; Schams and Berisha, 2001, 2002c) through inhibition of PGF2 secretion from the endometrium. The large amplitude pulses of PGF2 responsible for initiati on of luteolysis, result from decreasing P4 and increasing E2 concentrations (Goff, 2004). This is preceded by the increase in E2 and estrogen receptors (ER) which in turn up-regulates oxytocin receptor in the endometrium (Goff, 2004). At the end of the luteal phase, the num ber of ER increases, presumably due to E2 up-regulation of its own recepto r in endometrial cells (Spe ncer et al., 1996; Xiao and Goff, 1999; Ing and Tornesi, 1997). This increas e in ER is thought to initiate luteolysis by increasing oxytocin receptors (OTR) (Spe ncer et al., 1996; Xiao and Goff, 1999; Ing and Tornesi, 1997; Spencer and Bazer, 2002) ; however, up-regulation of OTR in the bovine endometrium precedes that of the ER (Robinson et al., 2001). Thus, it has been

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18 proposed that, even though increased concentr ation of OTR (~days 13-16) are primarily due to withdrawal of P4 inhibitory effects (Fairclough and Lau, 1992), increasing levels of E2 during this time can facilitate up-regulatio n of OTR gene expres sion (Vallet et al., 1990; Leavitt et al., 1985; Zhang et al., 1992). Th erefore, luteolysis is brought about by coordinated changes in both OTR and pros taglandin production, wh ich are regulated by changes in P4 and E2 concentrations. Acosta et al. (2002) de monstrated that the tr ansitory decrease of P4 may trigger the luteolytic cascade. Earlier studies have also shown a close relationship between the decrease in ovarian blood flow and systemic P4 concentration in the cow (Ford and Chenault, 1981; Wise et al., 1982). One of the main luteolytic actions of PGF2 is to decrease ovarian blood flow (Knickerbocker et al., 1988). Locally produced growth factors also medi ate the complex process of luteolysis. Members of the IGF system have been shown to play an important role in the process of PGF2 -induced luteolysis in cattle (Neuvians et al., 2003). Gene a nd protein expression of VEGF are known to decline after prosta glandin secretion (Neuvians et al., 2004a), while FGFs and their receptors are up-regulated during functional lute olysis (Berisha and Schams, 2005). Therefore, it has been sugge sted that cessation of VGEF-support of the CL plays a role during structural luteolysis, whereas FGFs seem to have a major impact in functional luteolysis (B erisha and Schams, 2005). There is also evidence that vasoconstric tive peptides, such as Ang II and ET-1 may play a role during physiological and induced luteolysis in cows (Girsh et al., 1996; Miyamoto et al., 1997; Hayashi and Mi yamoto, 1999; Hayashi et al., 2000). In vitro PGF2 potentiates the inhibitory activity of ET-1 on P4 secretion and stimulates Ang II

PAGE 35

19 release (Miyamoto et al., 1997). Hence, ET1 and Ang II may act as vasoconstrictors during functional luteolysis, as well as apoptosis indu cers during functional and/or structural luteolysis (S chams and Berisha, 2004). In the bovine ovary, cytokines such as tumor necrosis factor (TNF interferon (IFN and interleukin (IL-1 ) are up-regulated during induce d luteolysis (Neuvians et al., 2004b). There also is eviden ce that the combination of TNF and IFN are extremely cytotoxic (Petroff et al., 2001). Therefore, cytokines may be involved not only in structural, but also functional luteolysis in cattle (Berisha and Schams, 2005). Pregnancy Establishment in Domestic Ruminants The process of maternal recognition of pregnancy refers to the physiological window when the mother becomes cognizant of the embryo within her reproductive tract and prevents its elimination. If an embryo is present in th e bovine uterus between days 14 and 17 of the cycle, luteolysis does not take place and P4 secretion is maintained, resulting in establishment of pregnancy (Northey and French, 1980). This is achieved by a signal from the embryo that prevents the regression of the corpus luteum (CL) through inhibition of pulsatile PGF2 secretion from the endometrium (Bazer, 1992; Demmers et al., 2001). In domestic ruminants, the embryonic signal responsible for pregnancy establishment is a cytokine called interferon (IFN)(Lafrance and Goff, 1985; Spencer and Bazer, 1995). Interferonis synthesized by trophoblastic cells of bovine blastocyst (Lafrance and Goff, 1985; Roberts et al ., 1990; Spencer and Bazer, 1995), and its secretion is highest between days 15 and 17 of pregnancy (S tojkovic et al., 1995; Bazer et al., 1998).

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20 Pregnancy establishment is accomplished through several mechanisms. Interferoncan prevent luteolysis by down-regulation of endometrial oxytocin receptor, which prevents oxytocin-stimulated PGF2 secretion (Lafrance and Goff, 1985). Secretion of INFin ewes has been reported to reduce estradiol receptors, which prevents E2stimulation of OTR (Spencer and Bazer 1995). Moreover, production of PGF2 in cattle can be further suppressed by decreasing th e expression of PGHS-2 as well as PGFS (Lafrance and Goff, 1990; Bi nelli et al., 2000), enzymes which play key roles in the synthesis of this prostaglandin. Binelli et al. (2000) showed that INFblocked PGF2 production by reducing PGHS-2 and PLA2 gene expression. The suppression of PGHS-2 and PLA2 mRNA synthesis appears to be i ndependent of oxytocin-induced intracellular events (Pru et al., 2001). Another mechanism by which IFNmay inhibit luteolysis in cattle is by shifting prostaglandin biosynthesis from the luteolytic PGF2 to the luteotropic PGE2 (Okuda et al., 2002). Results from studies by Xi ao et al. (1998), showed that IFNinhibited PGHS-2 mRNA and attenuated pr ostaglandin secretion from epithelial cells, which are known to be the primary source of PGF2 while enhancing PGHS-2 mRNA and prostaglandin biosynthesis in stromal cells which are the primary source of PGE2 (Kim and Fortier, 1995; Asselin et al., 1996, 1998; Skarzynski et al., 2000). Therefore, achieving an optimal PGE2 to PGF2 ratio is essential for endometrial receptivity, myometrial quiescence, and mainte nance of a functional CL and P4 secretion, which are critical for successful establishmen t of pregnancy (Bazer et al., 1998).

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21 Energy Balance and Fertility Responses in Postpartum Dairy Cows In mammals, nutrition is critical for sust aining important biological processes that allows the animal to grow, survive and re produce. Food is consumed, digested, and broken down into nutrients, which are then absorbed and partiti oned throughout the body for utilization. Nutrients are utilized by tissues involved in maintenance of basic physiological processes, as we ll as establishing energy stores in the form of lipids and glycogen. The process of maintenance of physiological equilibrium or constant conditions of the internal milieu balance within a give n physiological state is under homeostatic control (B auman and Currie, 1980). However, in an animals life cycle, there are a series of physiological states th rough which it must go and adjust adequately. The orchestrated or coordina ted changes in metabolism of body tissues necessary to support the transition to a part icular physiological state are under homeorhetic regulation (Bauman and Currie, 1980). An example of such dramatic metabolic changes is represented by the onset of lactat ion in dairy cows. It is clea r then, that overall biological functions are governed by food intake, nut rient absorption and partitioning, and the resulting energy status of the animal within a given physiological state. Energy Balance in Transition Dairy Cows Energy balance of an animal is the diffe rence between energy intake and energy requirements within a given physiological st ate (Beam and Butler, 1999; Butler et al., 1981; Canfield and Butler, 1990). In dairy cows the onset of lactation cause an abrupt shift in nutritional requ irements in order to support milk production (Butler, 2000). This rapid increase in energy requirements and chan ges in the metabolic as well as endocrine status of the cow come a bout during the transition peri od (Bauman and Currie, 1980;

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22 Grummer, 1995). This is the result of the prio ritized status of lactation which allows it to proceed at the expense of any other physiolo gical processes (Bauman and Currie, 1980). The transition period extends from three weeks prepartum until three weeks postpartum, and refers to the period duri ng which endocrine and metabolic changes accommodate parturition and the onset of lactation (Grummer, 1995). A reduction in feed intake occurs during the final week s of pregnancy when nutrient demands for support of fetal growth and in itiation of milk synthesis ar e increasing (Grummer, 1995). As a result, there is a higher energy require ment than can be met or supported by dietary energy intake (Bell, 1995). The dietary ener gy that is consumed by the lactating animal is almost entirely used by the mammary tissue for milk production, leaving no energy for maintenance (Bell, 1995). To offset this en ergy deficit, the lactating animal mobilizes body energy reserves, which leads ultimately to a state of negative energy balance. Since the energy required for lactation a nd maintenance far exceeds energy intake, the resulting NEB promotes a massive mobili zation of fat from the adipose tissue and enhanced nutrient partitioni ng to the mammary gland for milk synthesis (Bauman and Currie, 1980). Consequently, the transition pe riod is characterized by increased plasma levels of non-esterified fatty acids (NEFA), indicative of onset of lactation. Once free fatty acids are released into blood, they are bound to albumin and other blood proteins to be transported to hepatic and non-hepatic tis sues. The uptake of NEFA into the liver takes place as blood flows though the liver. Once inside the liver, NEFAs can undergo three of the following metabolic fates as outlined by Drackley (1999): 1) complete oxidation to carbon dioxide to provide energy fo r the liver, 2) incomplete oxidation to

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23 produce ketone bodies as an alternate ener gy source, and 3) re-esterification into triacylglycerol (TAG). Normally, complete oxidation of fatty acid ta kes place, but first fatty acids must be translocated into the mitochondria. Mitoc hondrial uptake of FA is regulated by the activity of carnitine acyl transf erase (CAT-1). In spite of the central role of CAT-1 in liver lipid metabolism, little is known about it s regulation. However, studies have shown that high malonyl-CoA intracellular concen trations inhibit CAT-1 enzymatic activity (Brindle et al., 1985). Malonyl-CoA is pr oduced by the enzyme acetyl-CoA carboxylase and is an intermediate for FA synthesis. Zammit (1996) demonstrated that the sensitivity of CAT-1 to malonyl-CoA inhi bition is lessened during time s of low circulating insulin or insulin resistance in rodent s. Similarly, Brindle et al. (1985) found that malonyl-CoA concentrations were influenced by insulin and glucagon. Thus, it would appear that energy balance may play a role in CAT-1 regulation. Cows in NEB exhibit decreased capacity to metabolize fat, which may be linked to CAT1. Drackley et al. (1991) conducted a study with non-lactating cows to test the effects of carnitine and propionate on liver lipid metabolism. Fasti ng decreased oxidation of palmitate to CO2 and decreased palmitate esterification by bovine liver slices. Addition of carnitine in vitro increased oxidation of palmitate and also increased tota l utilization of palmitate. Similar results were obtained in early lactating cows (D rackley et al., 1991). An alternate pathway for hepatic NEFA metabolism is through partial oxidation that takes place in the peroxisome. This oxi dative pathway is similar to that in the mitochondria, with some exceptions. The in itial oxidation step is catalyzed by an oxydase, which results in produc tion of hydrogen peroxide ra ther than reduced NAD as

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24 seen in mitochondrial oxidat ion (Drackley, 1999). The next key difference is that peroxisomes do not contain respiratory chai n linked to ATP formation. As a result, peroxisomal oxidation is not subject to contro l by energy demands for the cell. Hence, these differences make the peroxisome well suit ed to partially oxidize fatty acids that are poor substrate for mitochondr ial oxidation (Drackley, 1999). The third metabolic fate of NEFAs entering the liver is reesterification into TAG. Resultant TAGs are either stored in the liver or packaged into very low density lipoproteins and exporte d into circulation. Fertility Responses in Postpartum Dairy Cows Negative energy balance during the first 3 weeks postpartum has been associated with extended interval to fi rst ovulation (Beam and Butler, 1999; Butler, 2001). The first ovulation occurs on average about 30 d postpar tum, with a range of 14-42 d (Butler and Smith, 1989; Staples et al., 1990). Conception rates in lactating da iry cows increases when the period of ovarian activity preceding insemination is longer and thus the number of preceding ovulatory cycles is greater (B utler and Smith, 1989; Thatcher and Wilcox, 1973). Since the number of ovulatory estrous cycles preceding insemination influences conception rate, the length of postpartum interv al to first ovulation provides an important measure for assessing the effects of NE B on reproductive performance (Butler, 2003). The severity and duration of NEB is vari able among cows and relates primarily to differences in dry matter intake and its rate of increase during early lactation (VillaGodoy et al., 1988; Staples et al., 1990). Fo r example, cows overc onditioned at calving exhibit decreased appetite and thus develop a more severe NEB than cows of moderate conditioning. These overconditioned animals mo bilize more fat from the adipose tissue and exhibit significant accumulation of TAG in the liver (Rukkwamsuk et al., 1999).

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25 Accumulation of TAG in the liver is, in turn, associated with a longe r interval to first ovulation and reduced fertility (Butler and Smith, 1989; Rukkwamsuk et al., 1999; Jorritsma et al., 2000). Fertility in postpartum cows is dire ctly dependent on a normal estrous cycle characterized by folliculogenesis, ovulation, corpus luteum (CL) formation and regression. However, the mechanisms by whic h endocrine and metabolic upsets result in reduced reproductive efficiency are not we ll understood. Many of the hormonal and metabolic changes that occur during the tran sition period can affect reproductive function by interacting with the hypothalamic-pituitary axis (Butler, 2000). The hypothalamic-pituitary axis is esse ntial in the modulation of gonadotropin secretion, which in turn plays a pivotal role in control of the estrous cycle. For example, in pubertal heifers, the establishment of LH pulsatility is responsibl e for the initiation of cyclicity (Schillo et al., 1992). Among the factors associated with onset of puberty, attainment of a critical le vel of body fat is important (Schillo et al., 1992), further stressing the importance of nutritional and metabolic status for normal reproductive function. The NEB experienced by the earl y postpartum cow decreases pulsatile LH secretion, which results in de layed resumption of ovarian cyclicity (Butler and Smith, 1989; Beam and Butler, 1999; Butler, 2000). The first ovulation postpartum reflect s the resumption and completion of preovulatory ovarian follicular development and recovery from the hormonal conditions of late pregnancy (Butler, 2000). Delayed tim e to first ovulation associated with NEB is presumably through inhibition of LH pulse frequency and low levels of blood glucose, insulin, and insulin-growth factor-I (IGF-I) that collectively prevent E2 production by

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26 dominant ovarian follicles (B utler, 2000). However, Beam and Butler (1999) reported that initiation of a follicular wave and formation of a large dominant follicle during NEB was not a limitation for first ovulation. In fact following parturition, a wave of follicular development takes place within 5-7 d in res ponse to elevated plasma FSH concentrations and regardless of NEB (Beam and Butler, 1999). Nonetheles s, ovulation of a dominant follicle during early lactation is dependent on the re-establishment of pulsatile LH secretion that is conducive to te rminal preovulatory growth and E2 production (Butler, 2000). Beam and Butler (1997) described th ree possible outcomes of follicular development during early postpartum period: 1) ovulation of the first dominant follicle around days 16-20 postpartum; 2) non-ovulation of the first domi nant follicle followed by turnover and a new follicular wave; 3) failu re of the dominant follicle to ovulate, becoming cystic and prolonging the interval to first ovulation to 40-50 days postpartum. As discussed by Jolly et al. (1995), the NE B experienced by the postpartum dairy cow represents a physiological state of under nutrition which impairs LH secretion and prevents ovulation. Consistent with this concept, Beam and Butle r (1997) observed that follicles emerging after the NEB nadir, rather than before, had greater diameter, enhanced E2 production, and were more likely to ovulate. NEB has also been shown to change the profile of metabolic hormones which may play an important local role in control of follic ular development in cattle. It is likely that these changes could alter the pattern of ovarian follicular growth and development, and subsequent CL function during the early postpartum period. Du ring early NEB period, the ability of ovarian follicles to produce sufficient E2 for ovulation appears to depend on the availability of insulin and IGF-I in se rum and the changing EB profile (Beam and

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27 Butler, 1999). Both insulin a nd IGF-I plasma concentrations are directly related to the energy status of the cow, and these hormones are essential for normal follicular development (Spicer et al., 1993; Simps on et al., 1994; Beam and Butler, 1999). Plasma glucose and insulin levels are decreased by energy deficit (Beam and Butler, 1999; Butler, 2000). In lactating dair y cows, there is high demand for glucose as the primary substrate for mammary lactose synt hesis (lactogenesis) (D iskin et al., 2003). Thus, reduced availability of glucose as a result of NEB ma y affect LH pulsatility, since it influences both tonic and surge modes of secretion, presumably through effects on GnRH (Diskin et al., 2003). Insulin also se rves as a metabolic signal influencing LH release from the pituitary (M onget and Martin, 1997). However, it also has been shown to increase ovarian response to gonadotropins and to stimulate recruitment of small follicles and enhanced follicular growth (Gong et al., 2001), suggesting a direct effect at the ovarian level. Moreover, insulin is known to stimulate bov ine follicular cells in vitro (Spicer et al., 1993) and in vivo (Simpson, et al., 1994). Energy balance also influences plasma le vels of insulin-like growth factor-I (IGFI), which is important for normal ovarian follicular development and activity (Beam and Butler, 1999). In postpartum dairy cows, IG F-I levels were 40-50% higher during the first two weeks in cows in which the domi nant follicle would ovul ate as compared to levels in cows with non-ovulatory follicles (Beam and Butler, 1997; 1998). Additionally, plasma E2 concentrations were highly correlated with plasma IGF-I levels (Beam and Butler, 1998). It has been shown that IG F-I directly stimulat e proliferation and steroidogenic capacity of thecal (Spicer and St ewart, 1996) and granul osa cells (Spicer et al., 1993) in vitro Consequently, during the postpartum period, the ability of follicles to

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28 produce sufficient E2 for ovulation seems to be dependent on availability of both insulin and IGF-I (Butler, 2000). In cattle, peripheral P4 concentration increases dur ing the first two to three postpartum ovulatory cycles (Vil la-Godoy et al., 1988; Spicer et al., 1990; Staples et al., 1990), and the rate of the increase in P4 levels is attenuated by NEB early postpartum (Villa-Godoy et al., 1988; Spicer et al., 1990). In this regard, Villa-Godoy and coworkers (1988) reported that cows with the most ne gative energy balance dur ing the first 9 days still had decreased P4 during their third estrous cycle, which corresponded to the start of the breeding period. The ability of a cow to produce and secrete optimum levels of P4 is important for fertility because plasma P4 concentrations are highly correlated with pregnancy outcomes in lactating dairy cows (Folman et al., 1990; Larson et al., 1997). In summary, it is well established that the energy state of the cow during early lactation can influence reproduc tive efficiency by modulating the estrous cycle. This may occur through modulation of gonadotropin secretion, specifically LH pulsatility (hypothalamic-pituitary axis). Alterna tively, local ovarian effects may involve modulation of follicular growth and devel opment by metabolic hormones (i.e. IGF-I and insulin), which could determine oocyte quality and viability and steroidogenic activity of the CL. However, energy-independent effects also have been observed after fatty acid supplementation. Thus, nutrition may influence reproductive efficiency in dairy cows not only by altering the energy status of the animal but also by influencing factors involved in the regulation of repro ductive processes like follicular dynamics, ovulation, CL function and embryo survival among others.

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29 Effects of Dietary Fats on Re productive Response in Cattle Fat supplementation of dairy rations is co mmonly used to allevi ate a portion of the dietary energy deficit experienced by early postpartum dairy cows (Butler, 2003). Supplemental long-chain fatty acids (LCFA) have been shown to increase conception rates (Schneider et al., 1988; Sklan et al., 1989; Ferguson et al., 1990), enhance pregnancy rates (Schneider et al,. 1988; Sklan et al., 1991), and reduce the interval to first estrus (Sklan et al., 1991). Dietary fats have also been shown to regulate eicosanoid synthesis (Abayasekara and Wathes, 1999; Cheng et al., 2001), modulate plasma P4 concentration (Carrol et al ., 1990; Lucy et al., 1993b; Ga rcia-Bojalil et al., 1998), stimulate ovarian follicular development (Lucy et al., 1993b; Thomas and Williams, 1996; Beam and Butler, 1997) and improve fertility (Staples et al., 1998). To understand the interaction of dietary fa ts and reproduction, it is essential to understand the basic nature and biology of fatty acids and their metabolism. Fatty acids belong to the family of lipids, which consis t of biological compounds that are soluble in organic solvents, such as chol esterol, TAG and phospholipids. Fatty acids are present in all cell types and contribute to cellular struct ure, provide fuel stor age and participate in many biological processes ranging from gene transcription to regulation of key metabolic pathways and physiological responses (Van Bilsen et al., 1997, 1998; Gurr et al., 2002). Fatty acids consist of a carbon chain that ends with a carboxyl group, varying in the chain length and the degree of unsaturati on or number of do uble bonds. Naturally occurring fatty acids can be saturated (no double bonds) or unsaturate d, consisting of one or more double bonds. Satura ted fatty acids (SFAs) have all the carbons holding the maximum number of hydrogens possible, thus re ferred as to be saturated with hydrogen. Some naturally occurring SFAs are palmitic acid (16:0), found in palm oil, and stearic

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30 acid (18:0), commonly present in animal fat (Jenkins, 2004). Unsaturated fatty acids can contain one (monounsaturated fa tty acids; MUFA) or more ( polyunsaturated fatty acids; PUFA) double bonds. Fatty acids are generally abbreviated by listing the number of carbons with the number of double bonds (i.e. 18:0; 18 carbons with no double bonds) (Jenkins, 2004). For unsaturated fatty acids, the omega system is used to identify the location of the terminal double bond relative to the methyl end of the carbon chain. In the omega system, carbon atoms in the chain are identified with Greek letters, wi th the last carbon of the chain, or the one farthest from th e carboxyl group, known as the omega (n)-carbon (Gurr et al., 2002). Thus, for PUFAs only the position closest to the omega carbon is given. For instance, a member of the n-6 family such as linoleic acid (18:2, n-6) has its first double bond at the 6 position (carbon number 6) counting from the omega end (Gurr et al., 2002). Fatty acids in mammals are either generated by de novo synthesis or provided by the diet. Dietary fatty acids may undergo elong ation and desaturation to generate isomers which may have different pr operties. Elongation involves the addition of two-carbon units to a chain through the act ion of enzymes known as elon gases. Desaturation, on the other hand, is catalyzed by desaturase enzyme s that insert a double bond into the acyl chain. These desaturase enzymes are classified according to the posit ion of insertion of the double bond, while the newly created double bonds are almost invariably separated from each other by a methylene group. However, desaturation of fatty acids in mammals does not occur at positions greater than 9 since the required desaturases are absent (Fischer, 1989; Cook, 1996;

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31 Mayes, 1996). Hence, the parent molecules for the n-6 and n-3 families, linoleic (LA; 18:2) and linolenic (LNA; 18:3) acids, respectively, cant be synthesized by the tissues and, therefore, must be supplied in the diet Mammals have been shown to have an absolute requirement for LA and LNA; therefore, these fatty acids are regarded as essential fatty acids (Burr and Burr, 1929; A aes-Jorgensen, 1961; Ho lman et al., 1982). Deficiency in these essential PUFAs may result in a variety of pathophysiologic effects in mammals that include reproductive inefficien cy (Burr and Burr, 1929). Nonetheless, the body has different requirements for n-6 and n-3 PUFAs, as they are involved in several, yet varied, essential functions. Even though the diet of ruminants contai ns predominantly PUFAs, fatty acids in blood, tissues and milk are highly saturated (A bayasekara and Wathes, 1999). This is the result of extensive biohydrogenation of PUFA s that takes place in the rumen through the activity of ruminal microorganisms (Ward et al., 1964). Biohydrogenation of unsaturated fatty acids consists of addition of hydr ogen by microbial enzy mes to the double bonds resulting in the saturation of dietary fatty acids. The principal pr oduct is stearic acid, a saturated fatty acid (18:0). However, even though ruminal biohydrogenation is extensive, it ranges from 60 90% (Murphy et al., 1987). In other words, incomplete biohydrogenation of unsaturated fatty acids resu lts in the formation of several isomers which will depend on the source of the dietary fatty acid fed. Some of the most common products of incomplete biohydrogenation are ol eic acid (18:1), conj ugated linoleic acid (CLA), and trans -vaccinic acid ( trans11; 18:1). Thus, in orde r for intact PUFAs to reach the small intestine for ab sorption and being transported to target tissues, they need to escape ruminal microbial hydrogenation process.

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32 Essential PUFAs may be made available for absorption by feeding ruminally inert fats. A number of techniques have been developed to protect fats which involve chemical treatment processes such as form aldehyde or calcium salts of fatty acids (Palmquist and Jenkins, 1980; As hes et al., 1992). Inclusi on of calcium soaps of longchain fatty acids (LCFA) in dairy cattle rations is commonly used to alleviate the dietary energy deficit experienced during early postpar tum. Cows fed calcium salts of LCFA have produced more milk and experienced improved fertility (Staples et al., 1998). Linoleic acid is abundant in nearly all commonly available unprocessed plant oils (e.g. corn, sunflower, safflower, and rape seed ) (Sargent, 1977). In cows, AA is provided by dietary intake of LA (Urich, 1994; Gu rr et al., 2002). However, dietary LA deprivation causes a general decrease in AA levels, although the pr oportion in different tissues can be diverse, suggesting tissue-sp ecific uptake of PUFA (Lefkowith et al., 1985). On the other hand, PUFAs of the n-3 fa mily, such as eicosa pentaenoic acid (EPA; 20:5) and DHA (22:6), are also essentia l for many bodily functions (Innis, 1991; Abayasekara and Wathes, 1999). They can be pr ovided in the diet or synthesized in the tissues from the parent molecule LNA. Linol enic acid is the predominant PUFA in most forage lipids (Palmquist and Jenkins, 1980) and high levels are also found in linseed oil, however, it also contains signi ficant amount of LA (Sarge nt, 1997). Fish oils, which contain low amounts of LNA, offer the most readily available dietary source of EPA and DHA (Neuringer et al., 1988). The potential mechanisms by which LCFAs affect reproductive responses in cattle include indirect effects of high energy inta ke on the overall energy state of the cow, as well as direct effects of dietary fatty acids on the pituitary, ovaries, a nd uterus (Staples et

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33 al., 1998; Mattos et al., 2000). The associa tion between fat content in the diet and fertility in lactating cows has being documented in a variety of studies. Improvement of the overall energy state provided by fatty acid supplementation (Staples et al., 1998; Jenkins and Palmquist, 198 4) may lead to re-establishment of LH pulsatility and ovarian cyclic ity in the lactating cow (Palmquist and Jenkins, 1980; Lucy et al., 1991). In fact, Sklan et al. (1994) reported that the energy provided by dietary fats increased LH secretion in dairy cows that co nsumed less energy than required. However, the mechanism by which LCFAs may affect LH secretion has not been described in ruminants. There is evidence that increase in consum ption of dietary fatty acids stimulates ovarian follicular growth in cattle through a mechanism that is independent from energy intake and weight gain (Stapl es et al., 1998). Increasing the dietary content of LCFAs in cattle increased both the number and size of follicles present in the ovary and shortened the interval to first ovulation postpartum (Hi ghtshoe et al., 1991; Lucy et al., 1991, 1992; Ryan et al., 1992; Thomas and Williams, 1996; Lammoglia et al., 1997; Beam and Butler, 1997). Lucy et al. (1993b) reported greater numbers of me dium-sized ovarian follicles (1.5 to 2.3 mm) in postpartum dairy cows fed a diet containing 2.2% calcium salts of LCFA compared to cows receiving an isocaloric control diet without calcium salts of LCFA. Several studies reported that supplemental fat increased not only the total number of ovarian follicles (Thomas and Williams, 1996; Beam and Butler, 1997; Lammoglia et al., 1997), but also the size of preovulatory follic les in cattle (Lucy et al., 1993b; Beam and Butler, 1997; Ol dick et al., 1997). Follicu lar growth also has been shown to be stimulated by LCFAs in cr ossbred beef cattle (Thomas et al., 1997).

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34 Moreover, lactating dairy cows fed calcium salts of fat enriched with LA or fish oil (high in n-3 PUFAs) had increased size of the dominant follicle compared to those fed calcium salts of oleic acid (18:1) (Mattos et al., 2000) Whether increased size of preovulatory follicles is due to exogenous LCFA-induced LH secretion from the pituitary, and how the altered ovarian follicular dynamics may im pact pregnancy outcome in dairy cows warrants further investigation. It is possible that increased serum concentr ations of insulin in response to feeding LCFAs to cattle (Palmquist and Moser, 1981; Thomas and Williams, 1996; Ryan et al., 1995) may play a role in medi ating increased follicular grow th. This insulin-mediated event may be by stimulation of granulosa ce ll IGF-I production (Yoshi mura et al., 1994). Stimulatory effects of dietary LCFAs on follicular development may be through enhanced steroidogenesis via increased choleste rol, thus increased s ubstrate availability for increased follicular steroid synthesis (Wehrman et al., 1991). In mature heifers and dairy cows, elevating fat intake increased both serum and follicular fluid cholesterol concentrations (Park et al., 1983; Talavera et al., 1985; Wehrman et al., 1991). Carroll and coworkers (1990) detected a 21% increase in plasma cholesterol concentrations in dairy cows fed ruminally inert fat compared to control cows. Si milarly, beef heifers supplemented with soybean oil had greater con centration of total chol esterol in serum and HDL cholesterol in follicular fluid when co mpared with control heifers (Ryan et al., 1992). Wehrman et al. (1991) showed that cows fed a high lipid diet had increased intrafollicular levels of androstendione as well as increased P4 output from the granulosa cells collected and cultured in vitro In addition, several stud ies have detected subtle increases in plasma P4 concentrations in cows fed hi gh-fat diets (Carroll et al., 1990;

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35 Lucy et al., 1993b; Garcia-Bojal il et al., 1998). Dietary inta ke of LCFAs in ruminants during luteal phase increased serum concentrations of P4 (Talavera et al., 1985; Carrol et al., 1990; Hawkins et al., 1995; Burke et al., 1996). Recent studies suggested that increase in plasma P4 in cows fed fat-supplemented diets may not be due to increased synthesi s but rather to redu ced clearance of P4 from circulation. In one study, when the CL of cows were removed by ovariectomy, P4 was cleared from blood at a much slower rate in cows fed supplemental fat compared to cows fed the control diet (Hawkins et al., 1 995). In addition, Sangsritavong et al. (2002) showed that fatty acids and/or TAG could increase circulating P4 and E2 concentrations by directly inhibiting liver cell metabolism of these steroid hormones. Because CL function and P4 is crucial for establishment and main tenance of pregnancy in ruminants, increased plasma P4 may result in improved pregnanc y rates in postpartum dairy cows fed supplemented fats. In fact, supplementa tion of LCFA has been shown to enhance luteal function as confirmed by reduced in cidence of short cycles (Williams, 1989). Dietary LCFAs may increase AA in the phospholipids pool of granulosa cells. The AA released upon gonadotropin s timulation (Cooke et al., 1991 ) had a direct effect on steroidogenesis in goldfish (Van der Kr aak and Chang, 1990) and hens (Johnson and Tilly, 1990). It can be used as the precur sor for prostaglandin production, which in turn may stimulate steroidogenesis as reported in granulosa cells for marmosets (Michael et al., 1993). This hypothesis is supported by th e observation that gonadotropins stimulate prostaglandin production in vitro (Tsang et al., 1988). Fertility responses may also be relate d to the effects of LCFAs on uterine eicosanoid production. Eicosanoi ds (i.e. prostaglandins, th romboxanes, leukotrienes and

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36 lipoxins) are synthesized from AA, which uses LA as the primary precursor (Kinsella et al., 1990). Prostaglandins (PG) of the 2 series (PGF2 PGE2) have been implicated in many reproductive processes including ovulation (Espey, 1980), follicular development (Wallach et al., 1975), corpus luteum func tion (Bazer and Thatcher, 1977; Auletta and Flint, 1988; Abayasekara et al., 1995; Poyser, 1995; Wathes and Lamming, 1995), parturition (Thorburn and Challis, 1979; Chal lis, 1980) and uterine involution (Hafez and Hafez, 2000). Hence, any influence that fatty acids might exert on PGF2 synthesis may affect overall reproductive performance. Ovulation was blocked by inhibition of PG synthesis in monkeys and restored by administration of PGF2 (Wallach et al., 1975; Tsafri ri et al., 1972). Experiments performed on rats showed that a diet high in n-3 PUFAs increased ovulation rate, whereas a diet high in n-6 PUFAs resulted in reduction of ovulati on rate (Trujillo and Broughton, 1995). Since both diets re sulted in increased levels of total PGE, the authors suggested that n-3 PUFAs may increas e ovulation by augmentation of the less biologically active PGE3 at the expense of PGE2. Long chain fatty acids may also influe nce luteal activity and function via modulation of uterine PGF2 production, which causes luteol ysis (Auletta and Flint, 1988; Bazer and Thatcher, 1977; Poyser, 1995). Essential PUFAs have been shown to inhibit PG secretion in seve ral cell types (Levine and Wort h, 1984; Achard et al., 1997) including bovine endometrial (BEND) cells (Mattos et al., 2003). Manipulation of dietary LCFA content may also influence synthesis of PGF2 as demonstrated by different studies. For example, dietary n6 and n-3 PUFA have the ability to alter gestational length and time of parturition thr ough modulation of PG synthesis in rats

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37 (Holman, 1971; Leaver et al., 1986), ewes (Baguma-Nibashek a et al., 1999), and humans (Olsen et al., 1986, 1992; Allen and Harris, 2001). Although there is some evidence indicating that n-6 PUFA, LA specifically, enhances PG production by providing more substrate for conversion to AA (Connolly et al ., 1996; Nakaya et al., 2001; Elmes et al., 2004; Petit et al., 2004), other studies have found an inhibito ry effect (Elattar and Lin, 1989; Pace-Asciak and Wolfe, 1968; Cheng et al., 2001). Abomasal infusion of cycling cattle with yellow grease (72% PUFAs and 17% LA) resulted in significant attenuation of oxytocin-induced secretion of 13, 14-dihydro-15-keto-PGF2 (PGFM), a metabolite of PGF2 (Oldick et al., 1997). A number of studies have demonstrated inhibitory effects of n-3 PUFAs on PGF2 production (Bezard et al., 1994; St aples et al., 1998; Abayasekara and Wathes, 1999; Mattos et al., 2000, 2001, 2002, 2004). Supplementation of dairy cattle with fish oil (high in n-3 PUFAs) reduc ed plasma concentrations of PGFM in early postpartum as well as in oxytoc in-induced cycling cows (M attos et al., 2002, 2004). It also has been suggested that fish oil ma y reduce the sensitivity of the CL to PGF2 since cycling cows fed fish meal had higher P4 plasma concentrations after injection of a luteolytic dose of PGF2 (Burke et al., 1997). Attenuation of uterine PGF2 secretion and decreased sensitivity of the CL to this PG caused by PUFAs may lead to improved fertility through enhanced luteal func tion, reduced embryonic loss and increased pregnancy rates. Positive effects of dietary fa ts on fertility response are not exclusively related to inhibition of PG biosynthesis. Prostagla ndins are key hormones in terms of cervical ripening and myometrial contractility, which are essential for parturition (Challis, 1980). Following parturition, fertility resumes after ut erine involution takes place, resulting in

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38 resumption of normal estrous cycles (Kiracofe, 1980). This process of uterine involution is caused by myometrial cont ractions stimulated by PGF2 A massive and sustained release of PGF2 takes place during the first two week s postpartum and is essential to reduce the uterine size and increase its t one (Hafez and Hafez, 2000). Plasma PGFM concentrations increase prior to calving (Sch indler et al., 1990), a nd generally decreases to basal levels within 21 d postpartum (Lewis et al., 1984; Del Vecchio et al., 1992a). Since the uterus seems to be the primary s ource of PGFM in postpartum cows, PGFM is considered to be an indicator of uterine PGF2 production in postpartum cows (Guilbault et al., 1984). Moreover, the dur ation of this postpartum PGF2 sustained release is negatively correlated with the number of days to complete uterine involution and the interval between parturition and resumption of normal ovarian activity (Lindell et al., 1982; Madej et al., 1984). For conception to occu r after calving, the uterus must return to its normal size. Thus, modulation of PG pr oduction by dietary fats may affect the timing of rebreeding postpartum. After parturition, the reproductive tract is also subject to ma ssive bacteriologic insult, and re-establishment of a sterile uterus is one of the requirements for pregnancy to take place in the subsequent postpartum period (Hafez and Hafez, 2000). Uterine infections, such as metritis, reduce reproducti ve efficiency by incr easing calving interval and preventing resumption of ovarian activity (Griffin et al., 1974; Arthur et al., 1989; Lewis, 1997). The incidence of uterine infec tions ranges from approximately 10-40% of postpartum dairy cattle (Arthur et al., 1989; Lewis, 1997). Multiparous cows may be more resistant to some uterine infections than are primiparous cows (Hussain, 1989).

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39 However, the physiological characteristics that make some animals more susceptible than others are not well understood. Eicosanoids are also known to have im portant immunomodulatory effects (Lewis, 2004). Recent studies have confirmed the effects of in vivo exposure to PGs on the in vitro response of lymphocytes (Ramadan et al ., 1997; Lewis, 2003; Wulster-Radcliffe et al.,. 2003). PGF2 has been reported to enhance immune function in vitro (Hoedemaker et al., 1992). In addition, PGF2 increased in vitro bactericidal activity of neutrophils from ovariectomized mares (Watson, 1988). Induced and spontaneous uterine infections increase of plasma PGFM concentrations (Del Vecchio et al., 1992a, 1994). However, Seals and coworkers (2002) reported that postpartum concentr ations of PGFM were invers ely related to emergence of uterine infections. They also reported that postpartum cows with depressed PGFM concentrations were more likely to develop uterine infections. In addition, aberrant PGF2 and PGE2 production has been associated with retained placenta (Gross et al., 1987; Heuwieser et al., 1992), which in turn is associated with increased incidence of uterine infections (Curtis et al., 1985). T hus, PGFM concentration in early postpartum cows may enhance uterine immune functions and be a good indicator of the likelihood of a cow to develop metritis. Regulation of Prostaglandin F2 Synthesis by Polyunsaturated Fatty Acids Evidence is accumulating that dietary ma nipulations of PUFA s can have major effects on eicosanoid production. Depending on the amount of particular fatty acids reaching the target tissues, supplemental PUFAs can either stimulate or inhibit prostanoids synthesis. In a human study, f eeding diets rich in AA increased plasma

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40 phospholipid levels of AA and urinary excretion of the stable metabolites of prostacyclin (PGI2) and TA2 (Sinclair and Mann, 1996). Consistent with these findings, ewes infused with either soybean oil ( 50% LA) or olive oil (16% LA) had greater serum PGFM concentrations than ewes infused with salin e (Burke et al., 1996). In postpartum beef heifers, infusion of lipid (20% soybean oil) through the jugular vein increased systemic concentrations of LA and PGFM after oxytocin injection (Filley et al., 1999). Recent reports in mammalian systems have shown reduced eicosanoid synthesis when PUFAs of the n-3 or n-6 families were fed in the diet. In a study with lactating dairy cows, Oldick et al. ( 1997) reported that OT-induced PGFM concentrations were greatly reduced in cows infused abomasally with yellow grease, compared with infusion of tallow, glucose or water. Additionally, it was demonstrated that supplementation of postpartum dairy cows with fish meal cont aining EPA and DHA cons iderably attenuated PGFM response to OT induction (Mattos et al ., 2002). In beef cows, whole cottonseed added to the diets doubled th e average lifespan of a GnRH-induced CL, compared with cows fed an isocaloric control diet (Williams 1989). Although PG concentrations were not measured in the latter study, a lipid-induced s uppression of PGF2 would be compatible with a longer CL life (Staples et al., 1998). These observations provide strong evidence for PUFA involvement in modulation of PG biosynthesis. It has been proposed that PUFAs may reduce PGF2 production through modulation of one or more steps of the PG biosyntheti c pathway. These include decreasing availability of the precursor AA, competing for PGHS-2 activity and directly inhibiting synthesis and/or act ivity of PGHS-2 (Mattos et al., 2000; Cheng et al., 2001).

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41 Reduced availability of AA as a precursor for conversion to PGF2 may result from the inhibition of AA synthesi s or its displacement from th e membrane phospholipid pool. It has been reported that di etary PUFAs may inhibit AA s ynthesis during the elongation and desaturation processes of LNA in th e liver (Bezard et al., 1994). In fact, supplementation of rats with n-3 PUFAs resulte d in reduced desaturation of LA and LNA by liver microsomes and lowered concentrati on of AA in liver phospholipids (Garg et al., 1988; Christiansen et al., 1991). Incubati on of rat hepatoma cells with n-3 PUFAs resulted in reduced 6 desaturase activity (Larsen et al., 1997). Sprecher (1981) observed that in rats there was a pref erential processing of n-3 PUFAs by 6 desaturase at the expense of n-6 PUFAs. This suggested that high levels of LNA in the diet could compete with LA for 6 desaturase activity, thus attenu ating the conversion of LA to AA. Reduced availability of AA will result in greater incorp oration of other fatty acids into the phospholipid pool of the plasma membrane and de creased availability of the substrate for PGHS-2 enzyme (German et al., 1988). Dietary n-3 PUFAs can also reduce PGF2 synthesis through competition with AA for PGHS-2 activity. The n-3 PUFAs, such as EPA, can serve as substrate for PGHS-2, thus leading to the synthesis of PGs of the 3 series at the e xpense of PGs of the 2 series. Indeed, feeding rats with diet s rich in n-3 PUFAs resulted in increased secretion of PGs of the 3 series from uterine tissues cultured in vitro (Leaver et al., 1991). Direct inhibition of PGHS -2 synthesis and/or activity by PUFAs may also contribute to atte nuation of PGF2 synthesis following dietary fatty acids supplementation. When bovine aortic endothe lial cells were incubated with n-3 PUFAs (EPA and DHA), PGHS-1 mRNA expression was reduced (Achard et al., 1997). In

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42 cultures of rat hepatoma cells, PGHS enzyme was almost completely inactivated with addition of EPA 30 seconds before addition of AA as substrate (Larsen et al., 1997). Furthermore, PUFAs such as AA, EPA, and DHA, have been also found to inhibit PGHS activity (Smith and Marnett, 1991). In addition to inhibiting PGHS-2 activit y, dietary PUFAs can also modulate the expression of PGHS-2 gene. The proposed me chanism for regulation of gene expression by PUFAs involves activation of nuclear transcription fact ors such as peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors belong to a family of nuclear receptor transc ription factors activated by specific fatty acids, eicosanoids, and peroxisome prolifer ators (Desvergne and Wahli, 1999). Three different isoforms, PPAR PPAR and PPAR have been identified and are encoded by different genes (Desvergne and Wahli, 1999 ). Upon activation, PPARs dimerize with the ubiquitous retinoid X r eceptor (RXR) and binds to a prescribed DNA sequence referred to as PPAR response element (PPRE) (Desvergne and Wahli, 1999). Previous studies have shown PPARs to be involved in regulation of genes modulating steroid and prostaglandin synthesis as well as mediati ng some of the growth hormone effects in hepatocytes (Lim et al., 1999; Zhou and Waxman, 1999; Schopee et al., 2002). In general, all the n-3 and n-6 PUFAs activate the three PPAR isoforms. However, their affinities for a given receptor vary, suggesting a role for tissue-specific availability and metabolism of particular fatty acids and differe nces in their affinity for a specific PPAR subtype (Sampath and Ntambi, 2005). Furthe rmore, it has been concluded that PUFAs are potent endogenous ligands since they ha ve been shown to activate PPARs at micromolar concentrations (Lehmann et al., 1997). MacLaren et al. (2003) documented

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43 expression of PPAR PPAR and PPAR in endometrium from cyclic and pregnant Holstein cows at day 17 following estrus. Thes e isoforms also have been detected in an immortalized bovine endome trial epithelia l cell line. Collectively, these findings suggest that select dietary n-3 fa tty acids can reduce PGF2 synthesis by decreasing AA concentratio n in tissue phospholipids by increasing the concentration of fatty acids that comp ete with AA for processing by PGHS-2, and by inhibiting PGHS-2 synthesis and/or activit y. Nutritional management that alters endometrial PGF2 biosynthesis may improve reproducti ve efficiency in high producing dairy cows in which fertility is impaired due to high metabolic demands associated with milk production. Conjugated Linoleic Acid and Reproduction Conjugated linoleic acid (CLA) refers to a group of geometrical and positional isomers of LA resulting from incomplete biohydrogenation in the rumen (Chin et al., 1992; Ma et al., 1999; Griinari et al., 2000). The number of double bonds remains the same as in the parent LA, but one of the double bonds is shifted to a new position by microbial isomerases. One or both of the double bonds are either in the cis or trans configurations separated by a single carbon-to-carbon linkage rather than by the normal methylene group. A broad number of cisand transCLA isomers have been identified in food; however, the most commonl y occurring CLA isomer is the cis -9, trans -11octadecadienoic acid with minor but significant proportions of trans -10, cis -12-18:2 (Parodi, 1977; 1997; Chin et al ., 1992; McGuire et al., 1998). After ruminal synthesis of cis -9, trans -11 CLA, it may be absorbed in the small intestine or further biohydrogenated into trans -11-octadecenoic acid ( trans vaccenic acid)

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44 by ruminal microorganisms. The trans -11 18:1 can be reduced to stearic acid (18:0) or transported to peripheral tissues as MUFA. In the peripheral tissues (i.e. mammary gland, muscle), this trans -isomer may be converted back into cis -9, trans -11 CLA by the action of 9 desaturase (Pollard et al., 1980; Holman and Mahfouz, 1981). This conversion appears to be a major source of cis -9, trans -11 CLA in the cows milk (Corl et al., 1998; Griinari and Bauma n, 1999; Santora et al., 2000). The presence of trans -10, cis -12 CLA in milk (Griinari and Bauman, 1999) suggests the existence of endogenous ruminal bact eria with the capability for synthesis of this isomer. In support of this hypothesis, LA has been shown to be converted to trans 10, cis -12 CLA by Propionobacter in vitro (Verhulst et al., 1987). However, the trans 10, cis -12 CLA detected in ruminal tissues (G riinari and Bauman, 1999; Dhiman et al., 1999) seem to originate solely from rumina l synthesis, since mammalian tissues do not have the 12 desaturase necessary for conversion of trans -10-octadecenoic acid (elaidic acid) back to trans -10, cis -12 CLA. A large number of beneficial effects have been attributed to CLA ranging from enhancing feed efficiency and growth in ra ts (Chin et al., 1994) and pigs (Bee, 2000), decreasing body fat in mice (DeLany et al., 1999) and pigs (Dugan et al., 1997; Ostrowska et al., 1999), and reduced milk fat synthesis in lactating dairy cows (Griinari et al., 1998; Romo et al., 2000; Baumgard et al., 2001). A lthough it is likely that some biological effects of CLA may be induced and/or enhanced synergistically by these isomers, there is evidence suggesting that numerous effects of CLA are due to separate actions of these biologically active isomers (P ariza et al., 2000). Ur uquhart et al. (2002) observed that after treatment of human vein endothelial cell s with 100 M of individual

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45 CLA isomers, differential effects were observed with cis -9, trans -11 CLA inhibiting, while trans -10, cis -12 CLA inducing Ca ionophore-stimul ated eicosanoid production. It seems that trans -10, cis -12 works preferentially through modulation of apoptosis and cell cycle, whereas cis -9, trans -12 may work through AA meta bolism (Ochoa et al., 2004). Nonetheless, beneficial actions of CLA have been linked to modulation of eicosanoid production (Sugano et al., 1998). Recent studies detected an inhibitory effect of CLA on eicosanoid synthesi s in various animal and cel l systems (Liu and Belury, 1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al, 2003). Feeding pregnant rats with dietary CLA re sulted in inhibition of uterine PGF2 independent of PUFA content (Harris et al., 2001). In a ddition, Cheng et al. ( 2003) reported that treatment of endometrial cells isolated from late pregnant ewes with CLA suppressed PGF2 in a dose dependent manner, while low doses of CLA stimulated PGE2 generation. Cheng and coworkers (2003) showed that tr eatment of intercotyl edonary endometrial cells with CLA resulted in a dos e-dependent inhibition of PGF2 production. Similarly, CLA also has been shown to inhibit PGF2 synthesis in rat placen tal and uterine tissues (Harris et al., 2001). Although the mechanism underlying this inhibiti on is not clear, it has been suggested that CLA may compete w ith AA for PGHS-2 activity as well as for incorporation to the membrane phospholip ids. Alternativel y, CLA may modulate conversion of PGF to PGE2 as reported by Gross and Williams (1988) in bovine placental cells. Therefore, CLA may regul ate reproductive processes through modulation of prostaglandin biosynthesis.

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46 CHAPTER 3 EFFECTS OF POLYUNSATURATED FA TTY ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS Introduction In the past decade, genetic selection fo r high milk production has been associated with a decrease in reproductive efficiency in lactating dairy cows (Butler, 2000). Poor reproductive efficiency includes early embryoni c loss (Thatcher et al., 1995), impaired ovarian cyclicity and low fertility rates (B utler, 2000), which colle ctively result in reduced life-long milk production (Plaizier et al., 1997). Early postpartum dairy cows have higher energy requirements than can be supported by dietary energy intake, which creates a negative energy state that can lead to impaired reproducti ve function (Butler, 2000). Polyunsaturated fatty acids (PUFA) are gene rally added to dairy rations to increase the energy density of the diet. In mammals the parent molecules for the n-6 and n-3 families, linoleic (LA; 18:2) and linolenic (LNA; 18:3) acids, respectively, cannot be synthesized by the tissues and, therefore, must be supplied in the diet Since there is an absolute requirement for these PUFAs, they are regarded as essent ial fatty acids (Burr and Burr, 1929; Aaes-Jorgense n, 1961; Holman et al., 1982). Supplemental polyunsaturated fatty acids (P UFA) have been reported to increase conception rates (Schneider et al., 1988; Sklan et al., 1989; Fe rguson et al., 1990), enhance pregnancy rates (Schneider et al,. 1988; Sklan et al., 1991), and reduce the interval to first estrus (Sklan et al., 1991) of lacta ting dairy cows. Select dietary fats have

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47 been also shown to regulate eicosanoid s ynthesis (Abayasekara and Wathes, 1999; Cheng et al., 2001), modulate plasma P4 concentration (Carrol et al., 1990; Lucy et al., 1993b; Garcia-Bojalil et al., 1998), stimulate ovarian follicular development (Lucy et al., 1993b; Thomas and Williams, 1996; Beam and Butler, 1997) and improve fertility (Staples et al., 1998) of lactating dairy cows. Prostaglandins are members of the eico sanoid family synthesized from 20 carbon PUFAs, such as arachidonic acid (AA). Ar achidonic acid is, in turn, synthesized from elongation and desaturation of LA. The firs t and rate limiting step of prostaglandin biosynthesis is the hydrolyti c release of AA by the action of cytosolic phospholipase A2 (cPLA2) enzyme (Lapetina, 1982). Following its release, AA is converted to prostaglandin H2 (PGH2) by the action of prostaglandin H synthase-2 (PGHS-2), also known as cyclooxygenase-2 (COX-2). Prostagl andin H synthase has cyclooxygenase and peroxidase activities that convert prostaglandin G (PGG) to PGH2 (Goff, 2004). After synthesis of PGH2, this endoperoxide is converted to one of several pos sible prostanoids by the action of specific terminal enzyme s. Biosynthesis of prostaglandin E2 (PGE2) and prostaglandin F2 (PGF2 ) are catalyzed by the acti on of prostaglandin E and prostaglandin F synthases, respectively (Goff, 2004). Prostaglandins of the 2-series, PGF2 and PGE2, are the most biologically active ei cosanoids and are involved in key reproductive processes such as follicular de velopment (Wallach et al., 1975), ovulation (Espey, 1980), luteolysis (Wathes and Lammi ng, 1995), and parturition (Challis, 1980). Essential PUFAs have been shown to inhi bit PG secretion in several cell types (Levine and Worth, 1984; Achard et al., 1997) including bovine e ndometrial (BEND) cells (Mattos et al., 2003). The BEND cells are a line of spontaneously replicating

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48 endometrial cells originati ng from cows in their 14th day of their estrous cycle (Staggs et al., 1998). Nutritional studies have shown that manipulatio n of dietary PUFA content may influence circulating concentrations of PGF2 For example, dietary n-6 and n-3 PUFA have the ability to alter gestati onal length and time of parturition through modulation of PG synthesis in rats (Holman, 1971; Leaver et al., 1986), ewes (BagumaNibasheka et al., 1999), and humans (Olsen et al., 1986; 1992; Allen and Harris, 2001). Although there is some evidence indicating that n-6 PUFAs, LA specifically, enhance PG production by providing more precursors for conversion to AA (Connolly et al., 1996; Nakaya et al., 2001; Elmes et al., 2004; Peti t et al., 2004), other studies have found an inhibitory effect of n-6 PUFAs on PG synt hesis (Elattar and Li n, 1989; Pace-Asciak and Wolfe, 1968; Cheng et al., 2001). A number of studies have demonstrated inhibitory effects of n-3 PUFAs on PGF2 production (Bezard et al., 1994; Staples et al., 1998; Abayasekara and Wathes, 1999; Mattos et al., 2000, 2001, 2002, 2004). Supplementation of dairy cattle with fish oil (high in n-3 PUFAs) greatly reduced plasma PGFM response to oxytocin (Mattos et al., 2002, 2004). It al so has been suggested that fish oil may reduce the sensitivity of the CL to PGF2 since cycling cows fed fish meal had higher P4 plasma concentrations after injec tion of a luteolytic dose of PGF2 (Burke et al., 1997). Attenuation of uterine PGF2 secretion and decreased sensit ivity of the CL to this PG caused by PUFAs may lead to improved fert ility through enhanced luteal function, reduced embryonic loss and increased pregnancy rates. The objective of this investigation was to examine the effects of n-6 and n-3 PUFAs on phorbol 13, 14-dibut yrate (PDBu)-induced PGF2 biosynthesis in BEND cells.

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49 We hypothesized that PUFAs may be more effective in inhibiting PGF2 production when compared to saturated fatty acids. Materials and Methods Materials Polystyrene tissue culture dishes (100 x 20 mm) were purchased from Corning (Corning Glass Works, Corning, NY). The Ham F-12 medium, antibiotic/antimycotic (ABAM), phorbol 13, 14-dibutyrate (PDBu), horse serum, D-valine, insulin, fatty acidfree bovine serum albumin (BSA), stearic acid (ST, C18:0), aprotinin, leupeptin, and pepstatin were from Sigma Chemical Co. (S t. Louis, MO). The Minimum Essential Medium (MEM) and fetal bovine serum (FBS) were from US Biologicals (Swampscott, MA) and Atlanta Biologicals (Norcross, GA), respectively. Linoleic acid (C18:2n-6), LNA (C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3), PGHS-2 and PGES antibodies, and PGF2 standard were from Cayman Chemicals (Ann Arbor, MI). The PPAR and secondary anti-rabbit IgG antibodies were fr om Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) kit was purchased from Perkin Elmer (Boston, MA). Hanks Balanced Salt Soluti on (HBSS) and TriZol reagent were from GIBCO BRL (Carlsbad, CA). Isotopically-labelled PGF2 (5, 6, 8, 9, 11, 12, 14, 15[n-3H] PGF2 ; 208 Ci/mmol) was from Amershan Bios ciences (Piscataway, NJ). The antiPGF2 antibody was purchased from Oxford Biom edicals (Oxford, MI). BioTrans nylon membrane and [ -32P]deoxycitidine triphosphate (SA 3000 Ci/nmol) were from MP Biomedicals (Atlanta, GA). The PGHS-2 cDNA probe was cloned from an ovarian follicular cDNA library (Liu et al., 1999), the PGES cDNA probe was cloned from

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50 endometrial cDNA (Guzeloglu et al., 2004), whereas the PPAR probe was generated from bovine endometrial R NA (Balaguer et al., 2005). Cell Culture and Treatment Bovine endometrial (BEND; American Type Culture Collection # CRL-2398; Manassas, VA) cells were plated on 100-mm tis sue culture plates in complete culture medium (40% Ham F-12, 40% MEM containing 10 ml ABAM/L, 0.0343g D-valine/L, 200 U insulin/L, 10% fetal bovine serum and 10% horse serum) and grown to confluence at 37C in a humidified at mosphere containing 95% O2 and 5% CO2. Cells then were rinsed twice with Hanks balanced salt solu tion (HBSS), and cultured in fresh serum-free medium containing appropriate tr eatments for an additional 24 h. To examine the effects of PDBu on PGF2 production, subconfluent BEND cells were incubated with medium alone (contro l, n=2) or medium supplemented with 100 ng/ml PDBu (n=2) for 6 h. After incubation, aliquots (1 ml) of ce ll-conditioned media were collected and stored at -20 C for subsequent analysis of PGF2 using RIA. The entire experiment was repeated five times. To investigate the effects of supplemental PUFAs on uterine PGF2 synthesis, BEND cells were treated with PDBu alone ( 100 ng/ml) or with a combination of PDBu and stearic (ST; 100 M), LA (100 M), LNA (100 M) or EPA (100 M). Subconfluent cells were incubated with se rum-free medium alone (PDBu) or with appropriate treatments (listed above) complexed with BSA (1:3 ratio) for a period of 24 h. Cells then were rinsed twice with HB SS, and challenged with phorbol ester 12, 13dibutyrate (PDBu, 100 ng/ml) for an additional 6 h. Control pl ates were cultured in the absence of PDBu. Samples of conditioned medi a (1 ml/plate) were collected after PDBu

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51 challenge and stored at -20C until analyzed for PGF2 concentration. The remaining cell monolayer was rinsed with HBSS, lysed in TriZol reagent, and stored at -80C for subsequent mRNA analysis. For analysis of protein abundance, cells were collected and lysed as described later in the section for We stern blot analysis. These experiments were repeated two times. PGF2 Radioimmunoassay The concentration of PGF2 in cell-conditioned media was measured as described by Danet-Desnoyers et al. (1994) and modified by Binelli et al. (2000). One hundred and fifty microliters of Tris buffer (50 mM Tris-HCl, 1 g /l sodium azide, pH 7.5), 50 l of conditioned media or standards (range 10 1000 pg/tube), 100 l of Tris buffer/serumfree medium, 100 l of anti-PGF2 antibody (1: 30,000 in Tris buffer) and 100 l of 3HPGF2 (18,000 dpm) were added sequentially to 12x75 mm disposable glass tubes, vortexed and incubated for 24 h at 4oC. After incubation, 500 l of dextran-coated charcoal solution (25 mg dextran and 250 mg charcoal into 100 ml RIA buffer) was added, and the mixture was incubated for an additional 4 min at 4oC. Assay tubes were then centrifuged (3200 rpm for 15 min at 4oC), decanted and counted in a beta counter. Assay sensitivity was 0.5 ng/ml and intraand inter-assay coefficients of variation were 8.2 and 15.8%, respectively. Final PGF2 concentrations were expressed as picograms per milliliter. RNA Isolation and Analysis Total RNA was isolated using TriZol r eagent, following the manufacturers instructions. Ten g of total RNA was fractioned in a 1.5 % agarose formaldehyde gel using the MOPS buffer (Fisher Scientific Pittsburgh, PA) (Ing et al., 1996) and

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52 transferred to a Biotrans nylon membrane by downward capillary transfer. The RNA was cross-linked to the nylon membrane by e xposure to a UV light source for 90 sec and baked for 1 h. Membranes were prehybridized for 2 h at 42C in ultrasensitive hybridization buffer (ULTRAhyb; Ambion, Au stin, TX) followed by an overnight incubation at 42C in the same ULTRAhyb solution containing the 32P-labeled bovine prostaglandin H synthase 2 (PGHS-2), prosta glandin E synthase (P GES), and peroxisome proliferator-activated receptor (PPAR cDNA probes. Filters were sequentially washed in 2X SSC (1X= 0.15 M sodium chloride, 0.015 M sodium citrate)-0.1% SDS and in 0.1x SSC-0.1% SDS two times each at 42 C and then exposed to X-ray film to detect radiolabeled bands. Equal loading of total RNA for each experimental sample was verified by comparison to 18S rR NA ethidium bromide staining. Western Blot Analysis of PGHS-2, PGES and PPAR For Western blot analyses, whole cell ly sates were prepared as described by Binnelli et al. (2000). Five hundred microliters of ice-cold whole cell extract buffer (50 mM Tris, pH 8.0, 300 mM NaCl, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P2O7, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 10% v/v glycerol, 0.5% v/v NP-40, and 10 g/ml each of Aprotinin, Leupeptin, a nd Pepstatin), were added to each dish and cells were collected in 1.5 ml micro centrifuge tubes and incubated for 30 min at 4C. Cell lysates then were centrifuged at 4C for 10 min (13000 rpm) to remove cell debris. Protein concentration in the supe rnatant was determined by the Lowry method (Lowry et al., 1951). Protein (25 g) from each dish was resolved on a 7.5% SDSPAGE, and electrophoretic ally transferred to a nitrocellulose membrane. The Membrane was blocked for 2 h in 5% (w/v) nonfat dr ied milk in Tris-buffered saline (TBS)

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53 containing 0.1% Tween-20 (TBST, pH 7.4), rinsed with TBST and hybridized with antibodies against either PGHS-2, PGES, or PPAR diluted (1:500) in 5% nonfat dried milk in TBS. The secondary antibody wa s anti-rabbit IgG (1:3000 in 5% nonfat dried milk in TBS). Target proteins were detected by enhanced chemiluminescence. Statistical Analyses Data were analyzed by Least-Squares an alysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the Statistical Analysis System (SAS, 2001). For PGF2 response, the mathematical model include d fixed effects of treatment (n = 5), experiment (n = 5), treatment by experiment interaction, and random effect of dish (n = 2/treatment) nested within treatment by expe riment interaction. The variance of dish nested within treatment by experiment interaction was used as the error term for all effects. For Northern and western blot data, hybridization volumes obtained from densitometric analysis were subjected to ANOVA using the GLM procedure. For Northern blot analysis, the mathematical model included independent fixed effects of treatment (n = 5), experiment (n = 2), trea tment by experiment interaction, and random residual error. Results are pr esented as ratios of densitome tric values for the target mRNA over those for 18S rRNA ethidium br omide staining, and are presented as LS means SEM. For Western blot analysis, the statistical model included only the main effect of treatment. Treatment effects were further analyzed usi ng preplanned orthogonal contrasts. These contrasts were constructed to compare PDBu vs. fa tty acids responses; SFA vs. PUFA; n-6 vs. n-3 PUFA; and n-3 LNA vs. longer chain n-3 EPA.

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54 Results To test the hypothesis that PUFAs may be more effectiv e in inhibiting endometrial PGF2 secretion, we examined endometrial PGF2 response to PDBu in the presence of n-6 (LA) and n-3 (LNA and EPA) PUFAs in comparison to saturated fatty acid (ST). In vitro systems designed to evaluate endometrial PGF2 secretion involved stimulation of cells with PDB u, an activator of protein ki nase C which induces PGHS-2 gene expression and secretion of PGF2 (Binelli et al., 2000). Treatment of BEND cells with PDBu resulted in a 25-fold induction ( P < 0.0001) of PGF2 secretion (Figure 3-1). The PDBu-induced PGF2 secretion coincided with in creased abundance of PGHS-2 mRNA ( P < 0.0001; Figure 32) and protein ( P = 0.0003; Figure 3-3). Stimulation of BEND cells with PDBu also resulted in induction ( P < 0.0001) of PGES gene expression (Figure 3-4), but no differences were observed for PGES pr otein abundance (Figure 3-5). Co-incubation of BEND cells with fatty acids decreased ( P < 0.0001) PGF2 response to PDBu (Figure 3-6). Analysis of individual fatty acid e ffects revealed that EPA greatly reduced PGF2 induction by PDBu. The other fatty acids had minimal effects on PDBu-induced prostaglandin production. To determine the molecular mechanism by which fatty acids altered PGF2 production in BEND cells, we examined PGHS-2, PGES and PPAR mRNA and protein responses to PDBu in the pres ence of various fatty acids. On average, long-chain fatty acids had no detectable eff ects on PGHS-2, PGES and PPAR mRNA responses to PDBu (Figures 3-7, 3-9 and 3-11). The long-chain fatty acids stimulated ( P = 0.0001) PGES protein expression, but failed to alter PGHS-2 or PPAR protein response to PDBu (Figures 3-8, 3-10 and 3-12).

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55 Discussion Polyunsaturated fatty acids have been asso ciated with improvement of reproductive efficiency in cattle, and modul ation of prostaglandin synthe sis has being suggested as a potential mechanism. In the present study, we examined the effects of selected longchain fatty acids on PDBu-induced PGF2 synthesis in BEND cells. On average, the long-chain fatty acids tested in this study decreased PGF2 response to PDBu in BEND cells. EPA decreased PGF2 production to a greater extent th an did the other fatty acids. However, both saturated and unsaturated fatt y acids had no detectable effects on PGHS2, PGES and PPAR mRNA responses to PDBu. Reports on the effects of feeding n6 PUFA on prostaglandin synthesis in vivo have been inconsistent. Recent studies showed that in vitro supplementation of LA to maternal intercotyledonary endometrial cells isolated fr om late pregnant ewes caused a significant reduction of 2-series prostagl andins (Cheng et at., 2003, 2004) Similarly, cyclic dairy cows fed a diet high in LA had reduced e ndometrial prostaglandi n production (Cheng et al., 2001). On the other hand, Cheng and others (2005) showed that late pregnant ewes fed a diet high in LA had increased endomet rial and placental prostaglandin production. In the present study, incubation of BEND ce lls with EPA caused a reduction of PGF2 secretion in response to PDBu stimulation. These findings are in ag reement with reports indicating that n-3 PUFAs inhi bit prostaglandin secretion in vitro (Achard et al., 1997; Levine and Worth, 1984; Matt os et al., 2001, 2003; Caldar i-Torres et al., 2006) and in vivo (Staples et al., 1998; Abayasekara and Wathes, 1999; Mattos et al., 2000, 2002, 2004).

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56 The mechanisms by which EPA alters PGF2 response to PDBu are not well understood. It has been proposed that PUFAs may reduce PGF2 production through modulation of one or more steps of the PG biosyntheti c pathway. These include decreasing availability of the precursor AA, competing for PGHS-2 activity and directly inhibiting synthesis and/or activ ity of PGHS-2 (Mattos et al ., 2000; Cheng et al., 2001). In the present study, no changes were observe d in PGHS-2 mRNA response to PDBu. This is consistent with a previous study (Mattos et al., 2003) and suggests that EPA may be competing with AA for the available bi nding sites on PGHS-2, thus shifting to production of 3-series prostaglandins (PGF3 and PGE3). This hypothesis does not rule out the possibility that EPA may also affect the PGHS-2 enzyme activity through posttranslational modifications. The observation that the long-chain fatty ac ids tested in this study had no effects on PPAR gene or protein expression indicates th at these fatty acids likely affect PGF2 production through mechanisms which do not involve PPAR induction. However, whether or not long-chain fatty acids alter PPAR activity warrants further investigation. Summary Phorbol-ester stimulated PGF2 production and up-regulated PGHS-2 gene and protein expression within 6 h in cultured BE ND cells. Priming of BEND cells with ST, LNA, and EPA reduced PGF2 response to PDBu by 17%, 14%, and 66%, respectively. Both saturated and unsaturated fatty acids had not detectable e ffects on PGHS-2, PGES or PPAR mRNA response to PDBu, suggesting that l ong-chain fatty acid s tested in this study likely affect PGF2 production through PGHS-2-, PGES-, and PPAR -independent mechanisms. Whether and how these fatty acid s affect the activitie s of various enzymes

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57 and transcription factor s involved in the PGF2 biosynthetic cascade warrants further information.

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58 Figure 3-1. Effect of phorbol 12, 13 dibutyrate (P DBu) on prostaglandin F2 (PGF2 ) secretion in bovine endometrial (BE ND) cells. Treatment by experiment interaction was significant ( P < 0.05). Data represents least square means SEM of five independent expe riments (treatment effect, P < 0.0001).

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59 Figure 3-2. Effect of phor bol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide synthase (PGHS-2) mRNA abundance in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Nort hern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment, P < 0.0001). There was no treatment by experiment interaction ( P = 0.06).

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60 Figure 3-3. Effect of phor bol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide synthase (PGHS-2) protein levels in bovine endometrial (BEND) cells. Twenty five micrograms of total cellula r protein extracted from control and treated cells were subjected to Wester n blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative We stern blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment, P = 0.0003).

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61 Figure 3-4. Effect of phor bol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase (PGES) mRNA abundance in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and re sulting densitometric values were analyzed by the GLM pro cedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experi ments (n = 4 for each treatment, P < 0.0001). Treatment by experiment interaction was significant ( P = 0.009).

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62 Figure 3-5. Effect of phor bol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase (PGES) protein levels in bovine endo metrial (BEND) cells. Twenty five micrograms of total cellular protein extracted from control and treated cells were subjected to Western blot analys is (A), and resulting densitometric values were analyzed by the GLM pro cedure of SAS (B). The top panel shows a representative Western blot whereas the bottom panel represents means SEM calculated over two experi ments (n = 4 for each treatment, P = 0.3).

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63 Figure 3-6. Effect of fatty acids on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometri al (BEND) cells. When treatment effects were detected ( P < 0.05), means were se parated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P < 0.0001. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.04. Contrast 3: (LA) vs. (LNA) + (EPA), P < 0.0001. Contrast 4: (LNA) vs. (EPA), P < 0.001. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. There was no treatment by experiment interaction ( P = 0.14). Treatments PDBuSTLALNAEPA PGF 2 (pg/ml) 2000 4000 6000 8000 10000 a b b c d

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64 Figure 3-7. Effect of fatty acids on pros taglandin endoperoxide synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells. Ten micrograms of tota l cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representa tive Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatme nt effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.1. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.06. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.06. Contrast 4: (LNA) vs. (EPA), P = 0.02. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by differe nt letters. There was no treatment by experiment interaction ( P = 0.38). Treatments PDBuSTLALNAEPA PGHS-2 mRNA (normalized to 18S rRNA) 0.00 0.05 0.10 0.15 18S ST LA LNA EPA PDBu rRNA PGHS-2 1 2 3 4 5 6 7 8 9 1(A) (B) a b b a,b a 4.4 kb

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65 Figure 3-8. Effect of fatty acids on pros taglandin endoperoxide synthase (PGHS-2) protein response to phorbol 12, 13 dibutyr ate (PDBu) in bovine endometrial (BEND) cells. Twenty five microgram s of total cellular protein extracted from control and treated cells were subject ed to Western blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representa tive Western blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatme nt effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.1. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.01. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.2. Contrast 4: (LNA) vs. (EPA), P = 0.5.

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66 Figure 3-9. Effect of fatty acids on prosta glandin E synthase (P GES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and re sulting densitometric values were analyzed by the GLM pro cedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experime nts (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contra st 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.09. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P < 0.0001. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.04. Contrast 4: (LNA) vs. (EPA), P = 0.003. When main treatment effect was significant ( P < 0.05), then differences between treatments are repres ented by different letters. Treatment by experiment interaction was significant ( P = 0.0003). Treatments PDBuSTLALNAEPA PGES mRNA (normalized to 18S rRNA) 0.00 0.08 0.16 0.24 0.32 0.40 1.3 kb 18S PDBu ST LA LNA EPA rRNA PGES 1 2 3 4 5 6 7 8 9 10 (A) (B) a b a c c

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67 Figure 3-10. Effect of fatty acids on prostaglandin E synt hase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Twenty five micrograms of total cellula r protein extracted from control and treated cells were subjected to Wester n blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative We stern blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. C ontrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.0001. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.02. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.05. Contrast 4: (LNA) vs. (EPA), P = 0.5. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. Treatments PDBuSTLALNAEPA PGES Protein Expression (arbitrary units) 75 100 125 150 ST LA LNA EPA PDBu (16 kDa) PGES 1 2 3 4 5 6 7 8 9 10 (A) (B) a b c b b,c

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68 Figure 3-11. Effect of fatty acids on peroxisome prolif erator-activated receptor (PPAR ) mRNA response to phorbol 12, 13 di butyrate (PDBu) in bovine endometrial (BEND) cells. Ten microgr ams of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a repr esentative Northern blot, whereas the bottom panel represents means SEM cal culated over two experiments (n = 4 for each treatment). When trea tment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.2. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.9. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.5. Contrast 4: (LNA) vs. (EPA), P = 0.2.

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69 Figure 3-12. Effect of fatty acids on peroxisome prolif erator-activated receptor (PPAR ) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Twenty five micrograms of total cellular protein extracted from control and treated ce lls were subjected to Western blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representati ve Western blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using or thogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.6. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.8. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.2. Contrast 4: (LNA) vs. (EPA), P = 0.9.

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70 CHAPTER 4 EFFECTS OF CONJUGATED LINO LEIC ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS Introduction Conjugated linoleic acid (CLA) refers to a group of geometrical and positional isomers of LA resulting from incomplete biohydrogenation in the rumen (Chin et al., 1992; Ma et al., 1999; Griinari et al., 2000). The number of double bonds remains the same as in the parent LA, but one of the double bonds is shifted to a new position by microbial isomerases. A broad number of cisand transCLA isomers have been identified in food; however, the most commonly occurring CLA isomer is the cis -9, trans -11-octadecadienoic acid with minor but significant proportions of trans -10, cis -1218:2 (Parodi, 1977, 1997; Chin et al ., 1992; McGuire et al., 1998). After ruminal synthesis of cis -9, trans -11 CLA, it may be absorbed in the small intestine or further biohydrogenated to trans -11-octadecenoic acid ( trans vaccenic acid) by rumen microorganisms. The trans -11 18:1 can be reduced to stearic acid (18:0) or transported to peripheral tissues as MUFA. In the peripheral tissues (i.e. mammary gland, muscle), this trans -isomer may be converted back into cis -9, trans -11 CLA by the action of 9 desaturase (Holman and Mahfouz, 1981; Pollard et al., 1980). This appears to be a major source of cis -9, trans -11 CLA in the cows milk (Corl et al., 1998; Griinari and Bauman, 1999; Santor a et al., 2000). The trans -10, cis -12 CLA detected in ruminal tissues (Griinari and Bauman, 1999; Dhiman et al., 1999) seem to originate solely from

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71 ruminal synthesis, since mammalian tissues do not have the 12 desaturase necessary for conversion of trans -10-octadecenoic acid (ela idic acid) back to trans -10, cis -12 CLA. A large number of beneficial effects have been attributed to CLA ranging from enhancing feed efficiency and growth (C hin et al., 1994; Bee, 2000), decreasing body fat in mice (DeLany et al., 1999) and pigs (Duga n et al., 1997; Ostrowska et al., 1999), and reducing milk fat synthesis in lactating dair y cows (Griinari et al., 1998; Romo et al., 2000; Baumgard et al., 2001). Although it is likely that some biological effects of CLA may be induced and/or enhanced synergistic ally by these isomers, there is evidence suggesting that the effects of CLA are due to separate actions of these biologically active isomers (Pariza et al., 2000). The anticarcinogenic effect of CLA has b een linked to its ability to modulate eicosanoid production (Sugano et al., 1998). Recent studies detected an inhibitory effect of CLA on eicosanoid synthesi s in various animal and cel l systems (Liu and Belury, 1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al, 2003). Feeding pregnant rats with dietary CLA re sulted in inhibition of uterine PGF2 independent of PUFA content (Harris et al., 2001). In a ddition, Cheng et al. ( 2003) reported that treatment of endometrial cells isolated from late pregnant ewes with CLA suppressed PGF2 in a dose dependent manner, while low doses of CLA stimulated PGE2 generation. Cheng and coworkers (2003) showed that tr eatment of intercotyl edonary endometrial cells with CLA resulted in a dos e-dependent inhibition of PGF2 production. Similarly, CLA has also been shown to inhibit PGF2 synthesis in rat placen tal and uterine tissues (Harris et al., 2001). Prostagl andins of the 2-series are involved in several reproductive processes such as ovulation (Espey, 1980), fo llicular growth and development (Wallach

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72 et al., 1975), and CL function (Abayaseka ra et al., 1995; Poyser, 1995; Wathes and Lamming, 1995). Hence, CLA isomers may regulate reproductive processes through modulation of PGF2 biosynthesis. The objective of this investigati on was to examine the effects of cis -9, trans -11 and trans -10, cis -12 CLA isomers on PBDu-induced PGF2 production in bovine endometrial (BEND) cells. We hypothesized that both CLA isomers woul d be equally effective in inhibiting PDBu-induced PGF2 production in BEND cells. Additionally, if these effects are specific to CLA, LA should have minimal effect on PGF2 production. Materials and Methods Materials Polystyrene tissue culture dishes (100 x 20 mm) were purchased from Corning (Corning Glass Works, Corning, NY). The Ham F-12 medium, antibiotic/antimycotic (ABAM), phorbol 13, 14-dibutyrate (PDBu), horse serum, D-valine, insulin, fatty acidfree bovine serum albumin (BSA), stearic acid (ST, C18:0), aprotinin, leupeptin, and pepstatin were from Sigma Chemical Co. (S t. Louis, MO). The Minimum Essential Medium (MEM) and fetal bovine serum (FBS) were from US Biologicals (Swampscott, MA) and Atlanta Biologicals (Norcross, GA), respectively. Linoleic acid (LA, C18:2n-6), cis -9, trans -11 conjugated linoleic acid (CLA, C18:2), trans -10, cis -12 CLA (C18:2), PGHS-2 and PGES antibodies, and PGF2 standard were from Cayman Chemicals (Ann Arbor, MI). The PPAR and secondary anti-rabbit IgG an tibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The e nhanced chemiluminescence (ECL) kit was purchased from Perkin Elmer (Boston, MA). Hanks Balanced Salt Solution (HBSS) and TriZol reagent were from GIBCO BRL (C arlsbad, CA). Isotopically-labelled PGF2 (5,

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73 6, 8, 9, 11, 12, 14, 15[n-3H] PGF2 ; 208 Ci/mmol) was from Amershan Biosciences (Piscataway, NJ). The anti-PGF2 antibody was purchased fro m Oxford Biomedicals (Oxford, MI). BioTrans nylon membrane and [ -32P]deoxycitidine triphosphate (SA 3000 Ci/nmol) were from MP Biomedicals (Atlanta, GA). The PGHS-2 cDNA probe was cloned from an ovarian follicular cDNA lib rary (Liu et al., 1999), the PGES cDNA probe was cloned from endometrial cDNA (Guzeloglu et al., 2004), whereas the PPAR probe was generated from bovine endo metrial RNA (Balaguer et al., 2005). Cell Culture and Treatment Bovine endometrial (BEND) cells were plat ed and cultured as de scribed in chapter 3. To investigate the effects of supplemental CLA acids on uterine PGF2 synthesis, BEND cells were treated with PDBu alone ( 100 ng/ml) or PDBu in combination with 100 M linoleic acid (LA), cis -9, trans -11 CLA or trans -10, cis -12 CLA. Fatty acids were complexed with BSA (1:3 ratio) for 2 h, and then treatments were applied to cells for a 24 h period. After treatment, cells were rins ed with HBSS and challenged with PDBu for another 6 h. The remaining cell monolayer was rinsed with HBSS, lysed in TriZol reagent, and stored at -80C for subseque nt mRNA analysis. This experiment was repeated two times. Radioimmunoassay of PGF2 and Northern and Western blot analyses were performed as described in chapter 3. Statistical Analyses Data were analyzed by Least-Squares an alysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the Statistical Analysis System (SAS, 2001). For PGF2 response, the mathematical model include d fixed effects of treatment (n = 4), experiment (n = 4), treatment by experiment interaction, and random effect of dish (n =

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74 2/trt) nested within treatmen t by experiment interaction. Th e variance of dish nested within treatment by experiment was used as th e error term for all upstream effects. For Northern and western blot data, hybridiza tion volumes obtained from densitometric analysis were subjected to ANOVA using th e GLM procedure. For Northern blot analysis, the statistical model included indepe ndent fixed effects of treatment (n = 4), experiment (n = 2), treatment by experiment interaction, and random residual error. Results are presented as ratios of densitometric values for target genes over those for 18S rRNA ethidium bromide staining, and are pres ented as LS means SEM. For Western blot analysis, the mathematical model incl uded only the main effect of treatment. Treatment effects were further analyzed us ing preplanned orthogonal contrasts. These contrasts were constructed to compare PD Bu vs. fatty acids responses; LA vs. CLA isomers; and cis -9, trans -11 CLA vs. trans -10, cis -12 CLA. Results In the present study we used bovine endomet rial (BEND) cells as a model to study CLA regulation of PGF2 production. BEND cells are a line of spontaneously replicating endometrial cells originating from d 14 cycli ng cows (Staggs et al ., 1998). Priming of BEND cells with cis -9, trans -11 or trans -10, cis -12 CLA isomers resulted in a 1.3-fold inhibition ( P < 0.0001) of PGF2 response to PDBu (Figure 4-1). Conversely, both CLA isomers stimulated ( P < 0.05) PGHS-2 mRNA response to PDBu (Figure 4-2). Coincubation with C18 fatty acids resu lted in a significant reduction ( P < 0.003) in PGES mRNA response to PDBu (Figure 4-3). In contrast with PGES gene expression, PPAR mRNA levels were increased ( P < 0.0008) by all the polyunsaturat ed fatty acids tested in

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75 this study (Figure 4-6). Average PPAR protein levels did not differ among treatments (Figure 4-7). Discussion Prostaglandins of the 2-series affect numerous processes in reproduction, including ovulation (Espey, 1980), follicular developmen t (Wallach et al., 1975), corpus luteum function (Poyser, 1995; Wathes and Lammi ng, 1995; Abayasekara and Wathes, 1999) and parturition (Challis et al., 1997). The anticarcinogeni c effect of CLA has been attributed partially to its i nhibitory effect on eicosanoid synthesis (Banni et al., 1999; Gregory and Kelly, 2001). In the present study, incubation of BEND cells with either cis -9, trans -11 or trans 10, cis -12 CLA isomers resulted in a 1.3-fold inhibition ( P < 0.0001) of PDBu-induced PGF2 secretion. This is in agreement with seve ral studies that have detected inhibitory effect of CLA on eicosanoid synthesis in vivo and in vitro (Liu and Belury, 1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Ed er et al., 2003). In addition, Cheng et al. (2003) reported that treatment of endometrial cells isolated from late pregnant ewes with CLA suppressed PGF2 production. However, CLA inhibition of endometrial PGF2 response to PDBu in BEND cells was no t mediated through repression of PGHS-2 gene expression. On the contrary, priming of BEND cells with CLA isomers resulted in increased PGHS-2 mRNA steady-state levels though no changes were observed at the protein level. The mechanism of CLA inhibition of PGF2 has not been fully elucidated. CLA may be competing with AA for incorporation into the membrane phospholipids as well as for PGHS-2 activity (Mattos et al., 2000; Cheng et al., 2001). Banni and others (1999)

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76 showed that CLA supplementation to rat mammary tissue increased accumulation of CLA metabolites and decreased LA metabolites, such as AA. These isomers may also be directly inhibiting the activit y of PGHS-2 (Mattos et al., 2000; Cheng et al., 2001). CLA metabolites have been shown to be powerful inhibitors of PGHS enzyme (Nugteren and Christ-Hazelhof, 1987). Alternatively, CLA may regul ate the conversion of PGF2 to PGE2, as reported by Gross and Williams (1988) in bovine placental cells. However, in this study, incubation of BEND cells with both CLA isomers re sulted in reduced PGES mRNA expression, with no changes in protein concentration. This finding provides no evidence that CLA favors PGE2 synthesis at the expense of PGF2 production. Whether CLA affects PGES activity was not documented in this study. CLA is a naturally occurring ligand of PPARs (Moya-Camarena et al., 1999). Priming of BEND cells with cis -9, trans -11 and trans -10, cis -12 CLA isomers induced PPAR gene expression, but no changes were observed in PPAR protein concentration. There was no correlation between PPAR mRNA levels and PGF2 concentration in cellconditioned medium. Collectively, these findi ngs indicate that CL A-induced attenuation of PGF2 secretion in BEND cells is not medi ated through modulation of PGHS-2 or PPAR gene expression. Whether and how CLA isomers affect PGHS-2 enzymatic activity warrants further study. Summary Evidence is rapidly accumulating that CLA isomers modulate eicosanoid biosynthesis in various cell systems. In the present study, priming of BEND cells with cis -9, trans -11 or trans -10, cis -12 CLA isomers greatly decreased PGF2 response to

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77 PDBu. Interestingly, co-incubation with both CLA isomers increased PGHS-2 mRNA abundance in PDBu-stimulated BEND cells, sugge sting that these fa tty acids alter PGF2 production through a mechanism that does not require repression of PGHS-2 gene expression. Further studies are needed to te st whether or not CLA isomers modulate the activity of various enzymes a nd transcription factors invo lved in the prostaglandin biosynthetic cascade.

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78 Figure 4-1. Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrate (PDB u) in bovine endometrial (BEND) cells. When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Cont rast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.0001. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans 10, cis -12), P < 0.0001. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.44. When main treatment effect was significant ( P < 0.05), then differences between treatments are repres ented by different letters. Treatment by experiment interaction was significant ( P < 0.0001). Treatments PDBuLACLA (c9,t11)CLA (t10,c12) PGF 2 (pg/ml) 0 1500 3000 4500 6000 a a b b

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79 Figure 4-2. Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin endoperoxide synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Te n micrograms of total cellular RNA isolated from control and treated BEND ce lls were subjected to Northern blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using or thogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.002. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.04. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.006. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. There was no tr eatment by experiment interaction ( P = 0.65). Treatments PDBuLACLA(c9,t11)CLA(t10,c12) PGHS-2 mRNA (normalized to 18S rRNA) 0.08 0.12 0.16 0.20 PDBu LA t 10, c 12 c 9 ,t 11 4.4 kb rRNA PGHS-2 18S 1 2 3 4 5 6 7 8 (A) (B) a b b c

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80 Figure 4-3. Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin E synthase (PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten microgr ams of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a repr esentative Northern blot, whereas the bottom panel represents means SEM cal culated over two experiments (n = 4 for each treatment). When trea tment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.003. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.2. Contrast 3: ( cis -9, trans -11) vs. ( trans 10, cis -12), P = 0.6. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by differe nt letters. There was no treatment by experiment interaction ( P = 0.10). Treatments PDBuLACLA(c9,t11)CLA(t10,c12) PGES mRNA (normalized to 18S rRNA) 0.06 0.08 0.10 0.12 0.14 1.3 kb PDBu LA t 10, c 12 c 9 ,t 11 rRNA PGES 18S 1 2 3 4 5 6 7 8 (A) (B) a b b b

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81 Figure 4-4. Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin endoperoxide synthase (PGHS-2) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Twen ty five micrograms of total cellular protein extracted from cont rol and treated cells were subjected to Western blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representati ve Western blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected (P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.2. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.2. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.3.

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82 Figure 4-5. Effect of c 9, t 11 and t 10, c 12 CLA isomers on prostaglandin E synthase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Twenty five micrograms of total cellular protein extracted from control and treated ce lls were subjected to Western blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representati ve Western blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using or thogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.6. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P = 0.05. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.2.

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83 Figure 4-6. Effect of c 9, t 11 and t 10, c 12 CLA isomers on peroxisome proliferatoractivated receptor (PPAR ) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulti ng densitometric values were analyzed by the GLM procedure of SAS (B). Th e top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.0008. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.02. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.01. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. There was no treatment by experiment interaction ( P = 0.08). Treatments PDBuLACLA(c9,t11)CLA(t10,c12) PPAR mRNA (normalized to 18S rRNA) 0.00 0.02 0.04 0.06 3.5 kb PDBu LA t 10, c 12 c 9 ,t 11 rRNA PPAR 1 2 3 4 5 6 7 8 (A) (B) a b c b 18S

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84 Figure 4-7. Effect of c 9, t 11 and t 10, c 12 CLA isomers on peroxisome proliferatoractivated receptor (PPAR ) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Twenty five micrograms of total cellular protein extracted from cont rol and treated cells were subjected to Western blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). Th e top panel shows a representative Western blot whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) + ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.4. Contrast 2: (LA) vs. ( cis -9, trans -11) + ( trans -10, cis -12), P < 0.2. Contrast 3: ( cis -9, trans -11) vs. ( trans -10, cis -12), P = 0.2.

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85 CHAPTER 5 EFFECTS OF CIS AND TRANS -OCTADECENOIC ACIDS ON PROSTAGLANDIN F2 PRODUCTION BY BOVINE ENDOMETRIAL CELLS Introduction Evidence is rapidly accumulating that dietar y manipulations of fatty acids can have major effects on eicosanoid synthesis in domestic mammals. Dietary supplementation of long-chain fatty acids, a method commonly used to increase the energy density of diets for lactating dairy cows, has been shown to attenuate eicosanoid synthesis (Abayasekara and Wathes, 1999; Cheng et al., 2001), increase serum P4 concentration (Carroll et al., 1990; Lucy et al., 1993; Garc ia-Bojalil et al., 1998), s timulate ovarian follicular development (Lucy et al., 1993; Thomas a nd Williams, 1996; Beam and Butler, 1997), and improve fertility (Staples et al., 1998) in cattle. Preliminary data in our laboratory indicate that trans -octadecenoic fatty acids shorten the postpartum interval to estrus in early postpartum Holstein cows, suggesting e nhanced ovarian activity and function after parturition. Naturally occurring monounsaturated fatty acids (MUFA) are generally 16-22 carbons in length and cont ain the double bond in the cis configuration. The most common naturally occurring MUFA is oleic acid (C18:1), which contains its double bond in the cis configuration. However, as a result of the biohydrogenati on process in the rumen, accumulation of trans C18:1 fatty acids (TFA) ta kes place by anaerobic microorganisms. Partial biohydrogenation of PUFAs generate ruminal conjugated linoleic acid (CLA) and trans vaccinic acid (C18:1; trans -11). As a result, cows on a

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86 typical forage diet accumulate trans -11, the major TFA present in ruminal contents (Jenkins, 2004). Moreover, Mosley et al. (2002) reported that bi ohydrogenation of oleic acid involves the formation of several TFA is omers rather than direct biohydrogenation to form stearic acid (ST; C18:0). In addition, the trans -9 isomer of octadecenoic acid can be converted to both ST and a series of TFA isomers (Proell et al., 2002). Since hydrogenation of trans -11 C18:1 to ST is less rapid than formation of trans -11 C18:1 (Tanaka and Shigeno, 1976; Singh and Hawke, 1979), it accumulates in the rumen making it more available fo r absorption (Keeney, 1970). The physiological mechanism(s) by which supplemental trans fatty acids may affect reproductive efficiency have not b een characterized. Kummerow and coworkers (2004) observed that dietary supplementation of pigs with TFA resulted in reduced concentration of plasma n-3 and n-6 PUFAs. In this study, TFA also were reported to inhibit conversion of linoleic acid (LA) to longer chain n-6 PUFAs. This agrees with earlier studies by Mahfouz and others, showi ng that TFAs had an inhibiting effect on 6 desaturase in vascular cells in vitro (1980) and in vivo (1984). Taken together, these studies indicate a possible role of octa decenoic fatty acids in reproduction through modulation of eicosanoid production. The objective of this study was to examine the effects of the cis and trans isomers of octadecenoic acid (C18:1) on phorbol 13, 14-dibutyrate (PDBu)-induced PGF2 production in cultured BEND cells. We hypothesized that MUFAs may modulate production of PGF2 in endometrial cells, and that these effects may differ among the isomers of octadecenoic acid tested.

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87 Materials and Methods Materials Polystyrene tissue culture dishes (100 x 20 mm) were purchased from Corning (Corning Glass Works, Corning, NY). The Ham F-12 medium, antibiotic/antimycotic (ABAM), phorbol 13, 14-dibutyrate (PDBu), horse serum, D-valine, insulin, fatty acidfree bovine serum albumin (BSA), stearic acid (ST, C18:0), aprotinin, leupeptin, and pepstatin were from Sigma Chemical Co. (S t. Louis, MO). The Minimum Essential Medium (MEM) and fetal bovine serum (FBS) were from US Biologicals (Swampscott, MA) and Atlanta Biologicals (Norcross, GA), respectively. Oleic acid ( cis -9 C18:1), elaidic acid ( trans -9 C18:1), vaccinic acid ( cis -11 C18:1), trans -vaccinic acid ( trans -11 C18:1), PGHS-2 and PGES antibodies, and PGF2 standard were from Cayman Chemicals (Ann Arbor, MI). The PPAR and secondary anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) kit was purchased from Perkin Elmer (Boston, MA). Hanks Balanced Salt Solution (HBSS) and TriZol reagent were from GIBCO BRL (C arlsbad, CA). Isotopically-labelled PGF2 (5, 6, 8, 9, 11, 12, 14, 15[n-3H] PGF2 ; 208 Ci/mmol) was from Amershan Biosciences (Piscataway, NJ). The anti-PGF2 antibody was purchased fro m Oxford Biomedicals (Oxford, MI). BioTrans nylon membrane and [ -32P]deoxycitidine triphosphate (SA 3000 Ci/nmol) were from MP Biomedicals (Atlanta, GA). The PGHS-2 cDNA probe was cloned from an ovarian follicular cDNA lib rary (Liu et al., 1999), the PGES cDNA probe was cloned from endometrial cDNA (Guzeloglu et al., 2004), whereas the PPAR probe was generated from bovine endo metrial RNA (Balaguer et al., 2005).

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88 Cell Culture and Treatment Bovine endometrial (BEND) cells were plat ed and cultured as de scribed in chapter 3. To investigate the effects of cis and trans -octadecenoic acids on uterine PGF2 synthesis, BEND cells were treated with PDBu alone (100 ng/ml) or PDBu in combination with 100 M of cis -9, trans -9, cis -11, or trans -11 isomers of octadecenoic acid. Fatty acids were complexed with BSA (1 :3 ratio) for 2 h, and then treatments were applied to cells for a period of 24 h. After treatment, cells then were rinsed with HBSS and challenged with PDBu for another 6 h. Samples of cell-conditioned media (1 mL/plate) were collected and stored at -20C for subsequent analysis for PGF2 using RIA. The remaining cell monolayer was rinsed with HBSS, lysed in TriZol reagent, and stored at -80C for subsequent mRNA analysis. This experiment was repeated two times. The PGF2 radioimmunoassay and Nort hern and Western blot an alyses were performed as described in chapter 3. Statistical Analyses Data were analyzed by Least-Squares an alysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the Statistical Analysis System (SAS, 2001). For PGF2 response, the mathematical model include d fixed effects of treatment (n = 5), experiment (n = 5), treatment by experiment interaction, and random effect of dish (n = 2/trt) nested within treatmen t by experiment interaction. Th e variance of dish nested within treatment by experiment was used as th e error term for all effects. For Northern and Western blot data, hybrid ization volumes obtained from densitometric analysis were subjected to ANOVA using the GLM procedur e. For Northern blot analysis, the statistical model included independent fixed eff ects of treatment (n = 5), experiment (n =

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89 2), treatment by experiment interaction, and ra ndom residual error. Results are presented as ratios of densitometric values for ta rget genes over those for 18S rRNA ethidium bromide staining, and are presented as LS mean s SEM. For Western blot analysis, the mathematical model included only the main eff ect of treatment. Treatment effects were further analyzed using preplanned ort hogonal contrasts. These contrasts were constructed to compare PDBu vs. fatty acids responses; isomers with double bond in position 9 vs. isomers with double bond in posi tion 11; geometric isomers in position 9 ( cis vs. trans ); geometric isomers in position 11 ( cis vs. trans ). Results To test the hypothesis that MU FAs may modulate endometrial PGF secretion, we examined endometrial PGF2 response to PDBu in the presence of cis -9, trans -9, cis -11 and trans -11 isomers of octadecenoic acid. Priming of BEND cells with cis and trans -fatty acids further enhanced PGF2 ( P < 0.0001; Figure 5-1) and PGHS-2 mRNA ( P = 0.001; Figure 5-2) responses to PDBu. Interestingly, priming of BEND ce lls with the MUFAs decreased ( P = 0.04) PGES mRNA response to PDBu (Figure 5-4). None of the fatty acid treatments altered PPAR mRNA (Figure 5-6) or protein (Figure 57) expression in cultured BEND cells. Discussion Evidence is rapidly accumulating that dietar y manipulations of fatty acids can have a major effect on eicosanoid synthesis, such as prostaglandins of the 2-series, in domestic animals. Prostaglandins of the 2-seri es affect numerous reproductive processes, including ovulation (Espey, 1980), follicular development (W allach et al., 1975), corpus

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90 luteum function (Poyser, 1995; Wathes a nd Lamming, 1995; Abayasekara and Wathes, 1999) and parturition (C hallis et al., 1997). In the present study, incubation of BEND cells with cis and trans -fatty acids resulted in significan t augmentation of PGF2 response to PDBu. Stimulation of endometrial PGF2 secretion by supplemental MUFA s coincided with significant induction of PGHS-2 mRNA. To our knowledge, th is is the first report of the effects of MUFAs on endometrial PGF2 secretion. Following parturition, fertility resumes af ter uterine involutio n and repair takes place, resulting in resumption of normal oestr ous cycles (Kiracofe, 1980). This process of uterine involution is caused by myom etrial contractions stimulated by PGF2 A massive and sustained release of PGF2 takes place during the fi rst two weeks postpartum and is essential to reduce the uterine size a nd increase its tone (H afez and Hafez, 2000). Moreover, the duration of this postpartum PGF2 sustained release is negatively correlated with the number of days to complete uterine involution and the interval between parturition and resumption of nor mal ovarian activity (Lindell et al., 1982; Madej et al., 1984). This is in agreement w ith preliminary data from our lab indicating higher estrous rates in transition Holstein cows fed trans fatty acids (Kurt Selberg, unpublished observations). The mechanism by which these MUFAs ma y be stimulating endometrial PGF2 is not well understood. Although we observed enha nced response of PGHS-2 mRNA to PDBu after incubation of BEND cells with MUFA, no changes were observed at the protein level (Figure 5-3; P = 0.32). Additionally, in the present study we did not measure PGHS-2 enzyme activity. Priming of BEND cells with the MUFAs decreased

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91 ( P = 0.04) PGES mRNA response to PDBu, indicating that MUFAs may favor PGF2 production over PGE2. However, no changes were obs erved on PGES protein levels (Figure 5-5; P = 0.10) and PGES enzyme activity was not documented in this study. Alternatively, dietary supplementation w ith TFA has been reported to reduce concentration of plasma n-3 and n-6 PUFAs in pigs (Kumme row et al., 2004). TFA were also reported to inhibit conve rsion of LA to longer chain n-6 PUFAs. This agrees with earlier studies by Mahfouz and others, showi ng that TFAs had an inhibiting effect on 6 desaturase in vascular cells in vitro (1980) and in vivo (1984). Additionally, essential PUFAs have been shown to inhibit PG secret ion in several cell types (Levine and Worth, 1984; Achard et al., 1997) including BEND cells (Mattos et al., 2003). Taken together, these studies indicate that isomers of octadecenoic fatty acids may induce PGF2 production through a mechanism involving inhibition of synthesis of PUFAs. Although fatty acids are naturally occurring ligands of PPARs, it appears that the effect of MUFAs on PGF2 does not require modulation of PPAR gene or protein since no changes were observed after incubation wi th the isomers of octadecenoic acid. Whether and how these fatty acids may control activity of this nuclear receptor is yet to be elucidated. Summary In the present study, priming of BEND cells with cis -9, trans -9, cis -11, or trans -11 isomers of octadecenoic acid further enhanced PGF2 and PGHS-2 mRNA response to PDBu. Interestingly, pre-incubation of BE ND cells with the MUFAs decreased PGES mRNA response to PDBu. None of the fatty acids altered PPAR mRNA or protein levels. Supplemental monoenoic fatt y acids appear to increase PGF2 through a

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92 mechanism that does not re quire induction of PPAR gene or protein expression. Whether and how these fatty acids may contro l activity of this nuclear receptor warrants further study.

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93 Figure 5-1. Effect of cis and trans isomers of octadecenoic acid on prostaglandin F2 (PGF2 ) response to phorbol 12, 13 dibutyrat e (PDBu) in bovine endometrial (BEND) cells. When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrast s. Contrast 1: (PDBu) vs. ( cis -9) + ( trans 9) + ( cis -11) + ( trans -11), P < 0.0001. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis 11) + ( trans -11), P < 0.0001. Contrast 3: ( cis -9) vs. ( trans -9), P < 0.0001. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.63. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. Treatment by expe riment interaction was significant ( P = 0.0002). Treatments PDBucis 9trans 9cis 11trans 11 PGF 2 (pg/ml) 3000 4500 6000 7500 a b c d d

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94 Figure 5-2. Effects of cis and trans isomers of octadecenoic acid on prostaglandin endoperoxide synthase (PGHS-2 ) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Cont rast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.001. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.44. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.39. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.06. When main treatmen t effect was significant ( P < 0.05), then differences between treatments are represented by differe nt letters. There was no treatment by experiment interaction ( P = 0.17). Treatments PDBucis 9trans 9cis 11trans 11 PGHS-2 mRNA (normalized to 18S rRNA) 0.1 0.2 0.3 0.4 4.4 kb 18S PDBu cis -9 trans 9 cis -11 trans -11 rRNA PGHS-2 1 2 3 4 5 6 7 8 9 10 (A) (B) a b b b b

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95 Figure 5-3. Effects of cis and trans isomers of octadecenoic acid on prostaglandin endoperoxide synthase (PGHS-2) pr otein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometri al (BEND) cells. Twenty five micrograms of total cellular protein extracted from control and treated cells were subjected to Western blot analys is (A), and resulting densitometric values were analyzed by the GLM pro cedure of SAS (B). The top panel shows a representative Western blot whereas the bottom panel represents means SEM calculated over two experi ments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Cont rast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.32. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.33. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.95. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.68.

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96 Figure 5-4. Effects of cis and trans isomers of octadecenoic acid on prostaglandin E synthase (PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Te n micrograms of total cellular RNA isolated from control and treated BEND ce lls were subjected to Northern blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using or thogonal contrasts. Contrast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.04. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.10. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.48. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.07. When main treatment effect was significant ( P < 0.05), then differences between treatments are represented by different letters. There was no treatment by experiment interaction ( P = 0.41). Treatments PDBucis 9trans 9cis 11trans 11 PGES mRNA (normalized to 18S rRNA) 0.20 0.24 0.28 0.32 0.36 * Treatments PDBucis 9trans 9cis 11trans 11 PGES mRNA (normalized to 18S rRNA) 0.20 0.24 0.28 0.32 0.36 1.3 kb 18S PDBu cis -9 trans 9 cis -11 trans -11 rRNA PGES 1 2 3 4 5 6 7 8 9 10 (A) (B) a b b,c a,c b,c

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97 Figure 5-5. Effects of cis and trans isomers of octadecenoic acid on prostaglandin E synthase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Te n micrograms of total cellular RNA isolated from control and treated BEND ce lls were subjected to Northern blot analysis (A), and resul ting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using or thogonal contrasts. Contrast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.10. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.72. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.13. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.18.

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98 Figure 5-6. Effects of cis and trans isomers of octadecenoic acid on peroxisome proliferator-activated receptor (PPAR ) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated from control and treated BEND cells were subjected to Northern blot analysis (A), and resulting densitometric values were analyzed by the GLM procedure of SAS (B). The top panel shows a representative Northern blot, whereas the bottom panel represents means SEM calculated over two experiments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Cont rast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.43. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.90. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.21. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.09. Treatment by experiment interaction was significant ( P = 0.006).

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99 Figure 5-7. Effects of cis and trans isomers of octadecenoic acid on peroxisome proliferator-activated receptor (PPAR ) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometri al (BEND) cells. Twenty five micrograms of total cellular protein extracted from control and treated cells were subjected to Western blot analys is (A), and resulting densitometric values were analyzed by the GLM pro cedure of SAS (B). The top panel shows a representative Western blot whereas the bottom panel represents means SEM calculated over two experi ments (n = 4 for each treatment). When treatment effects were detected ( P < 0.05), means were separated using orthogonal contrasts. Cont rast 1: (PDBu) vs. ( cis -9) + ( trans -9) + ( cis -11) + ( trans -11), P = 0.38. Contrast 2: ( cis -9) + ( trans -9) vs. ( cis -11) + ( trans -11), P = 0.05. Contrast 3: ( cis -9) vs. ( trans -9), P = 0.82. Contrast 4: ( cis -11) vs. ( trans -11), P = 0.14.

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100 CHAPTER 6 EFFECTS OF DIETARY TRANS FATTY ACIDS ON PROSTAGLANDIN F2 CONCENTRATIONS IN POSTPARTUM HOLSTEIN COWS Introduction Over the last five decades, continuous genetic progress for milk production has been associated to a decrease in reproductiv e efficiency of dairy cows (Butler, 2000). Poor reproductive performance affects the am ount of milk produced per cow per day of herd life, breeding costs, rates of volunt ary and involuntary culli ng, and the rate of genetic progress for traits of economic importance (Plaizier et al., 1997). Poor reproductive efficiency includes early embryoni c loss (Thatcher et al., 1995), impaired ovarian cyclicity and low fertility rates (B utler, 2000), which colle ctively result in reduced milk production (Plaizier et al., 1997) One of the factor s contributing to low breeding efficiency in high milking cows is the high energy deficit of early lactation, which delays re-establishment of ovarian cyclicity and impairs reproductive performance after calving (Butler, 2000). Lactating dairy cows in the first fe w weeks postpartum have higher energy requirements than can be supported by dietar y energy intake, which creates a negative energy state and can lead to impaired reproductive function (Butler, 2000). Energy balance of an animal is the difference be tween energy intake and energy requirements within a given physiological state (Beam and Butler, 1999; Butler et al., 1981; Canfield and Butler, 1990). In dairy cows, parturition and the onset of lactation cause an abrupt shift in nutritional requ irements in order to support milk production (Butler, 2000). This

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101 rapid increase in energy requirements and chan ges in the metabolic as well as endocrine status of the cow come a bout during the transition peri od (Bauman and Currie, 1980; Grummer, 1995). This is the result of the prio ritized status of lactation which allows it to proceed at the expense of any other physio logical process (Bauman and Currie, 1980). The transition period extends from three weeks prepartum until three weeks postpartum, and refers to the period duri ng which endocrine and metabolic changes accompany parturition and the onset of lactat ion (Grummer, 1995). A reduction in feed intake occurs during the fina l week of pregnancy when nutrient demands for support of fetal growth and initia tion of milk synthesis are increas ing (Grummer, 1995). The dietary energy that is consumed by the lactating animal is almost entirely used by the mammary tissue for milk production, leaving no energy for maintenance (Bell, 1995). To offset this energy deficit, the lactating animal m obilizes body energy reserves, which leads ultimately to a state of negative energy balan ce (NEB). This NEB represents a state of undernutrition, which results in massive m obilization of fat from adipose tissue, increasing plasma concentrations of non-este rified fatty acids (NEFA). Massive fat mobilization, in combination with reduced en ergy intake (dry matter intake), results in loss of body condition of lactating animals. Extensive data has shown that the aforementioned conditions (NEB and loss of body condition) invariably affect reproductive efficiency in lactating dairy cows. Fertility in postpartum cows is dire ctly dependent on a normal estrous cycle characterized by folliculogenesis, ovulation, corpus luteum (CL) formation and regression. However, the mechanisms by whic h endocrine and metabolic upsets result in reduced reproductive efficiency are not we ll understood. Many of the hormonal and

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102 metabolic changes that occur during the tran sition period can affect reproductive function by interacting with the hypotha lamic-pituitary axis (Butle r, 2000). Negative energy balance also has been shown to change th e profile of metabolic hormones which may play an important local role in control of follic ular development in cattle. It is likely that these changes could alter the pattern of ovarian follicular growth and development, and subsequent CL function during the earl y postpartum period. However, energyindependent effects also have been observ ed after fatty acid supplementation. Thus, nutrition may influence reproduc tive efficiency in dairy cows not only by altering the energy status of the animal but also by infl uencing factors involved in the regulation of reproductive processes like follicular dyna mics, ovulation, CL function, and embryo survival among others. Dietary supplementation of long chain fatty acids (LCFA), commonly used to increase energy density of diets for lactati ng dairy cows, has been shown to attenuate eicosanoid synthesis (Abaya sekara and Wathes, 1999; Ch eng et al., 2001), increase serum P4 concentration (Carroll et al., 1990; Lu cy et al., 1993; Garcia-Bojalil et al., 1998), stimulate ovarian follicular devel opment (Lucy et al., 1993; Thomas and Williams, 1996; Beam and Butler, 1997), and im prove fertility (Staples et al., 1998) in cattle. The potential mechanisms by which LC FAs affect reproductive responses in cattle include indirect effects of high energy inta ke on the overall energy state of the cow, as well as direct effects of dietary fatty acids on the pituitary, ovaries, a nd uterus (Staples et al., 1998; Mattos et al., 2000). Improvement of the overall energy state provided by fatty acid supplementation (Staples et al., 1998; Jenkins and Palmquist, 198 4) may lead to re-establishment of LH

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103 pulsatility and ovarian cyclicity in the lactating cow (Lucy et al., 1991). In fact, Sklan et al. (1994) reported that the en ergy provided by dietary fats increased LH secretion in dairy cows that consumed less energy than required. There is evidence that increase in consum ption of dietary fatty acids stimulates ovarian follicular growth in cattle through a mechanism that is independent from energy intake and weight gain (Stapl es et al., 1998). Increasing the dietary intake of LCFAs by cattle increased both the number and size of fo llicles present in the ovary and shortens the interval to first ovulation postpartum (Hightshoe et al ., 1991; Lucy et al., 1991, 1992; Ryan et al., 1992; Thomas and Williams, 1996; Lammoglia et al., 1997; Beam and Butler, 1997). Lucy et al. (1993b) reported greater numbers of me dium-sized ovarian follicles (6 9 mm) in postpartum dairy cows fed a diet containing 2.2% calcium salts of LCFA compared to cows receiv ing an isocaloric control diet Several studies reported that supplemental fat increased not only th e total number of ovarian follicles (Thomas and Williams, 1996; Beam and Butler, 1997; Lamm oglia et al., 1997), but also the size of preovulatory follicles in cattle (Lucy et al., 1993b; Beam and Butler, 1997; Oldick et al., 1997). Follicular growth also has been show n to be stimulated by LCFAs in crossbred beef cattle (Thomas et al., 1997). Moreover, la ctating dairy cows fed calcium salts of fat enriched in LA or fish oil (h igh in n-3 PUFAs) had increased size of the dominant follicle compared to those fed calcium salts of ol eic acid (18:1) (Staples et al., 1998). Fertility responses may also be relate d to the effects of LCFAs on uterine eicosanoid production. Eicosanoi ds (i.e. prostaglandins, th romboxanes, leukotrienes and lipoxins) are synthesized from AA, which uses LA as the primary precursor (Kinsella et al., 1990). Prostaglandins (PG) of the 2 series (PGF2 PGE2) have been implicated in

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104 many reproductive processes including ovulation (Espey, 1980), follicular development (Wallach et al., 1975), corpus luteum func tion (Bazer and Thatcher, 1977; Auletta and Flint, 1988; Abayasekara et al., 1995; Poyser, 1995; Wathes and Lamming, 1995), parturition (Thorburn and Challis, 1979; Chal lis, 1980) and uterine involution (Hafez and Hafez, 2000). Hence, any influence that fatty acids might exert on PGF2 synthesis may affect overall reproductive performance. Preliminary data in our labor atory indicate that feeding trans -octadecenoic fatty acids reduced fat mobilization (Selberg et al ., 2004) and shorten th e postpartum interval to estrus (Selberg et al., unpublished data) in early postpartum Holstein cows, suggesting enhanced ovarian activity and function after parturition. Mo reover, studies from our lab showed that supplementation of bovine endometrial (BEND) cells with trans octadecenoic fatty acids stimulated production of PGF2 (see Chapter 5). These observations collectively suggest that dietary trans fatty acids ( t FA) may improve reproductive efficiency in tran sition dairy cows by reducing the incidence of postpartum metabolic upsets such as fatty liver and stim ulating ovarian functi on after parturition. The objective of this investigation wa s to examine the effects of dietary t FAs on ovarian and PGF2 responses in early postpartum Hols tein cows. We hypothesized that supplementation of transition dairy cows with dietary t FAs may reduce periparturient fat mobilization and enhance PGFM production, th us stimulating ovarian function after parturition.

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105 Materials and Methods Materials A highly saturated fat product (ruminal bypass fat; RBF) mixed in the control diet was provided by Cargill (Minneapolis, MN). Calcium salts of trans -C18:1 fatty acid mix (EnerG TR) was provided by Virtus NutritionTM LLC (Fairlawn, OH). Kendall Monoject blood collection tubes (10 mL) containing EDTA as anticoagulant, and needles (20GA 1) were purchased from Webster Veterina ry (Alachua, FL). Borosilicate glass disposable culture tubes (12 x 75 mm), scin tillation vials (7 mL), flat bottom 96-well plates, Scintiverse II scintillation fluid, and serum filters were purchased from Fisher Scientific (Pittsburgh, PA). Polypropylene milk sample vials (2 mL) were from Capitol Vial, Inc (Auburn, AL). Autokit 3-HB, NEFA C, and Glucose C2 Autokit test kits were purchased from Wako Chemicals USA (Richmon d, VA). Activated charcoal, dextran, and authentic 13, 14-Dihydro-15-keto PGF2 were from Sigma Chemical Co. (St. Louis, MO). Isotopically-label led 13, 14-Dihydro-15-keto[n-3H] PGF2 ; 208 Ci/mmol was from Amershan Pharmacia Biotech (Piscataway, NJ ). The anti-13, 14-Dihydro-15-keto PGF2 antibody was kindly provided by Dr. William W. Thatcher (University of Florida, Gainesville, FL). Cows and Diets Holstein cows (18 multiparous and 12 prim iparous) were utilized in a completely randomized design to determine the e ffect of feeding calcium salts of trans -C18:1 fatty acids on production, metabolic and reproductive responses during the transition period. Primiparous heifers (n = 3) and multiparous cows (n = 5) that were diagnosed with displaced abomasum, enteritis, or that had metritis within 10 d after parturition were

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106 removed from production, metabolic and reproductive analyses (T able 6-1). Therefore, a total of nine heifers (4 C, 5 t FA) and 13 cows (7 C, 6 t FA) were used in statistical analyses. The experiment was conducted fr om March to July, 2005. All experimental animals were managed according to the guideli nes approved by the University of Florida Animal Care and Use Committee. Two dietary treatments were initiated a pproximately 28 d prior to estimated calving dates and continued through 21 d postpartum. The control diet (C) contained a highly saturated fat supplement (90 % saturated fatty acids; RBF) at 1.55% of dietary DM. The second experimental diet cont ained a Ca salt of primarily trans -C18:1 fatty acid ( t FA) at 1.8% of dietary DM. Diets were isolipid since the Ca salt pro duct was 15% Ca. The fatty acid profile of each fat supplement is in Table 6-2. Diets were formulated for intakes of approximately 150 to 200 g/d pr epartum and 250 to 300 g/d postpartum of supplemental lipid. Fat supplements were mi xed with the concentr ates and offered as part of the TMR to experimental animals. Prepartum cows were housed in pens with a sod base equipped with shaded Calan gates (American Calan Inc., Northwood, NH). Postpartum cows were housed in a freestall barn equipped with fans, sprinklers, and Calan gates. Intake of DM was measured daily. All experimental cows were offered ad libitum amounts of TMR to allow for 5 to 10% refusals (Table 6-3). Corn silage wa s the major forage component and ground corn was the primary concentrate. Dry matter of corn silage was determined weekly and the rations were adjusted accordingly to mainta in a constant forage: concentrate ratio on a DM basis. Samples of forages, dried at 55C and ground to pass 2-mm screen of a Wiley Mill (C.W. Brabender Instruments, Inc.), and concentr ate mixes were collected weekly,

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107 composited monthly, and analyzed by wet chemistry for fat (Soxhlet method) and minerals (Dairy One, Ithaca, NY), and CP (Elementar Analysensysteme, Hanau, Germany). Composite samples also were an alyzed for ADF and NDF with heat stable amylase (Termamyl 120L, Novo Nordisk Bioc hem, Franklinton, NC) as described by Van Soest et al. (1991). Detailed ingr edient and chemical composition of the experimental diets are listed in Tables 6-3 and 6-4, respectively. Postpartum cows were milked 3 times per day and milk weights were recorded at each milking. For each experimental cow, samples of milk from 2 consecutive morning (1000 h) and evening (1800 h) milkings were collected one day during wk 3 postpartum and analyzed for fat, protein, and SCC. Daily values were calculated by averaging morning and evening milk values. Body weights were measured and BCS assigned weekly by the same individual. Prepartum energy balance was calculated using the following equation: Energy balance (Mcal/d) = net energy of in take (net energy of maintenance + net energy of pregnancy). Net energy of intake was calculated by mu ltiplying the weekly average DMI (kg) by the calculated energy value of the diet (Mcal/kg) (NRC, 2001). Energy requirement for body maintenance was computed us ing the following equation (NRC, 2001): Net energy of maintenance (Mcal) = 0.08 x BW0.75

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108 Pregnancy requirements were estimated using the following equation (NRC, 2001): Net energy of pregnancy (Mcal) = [( 0.00318 x days pregnant 0.0352) x (calf BW/45)]/0.218 Postpartum energy balance was estimated using the following equation (NRC, 2001): Energy balance (Mcal/d) = net energy inta ke (net energy of maintenance + net energy of lactation) Milk energy was estimated by the following equation: Net energy of lactation (Mcal/d) = [( 0.0929 x % fat) + (0.0547 x % protein) + 0.192] x milk weight (kg) Collection of Blood Samples Blood (~20 mL) was collected once daily at 1730 h from d 14 before calculated due date until parturition and from d 15 until d 21 postpartum. Between the day of parturition (d 0) and d 14 postpartum, blood samples we re collected 2x per day at 0800 and 1730 h. Blood was collected via coccygeal arterio-ve nipuncture into evacuated blood tubes containing EDTA as an anticoagulant (10.5 mg ). Samples were placed immediately in ice until plasma was separate d by centrifugation at 3000 rpm for 30 min (4C). Plasma was separated and stored at -20C for subsequent metabolite and PGFM analyses. Metabolite and PGFM Assays Plasma concentrations of NEFA, BHBA, and glucose were measured in samples collected on days -14, -7, 7, 14, and 21, relativ e to the day of parturition. Plasma

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109 concentration of NEFA, BHBA, and glucose were measured with the NEFA C, Autokit 3-HB, and Glucose C2 Autok it test kits, respectively. Samples collected between d 5 prepartum and d 14 postpartum were analyzed for concentrations of PGFM using a modifi cation of the radioimmunoassay procedure described by Mitchell et al. (1976). The PGFM standard so lutions were made by serial dilutions in a buffer of a stock solution (1 g/mL in 10% ethanol and 90% PBS buffer) of authentic PGFM. Standards (100 L) were run in duplicat es at the following concentrations: 15.6, 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 pg/mL. The standard curve included 100 L of plasma containing low con centration of prostaglandin. Low PGFM plasma was obtained by pooling sa mples corresponding to wk 7 postpartum cows, known to have low to negligible pros taglandin concentrations. The PBS buffer contained 2.3 g/L of NaH2PO4H2O, 4.76 g/L of Na2HPO47H2O, 1 g/L of sodium azide, and 8.41 g/L of NaCL. The buffer pH was adjusted to 7.5 with NaOH. Activity and volume of radioactively labeled PGFM used were 18,000 dpm and 100 L, respectively. For unknown samples, the final assay volume was 400 L [i.e., 100 L of sample, 100 L of rabbit antiserum to PGFM (1:5000), 100 L of buffer, and 100 L of labeled PGFM]. After overnight in cubation at 4C, free PGFM was separated using 500 L of a solution of dextran-coated charcoal (50 mg dextran and 500 mg activated charcoal in 100 mL PBS buffer). After centr ifugation at 3500 rpm for 20 min (4C), the supernatant was tr ansferred to scinti llation vials and mixe d with 4.5 mL of Scintiverse II. Radioactivity was measured using a liquid scintillation counter (Packard, Canberra Company). Assay sensitivity wa s 15.6 pg/mL and intraand inter-assay

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110 coefficients of variation we re 6.5 and 5.9%, respectively. Final PGFM concentrations were expressed as picograms per milliliter. Ultrasonography Ultrasound examination of the ovaries wa s performed for each cow at days 7, 14 and 21 postpartum to determine the number of small (C1; 3 to 5 mm), medium (C2; 6 to 9 mm) and large (C3; > 9 mm) follicles. A real-time ultrasound scanner (Aloka SSD 500 V, Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5 MHz rectal probe was used. Statistical Analyses Milk production and intake responses were reduced to weekly means before statistical analysis. Milk composition was anal yzed by Least-Square analysis of variance (ANOVA) using the General Linear Model (GLM) procedure. All other dependent responses were evaluated using the MIXED pr ocedure for repeated measurement of the SAS software package (SAS, 2001). Fixed e ffects included treatment, parity, week relative to calving (for production responses) or days relative to calving (for metabolic and reproductive responses), and treatment by week interaction. The variance for cow nested within treatment by parity was used as ra ndom error term to test the main effect of treatment. Differential temporal responses to dietary treatments we re further examined using the SLICE option of the MIXED proce dure. Mean treatment and time (week or day relative to calving) effects are reported as least squares means. Results Production Responses Multiparous cows had greater ( P < 0.05) dry matter intake (15.5 0.7 kg/d), body weight (700 15.5 kg), and BCS (3.14 0.04) when compared to primiparous cows (11.9 0.8 kg/d, 539.7 18.7 kg, and 3.00 0.04, respectively) throughout the

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111 experimental period. However, these responses were similar for both treatment groups in either heifers or cows during th e prepartum and postpartum periods. Postpartum DMI in heifers and cows increased ( P < 0.0001) from 2.1% of BW at wk 1 to approximately 2.9% of BW at wk 3 of lactation, with no differences observed between treatment groups (Figure 6-1A, B). Patterns of BW response did not vary between the two dietary groups (Figure 6-2A B). Moreover, the amount of BW loss during the experimental period (wk -2 to wk 3) between control and t FA groups was similar in heifers (92 vs. 105 kg) and cows (95 vs. 99 kg). Changes in energy balance were similar for heifers and cows for bot h experimental diet s (Figure 6-3A, B). Heifers and cows fed control and t FA had comparable BCS and experienced similar patterns of change throughout the expe riment (Figure 6-4A, B). The main loss in BCS occurred between wk 2 prepartum and wk 1 postpartum in both heifers (3.26 vs. 2.98 0.1) and cows (3.40 vs. 3.00 0.1). Ho wever, during the postpartum period no detectable changes in BCS were observed. Average milk production during the 3-wk postpartum treatment period did not differ between dietary groups, although cows consistently produced more milk ( P < 0.0005) than heifers (35.8 vs. 44.5 kg/d) (Figur e 6-5A, B). The amount of milk produced as a function of consumption of DM (feed efficiency) increased with time in both treatment groups in heifers and cows (Figur e 6-6). At week 3 of lactation, heifers produced less milk than cows (35.8 2.2 kg/d vs. 44.5 1.8 kg/d) (Table 6-5). Mean milk fat and protein production and concentr ation and SCC did not differ between dietary treatments (Table 6-5).

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112 Metabolic Responses Both treatment groups had similar patterns of plasma NEFA concentrations, which were low during the prepartum period, dramatic ally increased at 1 to 2 wk after calving, then gradually declining over time (Figur e 6-7A, B). During the postpartum period, NEFA concentrations in heifers (491.9 93.4 Eq/L) and cows (420.0 70.7 Eq/L) peaked around d 7, then declining towa rds prepartum concentrations by d 21. Plasma BHBA concentrations during th e prepartum period were similar between treatment groups in heifers (2.03 1.1 mg/dL) and cows (2.66 0.8 mg/dL). Immediately after calving, a dramatic increas e in BHBA concentra tion was observed by d 7 postpartum in both heifers (4.91 1.1 mg/dL) and cows (4.53 0.8 mg/dL) (Figure 68A, B). After a plateau was reached between d 7 and 14, heifers from the control group further increased ( P = 0.035) plasma BHBA concentr ation at d 21 (6.68 1.1 mg/dL), while BHBA concentration in heifers from the t FA group continued to decline until d 21 (3.12 1.1 mg/dL) (Figure 6-8A). On the other hand, plasma BHBA concentration in control cows declined between d 7 and 21 (3.23 0.8 mg/dL). Cows fed t FA continued to gradually increase BHBA concentration through d 14 ( P = 0.04) (5.39 0.8 mg/dL) until reaching its highest concentration at d 21 ( P = 0.005) postpartum (5.90 0.8 mg/dL) (Figure 6-8B). Control and t FA-fed heifers and cows exhibited similar patterns of change in plasma glucose concentration throughout th e experimental period. Plasma glucose concentrations during the prepartum ( 60.9 3.3 vs. 61.6 2.4 mg/dL) and postpartum (56.0 3.3 vs. 52.4 2.4 mg/dL) periods were si milar between treatments in heifers and cows, respectively (Figure 6-9A, B). Con centrations of glucose in plasma were unaffected by dietary treatment (Figure 6-9A, B).

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113 Reproductive Responses The number and distribution of small (C1), medium (C2), and large (C3) follicles in the ovaries were not different between diet ary treatments for heifers and cows at d 7 and d 14 postpartum (Table 6-6). However, at d 21, cows tended ( P = 0.11) to have more C1 follicles (9.5 1.7) than heifer s (6.5 2.0), while heifers had ( P = 0.03) more C2 follicles (2.8 0.9) when compared to cows (0.8 0.9) (Table 6-6). The number of C3 follicles increased ( P < 0.05) from d 7 to d 21 postpartu m in heifers (0 vs. 1.2 0.4) and cows (0.3 vs. 1.1 0.3) (Table 6-6). Control and t FA-fed heifers had comparable pl asma PGFM concentrations and showed similar patterns of cha nge during the experimental period (Figure 6-10A). Pulses of plasma PGFM in heifers were characte rized by an initial ra ise around calving date (1125.7 285.8 pg/mL), persisting and reachi ng its highest concentration by d 4 (1155.1 285.8 pg/mL), then gradually decreasing and approaching basal concentration around d 10 postpartum (Figure 6-10A). Circulating PGFM concentra tion and patterns of change in control cows were similar to those observed in heifers. Initia l stimulation of PGFM secretion occurred one day prior to calving, peaking in the afte rnoon of d 1 postpartum (1501.7 322.3) and gradually decreasing to basal concentrati on by d 10 postpartum (Figure 6-10B). In t FAsupplemented cows, a dramatic increase ( P = 0.04) in PGFM concentration were observed by d 1 (2106.1 355.5) (Figure 6-10B). The t FA-stimulation of PGFM reached peak concentration at d 2 pm (3767.3 347.9), and persisted with higher concentration up to d 4 postpartum (2150.8 347.9) before de creasing to basal concentration by d 10 postpartum (Figure 6-10B). Even though greater PGFM concentrations were detected in t FA-fed cows between d 1 and 4 postpartum, the decrease in PGFM concentration was

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114 faster in t FA-fed cows (d 2 pm to d 10 pm = 458.2 pg/ mL per day) than control cows (d 1 pm to d 10 pm = 152.2 pg/mL per day). Discussion During the experimental period, DMI, BW, and BCS responses were similar between dietary groups. However, cows had c onsistently higher DMI (kg/d) and BW as well as better BCS when compared to heifers. Similarly, cows produced more milk than heifers, which is also related to the diffe rence in DMI between cows and heifers. Moreover, when DMI is expressed as a per centage of BW, no difference between cows and heifers were detected. Additionall y, milk production as a function of DMI (calculated feed efficiency) also was similar between cows and heifers. Taken together, these observations further support the fact of increased nutrient needs for growth of heifers taking place simultaneously with the demands of lactation and their lower feed intake capacity as described pr eviously (Rmond et al., 1991). The higher plasma NEFA and BHBA concentr ations detected in heifers and cows after parturition, regard less of dietary treatments, likely re flect the NEB of the animals, as previously observed (Invartsen and Anders en, 2000; Moorby et al., 2000). Additionally, induction of plasma NEFA and BHBA concentr ations after parturit ion in heifers and cows results from decreased DMI prior to calving and periparturient hormonal changes that stimulate fat mobilization from the adi pose tissue to provide energy for lactogenesis (Vazquez-Aon et al., 1994; Grum et al., 1996). This is further supported by the observation that plasma NEFA concentration is a reliable index of the magnitude of adipose fat mobilization (Bauman et al., 1988). In the present study, plasma NEFA concentrat ions were not affected by either diet ( P = 0.44) or parity ( P = 0.94). The lack of parity eff ect contrasts with several cattle

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115 studies in which heifers mobilized more fat fr om adipose tissue than cows as indicated by plasma NEFA concentrations (Belyea et al., 1975; Drackley et al., 2003; Meikle et al., 2004; Cavestany et al., 2005). In the aforementioned studies, differences between heifers and cows were attributed to higher ener getic demands (e.g. growth) experienced by heifers. Thus, results from the present study indicate that suppleme ntation with either saturated or t FA may assist heifers to reduce the dramatic energy deficit generally experienced due to the combina tion of growth and lactation. While the patterns of change of NEFA concentration postpartum in the present study were similar to several published reports (Garcia-Bojalil et al., 1998; Mattos et al., 2004; Meikle et al., 2004; Cavestany et al., 2005), NEFA concentrations, in general, appeared to be slightly lower. This diffe rence may be due to different experimental conditions as well as dietary supplementa tion. However, cows supplemented with t FA in this study exhibited a similar profile a nd NEFA concentratio ns during the early postpartum period compared to NEFA pr ofile and concentrations observed on t FA-fed cows in the study by Selberg et al. (2004). Taken together, these observations indicate that dietary supplementation w ith either saturated fat or t FA may reduce the fat mobilization observed in cows and heifers during the transition period. This is in agreement with the study by Selberg et al. (2004) indicat ing that dietary trans octadecenoic fatty acids reduced fat mobiliz ation by early postpartum Holstein cows. Plasma BHBA concentrations in heifers a nd cows were similar to those previously reported (Meikle et al., 2004; Ca vestany et al., 2005; Selberg et al., 2004). The greater BHBA concentration detected in heifers and cows soon afte r calving likely reflects the NEB experienced by these animals around parturition. Although not statistically

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116 significant, NEFA concentrations were numer ically greater in control heifers than t FAsupplemented heifers at d 7 (564.0 vs. 419.8 84.2). When compared to the control group, t FA-fat cows exhibited numerically grea ter blood NEFA concentration during the postpartum period. Hence, increased plasma BHBA levels experien ced by control heifers and t FA cows may be the product of NEFA accumulation in the liver, thus shifting metabolism towards partial oxidation. The observation that supplemental t FA failed to alter peripheral glucose concentration in periparturient animals is cons istent with a recent ca ttle study (Selberg et al., 2004). Although Selberg and coworker s (2004) also found that dietary trans fatty acids induced steady state mRNA levels of enzymes involved in hepatic gluconeogenesis, no information on enzyme activity was availa ble. Hence, they concluded that the discrepancy between the amount of an enzyme and the concentrati on of its metabolic product may reflect differences between periphe ral processing of the metabolite and the enzymatic activity. In the present study, we examined the e ffects of the dietary supplementation on follicular dynamics by monitoring the proportion of small (C1), medi um (C2) and large (C3) ovarian follicles at d 7, 14 and 21 postpartu m. There was no parity or diet effect on the number of smalland medium-size follicles (3 to 9 mm) within the first three weeks of lactation. The increased number of larg e follicles between d 7 and d 21 observed in heifers and cows reflects the normal movement of follicles from class 2 to class 3 between wk 1 and 3 postpartum. Several studies have shown that manipula tions of dietary fatty acids may have major effects on PGFM concentration in ruminants. A number of studies have

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117 demonstrated inhibitory e ffects of n-3 PUFAs on PGF2 production (Bezard et al., 1994; Staples et al., 1998; Abayasekara an d Wathes, 1999; Mattos et al., 2000, 2001, 2002, 2004). Supplementation of dairy cattle with fish m eal (high in n-3 PUFAs) reduced basal and OT-induced plasma PGFM concentrations in early postpartum dairy cows (Mattos et al., 2002, 2004). In the present stud y, cows supplemented with dietary t FA had higher plasma PGFM concentration than control cows. The increased PGFM concentration observed in cows supplemented with t FA is in agreement with in vitro studies from our laboratory showing that supplementation of bovine endometrial (BEND) cells with trans isomers of octadecenoic fatty acids resu lted in significant augmentation of PGF2 response to PDBu (Chapter 5). Positive effects of dietary fa ts on fertility response are not related exclusively to inhibition of PG biosynthesis. For example, preliminary data in our laboratory indicated that trans -octadecenoic fatty acids may shorten the pos tpartum interval to estrus in early postpartum Holstein cows, suggesting enha nced ovarian activity and function after parturition (Selberg et al., unpublished data ). Following partur ition, resumption of normal estrous cycles is dependent on uterin e involution and repair caused by myometrial contractions stimulated by PGF2 (Kiracofe, 1980). In additi on, application of exogenous PGF2 early postpartum, increased myoelectrical activity and contrac tion of the uterus (Patil et al., 1980; Gajewski et al., 1999). Thus, enhanced ovarian activity and function may be related to elevated levels of PGFM since the present study showed that dietary t FA greatly increased and sustained higher pl asma PGFM concentration between days 1 and 4 postpartum when compared to cont rol cows. In addition, the duration of postpartum PGF2 sustained release is negatively co rrelated with the num ber of days to

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118 complete uterine involution and the interv al between parturition and resumption of normal ovarian activity (Lindell et al., 1982; Madej et al., 1984). While the uterus reaches the size of the nonpregnant uterus at about three weeks postpartum, Lindell and Kindahl (1983) reported that exogenous application of PGF2 between days 3 and 10 postpartum decreased involu tion time by about one week. Increased plasma PGFM also may have beneficial effects on immune competency since, in this study, the proportion of cows w ith metritis was higher in control compared to t FA dietary group (22 % vs. 0 %; Table 6-1). A study by S eals et al. (2002) supports this observation as they reported that postpar tum concentrations of PGFM were inversely related to emergence of uter ine infections. Since PGF2 stimulates myometrial contraction (Lindell and Kindahl 1983; Patil et al., 1980; Gaje wski et al., 1999), this may be a mechanism to expel debris and micro-or ganisms that contaminate the uterine lumen after calving (Dhaliwal et al., 2001). Moreover, PGF2 may have a stimulatory effect on the phagocytic activity of uterine polymorphonuc lear inflammatory cells (Paisley et al., 1986). Summary Feeding trans fatty acids did not induce any signif icant alterations in production as well as metabolic responses when compared to supplementation with saturated fatty acids in either heifers or cows. Although no di fferences were observed in PGFM levels between dietary groups in heifers, di etary supplementation of cows with t FA, increased plasma PGFM concentration within 4 d postp artum when compared to saturated fatty acids. Whether this augmentation in PGF2 production results in enhanced ovarian activity and fertility as well as immune competency, warrants further research.

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119 Table 6-1. Incidence of health disorders of heifers and cows fed diets containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA). Disorder Heifers Cows Control t FA Control t FA Calving Difficulty1 1/6 (17%) 2/6 (33%) 1/9 (11%) 0/9 (0%) Retained Placenta2 0/6 (0%) 1/6 (17%) 2/9 (22%) 2/9 (22%) Enteritis3 0/6 (0%) 0/6 (0%) 0/9 (0%) 1/9 (11%) Metritis4 2/6 (33%) 1/6 (17%) 2/9 (22%) 0/9 (0%) Displaced Abomasum5 0/6 (0%) 0/6 (0%) 1/9 (11%) 2/9 (22%) 1 Calving difficulty refers to animals that scored greater than 2 in a 5 point calving difficulty scale. 2 Retained placenta refers to animals that did not expel fetal memb ranes within 12 h of calving. 3 Enteritis refers to inflammation of the intestinal cells (enterocytes). 4 Metritis refers to inflammation of the uter us with purulent discharge from the vagina and a fever greater than 102 C for two consecutive days. 5 Displaced abomasum refers to cases that required surgery.

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120 Table 6-2. Fatty acid profile according to the manufacturer s of a highly saturated fat (RBF; Cargill, Minneapolis, MN) and a Ca salt lipid enriched in trans C18:1 ( t FA) (Virtus Nutrition LLC, Fairlaw n, OH) fed during the prepartum and postpartum periods to Holstein heifers and cows. Fatty acid RBF t FA --------(g/100 g)-------C12:0 0.04 C14:0 5.60 0.31 C16:0 37.80 12.21 C16:1 0.15 C18:0 48.00 6.70 C18:1, trans 6-8 20.62 C18:1, tran -9 10.47 C18:1, trans -10 10.62 C18:1, trans -11 7.05 C18:1, trans -12 8.73 C18:1, cis -9 4.80 10.04 C18:1, cis -9, cis -12 1.97 Unknown 3.80 11.07

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121 Table 6-3. Ingredient composition of prepartum and postpartum diets. Ingredient Prepartum Postpartum --------% of DM-------Corn silage 45.0 37.5 Bermudagrass hay 15.0 Alfalfa hay 11.9 Corn meal 14.9 20.3 Soy Plus1 13.4 Soybean meal 12.0 7.6 Citrus pulp 5.0 Cottonsead hulls 2.5 Fat supplement2 1.5 1.8 1.5 1.8 Mineral and vitamin mix3 6.5 Mineral and vitamin mix4 4.8 Trace mineralized salt5 0.1 Biophos6 0.3 1 West Central Soy, Ralston, IA. 2 Fat source consisted of 1.5% and 1.8% of a highly saturated fat (RBF) and a Ca salt lipid enriched in trans C18:1 ( t FA), respectively. 3 Mineral and vitamin mix contained 22.8% CP, 2.1% fat, 22.89% Ca, 0.16% P, 2.77% Mg, 0.75% Na, 0.20% K, 2.42% S, 8.03% Cl, 150 mg/kg of Mn, 97.0 mg/kg of Zn, 168 mg/kg of Fe, 186 mg/kg of Cu, 11 mg/kg of Co, 8.4 mg/kg of I, 6.9 mg/kg of Se, 268,130 IU/kg of vitamin A, 40,000 IU/kg of vitamin D, and 1,129 IU/kg of vitamin E (DM basis). 4 Mineral and vitamin mix contained 26.4% CP, 1. 74% fat, 10.15% Ca, 0.90% P, 3.1% Mg, 8.6% Na, 5.1% K, 1.5% S, 4.1% Cl, 2,231 mg/kg of Mn, 1,698 mg/kg of Zn, 339 mg/kg of Fe, 512 mg/kg of Cu, 31 mg/kg of Co, 26 mg/kg of I, 7.9 mg/kg of Se, 147,756 IU/kg of vitamin A, 43,750 IU/kg of vitamin D, and 787 IU/kg of vitamin E (DM basis). 5 Minimum concentrations of 40% Na, 55% Cl, 0.25% Mn, 0.2% Fe, 0.033% Cu, 0.007% I, 0.005% Zn, and 0.0025% Co (DM basis). 6 IMC-Agrico, Bannockburn, IL.

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122 Table 6-4. Chemical composition of prepartum and postpartum diets. Prepartum Postpartum Ingredient Control t FA Control t FA DM, % 47.2 47.2 50.9 50.9 CP, % DM 13.8 14.0 17.2 16.9 ADF, % DM 22.1 22.3 17.8 17.8 NDF, % DM 37.4 37.7 31.1 31.2 Lipid, % DM 3.89 4.05 4.64 5.45 NEL, Mcal/kg 1.53 1.53 1.67 1.67 Ca, % DM 1.78 1.83 1.02 1.11 P, % DM 0.33 0.34 0.48 0.47 Mg, % DM 0.33 0.34 0.34 0.34 K, % DM 1.30 1.32 1.56 1.57 Na, % DM 0.20 0.19 0.45 0.43 Fe, mg/kg 563 541 417 425 Zn, mg/kg 65 58 92 90 Cu, mg/kg 27 23 32 37 Mn, mg/kg 67 61 92 88 Mo, mg/kg 0.77 0.97 1.4 1.1

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123 Table 6-5. Performance of l actating heifers and cows fed a diet containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA) at wk 3 postpartum. Heifers Cows P -value Variable Control t FA Control t FA SEM Diet Parity D x P Milk, kg/d 37.0 34.6 45.2 43.8 2.50 0.34 0.0001 0.8420 3.5% FCM1, kg 29.5 27.8 36.7 35.5 2.39 0.55 0.0053 0.95 Milk fat % 3.36 3.48 3.48 3.31 0.24 0.90 0.92 0.56 kg/d 0.83 0.79 1.06 1.02 0.11 0.73 0.04 0.98 Milk true protein % 2.72 2.60 2.81 2.85 0.09 0.69 0.08 0.39 kg/d 0.65 0.60 0.86 0.88 0.07 0.84 0.001 0.56 SCC (x 1000) 116 135 65 87 29 0.80 0.55 0.94 1 3.5% Fat-corrected milk = (0.4324)(kg milk) + (kg milk fat)(16.216)

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124 Table 6-6. Distribution of follicles* in lactat ing heifers and cows fed a diet containing a highly saturated fat (Control) or a Ca salt enriched in trans C18:1 ( t FA). C1 = 3 5 mm; C2 = 6 9 mm; C3 = > 9 mm. Variable Heifers Cows SEM P -value Control t FA Control t FA Diet Parity D x P Day 7 postpartum C1 9.5 9.8 8.4 7.3 1.8 0.83 0.34 0.77 C2 1.3 2.2 0.7 2.2 0.9 0.17 0.74 0.49 C3 0.0 0.0 0.3 0.3 0.3 0.94 0.36 0.84 Day 14 postpartum C1 8.0 10.2 8.4 6.5 1.8 0.94 0.38 0.55 C2 1.8 1.2 1.3 1.2 0.9 0.70 0.78 0.97 C3 0.8 0.8 0.7 1.2 0.3 0.46 0.63 0.73 Day 21 postpartum C1 5.8 7.2 10.0 9.0 1.8 0.90 0.11 0.39 C2 3.8 1.8 0.4 1.2 0.9 0.49 0.03 0.07 C3 1.3 1.0 1.0 1.2 0.3 0.90 0.90 0.94

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125 Figure 6-1. Average dry matter intake (DMI) as a percentage of body weight (BW) of periparturient Holstein heifers (A) and cows (B) fed a control (C) or trans C18:1 ( t FA)-supplemented diet. Data repr esents least square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.3750). Dry Matter Intake (% BW) 0.5 1.0 1.5 2.0 2.5 3.0 Control t FA Week relative to calving -2-1123 Dry Matter Intake (% BW) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 (A) (B)

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126 Figure 6-2. Average body weight (BW) of periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet. Data represents least square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.9559). Body Weight (Kg) 150 300 450 600 750 Control t FA Week relative to calving -2-1123 Body Weight (Kg) 0 150 300 450 600 750 (A) (B)

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127 Figure 6-3. Calculated energy ba lance by week relative to parturition for Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet. Data represents least square m eans SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.1959). Energy Balance (Mcal/d) -8 -4 0 4 8 Control t FA Day relative to calving -2-1123 Energy Balance (Mcal/d) -12 -8 -4 0 4 8 (A) (B)

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128 Figure 6-4. Average body conditi on score (BCS) of peripartur ient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet. Data represents least square m eans SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.8333). Body Condition Score 0.6 1.2 1.8 2.4 3.0 3.6 Control t FA Week relative to calving -2-1123 Body Condition Score 0.0 0.6 1.2 1.8 2.4 3.0 3.6 (A) (B)

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129 Figure 6-5. Temporal patterns of milk yield by periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet. Data represents least square m eans SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.9203). Milk (Kg/d) 10 20 30 40 50 Control t FA Week relative to calving 123 Milk (Kg/d) 0 10 20 30 40 50 (A) (B)

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130 Figure 6-6. Average feed efficiency as a function of milk yi eld over intake of periparturient Holstein heifers (A ) and cows (B) fed a control or trans -C18:1 ( t FA)-supplemented diet. Data represents least square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was no trt x par x wk interaction ( P = 0.1818). Feed Efficiency (Kg milk/Kg DMI) 0.5 1.0 1.5 2.0 2.5 3.0 Control t FA Week relative to calving 123 Feed Efficiency (Kg milk/Kg DMI) 0.0 0.5 1.0 1.5 2.0 2.5 ( A ) ( B )

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131 Figure 6-7. Plasma NEFA concentrations by week relative to calving in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet. Data represents l east square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was a par x day interaction ( P = 0.04). NEFA ( Eq/L) 100 200 300 400 500 600 700 Control t FA Day relative to calving -14-771421 NEFA ( Eq/L) 0 100 200 300 400 500 600 (A) (B)

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132 Figure 6-8. Plasma BHBA concentrations by week relative to calvi ng in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet. Data represents l east square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). Asterisks indicate sign ificant treatment differences ( P < 0.05). There was a trt x par x day interaction ( P = 0.0244). BHBA (mg/dL) 1.5 3.0 4.5 6.0 7.5 9.0 Control t FA Day relative to calving -14-771421 BHBA (mg/dL) 0.0 1.5 3.0 4.5 6.0 7.5 (A) (B)

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133 Figure 6-9. Plasma glucose concentrations by week relative to calvi ng in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet. Data represents l east square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). There was a trt x par x day interaction ( P = 0.0124). Glucose (mg/dL) 15 30 45 60 75 Control t FA Day relative to calving -14-771421 Glucose (mg/dL) 0 15 30 45 60 (A) (B)

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134 Figure 6-10. Plasma PGFM concentrations by week relative to calvi ng in periparturient Holstein heifers (A) and cows (B) fed a control or trans -C18:1 ( t FA)supplemented diet. Data represents l east square means SEM (Heifers: 4 C, 5 t FA; Cows: 7 C, 6 t FA). Asterisks indicate sign ificant treatment differences ( P < 0.05). Plasma PGFM (pg/ml) 750 1500 2250 3000 3750 Control t FA Day relative to calving -5151015 Plasma PGFM (pg/ml) 0 750 1500 2250 3000 3750 (A) (B)

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135 CHAPTER 7 GENERAL DISCUSSION In the past decade, genetic selection fo r high milk production has been associated with a decrease in reproductive efficiency in lactating dairy cows. This reduction in reproductive efficiency stems from early embr yonic loss, impaired ov arian cyclicity and low fertility rates in high producing dairy cows. The transition to lactation, which extends from 3 weeks before calving to 3 w eeks after parturition, is characterized by an abrupt shift in nutritional requirements, follo wed by dramatic metabolic changes in order to support lactation. Essentia lly all of the energy that is consumed is used by the mammary tissue for milk production, leavi ng no energy for maintenance. Hence, lactating dairy cows experience a state of negative energy balance (NEB), resulting in massive mobilization of fat from the adipose tissue. The mechanisms by which the periparturie nt metabolic upsets reduce reproductive efficiency are not well understood. Howe ver, available evidence indicates that reproductive efficiency is dependent on the gr owth and development of a viable oocyte that can then be fertilized. This is directly dependent on a normal estrous cycle characterized by folliculogenesis, ovulation, and corpus luteum (CL) formation and regression (~21 days). It has been shown that with inappropri ate nutrition, ovarian cyclicity may cease. Thus the nutritional and energy state of the cow may influence reproductive efficiency by modulating the es trous cycle. This could be through the neuroendocrine modulation of gonadotropin secr etion, although more consistent evidence is needed. Alternatively, local ovarian e ffects could take place through modulation of

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136 follicular growth and development, which might determine oocyte quality and viability and steroidogenic activity of the CL. Howeve r, energy-independent effects may also be observed after fatty acid supplementati on, which could involve prostaglandin biosynthesis. Therefore, nutri tion can influence reproductive e fficiency in dairy cows not only by altering the energy status of the animal, but also by influencing factors involved in the regulation of repro ductive processes like follicular dynamics, ovulation, CL function and embryo survival. Long-chain fatty acids (LCFA) are generally added to dairy rations to increase the energy density of the diet. It is expected that supplementati on of the diet with fatty acids may enhance reproductive efficiency by enhanci ng the energy status of the dairy cow. However, recent studies indi cate that dietary fatty aci ds may affect reproductive efficiency in farm animals through an ener gy-independent mechanism. One way that fatty acids could enhance reproductive e fficiency would be through regulation of prostaglandin biosynthesis. Prostaglandin production can be influenced by nutrition since the precursor for the biologically ac tive prostaglandin of the two series is arachidonic acid (AA), an n-6 fatty acid s ynthesized from elongation/desaturation of linoleic acid (LA). Prostaglandi ns (PG) of the 2 series (PGF2 PGE2) have been implicated in the process of reproduction; including ovulation, follicular development, and CL function. Hence, any effects of fatty acids on PGF2 synthesis is likely to affect overall reproductive performance. There are reports that indicate that di etary supplementation of fat has positive effects on reproduction. Many speculate that these positive eff ects are a result of inhibition of prostagladin bi osynthesis, specifically PGF2 which results in prevention of

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137 CL regression and increased progester one secretion around the time of embryo recognition. The CL function is very importa nt for reproductive efficiency since it is critical for establishment and maintenance of pregnancy. Alternatively, stimulation of PGF2 during the first 10 d of lact ation also would be benefici al since this prostaglandin is important for uterine i nvolution after parturition. We first set out to examine the effects of PUFAs on PGF2 responses to PDBu in cultured bovine endometrial (BEND) cells (Cha pters 3 and 4). Phorbol-ester stimulated PGF2 production and up-regulated PGHS-2 gene and protein expression within 6 h in cultured BEND cells. Priming of BEND cells with EPA and both, cis -9, trans -11 and trans -10, cis -12, CLA isomers decreased PGF2 response to PDBu by 66% and 24%, respectively. This is in agreement with several in vivo and in vitro studies that have detected inhibitory effects of EPA (Achar d et al., 1997; Levine and Worth, 1984; Mattos et al., 2000, 2001, 2002, 2003, 2004; Caldari-Torres et al., 2006; Staples et al., 1998; Abayasekara and Wathes, 1999) and CLA (Liu and Belury, 1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al., 2003) on eicosanoid synthesis. While EPA did not have detectable effects on PGHS-2, both CLA isomers increased PGHS-2 mRNA abundance in PDBu -stimulated BEND cells, indicating that EPA or CLA likely affected PGF2 production through a mechanism that does not require repression of PGHS-2 gene expression. Moreover, even tho ugh CLA increased PPAR mRNA response to PDBu, no changes were de tected at the protein levels. These observations collectively suggest th at modulation of endometrial PGF2 production by supplemental fatty acids is through PGHS-2-, PGES-, and PPAR -independent mechanisms. Whether and how these fatty ac ids may affect the act ivities of various

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138 enzymes and transcription f actors involved in the PGF2 biosynthetic cascade warrants further information. Next, we evaluated the effects of monouns aturated fatty acids (MUFA) on bovine endometrial PGF2 production. The most common natu rally occurring MUFA is oleic acid (C18:1), which contains its double bond in the cis configuration. However, as a result of the biohydrogenation process by microor ganisms in the rumen, accumulation of trans C18:1 fatty acids ( t FA) may occur in ruminants. More over, Mosley et al. (2002) showed that biohydrogenation of oleic acid i nvolves the formation of several t FA isomers rather than direct biohydrogenation to form stearic acid (ST; C18:0). In addition, the trans -9 isomer of octadecenoic acid can be c onverted to both ST and a series of t FA isomers (Proell et al., 2002). Results indi cated that supplementation with trans fatty acids resulted in enhanced endometrial PGF2 production in vitro (Chapter 5) as well as in vivo (Chapter 6). The mechanism by which t FA stimulates endometrial PGF2 is not well understood. Although we observed enhanced re sponse of PGHS-2 mR NA to PDBu after incubation of BEND cells with t FA, no changes were observed at the protein level. Additional studies are needed to examine if a nd to what extent these fatty acids modulate the activity of various enzymes involved in the prostaglandin bi osynthetic cascade. Dietary supplementation with t FA has been shown to reduce plasma concentration of n-3 and n-6 PUFAs in pigs (Kummerow et al., 2004). Trans fatty acids also have been shown to inhibit conversion of LA to longer chain n-6 PUFAs in vitro (Mahfouz et al., 1980) and in vivo (Mahfouz et al., 1984). Additi onally, essential PUFAs have been reported to inhibit prostagla ndin secretion in several cell types (Levine and Worth, 1984; Achard et al., 1997), including BEND cells (Mat tos et al., 2003). Ta ken together, these

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139 studies indicate that isomers of oc tadecenoic fatty acids may induce PGF2 production through a mechanism involving inhibition of synthesis of PUFAs. Although fatty acids are naturally occurring ligands of PPARs, it appears that the effect of t FA on PGF2 does not require modulation of PPAR gene or protein since no changes were observed after incubation with t FA. Whether and how these fatty acids may control activity of this nuclear receptor is yet to be elucidated. The present study indicate d that supplemental t FA induced PGF2 production by BEND cells. However, the physiological significance of this increase in PGF2 production is yet to be elucidated. Followi ng parturition, resumpti on of normal estrous cycles is dependent on uter ine involution and repair re sulting from myometrial contractions stimulated by PGF2 (Kiracofe, 1980). In additi on, application of exogenous PGF2 early postpartum, increased myoelectrical activity and contrac tion of the uterus (Patil et al., 1980; Gajewski et al., 1999). Thus, enhanced ovarian activity and function may be related to elevated PGF2 concentration since we showed that dietary t FA greatly increased and sustained higher plasma PGFM concentration within the first week of lactation. While the uterus reaches the size of the nonpregna nt uterus at about three weeks postpartum, Lindell and Kindahl (1983) showed that exoge nous application of PGF2 between days 3 and 10 postpartum decreased involution time by about one week. Increased plasma PGFM also may have beneficial effects on immune competency because in this study, the proportion of cows with metritis was higher in control when compared to the t FA dietary group (22 vs. 0%). A si milar observation was made by Seals et al. (2002), who reported that postpartum concentrations of PGFM were inversely related to emergence of uterin e infections. Moreover, PGF2 may have a stimulatory

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140 effect on the phagocytic activity of uter ine polymorphonuclear inflammatory cells (Paisley et al., 1986). Whether or not this augmentation in PGF2 production results in improved fertility warrants further research. Further understanding on how t FA may affect reproductive performance in postpartum dairy cows will lead to the deve lopment of novel nutritional strategies to increase embryo survival and pregnancy ra tes in high producing dairy cows in which fertility is impaired due to high metabolic de mands associated with milk production. For example, modulation of endometrial PGF2 production by manipulation of dietary longchain fatty acids, such as dietary supplementation with t FA (during transition period) or n-3 PUFAs (during the breeding period), may reduce the postpartum interval to estrus and increase pregnancy rates in high producing dairy cows.

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171 BIOGRAPHICAL SKETCH Carlos Jess Rodrguez Sallaberry wa s born on December 2, 1971, to Fritz I. Rodrguez Munoz and Lourdes Sallaberry Morcig lio in Mayagez, Puerto Rico. He is the youngest of seven children. He received his bachelors degree in molecular genetics from The Ohio State University, Columbus, OH, in December 1994. He attended the University of Puerto Rico, Mayagez Campus for a year to complete a postbachalaurate degree in the Department of Animal Industr y, and subsequently en tered the Graduate School in the Department of Animal Sciences at the University of Florida in 1997 where he completed his masters program under the supervision of Dr. Rosalia C.M. Simmen in the Fall of 1999. He continued his PhD progr am with Dr. Simmen, which he was forced to postpone for personal reasons. During th e period between March of 2001 and April of 2003 he worked as a technician in the labor atory of Dr. Karen Moore, Department of Animal Sciences, University of Florida. He then resumed his PhD degree under the supervision of Dr. Lokenga Badinga. After completion of the PhD degree, he plans to work as a postdoctoral fellow in the laborator y of Dr. Andrs Kowalski in Universidad Centrooccidental Lisandro Alva rado, Barquisimeto, Venezuela.


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Physical Description: Mixed Material
Copyright Date: 2008

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REGULATION OF PROSTAGLANDIN Fza BIOSYNTHESIS BY LONG CHAIN
FATTY ACIDS INT CATTLE















By

CARLOS J. RODRIGUEZ SALLABERRY


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

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Carlos J. Rodriguez Sallaberry



































Este trabajo se lo quiero dedicar a las dos Lourdes de mi vida: Lourdes Antonia, fuente
de inspiraci6n, lucha y sacrificio; Lourdes Paola, quien con su mera existencia me da
fuerzas para seguir adelante todos los dias y represent lo mej or de mi.
















ACKNOWLEDGMENTS

I like to express my gratitude to Dr. Lokenga Badinga, for his guidance throughout

the process of completing this proj ect, including reading and editing this dissertation, and

for the excellent scientific and intellectual training I received in his laboratory during this

time. I thank him for being an excellent mentor and advisor, by being there every time I

needed advice and setting a great example for the perfect balance between work and

family. Acknowledgements are extended to Dr. Charles Staples, Dr. William Buhi, and

Dr. Ramon Littell for serving as members of my supervisory committee and for their

suggestions and insights that helped towards the completion of this study.

I would also like to thank David Armstrong and the whole crew of the DRU,

especially Mary, Eric, and Jerry; without their assistance and cooperation the trial would

not have been a success. I want also to thank Werner Collante for his help in the

laboratory and throughout the proj ect. I wish to give special thanks to Dr. Andres

Kowalski, Jeremy Block, Dervin Dean, Maria B. Padua, and Elizabeth Johnson-Greene;

their help and friendship is going be eternally appreciated.

Gratitude and love are extended to my daughter, Lourdes, and my family for their

neverending support in all my endeavors.

Lastly, I want to thank my best friend and love, Cristina, for being there for me

every time I needed a push and words of encouragement.



















TABLE OF CONTENTS


page

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

LI ST OF T ABLE S .........__.. ..... .__. .............._ viii.

LIST OF FIGURES .............. .................... ix

ABBREVIATION KEY .............. ....................xii

AB S TRAC T ......_ ................. ............_........x

CHAPTER

1 INTRODUCTION ................. ...............1.......... ......

2 LITERATURE REVIEW ................. ...............5................


Estrous Cycle .................. ........... ...............5.......
Neuro-Endocrine System ................. ...............6.................
Stages of the Estrous Cycle .................. ............ .. ...............7.....
Follicular Development during the Estrous Cycle .............. ....................8
Ovulation and Development of the Corpus Luteum ................. .....................1 1
Prostaglandin Biosynthesis and Luteolysis in Cattle................. ...............1
Prostaglandin Bi osynthesi s ................ ...............16........... ...
Luteolysis in Cattle............... ..... ..... ............1
Pregnancy Establishment in Domestic Ruminants..........._.._. .. ..............._ ...._.19
Energy Balance and Fertility Responses in Postpartum Dairy Cows ................... ......21
Energy Balance in Transition Dairy Cows ................. ................ ......... .21
Fertility Responses in Postpartum Dairy Cows ................. ........................24
Effects of Dietary Fats on Reproductive Response in Cattle ........._.._... ........._.......29
Regulation of Prostaglandin Fza Synthesis by Polyunsaturated Fatty Acids ......39
Conjugated Linoleic Acid and Reproduction ................. ......__. ........._.._.43

3 EFFECTS OF POLYNAUNSATUAED FATTY ACIDS ON
PROSTAGLANDIN Fza PRODUCTION BY BOVINE ENDOMETRIAL
CELLS .............. ...............46....

Introducti on ................. ...............46.................
M materials and M ethods .............. ...............49....












M materials ................. .. ...___. ...............49.......
Cell Culture and Treatment .............. ...............50....

PGF 2a Radioimmunoassay .............. ...............5 1....
RNA AIsol ation and Analy si s.................... .....................51

Western Blot Analysis of PGHS-2, PGES and PPARS ................. ................ .52
Statistical Analyses............... ...............53
Re sults ................ ...............54.................
Discussion ................. ...............55.................

Sum m ary ................. ...............56.......... ......


4 EFFECTS OF CONJUGATED LINOLEIC ACIDS ON PROSTAGLANDIN Fza
PRODUCTION BY BOVINE ENDOMETRIAL CELLS............. ... .........___...70


Introducti on ...._ _. ................. ...............70.......
Materials and Methods .............. ...............72....
M materials ................. .. ...___. ...............72.......
Cell Culture and Treatment .............. ...............73....

Statistical Analyses............... ...............73
Re sults........._...... ...............74..._. ._ ......
Discussion ........._...... ...............75..._.._. ......

Summary ........._...... ...............76..._.._. ......


5 EFFECTS OF CIS- AND 7RANS-OCTADECENOIC ACIDS ON

PROSTAGLANDIN Fza PRODUCTION BY BOVINE ENDOMETRIAL
CELLS .............. ...............85....


Introducti on ................. ...............85.................
Materials and Methods .............. ...............87....
M materials ................. .. ......... ...............87......
Cell Culture and Treatment .............. ...............88....

Statistical Analy ses............... ...............88
Re sults ................ ...............89.................
Discussion ................. ...............89.................

Sum m ary ................. ...............9.. 1..............


6 EFFECTS OF DIETARY 7RANS FATTY ACIDS ON PROSTAGLANDIN Fza
CONCENTRATIONS IN POSTPARTUM HOLSTEIN COWS ............................100


Introducti on ........._.__....... ._ __ ...............100....
Materials and Methods .............. ...............105....
M materials ........._.__........_. ...............105....
Cows and Diets .........._.... ....._ __ ...............105....

Collection of Blood Samples............... ...............108
Metabolite and PGFM Assays .....__.....___ ..........._ ............10
Ultrasonography ................. ...............110......... ......
Statistical Analyses ................. ...............110................
Re sults ................ ...............110................












Production Responses ................. ...............110................
M etabolic Responses ................. ...............112................

Reproductive Responses ................. ...............113................
Discussion ................. ...............114................

Summary ................. ...............118................


7 GENERAL DI SCU SSION ................. ................. 13......... 5...


LIST OF REFERENCES ................. ...............141................


BIOGRAPHICAL SKETCH ................. ...............171......... ......
















LIST OF TABLES


Table pg

6-1 Incidence of health disorders of heifers and cows fed diets containing a highly
saturated fat (Control) or a Ca salt enriched in transrt~t~rt~t~rt~t~rt~ Cls I (tFA). ................... .......119

6-2 Fatty acid profile according to the manufacturers of a highly saturated fat (RBF;
Cargill, Minneapolis, MN) and a Ca salt lipid enriched in trans Cls I (tFA).........120

6-3 Ingredient composition of prepartum and postpartum diets ................. ............... 121

6-4 Chemical composition of prepartum and postpartum diets. ................. ............... 122

6-5 Performance of lactating heifers and cows fed a diet containing a highly
saturated fat (Control) or a Ca salt enriched in transrt~t~rt~t~rt~t~rt~ Cls I (tFA) at wk 3
postpartum ................. ...............123................

6-6 Distribution of follicles* in lactating heifers and cows fed a diet containing a
highly saturated fat (Control) or a Ca salt enriched in transrt~t~rt~t~rt~t~rt~ Cls:1 (tFA)................124

















LIST OF FIGURES


Figure pg

3-1 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin Fzca (PGF2ac)
secretion in bovine endometrial (BEND) cells. ............. ...............58.....

3-2 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide
synthase (PGHS-2) mRNA abundance in bovine endometrial (BEND) cells. ........59

3-3 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide
synthase (PGHS-2) protein levels in bovine endometrial (BEND) cells. ................60

3-4 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase (PGES)
mRNA abundance in bovine endometrial (BEND) cells. ............. ....................61

3-5 Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase (PGES)
protein levels in bovine endometrial (BEND) cells. ............. .....................6

3-6 Effect of fatty acids on prostaglandin Fza (PGF2a) TOSponse to phorbol 12, 13
dibutyrate (PDBu) in bovine endometrial (BEND) cells. ............. ....................63

3-7 Effect of fatty acids on prostaglandin endoperoxide synthase (PGHS-2) mRNA
response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND)
cells............... ...............64.

3-8 Effect of fatty acids on prostaglandin endoperoxide synthase (PGHS-2) protein
response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND)
cell s............... ...............65.

3-9 Effect of fatty acids on prostaglandin E synthase (PGES) mRNA response to
phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. ................66

3-10 Effect of fatty acids on prostaglandin E synthase (PGES) protein response to
phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. ................67

3-11 Effect of fatty acids on peroxisome proliferator-activated receptor 6 (PPARS)
mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cell s. ............. ...............68.....










3-12 Effect of fatty acids on peroxisome proliferator-activated receptor 6 (PPARS)
protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cell s. ............. ...............69.....

4-1 Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin Fzca (PGF2ac)
response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND)
cells............... ...............78.

4-2 Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin endoperoxide
synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............79.....

4-3 Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin E synthase (PGES)
mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cell s. ............. ...............8 0....

4-4 Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin endoperoxide
synthase (PGHS-2) protein response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............81.....

4-5 Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin E synthase (PGES)
protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cell s. ............. ...............8 2....

4-6 Effect of c9, t11 and tl0, cl2 CLA isomers on peroxisome proliferator-activated
receptor 6 (PPARS) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............83.....

4-7 Effect of c9, t11 and tl0, cl2 CLA isomers on peroxisome proliferator-activated
receptor 6 (PPARS) protein response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............84.....

5-1 Effect of cis- and trans- isomers of octadecenoic acid on prostaglandin Fza
(PGF2a) TOSponse to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cell s. ............. ...............93.....

5-2 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on prostaglandin
endoperoxide synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate
(PDBu) in bovine endometrial (BEND) cells. ............. ...............94.....

5-3 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on prostaglandin
endoperoxide synthase (PGHS-2) protein response to phorbol 12, 13 dibutyrate
(PDBu) in bovine endometrial (BEND) cells. ............. ...............95.....

5-4 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on prostaglandin E
synthase (PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............96.....










5-5 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on prostaglandin E
synthase (PGES) protein response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. ............. ...............97.....

5-6 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on peroxisome
proliferator-activated receptor 6 (PPARS) mRNA response to phorbol 12, 13
dibutyrate (PDBu) in bovine endometrial (BEND) cells. ............. ....................98

5-7 Effects of cis- and trans-t~t~rt~t~rt~t~rt~ isomers of octadecenoic acid on peroxisome
proliferator-activated receptor 6 (PPARS) protein response to phorbol 12, 13
dibutyrate (PDBu) in bovine endometrial (BEND) cells. ............. ....................99

6-1 Average dry matter intake (DMI) as a percentage of body weight (BW) of
periparturient Holstein heifers (A) and cows (B) fed a control or trans-Cis:1r~rtrtrtrt~t~t~
(tFA)-supplemented diet. ............. ...............125....

6-2 Average body weight (BW) of periparturient Holstein heifers (A) and cows (B)
fed a control or trans-Cls:1 (tFA)-supplemented diet. .............. .....................2

6-3 Calculated energy balance by week relative to parturition for Holstein heifers
(A) and cows (B) fed a control or trans-Cls:1 (tFA)-supplemented diet. ...............127

6-4 Average body condition score (BCS) of periparturient Holstein heifers (A) and
cows (B) fed a control or trans-Cis:Ir~rtrtrtrt~t~t~ (tFA)-supplemented diet. ............................128

6-5 Temporal patterns of milk yield by periparturient Holstein heifers (A) and cows
(B) fed a control or trans-Cls:1 (tFA)-supplemented diet. ................. ..................129

6-6 Average feed efficiency as a function of milk yield over intake of periparturient
Holstein heifers (A) and cows (B) fed a control or trans-Cis Ir~rtrtrtrt~t~t~ (tFA)-
supplemented diet ................. ...............130................

6-7 Plasma NEFA concentrations by week relative to calving in periparturient
Holstein heifers (A) and cows (B) fed a control or trans-Cis Ir~rtrtrtrt~t~t~ (tFA)-
supplemented diet ................. ................. 13......... 1....

6-8 Plasma BHBA concentrations by week relative to calving in periparturient
Holstein heifers (A) and cows (B) fed a control or trans-Cis Ir~rtrtrtrt~t~t~ (tFA)-
supplemented diet ................. ...............132................

6-9 Plasma glucose concentrations by week relative to calving in periparturient
Holstein heifers (A) and cows (B) fed a control or trans-Cis Ir~rtrtrtrt~t~t~ (tFA)-
supplemented diet ................. ...............133................

6-10 Plasma PGFM concentrations by week relative to calving in periparturient
Holstein heifers (A) and cows (B) fed a control or trans-Cis Ir~rtrtrtrt~t~t~ (tFA)-
supplemented diet ................. ...............134................
















ABBREVIATION KEY

Arachidonic acid

Angiotensin

Angiopoietin

Bovine endometrial cells

Beta hydroxybutyric acid

Carnitine acyl transferase

Corpus luteum

Conjugated linoleic acid

Docosahexaenoic acid

Estrogen

Endothelial cells

Eicosapentaenoic acid

Estrogen receptor

Endothelin-1

Fibroblast growth factor

Follicle-stimulating hormone

Growth hormone

Gonadotropin-releasing hormone

Insulin-like growth factor

Interferon gamma


AA

Ang

ANPT

BEND cells

BHBA

CAT-1

CL

CLA

DHA

E2

EC

EPA

ER

ET-1

FGF

FSH

GH

GnRH

IGF

IFN-y









IFN-T

IL-1

JAK/STAT



LA

LCFA

LH

LNA

MUFA

NEB

NEFA

OT

OTR

P4

PDBu

PG

PGG

PGE2

PGES

PGF2a

PGFS

PGFM


Interferon tau

Interleukin

Janus kinase signal transducer and activator

of transcription

Linoleic acid

Long-chain fatty acid

Luteinizing hormone

Linolenic acid

Monounsaturated fatty acid

Negative energy balance

Non-esterified fatty acid

Oxytocin

Oxytocin receptor

Progesterone

Phorbol-12, 13-dibutyrate

Prostaglandin

Prostaglandin G

Prostaglandin E2

Prostaglandin E synthase

Prostaglandin Fza

Prostaglandin F synthase

PGF2a metabolite (13,1 4-dihydro-15-keto

prostaglandin Fza)









PGH2 Prostaglandin H2

PGHS Prostaglandin endoperoxide synthase

PGI2 Prostaglandin 12 (Prostacyclin)

PKC Protein kinase C

cPLA2 Cytosolic phospholipase A2

PLC Phospholipase C

PPARs Peroxisome proliferators-activated receptors

PPRE PPAR response element

PUFA Polyunsaturated fatty acid

SFA Saturated fatty acid

TAG Triacylglycerol

TNF-a Tumor necrosis factor a

VEGF Vascular endothelial growth factor
















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

REGULATION OF PROSTAGLANDIN Fza BIOSYNTHESIS BY LONG CHAIN
FATTY ACIDS INT CATTLE

By

Carlos J. Rodriguez Sallaberry

May, 2006

Chair: Lokenga Badinga
Major Department: Animal Sciences

A series of in vitro experiments and an in vivo experiment were conducted to

examine the effects of long-chain fatty acids (LCFA) on PGF2a biosynthesis in cattle.

Treatment of bovine endometrial (BEND) cells with phorbol 13, 14-dibutyrate (PDBu)

resulted in induction of PGFza Secretion. The PDBu-induced PGF2a Secretion coincided

with increased PGHS-2 mRNA and protein expression. There was no evidence for PDBu

modulation of PPARS mRNA or protein synthesis in cultured BEND cells.

Priming of BEND cells with ST, LNA, and EPA reduced PGF2a TOSponse to PDBu

by 17%, 14%, and 66%, respectively. Both saturated and unsaturated fatty acids had no

detectable effects on PGHS-2, PGES or PPARS mRNA response to PDBu. Similarly,

supplementation of BEND cells with cis-9, trans-11 or trans-10,rtrt~t~r~rtrt~t~ cis-12 CLA isomers

greatly decreased PGF2a TOSponse to PDBu. Co-incubation with both CLA isomers

increased PGHS-2 and PPARS mRNA abundance in PDBu-stimulated BEND cells,

suggesting that these fatty acids alter PGF2a prOduction through a mechanism that does










not require repression of PGHS-2 or PPARS gene expression. Priming of BEND cells

with cis-9, trans-9,t~t~rtrt~t~rtrt~ cis-11i, and trans-trt~r~rt~r~rt~t~rt 11 isomers of octadecenoic acid further enhanced

PGF2a and PGHS-2 mRNA response to PDBu. Pre-incubation of BEND cells with cis

and transrt~t~rt~t~rt~t~rt~ monounsaturated fatty acids decreased PGES mRNA response to PDBu. None

of the fatty acids studied altered PPARS mRNA or protein levels.

Feeding transrt~t~rt~t~rt~t~rt~ fatty acids to primiparous or multiparous Holstein cows did not

induce any significant alterations in production or metabolic responses when compared to

supplementation with saturated fatty acids. Dietary supplementation of cows with tFA

significantly increased plasma PGFM concentration within the first week of lactation.

Whether this augmentation in PGF2a prOduction results in improved fertility and immune

competency warrants further research.















CHAPTER 1
INTTRODUCTION

In recent years, the effect of nutrition on reproduction has generated much attention

and has become increasingly important in the dairy industry. In the past decade, genetic

selection for high milk production has been associated to a decrease in reproductive

efficiency in lactating dairy cows (Butler, 2000). Poor reproductive efficiency includes

early embryonic loss (Thatcher et al., 1995), impaired ovarian cyclicity and low fertility

rates (Butler, 2000), which collectively result in reduced milk production (Plaizier et al.,

1997). Early lactating dairy cows have higher energy requirements than can be supported

by dietary energy intake, which creates a negative energy state and leads to impaired

reproductive function (Butler, 2000).

To discuss the effects of nutrition on reproduction during early pregnancy, it is very

important to understand the changes brought about by lactation. The most critical period

of lactation is considered to be the transition period, which extends from 3 weeks before

calving to 3 weeks after parturition. During this period, dramatic metabolic changes

(homeorhesis) take place in order to support lactation. Parturition and the onset of

lactation cause an abrupt shift in nutritional requirements. Essentially all of the energy

that is consumed by the lactating animal is used by mammary tissue for milk production,

leaving an insignificant amount of energy to be distributed for other physiological

processes (Bell, 1995). Hence, the lactating animal experiences a state of negative

energy balance (NEB). This NEB represents a state of undernutrition, which results in

massive mobilization of fat from adipose tissue, increasing plasma levels of non-









esterified fatty acids (NEFA). This massive fat mobilization, in combination with

reduced energy intake (dry matter intake), results in loss of body condition of lactating

animals. Extensive data have shown that the aforementioned conditions (NEB and loss

of body condition) invariably affect reproductive efficiency in lactating dairy cows.

The mechanisms by which the periparturient metabolic upsets result in reduced

reproductive efficiency are not well understood. However, available evidence indicates

that reproductive efficiency is dependent on the growth and development of a viable

oocyte that can then be fertilized. This is directly dependent on a normal estrous cycle

characterized by folliculogenesis, ovulation, corpus luteum (CL) formation and

regression (~21 days). It has been shown that with inappropriate nutrition, ovarian

cyclicity may cease. The estrous cycle consists of two major phases: the follicular phase

(formation of preovulatory follicles) and luteal phase (CL formation and regression).

These two phases are controlled through the action of gonadotropins (FSH and LH),

which in turn are regulated by the action of hypothalamic gonadotropin releasing

hormone (GnRH) on the pituitary. Therefore, nutrition can affect reproductive efficiency

through modulation of the hypothalamic-pituitary axis and/or direct effects at the ovarian

level .

The hypothalamic-pituitary axis is essential in the modulation of gonadotropins

secretion, which in turn plays a pivotal role in control of the estrous cycle. Even though

FSH is critical for follicular growth, LH pulsatility is responsible for normal ovarian

activity since it is critical for ovulation. However, results from numerous studies have

been inconsistent establishing the relationship between nutritional status and secretion of

gonadotropins.









Metabolic upsets, like NEB, have been shown to change the profile of metabolic

hormones such as growth hormone (GH), insulin, and insulin-like growth factor I (IGF-

I), all of which play an important role in control of follicular development in cattle.

Therefore, it is likely that changes in any of these factors due to NEB could alter the

pattern of ovarian follicular growth and development during early postpartum period

resulting in reduced reproductive efficiency. Several studies have shown that animals in

a less severe NEB state have increased levels of plasma insulin and IGF-I, which

coincide with a gonadotropin-independent enhancement of follicular growth. These

hormones can also influence steroidogenic activity of growing follicles, which is critical

for recruitment, selection, dominance and ovulation.

Long-chain fatty acids (LCFA) are generally added to dairy rations to increase the

energy density of the diet. It is expected that supplementation of the diet with fatty acids

may enhance reproductive efficiency by enhancing the energy status of the dairy cow.

However, recent studies indicate that dietary fatty acids may affect reproductive

efficiency in farm animals through an energy-independent mechanism. One way that

fatty acids could enhance reproductive efficiency would be through regulation of

prostaglandin biosynthesis. Prostaglandin production can be influenced by nutrition

since the precursor for the biologically active prostaglandin of the two series is

arachidonic acid (AA), an n-6 fatty acid synthesized from elongation/desaturation of

linoleic acid (LA). Prostaglandins (PG) of the 2 series (PGF2a, PGE2) have been

implicated in the process of reproduction, including ovulation, follicular development,

and corpus luteum functions. Hence, any effects of fatty acids on PGF2a Synthesis are

likely to affect overall reproductive performance.









In summary, it is well established that the energy state of the cow during early

lactation can influence reproductive efficiency by modulating the estrous cycle. This

could be through the neuroendocrine modulation of gonadotropin secretion, specifically

LH pulsatility (hypothalamic-pituitary axis), although more consistent evidence is

needed. Alternatively, local ovarian effects could take place through modulation of

follicular growth and development by metabolic hormones (IGF-I and insulin), which

could determine oocyte quality and viability and steroidogenic activity of the CL.

However, energy-independent effects may also be observed after fatty acid

supplementation, which could involve prostaglandin biosynthesis. Therefore, nutrition

can influence reproductive efficiency in dairy cows not only by altering the energy status

of the animal but also by influencing factors involved in the regulation of reproductive

processes like follicular dynamics, ovulation, CL function and embryo survival among

others.

The goal of this research proj ect was to examine the physiological effects of

supplemental fatty acids on endometrial PGF2a prOduction in cattle. This dissertation

begins with a brief overview of the physiology of reproduction and how dietary fatty

acids affect energy balance and reproductive processes in cattle (Chapter 2).

Experiments described in Chapters 3 and 4 were designed to elucidate endometrial PGF2a

responses to omega-6 and omega-3 fatty acids (Chapter 3) and to cis and trans

conjugated isomers of linoleic acid (Chapter 4). Experiments described in Chapters 5 and

6 evaluated the effects of transrt~t~rt~t~rt~t~rt~ fatty acids on bovine endometrial PGF2a prOduction in

vitro (Chapter 5) and in vivo (Chapter 6). The dissertation is concluded with a general

discussion of the maj or findings of this research proj ect (Chapter 7).















CHAPTER 2
LITERATURE REVIEW

Estrous Cycle

The onset of puberty is regarded as the start of the reproductive life of a female,

when she enters a period of reproductive cyclicity that continues throughout most of her

productive life. This period of reproductive cyclicity is known as the estrous cycle and

its importance is stressed by the fact that it provides the female with several opportunities

throughout life to be bred and become pregnant in order to perpetuate the existence of her

species.

The estrous cycle consists of a series of predictable events, which in general terms

are estrus, ovulation, and the beginning of a new cycle with the onset of a subsequent

estrus. At the beginning of the estrous cycle, the female enters a state of sexual

receptivity, known as estrus, which is followed by mating. Usually, mating takes place

prior to ovulation to increase the chances of the newly released oocyte to be fertilized.

However, if conception does not occur, another cycle begins, providing the female with

another opportunity to reproduce. On the other hand, if conception does occur, the

female enters a period in which she will not exhibit regular estrous cycles during

pregnancy. This is known as a period of gestational anestrus, which allows for fetal

growth and development, and ends after parturition and uterine involution. This and

other examples of anestrus or the absence of a regular estrous cycle will be discussed

later in this chapter.









The estrous cycle and the events governing reproductive cyclicity are tightly

regulated by a series of hormones and factors that will eventually determine normal

reproductive efficiency.

Neuro-Endocrine System

All reproductive processes, including the estrous cycle, are under the control of two

systems: the central nervous system and the endocrine system. These two systems

interact with each other through the hypothalamus, resulting in a neuro-endocrine

network responsible for the initiation, coordination and regulation of the functions of the

reproductive system (Senger, 1997; Hafez and Hafez, 2000).

The hypothalamus consists of clusters of nerve cell bodies, which are responsible

for production of gonadotropin releasing hormone (GnRH), and the paraventricular

nucleus, which produces oxytocin (Senger, 1997). Each set of hypothalamic nuclei has

different functions and is stimulated under different conditions. However, the

communication between the hypothalamus and the pituitary is necessary for these

hormones to exert their action. This communication between the hypothalamus and the

pituitary is referred to as the hypothalamic-pituitary axis.

The pituitary gland is comprised of the posterior and the anterior lobe. The

hypothalamus communicates with the posterior lobe through neural connections, and

therefore the posterior pituitary is referred to as neurohypophysis. For example, oxytocin

is synthesized in neurons in the paraventricular nucleus and it is transported down the

axon to the posterior pituitary where it is released into the blood (Senger, 1997). On the

other hand, the hypothalamus communicates with the anterior lobe adenohypophysiss)

through the hypothalamo-hypophyseal portal system, which prevents hormones

synthesized in the hypothalamus from entering and being diluted by the systemic









circulation. Gonadotropin-releasing hormone is synthesized in either the surge or tonic

center of the hypothalamus and is released into the primary capillary plexus at the stalk of

the pituitary. Blood enters this capillary system from the superior hypophyseal artery and

transports GnRH to a secondary capillary plexus in the anterior pituitary where it acts on

target cells to release either follicle stimulating hormone (FSH) or luteinizing hormone

(LH) (Senger, 1997; Hafez and Hafez, 2000).

Hormones that are secreted by the pituitary are then transported through systemic

circulation to the target tissue to elicit the physiological response. This is where the

neural and the endocrine systems interact to modulate reproductive functions such as the

estrous cycle.

Stages of the Estrous Cycle

The estrous cycle is divided into four stages: proestrus, estrus, metestrus and

diestrus. Proestrus begins when circulating concentration of progesterone (P4) declines as

a result of the regression of the corpus luteum from the previous cycle. It lasts from 2 to

5 days, depending on species, and terminates with the onset of estrus. Proestrus is

characterized by an endocrine transition from a period of P4 dominance to a period of

estradiol (E2) dominance under the control of FSH and LH. During this period,

recruitment of follicles takes place and the reproductive tract prepares for the next stage,

estrus (Senger, 1997).

Estrus is the stage of the estrous cycle during which the female undergoes

behavioral as well as endocrine changes characterized by sexual receptivity. These

behavioral and physiological changes in the female are induced by E2, which is the

dominant hormone during this stage. Sexual receptivity during estrus increases gradually









until the animal reaches the typical standing estrus or lordosis. In cattle, estrus can

extend from 6 to 24 h, with an average of 15 h (Senger, 1997).

Metestrus is characterized by the transition from E2 dominance to P4 Secretion as a

result of the formation of a functional CL. At the beginning of this stage, concentration

of both E2 and P4 is relatively low. However, the newly ovulated follicle undergoes

cellular and structural changes that result in luteinization of the follicle and formation of

the CL, which starts producing P4 Soon after ovulation (Senger, 1997).

Diestrus is the longest stage of the estrous cycle, encompassing maximal CL

function and sustained P4 Secretion, and ending with the regression of the CL. High P4

levels prepare the uterus and its microenvironment for early embryo development and

implantation. The duration of diestrus (10 to 14 days) is directly related to CL function

and P4 Secretion.

Under certain physiological conditions the female does not exhibit regular estrous

cycles and is said to be in anestrus. Anestrus, or lack of estrous cyclicity, is the result of

insufficient GnRH release from the hypothalamus to stimulate and maintain gonadotropin

secretion. This can be caused by pregnancy, lactation, season or stress (Senger, 1997).

Follicular Development during the Estrous Cycle

The follicular phase is the stage of the estrous cycle when preovulatory follicle

development is stimulated by increasing concentrations of FSH and LH in plasma. This

phase coincides with a marked reduction in plasma P4, which leads to significant increase

in tonic GnRH secretion. Increased basal concentration of GnRH stimulates FSH and LH

secretion from the pituitary and promotes follicular development and E2 prOduction.

In cattle, growth of early antral follicles (~3 mm) is considered to be gonadotropin-

independent (Scaramuzzi et al., 1993). The transient rise in FSH at the time of luteal









regression initiates the process of recruitment, which consists of stimulation of a cohort

of small antral follicles to grow beyond 4 mm in diameter around days 1-2 of the cycle

(Adams et al., 1992; Sunderland et al., 1994; Ginther et al., 1997). Follicle-stimulating

hormone stimulates ovarian follicles to acquire key properties associated with E2

synthesis such as increased cytochrome P450 side-chain cleavage and cytochrome P450

aromatase enzyme activities (Bao and Garverick, 1998; Garverick et al., 2002).

The process of recruitment is then followed by a decline in FSH due to the negative

feedback by E2 and inhibin from the dominant follicle (Ginther et al., 1998; Hunter et al.,

2004). This decline in FSH has been identified as an important component of the

selection process (Mihm et al., 1997). Follicle selection then results in a decrease in the

number of growing follicles and is thought to end with growth divergence or deviation of

a dominant follicle from the subordinate follicles (Evans, 2003). During this time, the

dominant follicle can use LH for its continued growth, and this is supported by the fact

that there is a switch from FSH- to LH-dependency as the follicle matures and FSH

concentration declines in cattle (Gong et al., 1996; Webb et al., 2003). Additionally,

there are reports of a transient increase in circulating LH (Kulick et al., 1999, 2001) as

well as increased expression of LH receptors in granulosa cells (Xu et al., 1995; Goudet

et al., 1999; Beg et al, 2001) surrounding deviation of the dominant follicle in cattle.

Acquisition of LH receptors in granulosa cells might allow the transient increase in LH to

have a functional effect in follicle selection, since LH is known to increase E2

concentration in follicular fluid, thus facilitating the establishment of dominance (Ginther

et al., 2000b). The freshly selected dominant follicle grows to a much larger size (12-20

mm) than all other subordinate follicles, resulting in enhanced E2 Secretion (Ginther et al.,










1997). This enhanced E2 Secretion is associated with increased expression of the genes

encoding aromatase, 3 P-hydroxy-steroid dehydrogenase and FSH-receptor, as well as the

acquisition of LH-receptors in granulosa cells (Ireland and Roche, 1983; Xu et al., 1995;

Bao et al., 1997a, b; Evans and Fortune, 1997). High E2 Secretion is also responsible for

maintaining low FSH concentrations to prevent growth of another cohort of follicles

(Ireland et al., 1984, Ginther et al., 1999, 2000a, b).

The growth of the first wave dominant follicle does not take place for more than 3-

4 days, as P4 Secreted from the developing CL inhibits LH pulse frequency and the LH-

dependent dominant follicle becomes atretic (Sunderland et al., 1994; Evans et al., 1997).

As a result, the first wave dominant follicle loses its ability to produce E2 between days 7

and 9 of the estrous cycle. The loss of dominance is followed by another transient rise in

FSH, resulting in the emergence of a new follicular wave (Sunderland et al., 1994). If the

CL fails to regress, the second dominant follicle will undergo atresia and a third wave of

follicles emerges. However, if luteolysis occurs during this second wave of follicular

development, then the dominant follicle will ovulate (Cooke et al., 1997).

In cattle, most estrous cycles consist of two to three waves of follicular

development (Fortune et al., 1988; Savio et al., 1988; Ginther et al., 1989a) that emerge

on about days 2 and 11, or days 2, 9 and 11 for animals with two or three follicular

waves, respectively (Sirois and Fortune, 1988). Lactating Holstein cows tend to have two

waves per cycle (Taylor and Raj amahendran, 1991, Townson et al., 2002), whereas beef

and dairy heifers tend to have two or three waves per cycle (Savio et al., 1988; Sirois and

Fortune, 1988; Ginther et al., 1989c). Cattle with two follicular waves tend to have









shorter estrous cycles, ovulate larger and older follicles and to be less fertile than those

with three waves (Townson et al., 2002).

Ovulation and Development of the Corpus Luteum

The luteal phase encompasses about 80% of the estrous cycle and covers the period

of time that includes ovulation of the dominant preovulatory follicle, CL formation, and

regression of the CL.

A landmark event that precedes ovulation is the preovulatory LH surge, which is

stimulated by increased concentrations of E2 in plaSma (Senger, 1997). The LH surge

triggers a cascade of events that cause biochemical and structural changes in the

preovulatory follicle that lead to the rupture of the follicle wall, resulting in the release of

the oocyte and subsequent development of the CL (Berisha and Schams, 2005; Senger,

1997). Ovarian blood flow increases at the time of ovulation and subsequent CL

formation (Senger, 1997), which emphasizes the importance of angiogenesis during

metestrus and diestrus.

Angiogenesis is defined as the generation of new blood vessels through sprouting

from already existing ones. This process involves degradation of the capillary vessel

membrane, through which endothelial cells (EC) from pre-existing vessels migrate and

proliferate to create a new lumen and further vessel maturation (Abulafia and Sherer,

2000). Angiogenesis is critical for final development and differentiation of the

preovulatory follicle as an increase in both vascular area and blood flow has been

demonstrated during this stage (Acosta et al., 2003). Acosta et al. (2003) showed that

there was a marked increase in blood flow and volume in the subsequent CL, which is

closely associated with increased plasma P4 COncentrations. Moreover, it has been

reported that follicular diameter, E2 COncentration in the follicular fluid, and vascular area









are highly correlated (Mattioli et al., 2001). Thus, ovulation is the result of an interaction

between LH surge and local factors that include steroids, prostaglandins and vasoactive

peptides (Acosta and Miyamoto, 2004). Luteinizing hormone has been shown to increase

ovarian blood flow in rats (Varga et al., 1985), rabbits (Janson, 1975), and sheep

(Niswender et al., 1976). Thus, increased vascular dilatation and permeability, together

with degradation of collagen layers that provide strength to the follicular wall, are

necessary for facilitating follicular rupture (Murdoch et al., 1986; Abisogun et al., 1988)

and oocyte release.

Increased ovarian blood flow may increase the availability of gonadotropins,

nutrients, hormonal substrates and other blood components that are necessary for

ovulation (Acosta and Miyamoto, 2004). Hence, vasoactive peptides released within the

follicular wall may modulate local changes in blood flow observed in the ovulatory

follicle (Brannstrom et al., 1998; Acosta et al., 2003). This cascade could then mediate

LH action to increase prostaglandin production during ovulation. Vasoactive peptides

have been shown to play important roles in the ovulation process as well as early luteal

development, through modulation of local secretion of prostaglandins and steroid

hormones (Acosta et al., 1999, 2000; Kobayashi et al., 2002). Although several

promoters of angiogenesis have been identified, vascular endothelial growth factors

(VEGF), fibroblast growth factors (FGF), insulin-like growth factors (IGF) and

angiopoietin (ANPT) are thought to be the most important factors modulating follicle

maturation and CL formation (Berisha and Schams, 2005).

Normal development of the CL and its capacity to produce P4, grOwth factors,

angiogenic factors, and vasoactive substances is dependent on its vascularization (Acosta









and Miyamoto, 2004). Ovarian blood flow decreases shortly after ovulation, but

gradually increases from day 2 to day 5 of the estrous cycle. This period also coincides

with marked increases in CL volume and peripheral P4 COncentration (Acosta et al.,

2003). Angiogenesis within the CL reaches a peak 2-3 days after ovulation (Reynolds et

al., 2000) and appears to be locally modulated by angiotensin (Ang II) and growth

factors, which also support P4 Synthesis in luteal cells (Kobayashi et al., 2001b).

Immediately after ovulation, the follicle walls collapse, allowing theca internal and

granulosa cells to mix with one another. At the same time, these cells undergo a dramatic

transformation during which they become luteinized after stimulation by preovulatory LH

surge (Senger, 1997). Theca internal cells become the small luteal cells, which contain

numerous lipid droplets, while granulosa cells become the large luteal cells with

secretary granules containing oxytocin (Senger, 1997). Within the developing CL, large

luteal cells increase in size, while small luteal cells increase in number (Senger, 1997).

The process of luteinization is characterized by increased synthesis and activity of

enzymes responsible of switching steroid production from E2 to P4 (Juengel and

Niswender, 1999). Progesterone exerts a strong negative feedback on the hypothalamus,

reducing GnRH pulses, thus preventing development of the dominant follicle, E2

synthesis and ovulation (Senger, 1997). It also promotes endometrial secretions and

quiescence, which ultimately favors embryo implantation.

In cattle, LH and growth hormone (GH) are the primary hormones regulating

development and function of the CL, and this is supported by expression of mRNA for

both LH and GH receptors in the CL during the estrous cycle (Schams and Berisha,

2004). Luteinizing hormone is the principal hormone stimulating P4 Synthesis by the









small luteal cells (Niswender and Nett, 1988) and this is further supported by the fact that

most of LH receptors are found in these cells (Schams and Berisha, 2004). On the other

hand, receptors for growth hormone are mainly located on large luteal cells (Lucy et al.,

1993; Kirby et al., 1996; Koelle et al., 1998) which are responsible for 80% of total P4

production by the CL (Niswender et al., 1985). Growth hormone has been shown to

stimulate P4 and oxytocin secretion by the bovine CL in vitro (Liebermann and Schams,

1994) and to support CL development in vivo (Lucy et al., 1994; Juengel et al., 1997).

Furthermore, GH is a more powerful stimulator of prostaglandin Fza and P4 prOduction

than LH in vitro (Kobayashi et al., 2001a).

There are many other local autocrine and paracrine regulators that play an

important modulatory role during the lifespan of the CL. Growth factors involved in

angiogenesis, such as IGFs and FGFs, induce CL function by stimulating secretion of P4

and oxytocin (Einspanier et al., 1990; Sauerewein et al., 1992). Insulin-like growth

factor-1 has been localized in the cytoplasm of both small and large luteal cells

(Amselgruber et al., 1994). Fibroblast growth factor-2 gene and protein expression also

have been demonstrated in large luteal cells of the bovine CL (Schams et al., 1994).

Ovarian peptides such as oxytocin (OT), angiotensin II (Ang II), endothelin-1 (ET-1),

progesterone and prostaglandin also are important in CL function. It is well established

that oxytocin is highly expressed in the ruminant CL (Wathes et al., 1983; Schams,

1992), and it has been localized in both small and large luteal cells in cattle (Kruip et al.,

1985). The LH surge appears to stimulate ovarian production and secretion of OT

together with P4 (Schams and Berisha, 2004). There are reports that support a potent

luteotropic role of OT during the developing phase of the bovine CL (Schams, 1989;









Schams, 1996). Moreover, OT potentiates the luteotrophic effects of LH on P4 Secretion

in vitro (Schams et al., 1995). On the other hand, the vasoactive peptides Ang II and ET-

1 have been shown to directly inhibit P4 prOduction in bovine luteal cells (Girsh et al.,

1996; Miyamoto et al., 1997; Hayashi and Miyamoto, 1999).

Prostaglandin Biosynthesis and Luteolysis in Cattle

Prostaglandins are members of the eicosanoid family of fatty acids. They are

derived from 20 carbon polyunsaturated fatty acids, such as arachidonic acid (AA).

Prostaglandins of the 2-series, namely prostaglandin Fza (PGF2a) and prostaglandin E2

(PGE2), are the most biologically active eicosanoids and are involved in key reproductive

processes such as follicular development (Wallach et al., 1975), ovulation (Espey, 1980),

luteolysis (Wathes and Lamming, 1995), and parturition (Challis, 1980). Prostaglandin

Fza is an important factor modulating luteal function in cattle. There are two different

sources of PGF2a within the reproductive tract. Temporal expression and site of

synthesis then determine its modulatory role during luteal function. In contrast to the

luteolytic effects of endometrium-derived PGF2a, luteal PGF2a Seems to be luteotrophic

during early and mid-luteal phases (Miyamoto et al., 1993). Bovine CL produces high

amounts of PGF2a during early luteal phase, but PGF2a COncentrations decrease as the

luteal cycle progresses (Milvae and Hansel, 1983; Schams et al., 1995; Skarzynski and

Okuda, 1999). Furthermore, in an in vitro study, infusion of Ang II stimulated P4 and

PGF2a TeleaSe from the developing CL (Kobayashi et al., 2001b). Moreover, Kobayashi

et al. (2001a) showed that Ang II together with PGF2a highly stimulated P4 Secretion

from developing bovine CL. In mid-cycle luteal cells, P4 inhibits luteal PGF2a Secretion

(Pate, 1988; Skarzynski and Okuda, 1999). The other source of PGF2a biosynthesis and









secretion is the endometrial tissue of the bovine uterus and this is associated with the

luteolytic cascade.

Prostaglandin Biosynthesis

Prostaglandins of the 2-series are synthesized from arachidonic acid (AA), which

uses linoleic acid as the primary precursor. Arachidonic acid is store in an esterified form

at the sn2 position of the membrane phospholipids bilayer (Crofford, 2001). The first and

rate limiting step of prostaglandin biosynthesis is the hydrolytic release of AA by the

action of cytosolic phospholipase A2 (cPLA2) enzyme (Lapetina, 1982). This enzyme has

a preference for phospholipids containing AA at the sn2 position. Upon cell activation,

release of intracellular Ca2+ Stimulate cPLA translocation and binding to the membrane,

which is a prerequisite for its enzymatic activity (Murakami et al., 1997; Leslie, 1997).

Following its release, AA is converted to prostaglandin H2 (PGH2) by the action of

prostaglandin H synthase (PGHS), also known as cyclooxygenase (COX). Prostaglandin

H synthase has cyclooxygenase and peroxidase activities that convert prostaglandin G

(PGG) to PGH2 (Goff, 2004). This enzyme consists of two isomers (PGHS-1 and PGHS-

2) (Goff, 2004), which are primarily located on the luminal surface of the endoplasmic

reticulum and the inner and outer membranes of the nuclear envelope (Spencer et al.,

1998). The constitutively expressed PGHS-1 is considered to play a housekeeping role,

whereas PGHS-2 is the inducible form, hence stimulated by hormones, growth factors,

etc. in a variety of tissues (Goff, 2004).

After synthesis of PGH2, this endoperoxide is converted to one of several possible

prostanoids by the action of specific terminal enzymes. Biosynthesis of prostaglandin E2










(PGE2) and prostaglandin Fza (PGF2a) are catalyzed by the action of prostaglandin E and

prostaglandin F synthases, respectively (Goff, 2004).

Luteolysis in Cattle

In ruminants, ovarian E2, Oxytocin (OT), and P4 Seem to be the physiological

regulators of synthesis and secretion of uterine PGF2a during the estrous cycle.

Prostaglandin Fza TeleaSed from the endometrium is the principal luteolytic agent in

ruminants (Lukaszewska and Hansel, 1970; McCracken, 1971; McCracken et al., 1972).

There is clear evidence that luteal regression at the end of the estrous cycle is caused by

episodic release of endometrial PGF2a that reaches the CL through a counter current

mechanism between the uterine vein and ovarian artery (Senger, 1997, Krzymowski and

Stefaniczyk-Krzymowska, 2004; Schams and Berisha, 2004).

It has been demonstrated that P4 regulates the lifespan of the CL (Ottobre et al.,

1980; Schams et al., 1998; Schams and Berisha, 2001, 2002c) through inhibition of

PGF2a Secretion from the endometrium. The large amplitude pulses of PGFa,,

responsible for initiation of luteolysis, result from decreasing P4 and increasing E2

concentrations (Goff, 2004). This is preceded by the increase in E2 and estrogen

receptors (ER) which in turn up-regulates oxytocin receptor in the endometrium (Goff,

2004). At the end of the luteal phase, the number of ER increases, presumably due to E2

up-regulation of its own receptor in endometrial cells (Spencer et al., 1996; Xiao and

Goff, 1999; Ing and Tornesi, 1997). This increase in ER is thought to initiate luteolysis

by increasing oxytocin receptors (OTR) (Spencer et al., 1996; Xiao and Goff, 1999; Ing

and Tornesi, 1997; Spencer and Bazer, 2002); however, up-regulation of OTR in the

bovine endometrium precedes that of the ER (Robinson et al., 2001). Thus, it has been










proposed that, even though increased concentration of OTR (~days 13-16) are primarily

due to withdrawal of P4 inhibitory effects (Fairclough and Lau, 1992), increasing levels

of E2 during this time can facilitate up-regulation of OTR gene expression (Vallet et al.,

1990; Leavitt et al., 1985; Zhang et al., 1992). Therefore, luteolysis is brought about by

coordinated changes in both OTR and prostaglandin production, which are regulated by

changes in P4 and E2 COncentrations.

Acosta et al. (2002) demonstrated that the transitory decrease of P4 may trigger the

luteolytic cascade. Earlier studies have also shown a close relationship between the

decrease in ovarian blood flow and systemic P4 COncentration in the cow (Ford and

Chenault, 1981; Wise et al., 1982). One of the main luteolytic actions of PGFza is to

decrease ovarian blood flow (Knickerbocker et al., 1988).

Locally produced growth factors also mediate the complex process of luteolysis.

Members of the IGF system have been shown to play an important role in the process of

PGFza-induced luteolysis in cattle (Neuvians et al., 2003). Gene and protein expression

of VEGF are known to decline after prostaglandin secretion (Neuvians et al., 2004a),

while FGFs and their receptors are up-regulated during functional luteolysis (Berisha and

Schams, 2005). Therefore, it has been suggested that cessation of VGEF-support of the

CL plays a role during structural luteolysis, whereas FGFs seem to have a maj or impact

in functional luteolysis (Berisha and Schams, 2005).

There is also evidence that vasoconstrictive peptides, such as Ang II and ET-1 may

play a role during physiological and induced luteolysis in cows (Girsh et al., 1996;

Miyamoto et al., 1997; Hayashi and Miyamoto, 1999; Hayashi et al., 2000). In vitro,

PGF2a pOtentiates the inhibitory activity of ET-1 on P4 Secretion and stimulates Ang II









release (Miyamoto et al., 1997). Hence, ET-1 and Ang II may act as vasoconstrictors

during functional luteolysis, as well as apoptosis inducers during functional and/or

structural luteolysis (Schams and Berisha, 2004).

In the bovine ovary, cytokines such as tumor necrosis factor (TNFu), interferon

(IFNy), and interleukin (IL-1P) are up-regulated during induced luteolysis (Neuvians et

al., 2004b). There also is evidence that the combination of TNFu and IFNy are extremely

cytotoxic (Petroff et al., 2001). Therefore, cytokines may be involved not only in

structural, but also functional luteolysis in cattle (Berisha and Schams, 2005).

Pregnancy Establishment in Domestic Ruminants

The process of maternal recognition of pregnancy refers to the physiological

window when the mother becomes cognizant of the embryo within her reproductive tract

and prevents its elimination. If an embryo is present in the bovine uterus between days

14 and 17 of the cycle, luteolysis does not take place and P4 Secretion is maintained,

resulting in establishment of pregnancy (Northey and French, 1980). This is achieved by

a signal from the embryo that prevents the regression of the corpus luteum (CL) through

inhibition of pulsatile PGF2a Secretion from the endometrium (Bazer, 1992; Demmers et

al., 2001).

In domestic ruminants, the embryonic signal responsible for pregnancy

establishment is a cytokine called interferon (IFN)-z (Lafrance and Goff, 1985; Spencer

and Bazer, 1995). Interferon-z is synthesized by trophoblastic cells of bovine blastocyst

(Lafrance and Goff, 1985; Roberts et al., 1990; Spencer and Bazer, 1995), and its

secretion is highest between days 15 and 17 of pregnancy (Stojkovic et al., 1995; Bazer et

al., 1998).










Pregnancy establishment is accomplished through several mechanisms. Interferon-

z can prevent luteolysis by down-regulation of endometrial oxytocin receptor, which

prevents oxytocin-stimulated PGF2a Secretion (Lafrance and Goff, 1985). Secretion of

INF-z in ewes has been reported to reduce estradiol receptors, which prevents E2-

stimulation of OTR (Spencer and Bazer, 1995). Moreover, production of PGF2a in cattle

can be further suppressed by decreasing the expression of PGHS-2 as well as PGFS

(Lafrance and Goff, 1990; Binelli et al., 2000), enzymes which play key roles in the

synthesis of this prostaglandin. Binelli et al. (2000) showed that INF-z blocked PGF2a

production by reducing PGHS-2 and PLA2 gene expression. The suppression of PGHS-2

and PLA2 mRNA synthesis appears to be independent of oxytocin-induced intracellular

events (Pru et al., 2001).

Another mechanism by which IFN-z may inhibit luteolysis in cattle is by shifting

prostaglandin biosynthesis from the luteolytic PGF2a to the luteotropic PGE2 (Okuda et

al., 2002). Results from studies by Xiao et al. (1998), showed that IFN-z inhibited

PGHS-2 mRNA and attenuated prostaglandin secretion from epithelial cells, which are

known to be the primary source of PGF2a, while enhancing PGHS-2 mRNA and

prostaglandin biosynthesis in stromal cells, which are the primary source of PGE2 (Kim

and Fortier, 1995; Asselin et al., 1996, 1998; Skarzynski et al., 2000). Therefore,

achieving an optimal PGE2 to PGF2a ratio is essential for endometrial receptivity,

myometrial quiescence, and maintenance of a functional CL and P4 Secretion, which are

critical for successful establishment of pregnancy (Bazer et al., 1998).










Energy Balance and Fertility Responses in Postpartum Dairy Cows

In mammals, nutrition is critical for sustaining important biological processes that

allows the animal to grow, survive and reproduce. Food is consumed, digested, and

broken down into nutrients, which are then absorbed and partitioned throughout the body

for utilization. Nutrients are utilized by tissues involved in maintenance of basic

physiological processes, as well as establishing energy stores in the form of lipids and

glycogen. The process of maintenance of physiological equilibrium or constant

conditions of the internal milieu balance within a given physiological state is under

homeostatic control (Bauman and Currie, 1980). However, in an animal's life cycle,

there are a series of physiological states through which it must go and adjust adequately.

The orchestrated or coordinated changes in metabolism of body tissues necessary to

support the transition to a particular physiological state are under homeorhetic regulation

(Bauman and Currie, 1980). An example of such dramatic metabolic changes is

represented by the onset of lactation in dairy cows. It is clear then, that overall biological

functions are governed by food intake, nutrient absorption and partitioning, and the

resulting energy status of the animal within a given physiological state.

Energy Balance in Transition Dairy Cows

Energy balance of an animal is the difference between energy intake and energy

requirements within a given physiological state (Beam and Butler, 1999; Butler et al.,

1981; Canfield and Butler, 1990). In dairy cows, the onset of lactation cause an abrupt

shift in nutritional requirements in order to support milk production (Butler, 2000). This

rapid increase in energy requirements and changes in the metabolic as well as endocrine

status of the cow come about during the transition period (Bauman and Currie, 1980;









Grummer, 1995). This is the result of the prioritized status of lactation which allows it to

proceed at the expense of any other physiological processes (Bauman and Currie, 1980).

The transition period extends from three weeks prepartum until three weeks

postpartum, and refers to the period during which endocrine and metabolic changes

accommodate parturition and the onset of lactation (Grummer, 1995). A reduction in

feed intake occurs during the final weeks of pregnancy when nutrient demands for

support of fetal growth and initiation of milk synthesis are increasing (Grummer, 1995).

As a result, there is a higher energy requirement than can be met or supported by dietary

energy intake (Bell, 1995). The dietary energy that is consumed by the lactating animal

is almost entirely used by the mammary tissue for milk production, leaving no energy for

maintenance (Bell, 1995). To offset this energy deficit, the lactating animal mobilizes

body energy reserves, which leads ultimately to a state of negative energy balance.

Since the energy required for lactation and maintenance far exceeds energy intake,

the resulting NEB promotes a massive mobilization of fat from the adipose tissue and

enhanced nutrient partitioning to the mammary gland for milk synthesis (Bauman and

Currie, 1980). Consequently, the transition period is characterized by increased plasma

levels of non-esterified fatty acids (NEFA), indicative of onset of lactation. Once free

fatty acids are released into blood, they are bound to albumin and other blood proteins to

be transported to hepatic and non-hepatic tissues. The uptake of NEFA into the liver

takes place as blood flows though the liver. Once inside the liver, NEFAs can undergo

three of the following metabolic fates as outlined by Drackley (1999): 1) complete

oxidation to carbon dioxide to provide energy for the liver, 2) incomplete oxidation to









produce ketone bodies as an alternate energy source, and 3) re-esterification into

triacylglycerol (TAG).

Normally, complete oxidation of fatty acid takes place, but first fatty acids must be

translocated into the mitochondria. Mitochondrial uptake of FA is regulated by the

activity of camitine acyl transferase (CAT-1). In spite of the central role of CAT-1 in

liver lipid metabolism, little is known about its regulation. However, studies have shown

that high malonyl-CoA intracellular concentrations inhibit CAT-1 enzymatic activity

(Brindle et al., 1985). Malonyl-CoA is produced by the enzyme acetyl-CoA carboxylase

and is an intermediate for FA synthesis. Zammit (1996) demonstrated that the sensitivity

of CAT-1 to malonyl-CoA inhibition is lessened during times of low circulating insulin

or insulin resistance in rodents. Similarly, Brindle et al. (1985) found that malonyl-CoA

concentrations were influenced by insulin and glucagon.

Thus, it would appear that energy balance may play a role in CAT-1 regulation.

Cows in NEB exhibit decreased capacity to metabolize fat, which may be linked to CAT-

1. Drackley et al. (1991) conducted a study with non-lactating cows to test the effects of

carnitine and propionate on liver lipid metabolism. Fasting decreased oxidation of

palmitate to CO2 and decreased palmitate esterification by bovine liver slices. Addition

of camitine in vitro increased oxidation of palmitate and also increased total utilization of

palmitate. Similar results were obtained in early lactating cows (Drackley et al., 1991).

An alternate pathway for hepatic NEFA metabolism is through partial oxidation

that takes place in the peroxisome. This oxidative pathway is similar to that in the

mitochondria, with some exceptions. The initial oxidation step is catalyzed by an

oxydase, which results in production of hydrogen peroxide rather than reduced NAD as









seen in mitochondrial oxidation (Drackley, 1999). The next key difference is that

peroxisomes do not contain respiratory chain linked to ATP formation. As a result,

peroxisomal oxidation is not subject to control by energy demands for the cell. Hence,

these differences make the peroxisome well suited to partially oxidize fatty acids that are

poor substrate for mitochondrial oxidation (Drackley, 1999).

The third metabolic fate ofNEFAs entering the liver is reesterification into TAG.

Resultant TAGs are either stored in the liver or packaged into very low density

lipoproteins and exported into circulation.

Fertility Responses in Postpartum Dairy Cows

Negative energy balance during the first 3 weeks postpartum has been associated

with extended interval to first ovulation (Beam and Butler, 1999; Butler, 2001). The first

ovulation occurs on average about 30 d postpartum, with a range of 14-42 d (Butler and

Smith, 1989; Staples et al., 1990). Conception rates in lactating dairy cows increases

when the period of ovarian activity preceding insemination is longer and thus the number

of preceding ovulatory cycles is greater (Butler and Smith, 1989; Thatcher and Wilcox,

1973). Since the number of ovulatory estrous cycles preceding insemination influences

conception rate, the length ofpostpartum interval to first ovulation provides an important

measure for assessing the effects of NEB on reproductive performance (Butler, 2003).

The severity and duration of NEB is variable among cows and relates primarily to

differences in dry matter intake and its rate of increase during early lactation (Villa-

Godoy et al., 1988; Staples et al., 1990). For example, cows overconditioned at calving

exhibit decreased appetite and thus develop a more severe NEB than cows of moderate

conditioning. These overconditioned animals mobilize more fat from the adipose tissue

and exhibit significant accumulation of TAG in the liver (Rukkwamsuk et al., 1999).









Accumulation of TAG in the liver is, in turn, associated with a longer interval to first

ovulation and reduced fertility (Butler and Smith, 1989; Rukkwamsuk et al., 1999;

Jorritsma et al., 2000).

Fertility in postpartum cows is directly dependent on a normal estrous cycle

characterized by folliculogenesis, ovulation, corpus luteum (CL) formation and

regression. However, the mechanisms by which endocrine and metabolic upsets result in

reduced reproductive efficiency are not well understood. Many of the hormonal and

metabolic changes that occur during the transition period can affect reproductive function

by interacting with the hypothalamic-pituitary axis (Butler, 2000).

The hypothalamic-pituitary axis is essential in the modulation of gonadotropin

secretion, which in turn plays a pivotal role in control of the estrous cycle. For example,

in pubertal heifers, the establishment of LH pulsatility is responsible for the initiation of

cyclicity (Schillo et al., 1992). Among the factors associated with onset of puberty,

attainment of a critical level of body fat is important (Schillo et al., 1992), further

stressing the importance of nutritional and metabolic status for normal reproductive

function. The NEB experienced by the early postpartum cow decreases pulsatile LH

secretion, which results in delayed resumption of ovarian cyclicity (Butler and Smith,

1989; Beam and Butler, 1999; Butler, 2000).

The first ovulation postpartum reflects the resumption and completion of

preovulatory ovarian follicular development and recovery from the hormonal conditions

of late pregnancy (Butler, 2000). Delayed time to first ovulation associated with NEB is

presumably through inhibition of LH pulse frequency and low levels of blood glucose,

insulin, and insulin-growth factor-I (IGF-I) that collectively prevent E2 prOduction by









dominant ovarian follicles (Butler, 2000). However, Beam and Butler (1999) reported

that initiation of a follicular wave and formation of a large dominant follicle during NEB

was not a limitation for first ovulation. In fact, following parturition, a wave of follicular

development takes place within 5-7 d in response to elevated plasma FSH concentrations

and regardless of NEB (Beam and Butler, 1999). Nonetheless, ovulation of a dominant

follicle during early lactation is dependent on the re-establishment of pulsatile LH

secretion that is conducive to terminal preovulatory growth and E2 prOduction (Butler,

2000). Beam and Butler (1997) described three possible outcomes of follicular

development during early postpartum period: 1) ovulation of the first dominant follicle

around days 16-20 postpartum; 2) non-ovulation of the first dominant follicle followed by

turnover and a new follicular wave; 3) failure of the dominant follicle to ovulate,

becoming cystic and prolonging the interval to first ovulation to 40-50 days postpartum.

As discussed by Jolly et al. (1995), the NEB experienced by the postpartum dairy cow

represents a physiological state of undernutrition which impairs LH secretion and

prevents ovulation. Consistent with this concept, Beam and Butler (1997) observed that

follicles emerging after the NEB nadir, rather than before, had greater diameter, enhanced

E2 prOduction, and were more likely to ovulate.

NEB has also been shown to change the profile of metabolic hormones which may

play an important local role in control of follicular development in cattle. It is likely that

these changes could alter the pattern of ovarian follicular growth and development, and

subsequent CL function during the early postpartum period. During early NEB period,

the ability of ovarian follicles to produce sufficient E2 foT OVulation appears to depend on

the availability of insulin and IGF-I in serum and the changing EB profile (Beam and









Butler, 1999). Both insulin and IGF-I plasma concentrations are directly related to the

energy status of the cow, and these hormones are essential for normal follicular

development (Spicer et al., 1993; Simpson et al., 1994; Beam and Butler, 1999).

Plasma glucose and insulin levels are decreased by energy deficit (Beam and

Butler, 1999; Butler, 2000). In lactating dairy cows, there is high demand for glucose as

the primary substrate for mammary lactose synthesis (lactogenesis) (Diskin et al., 2003).

Thus, reduced availability of glucose as a result of NEB may affect LH pulsatility, since

it influences both tonic and surge modes of secretion, presumably through effects on

GnRH (Diskin et al., 2003). Insulin also serves as a metabolic signal influencing LH

release from the pituitary (Monget and Martin, 1997). However, it also has been shown

to increase ovarian response to gonadotropins and to stimulate recruitment of small

follicles and enhanced follicular growth (Gong et al., 2001), suggesting a direct effect at

the ovarian level. Moreover, insulin is known to stimulate bovine follicular cells in vitro

(Spicer et al., 1993) and in vivo (Simpson, et al., 1994).

Energy balance also influences plasma levels of insulin-like growth factor-I (IGF-

I), which is important for normal ovarian follicular development and activity (Beam and

Butler, 1999). In postpartum dairy cows, IGF-I levels were 40-50% higher during the

first two weeks in cows in which the dominant follicle would ovulate as compared to

levels in cows with non-ovulatory follicles (Beam and Butler, 1997; 1998). Additionally,

plasma E2 COncentrations were highly correlated with plasma IGF-I levels (Beam and

Butler, 1998). It has been shown that IGF-I directly stimulate proliferation and

steroidogenic capacity of thecal (Spicer and Stewart, 1996) and granulosa cells (Spicer et

al., 1993) in vitro. Consequently, during the postpartum period, the ability of follicles to









produce sufficient E2 foT OVulation seems to be dependent on availability of both insulin

and IGF-I (Butler, 2000).

In cattle, peripheral P4 COncentration increases during the first two to three

postpartum ovulatory cycles (Villa-Godoy et al., 1988; Spicer et al., 1990; Staples et al.,

1990), and the rate of the increase in P4 leVOIS is attenuated by NEB early postpartum

(Villa-Godoy et al., 1988; Spicer et al., 1990). In this regard, Villa-Godoy and coworkers

(1988) reported that cows with the most negative energy balance during the first 9 days

still had decreased P4 during their third estrous cycle, which corresponded to the start of

the breeding period. The ability of a cow to produce and secrete optimum levels of P4 is

important for fertility because plasma P4 COncentrations are highly correlated with

pregnancy outcomes in lactating dairy cows (Folman et al., 1990; Larson et al., 1997).

In summary, it is well established that the energy state of the cow during early

lactation can influence reproductive efficiency by modulating the estrous cycle. This

may occur through modulation of gonadotropin secretion, specifically LH pulsatility

(hypothalamic-pituitary axis). Alternatively, local ovarian effects may involve

modulation of follicular growth and development by metabolic hormones (i.e. IGF-I and

insulin), which could determine oocyte quality and viability and steroidogenic activity of

the CL. However, energy-independent effects also have been observed after fatty acid

supplementation. Thus, nutrition may influence reproductive efficiency in dairy cows not

only by altering the energy status of the animal but also by influencing factors involved

in the regulation of reproductive processes like follicular dynamics, ovulation, CL

function and embryo survival among others.









Effects of Dietary Fats on Reproductive Response in Cattle

Fat supplementation of dairy rations is commonly used to alleviate a portion of the

dietary energy deficit experienced by early postpartum dairy cows (Butler, 2003).

Supplemental long-chain fatty acids (LCFA) have been shown to increase conception

rates (Schneider et al., 1988; Sklan et al., 1989; Ferguson et al., 1990), enhance

pregnancy rates (Schneider et al,. 1988; Sklan et al., 1991), and reduce the interval to first

estrus (Sklan et al., 1991). Dietary fats have also been shown to regulate eicosanoid

synthesis (Abayasekara and Wathes, 1999; Cheng et al., 2001), modulate plasma P4

concentration (Carrol et al., 1990; Lucy et al., 1993b; Garcia-Bojalil et al., 1998),

stimulate ovarian follicular development (Lucy et al., 1993b; Thomas and Williams,

1996; Beam and Butler, 1997) and improve fertility (Staples et al., 1998).

To understand the interaction of dietary fats and reproduction, it is essential to

understand the basic nature and biology of fatty acids and their metabolism. Fatty acids

belong to the family of lipids, which consist of biological compounds that are soluble in

organic solvents, such as cholesterol, TAG and phospholipids. Fatty acids are present in

all cell types and contribute to cellular structure, provide fuel storage and participate in

many biological processes ranging from gene transcription to regulation of key metabolic

pathways and physiological responses (Van Bilsen et al., 1997, 1998; Gurr et al., 2002).

Fatty acids consist of a carbon chain that ends with a carboxyl group, varying in the

chain length and the degree of unsaturation or number of double bonds. Naturally

occurring fatty acids can be saturated (no double bonds) or unsaturated, consisting of one

or more double bonds. Saturated fatty acids (SFAs) have all the carbons holding the

maximum number of hydrogens possible, thus referred as to be saturated with hydrogen.

Some naturally occurring SFAs are palmitic acid (16:0), found in palm oil, and stearic









acid (18:0), commonly present in animal fat (Jenkins, 2004). Unsaturated fatty acids can

contain one (monounsaturated fatty acids; MUFA) or more (polyunsaturated fatty acids;

PUFA) double bonds.

Fatty acids are generally abbreviated by listing the number of carbons with the

number of double bonds (i.e. 18:0; 18 carbons with no double bonds) (Jenkins, 2004).

For unsaturated fatty acids, the omega system is used to identify the location of the

terminal double bond relative to the methyl end of the carbon chain. In the omega

system, carbon atoms in the chain are identified with Greek letters, with the last carbon of

the chain, or the one farthest from the carboxyl group, known as the omega (n)-carbon

(Gurr et al., 2002). Thus, for PUFAs only the position closest to the omega carbon is

given. For instance, a member of the n-6 family such as linoleic acid (18:2, n-6) has its

first double bond at the A6 position (carbon number 6) counting from the omega end

(Gurr et al., 2002).

Fatty acids in mammals are either generated by de novo synthesis or provided by

the diet. Dietary fatty acids may undergo elongation and desaturation to generate isomers

which may have different properties. Elongation involves the addition of two-carbon

units to a chain through the action of enzymes known as elongases. Desaturation, on the

other hand, is catalyzed by desaturase enzymes that insert a double bond into the acyl

chain. These desaturase enzymes are classified according to the position of insertion of

the double bond, while the newly created double bonds are almost invariably separated

from each other by a methylene group.

However, desaturation of fatty acids in mammals does not occur at positions

greater than A9 since the required desaturases are absent (Fischer, 1989; Cook, 1996;










Mayes, 1996). Hence, the parent molecules for the n-6 and n-3 families, linoleic (LA;

18:2) and linolenic (LNA; 18:3) acids, respectively, can't be synthesized by the tissues

and, therefore, must be supplied in the diet. Mammals have been shown to have an

absolute requirement for LA and LNA; therefore, these fatty acids are regarded as

essential fatty acids (Burr and Burr, 1929; Aaes-Jorgensen, 1961; Holman et al., 1982).

Deficiency in these essential PUFAs may result in a variety of pathophysiologic effects in

mammals that include reproductive inefficiency (Burr and Burr, 1929). Nonetheless, the

body has different requirements for n-6 and n-3 PUFAs, as they are involved in several,

yet varied, essential functions.

Even though the diet of ruminants contains predominantly PUFAs, fatty acids in

blood, tissues and milk are highly saturated (Abayasekara and Wathes, 1999). This is the

result of extensive biohydrogenation of PUFAs that takes place in the rumen through the

activity of rminal microorganisms (Ward et al., 1964). Biohydrogenation of unsaturated

fatty acids consists of addition of hydrogen by microbial enzymes to the double bonds

resulting in the saturation of dietary fatty acids. The principal product is stearic acid, a

saturated fatty acid (18:0). However, even though ruminal biohydrogenation is extensive,

it ranges from 60 90% (Murphy et al., 1987). In other words, incomplete

biohydrogenation of unsaturated fatty acids results in the formation of several isomers

which will depend on the source of the dietary fatty acid fed. Some of the most common

products of incomplete biohydrogenation are oleic acid (18:1), conjugated linoleic acid

(CLA), and trans-vaccinic~t~t~t~t~t~t~ acid (tranzs-11; 18:1). Thus, in order for intact PUFAs to

reach the small intestine for absorption and being transported to target tissues, they need

to escape ruminal microbial hydrogenation process.









Essential PUFAs may be made available for absorption by feeding ruminally inert

fats. A number of techniques have been developed to protect fats which involve

chemical treatment processes such as formaldehyde or calcium salts of fatty acids

(Palmquist and Jenkins, 1980; Ashes et al., 1992). Inclusion of calcium soaps of long-

chain fatty acids (LCFA) in dairy cattle rations is commonly used to alleviate the dietary

energy deficit experienced during early postpartum. Cows fed calcium salts ofLCFA

have produced more milk and experienced improved fertility (Staples et al., 1998).

Linoleic acid is abundant in nearly all commonly available unprocessed plant oils

(e.g. corn, sunflower, safflower, and rape seed) (Sargent, 1977). In cows, AA is provided

by dietary intake of LA (Urich, 1994; Gurr et al., 2002). However, dietary LA

deprivation causes a general decrease in AA levels, although the proportion in different

tissues can be diverse, suggesting tissue-specific uptake of PUFA (Lefkowith et al.,

1985). On the other hand, PUFAs of the n-3 family, such as eicosapentaenoic acid (EPA;

20:5) and DHA (22:6), are also essential for many bodily functions (Innis, 1991;

Abayasekara and Wathes, 1999). They can be provided in the diet or synthesized in the

tissues from the parent molecule LNA. Linolenic acid is the predominant PUFA in most

forage lipids (Palmquist and Jenkins, 1980) and high levels are also found in linseed oil,

however, it also contains significant amount ofLA (Sargent, 1997). Fish oils, which

contain low amounts of LNA, offer the most readily available dietary source of EPA and

DHA (Neuringer et al., 1988).

The potential mechanisms by which LCFAs affect reproductive responses in cattle

include indirect effects of high energy intake on the overall energy state of the cow, as

well as direct effects of dietary fatty acids on the pituitary, ovaries, and uterus (Staples et










al., 1998; Mattos et al., 2000). The association between fat content in the diet and

fertility in lactating cows has being documented in a variety of studies.

Improvement of the overall energy state provided by fatty acid supplementation

(Staples et al., 1998; Jenkins and Palmquist, 1984) may lead to re-establishment of LH

pulsatility and ovarian cyclicity in the lactating cow (Palmquist and Jenkins, 1980; Lucy

et al., 1991). In fact, Sklan et al. (1994) reported that the energy provided by dietary fats

increased LH secretion in dairy cows that consumed less energy than required. However,

the mechanism by which LCFAs may affect LH secretion has not been described in

rummnants .

There is evidence that increase in consumption of dietary fatty acids stimulates

ovarian follicular growth in cattle through a mechanism that is independent from energy

intake and weight gain (Staples et al., 1998). Increasing the dietary content of LCFAs in

cattle increased both the number and size of follicles present in the ovary and shortened

the interval to first ovulation postpartum (Hightshoe et al., 1991; Lucy et al., 1991, 1992;

Ryan et al., 1992; Thomas and Williams, 1996; Lammoglia et al., 1997; Beam and

Butler, 1997). Lucy et al. (1993b) reported greater numbers of medium-sized ovarian

follicles (1.5 to 2.3 mm) in postpartum dairy cows fed a diet containing 2.2% calcium

salts of LCFA compared to cows receiving an isocaloric control diet without calcium

salts of LCFA. Several studies reported that supplemental fat increased not only the total

number of ovarian follicles (Thomas and Williams, 1996; Beam and Butler, 1997;

Lammoglia et al., 1997), but also the size of preovulatory follicles in cattle (Lucy et al.,

1993b; Beam and Butler, 1997; Oldick et al., 1997). Follicular growth also has been

shown to be stimulated by LCFAs in crossbred beef cattle (Thomas et al., 1997).









Moreover, lactating dairy cows fed calcium salts of fat enriched with LA or fish oil (high

in n-3 PUFAs) had increased size of the dominant follicle compared to those fed calcium

salts of oleic acid (18:1) (Mattos et al., 2000). Whether increased size of preovulatory

follicles is due to exogenous LCFA-induced LH secretion from the pituitary, and how the

altered ovarian follicular dynamics may impact pregnancy outcome in dairy cows

warrants further investigation.

It is possible that increased serum concentrations of insulin in response to feeding

LCFAs to cattle (Palmquist and Moser, 1981; Thomas and Williams, 1996; Ryan et al.,

1995) may play a role in mediating increased follicular growth. This insulin-mediated

event may be by stimulation of granulosa cell IGF-I production (Yoshimura et al., 1994).

Stimulatory effects of dietary LCFAs on follicular development may be through

enhanced steroidogenesis via increased cholesterol, thus increased substrate availability

for increased follicular steroid synthesis (Wehrman et al., 1991). In mature heifers and

dairy cows, elevating fat intake increased both serum and follicular fluid cholesterol

concentrations (Park et al., 1983; Talavera et al., 1985; Wehrman et al., 1991). Carroll

and coworkers (1990) detected a 21% increase in plasma cholesterol concentrations in

dairy cows fed ruminally inert fat compared to control cows. Similarly, beef heifers

supplemented with soybean oil had greater concentration of total cholesterol in serum and

HDL cholesterol in follicular fluid when compared with control heifers (Ryan et al.,

1992). Wehrman et al. (1991) showed that cows fed a high lipid diet had increased

intrafollicular levels of androstendione as well as increased P4 Output from the granulosa

cells collected and cultured in vitro. In addition, several studies have detected subtle

increases in plasma P4 COncentrations in cows fed high-fat diets (Carroll et al., 1990;










Lucy et al., 1993b; Garcia-Bojalil et al., 1998). Dietary intake of LCFAs in ruminants

during luteal phase increased serum concentrations of P4 (Talavera et al., 1985; Carrol et

al., 1990; Hawkins et al., 1995; Burke et al., 1996).

Recent studies suggested that increase in plasma P4 in COws fed fat-supplemented

diets may not be due to increased synthesis but rather to reduced clearance of P4 frOm

circulation. In one study, when the CL of cows were removed by ovariectomy, P4 WAS

cleared from blood at a much slower rate in cows fed supplemental fat compared to cows

fed the control diet (Hawkins et al., 1995). In addition, Sangsritavong et al. (2002)

showed that fatty acids and/or TAG could increase circulating P4 and E2 COncentrations

by directly inhibiting liver cell metabolism of these steroid hormones. Because CL

function and P4 is crucial for establishment and maintenance of pregnancy in ruminants,

increased plasma P4 may result in improved pregnancy rates in postpartum dairy cows

fed supplemented fats. In fact, supplementation of LCFA has been shown to enhance

luteal function as confirmed by reduced incidence of short cycles (Williams, 1989).

Dietary LCFAs may increase AA in the phospholipids pool of granulosa cells. The AA

released upon gonadotropin stimulation (Cooke et al., 1991) had a direct effect on

steroidogenesis in goldfish (Van der Kraak and Chang, 1990) and hens (Johnson and

Tilly, 1990). It can be used as the precursor for prostaglandin production, which in turn

may stimulate steroidogenesis as reported in granulosa cells for marmosets (Michael et

al., 1993). This hypothesis is supported by the observation that gonadotropins stimulate

prostaglandin production in vitro (Tsang et al., 1988).

Fertility responses may also be related to the effects of LCFAs on uterine

eicosanoid production. Eicosanoids (i.e. prostaglandins, thromboxanes, leukotrienes and









lipoxins) are synthesized from AA, which uses LA as the primary precursor (Kinsella et

al., 1990). Prostaglandins (PG) of the 2 series (PGF2u, PGE2) have been implicated in

many reproductive processes including ovulation (Espey, 1980), follicular development

(Wallach et al., 1975), corpus luteum function (Bazer and Thatcher, 1977; Auletta and

Flint, 1988; Abayasekara et al., 1995; Poyser, 1995; Wathes and Lamming, 1995),

parturition (Thorburn and Challis, 1979; Challis, 1980) and uterine involution (Hafez and

Hafez, 2000). Hence, any influence that fatty acids might exert on PGF2u Synthesis may

affect overall reproductive performance.

Ovulation was blocked by inhibition of PG synthesis in monkeys and restored by

administration of PGFza (Wallach et al., 1975; Tsafriri et al., 1972). Experiments

performed on rats showed that a diet high in n-3 PUFAs increased ovulation rate,

whereas a diet high in n-6 PUFAs resulted in reduction of ovulation rate (Trujillo and

Broughton, 1995). Since both diets resulted in increased levels of total PGE, the authors

suggested that n-3 PUFAs may increase ovulation by augmentation of the less

biologically active PGE3 at the expense of PGE2.

Long chain fatty acids may also influence luteal activity and function via

modulation of uterine PGF2a prOduction, which causes luteolysis (Auletta and Flint,

1988; Bazer and Thatcher, 1977; Poyser, 1995). Essential PUFAs have been shown to

inhibit PG secretion in several cell types (Levine and Worth, 1984; Achard et al., 1997)

including bovine endometrial (BEND) cells (Mattos et al., 2003). Manipulation of

dietary LCFA content may also influence synthesis of PGF2 a S demonstrated by

different studies. For example, dietary n-6 and n-3 PUFA have the ability to alter

gestational length and time of parturition through modulation of PG synthesis in rats










(Holman, 1971; Leaver et al., 1986), ewes (Baguma-Nibasheka et al., 1999), and humans

(Olsen et al., 1986, 1992; Allen and Harris, 2001). Although there is some evidence

indicating that n-6 PUFA, LA specifically, enhances PG production by providing more

substrate for conversion to AA (Connolly et al., 1996; Nakaya et al., 2001; Elmes et al.,

2004; Petit et al., 2004), other studies have found an inhibitory effect (Elattar and Lin,

1989; Pace-Asciak and Wolfe, 1968; Cheng et al., 2001). Abomasal infusion of cycling

cattle with yellow grease (72% PUFAs and 17% LA) resulted in significant attenuation of

oxytocin-induced secretion of 13, 14-dihydro-15-keto-PGF2a (PGFM), a metabolite of

PGF2a (Oldick et al., 1997). A number of studies have demonstrated inhibitory effects of

n-3 PUFAs on PGF2a prOduction (Bezard et al., 1994; Staples et al., 1998; Abayasekara

and Wathes, 1999; Mattos et al., 2000, 2001, 2002, 2004). Supplementation of dairy

cattle with fish oil (high in n-3 PUFAs) reduced plasma concentrations of PGFM in early

postpartum as well as in oxytocin-induced cycling cows (Mattos et al., 2002, 2004). It

also has been suggested that fish oil may reduce the sensitivity of the CL to PGF2a SinCe

cycling cows fed fish meal had higher P4 plaSma concentrations after inj section of a

luteolytic dose of PGFza (Burke et al., 1997). Attenuation of uterine PGF2a Secretion and

decreased sensitivity of the CL to this PG caused by PUFAs may lead to improved

fertility through enhanced luteal function, reduced embryonic loss and increased

pregnancy rates.

Positive effects of dietary fats on fertility response are not exclusively related to

inhibition of PG biosynthesis. Prostaglandins are key hormones in terms of cervical

ripening and myometrial contractility, which are essential for parturition (Challis, 1980).

Following parturition, fertility resumes after uterine involution takes place, resulting in









resumption of normal estrous cycles (Kiracofe, 1980). This process of uterine involution

is caused by myometrial contractions stimulated by PGF2a. A massive and sustained

release of PGF2a takes place during the first two weeks postpartum and is essential to

reduce the uterine size and increase its tone (Hafez and Hafez, 2000). Plasma PGFM

concentrations increase prior to calving (Schindler et al., 1990), and generally decreases

to basal levels within 21 d postpartum (Lewis et al., 1984; Del Vecchio et al., 1992a).

Since the uterus seems to be the primary source of PGFM in postpartum cows, PGFM is

considered to be an indicator of uterine PGF2a prOduction in postpartum cows (Guilbault

et al., 1984). Moreover, the duration of this postpartum PGF2a Sustained release is

negatively correlated with the number of days to complete uterine involution and the

interval between parturition and resumption of normal ovarian activity (Lindell et al.,

1982; Madej et al., 1984). For conception to occur after calving, the uterus must return to

its normal size. Thus, modulation of PG production by dietary fats may affect the timing

of rebreeding postpartum.

After parturition, the reproductive tract is also subj ect to massive bacteriologic

insult, and re-establishment of a sterile uterus is one of the requirements for pregnancy to

take place in the subsequent postpartum period (Hafez and Hafez, 2000). Uterine

infections, such as metritis, reduce reproductive efficiency by increasing calving interval

and preventing resumption of ovarian activity (Griffin et al., 1974; Arthur et al., 1989;

Lewis, 1997). The incidence of uterine infections ranges from approximately 10-40% of

postpartum dairy cattle (Arthur et al., 1989; Lewis, 1997). Multiparous cows may be

more resistant to some uterine infections than are primiparous cows (Hussain, 1989).









However, the physiological characteristics that make some animals more susceptible than

others are not well understood.

Eicosanoids are also known to have important immunomodulatory effects (Lewis,

2004). Recent studies have confirmed the effects of in vivo exposure to PGs on the in

vitro response of lymphocytes (Ramadan et al., 1997; Lewis, 2003; Wulster-Radcliffe et

al.,. 2003). PGF2a has been reported to enhance immune function in vitro (Hoedemaker

et al., 1992). In addition, PGF2a inCreaSed in vitro bactericidal activity of neutrophils

from ovariectomized mares (Watson, 1988).

Induced and spontaneous uterine infections increase of plasma PGFM

concentrations (Del Vecchio et al., 1992a, 1994). However, Seals and coworkers (2002)

reported that postpartum concentrations of PGFM were inversely related to emergence of

uterine infections. They also reported that postpartum cows with depressed PGFM

concentrations were more likely to develop uterine infections. In addition, aberrant

PGF2a and PGE2 prOduction has been associated with retained placenta (Gross et al.,

1987; Heuwieser et al., 1992), which in turn is associated with increased incidence of

uterine infections (Curtis et al., 1985). Thus, PGFM concentration in early postpartum

cows may enhance uterine immune functions and be a good indicator of the likelihood of

a cow to develop metritis.

Regulation of Prostaglandin F~a Synthesis by Polyunsaturated Fatty Acids

Evidence is accumulating that dietary manipulations of PUFAs can have maj or

effects on eicosanoid production. Depending on the amount of particular fatty acids

reaching the target tissues, supplemental PUFAs can either stimulate or inhibit

prostanoids synthesis. In a human study, feeding diets rich in AA increased plasma









phospholipid levels of AA and urinary excretion of the stable metabolites of prostacyclin

(PGI2) and TA2 (Sinclair and Mann, 1996). Consistent with these findings, ewes infused

with either soybean oil (50% LA) or olive oil (16% LA) had greater serum PGFM

concentrations than ewes infused with saline (Burke et al., 1996). In postpartum beef

heifers, infusion of lipid (20% soybean oil) through the jugular vein increased systemic

concentrations of LA and PGFM after oxytocin inj section (Filley et al., 1999).

Recent reports in mammalian systems have shown reduced eicosanoid synthesis

when PUFAs of the n-3 or n-6 families were fed in the diet. In a study with lactating

dairy cows, Oldick et al. (1997) reported that OT-induced PGFM concentrations were

greatly reduced in cows infused abomasally with yellow grease, compared with infusion

of tallow, glucose or water. Additionally, it was demonstrated that supplementation of

postpartum dairy cows with fish meal containing EPA and DHA considerably attenuated

PGFM response to OT induction (Mattos et al., 2002). In beef cows, whole cottonseed

added to the diets doubled the average lifespan of a GnRH-induced CL, compared with

cows fed an isocaloric control diet (Williams, 1989). Although PG concentrations were

not measured in the latter study, a lipid-induced suppression of PGF2a WOuld be

compatible with a longer CL life (Staples et al., 1998). These observations provide

strong evidence for PUFA involvement in modulation of PG biosynthesis.

It has been proposed that PUFAs may reduce PGF2a prOduction through

modulation of one or more steps of the PG biosynthetic pathway. These include

decreasing availability of the precursor AA, competing for PGHS-2 activity and directly

inhibiting synthesis and/or activity of PGHS-2 (Mattos et al., 2000; Cheng et al., 2001).









Reduced availability of AA as a precursor for conversion to PGF2a may result from

the inhibition of AA synthesis or its displacement from the membrane phospholipid pool.

It has been reported that dietary PUFAs may inhibit AA synthesis during the elongation

and desaturation processes of LNA in the liver (Bezard et al., 1994). In fact,

supplementation of rats with n-3 PUFAs resulted in reduced desaturation of LA and LNA

by liver microsomes and lowered concentration of AA in liver phospholipids (Garg et al.,

1988; Christiansen et al., 1991). Incubation of rat hepatoma cells with n-3 PUFAs

resulted in reduced A6 desaturase activity (Larsen et al., 1997). Sprecher (1981)

observed that in rats there was a preferential processing of n-3 PUFAs by A6 desaturase

at the expense of n-6 PUFAs. This suggested that high levels of LNA in the diet could

compete with LA for A6 desaturase activity, thus attenuating the conversion of LA to

AA. Reduced availability of AA will result in greater incorporation of other fatty acids

into the phospholipid pool of the plasma membrane and decreased availability of the

substrate for PGHS-2 enzyme (German et al., 1988).

Dietary n-3 PUFAs can also reduce PGF2a Synthesis through competition with AA

for PGHS-2 activity. The n-3 PUFAs, such as EPA, can serve as substrate for PGHS-2,

thus leading to the synthesis of PGs of the 3 series at the expense of PGs of the 2 series.

Indeed, feeding rats with diets rich in n-3 PUFAs resulted in increased secretion of PGs

of the 3 series from uterine tissues cultured in vitro (Leaver et al., 1991).

Direct inhibition of PGHS-2 synthesis and/or activity by PUFAs may also

contribute to attenuation of PGF2a Synthesis following dietary fatty acids

supplementation. When bovine aortic endothelial cells were incubated with n-3 PUFAs

(EPA and DHA), PGHS-1 mRNA expression was reduced (Achard et al., 1997). In









cultures of rat hepatoma cells, PGHS enzyme was almost completely inactivated with

addition of EPA 30 seconds before addition of AA as substrate (Larsen et al., 1997).

Furthermore, PUFAs such as AA, EPA, and DHA, have been also found to inhibit PGHS

activity (Smith and Marnett, 1991).

In addition to inhibiting PGHS-2 activity, dietary PUFAs can also modulate the

expression of PGHS-2 gene. The proposed mechanism for regulation of gene expression

by PUFAs involves activation of nuclear transcription factors such as peroxisome

proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors

belong to a family of nuclear receptor transcription factors activated by specific fatty

acids, eicosanoids, and peroxisome proliferators (Desvergne and Wahli, 1999). Three

different isoforms, PPARot, PPARy, and PPARS, have been identified and are encoded

by different genes (Desvergne and Wahli, 1999). Upon activation, PPARs dimerize with

the ubiquitous retinoid X receptor (RXR) and binds to a prescribed DNA sequence

referred to as PPAR response element (PPRE) (Desvergne and Wahli, 1999). Previous

studies have shown PPARs to be involved in regulation of genes modulating steroid and

prostaglandin synthesis as well as mediating some of the growth hormone effects in

hepatocytes (Lim et al., 1999; Zhou and Waxman, 1999; Schopee et al., 2002). In

general, all the n-3 and n-6 PUFAs activate the three PPAR isoforms. However, their

affinities for a given receptor vary, suggesting a role for tissue-specific availability and

metabolism of particular fatty acids and differences in their affinity for a specific PPAR

subtype (Sampath and Ntambi, 2005). Furthermore, it has been concluded that PUFAs

are potent endogenous ligands since they have been shown to activate PPARs at

micromolar concentrations (Lehmann et al., 1997). MacLaren et al. (2003) documented










expression of PPARot, PPARy, and PPARS in endometrium from cyclic and pregnant

Holstein cows at day 17 following estrus. These isoforms also have been detected in an

immortalized bovine endometrial epithelial cell line.

Collectively, these Eindings suggest that select dietary n-3 fatty acids can reduce

PGF2a Synthesis by decreasing AA concentration in tissue phospholipids by increasing

the concentration of fatty acids that compete with AA for processing by PGHS-2, and by

inhibiting PGHS-2 synthesis and/or activity. Nutritional management that alters

endometrial PGF2a biosynthesis may improve reproductive efficiency in high producing

dairy cows in which fertility is impaired due to high metabolic demands associated with

milk production.

Conjugated Linoleic Acid and Reproduction

Conjugated linoleic acid (CLA) refers to a group of geometrical and positional

isomers of LA resulting from incomplete biohydrogenation in the rumen (Chin et al.,

1992; Ma et al., 1999; Griinari et al., 2000). The number of double bonds remains the

same as in the parent LA, but one of the double bonds is shifted to a new position by

microbial isomerases. One or both of the double bonds are either in the cis or trans

configurations separated by a single carbon-to-carbon linkage rather than by the normal

methylene group. A broad number of cis- and trans-t~t~rt~t~rt~t~rt~ CLA isomers have been identified

in food; however, the most commonly occurring CLA isomer is the cis-9, trans-1rt~r~rt~r~rt~t~rt 1-

octadecadienoic acid with minor but significant proportions of trans-10, cis-12-18:2

(Parodi, 1977; 1997; Chin et al., 1992; McGuire et al., 1998).

After ruminal synthesis of cis-9, trans-11 CLA, it may be absorbed in the small

intestine or further biohydrogenated into trans-11 -octadecenoic acid (trans vaccenic acid)










by ruminal microorganisms. The trans-11 18:1 can be reduced to stearic acid (18:0) or

transported to peripheral tissues as MUJFA. In the peripheral tissues (i.e. mammary

gland, muscle), this trans-isomer may be converted back into cis-9, trans-11 CLA by the

action of A9 desaturase (Pollard et al., 1980; Holman and Mahfouz, 1981). This

conversion appears to be a maj or source of cis-9, trans-11 CLA in the cow' s milk (Corl

et al., 1998; Griinari and Bauman, 1999; Santora et al., 2000).

The presence of trans-1rt~r~rt~r~rt~t~rt 0, cis-12 CLA in milk (Griinari and Bauman, 1999)

suggests the existence of endogenous ruminal bacteria with the capability for synthesis of

this isomer. In support of this hypothesis, LA has been shown to be converted to trans-

10, cis-12 CLA by Propionobacter in vitro (Verhulst et al., 1987). However, the trans-

10, cis-12 CLA detected in ruminal tissues (Griinari and Bauman, 1999; Dhiman et al.,

1999) seem to originate solely from ruminal synthesis, since mammalian tissues do not

have the Al2 desaturase necessary for conversion of trans-trt~r~rt~r~rt~t~rt 10-octadecenoic acid (elaidic

acid) back to trans-1rt~r~rt~r~rt~t~rt 0, cis-12 CLA.

A large number of beneficial effects have been attributed to CLA ranging from

enhancing feed efficiency and growth in rats (Chin et al., 1994) and pigs (Bee, 2000),

decreasing body fat in mice (DeLany et al., 1999) and pigs (Dugan et al., 1997;

Ostrowska et al., 1999), and reduced milk fat synthesis in lactating dairy cows (Griinari

et al., 1998; Romo et al., 2000; Baumgard et al., 2001). Although it is likely that some

biological effects of CLA may be induced and/or enhanced synergistically by these

isomers, there is evidence suggesting that numerous effects of CLA are due to separate

actions of these biologically active isomers (Pariza et al., 2000). Uruquhart et al. (2002)

observed that after treatment of human vein endothelial cells with 100 CIM of individual









CLA isomers, differential effects were observed with cis-9, trans-1rt~r~rt~r~rt~t~rt 1 CLA inhibiting,

while trans-10,rtrt~t~r~rtrt~t~ cis-12 CLA inducing Ca ionophore-stimulated eicosanoid production. It

seems that trans-10, cis-12 works preferentially through modulation of apoptosis and cell

cycle, whereas cis-9, tranzs-12 may work through AA metabolism (Ochoa et al., 2004).

Nonetheless, beneficial actions of CLA have been linked to modulation of

eicosanoid production (Sugano et al., 1998). Recent studies detected an inhibitory effect

of CLA on eicosanoid synthesis in various animal and cell systems (Liu and Belury,

1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al, 2003). Feeding

pregnant rats with dietary CLA resulted in inhibition of uterine PGF2a independent of

PUFA content (Harris et al., 2001). In addition, Cheng et al. (2003) reported that

treatment of endometrial cells isolated from late pregnant ewes with CLA suppressed

PGF2a in a dose dependent manner, while low doses of CLA stimulated PGE2 generation.

Cheng and coworkers (2003) showed that treatment of intercotyledonary endometrial

cells with CLA resulted in a dose-dependent inhibition of PGF2a prOduction. Similarly,

CLA also has been shown to inhibit PGF2a Synthesis in rat placental and uterine tissues

(Harris et al., 2001). Although the mechanism underlying this inhibition is not clear, it

has been suggested that CLA may compete with AA for PGHS-2 activity as well as for

incorporation to the membrane phospholipids. Alternatively, CLA may modulate

conversion of PGF2a to PGE2 aS reported by Gross and Williams (1988) in bovine

placental cells. Therefore, CLA may regulate reproductive processes through modulation

of prostaglandin biosynthesis.















CHAPTER 3
EFFECTS OF POLYUNSATURATED FATTY ACIDS ON PROSTAGLANDIN Fza
PRODUCTION BY BOVINTE ENDO1VETRIAL CELLS

Introduction

In the past decade, genetic selection for high milk production has been associated

with a decrease in reproductive efficiency in lactating dairy cows (Butler, 2000). Poor

reproductive efficiency includes early embryonic loss (Thatcher et al., 1995), impaired

ovarian cyclicity and low fertility rates (Butler, 2000), which collectively result in

reduced life-long milk production (Plaizier et al., 1997). Early postpartum dairy cows

have higher energy requirements than can be supported by dietary energy intake, which

creates a negative energy state that can lead to impaired reproductive function (Butler,

2000).

Polyunsaturated fatty acids (PUFA) are generally added to dairy rations to increase

the energy density of the diet. In mammals, the parent molecules for the n-6 and n-3

families, linoleic (LA; 18:2) and linolenic (LNA; 18:3) acids, respectively, cannot be

synthesized by the tissues and, therefore, must be supplied in the diet. Since there is an

absolute requirement for these PUFAs, they are regarded as essential fatty acids (Burr

and Burr, 1929; Aaes-Jorgensen, 1961; Holman et al., 1982).

Supplemental polyunsaturated fatty acids (PUFA) have been reported to increase

conception rates (Schneider et al., 1988; Sklan et al., 1989; Ferguson et al., 1990),

enhance pregnancy rates (Schneider et al,. 1988; Sklan et al., 1991), and reduce the

interval to first estrus (Sklan et al., 1991) of lactating dairy cows. Select dietary fats have









been also shown to regulate eicosanoid synthesis (Abayasekara and Wathes, 1999; Cheng

et al., 2001), modulate plasma P4 COncentration (Carrol et al., 1990; Lucy et al., 1993b;

Garcia-Bojalil et al., 1998), stimulate ovarian follicular development (Lucy et al., 1993b;

Thomas and Williams, 1996; Beam and Butler, 1997) and improve fertility (Staples et al.,

1998) of lactating dairy cows.

Prostaglandins are members of the eicosanoid family synthesized from 20 carbon

PUFAs, such as arachidonic acid (AA). Arachidonic acid is, in turn, synthesized from

elongation and desaturation of LA. The first and rate limiting step of prostaglandin

biosynthesis is the hydrolytic release of AA by the action of cytosolic phospholipase A2

(cPLA2) enzyme (Lapetina, 1982). Following its release, AA is converted to

prostaglandin H2 (PGH2) by the action of prostaglandin H synthase-2 (PGHS-2), also

known as cyclooxygenase-2 (COX-2). Prostaglandin H synthase has cyclooxygenase and

peroxidase activities that convert prostaglandin G (PGG) to PGH2 (Goff, 2004). After

synthesis of PGH2, this endoperoxide is converted to one of several possible prostanoids

by the action of specific terminal enzymes. Biosynthesis of prostaglandin E2 (PGE2) and

prostaglandin Fza (PGFza) are catalyzed by the action of prostaglandin E and

prostaglandin F synthases, respectively (Goff, 2004). Prostaglandins of the 2-series,

PGF2a and PGE2, are the most biologically active eicosanoids and are involved in key

reproductive processes such as follicular development (Wallach et al., 1975), ovulation

(Espey, 1980), luteolysis (Wathes and Lamming, 1995), and parturition (Challis, 1980).

Essential PUFAs have been shown to inhibit PG secretion in several cell types

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

cells (Mattos et al., 2003). The BEND cells are a line of spontaneously replicating









endometrial cells originating from cows in their 14th day of their estrous cycle (Staggs et

al., 1998). Nutritional studies have shown that manipulation of dietary PUFA content

may influence circulating concentrations of PGFza. For example, dietary n-6 and n-3

PUFA have the ability to alter gestational length and time of parturition through

modulation of PG synthesis in rats (Holman, 1971; Leaver et al., 1986), ewes (Baguma-

Nibasheka et al., 1999), and humans (Olsen et al., 1986; 1992; Allen and Harris, 2001).

Although there is some evidence indicating that n-6 PUFAs, LA specifically, enhance PG

production by providing more precursors for conversion to AA (Connolly et al., 1996;

Nakaya et al., 2001; Elmes et al., 2004; Petit et al., 2004), other studies have found an

inhibitory effect of n-6 PUFAs on PG synthesis (Elattar and Lin, 1989; Pace-Asciak and

Wolfe, 1968; Cheng et al., 2001). A number of studies have demonstrated inhibitory

effects of n-3 PUFAs on PGF2a prOduction (Bezard et al., 1994; Staples et al., 1998;

Abayasekara and Wathes, 1999; Mattos et al., 2000, 2001, 2002, 2004). Supplementation

of dairy cattle with fish oil (high in n-3 PUFAs) greatly reduced plasma PGFM response

to oxytocin (Mattos et al., 2002, 2004). It also has been suggested that fish oil may

reduce the sensitivity of the CL to PGF2a Since cycling cows fed fish meal had higher P4

plasma concentrations after inj section of a luteolytic dose of PGF2a (Burke et al., 1997).

Attenuation of uterine PGF2a Secretion and decreased sensitivity of the CL to this PG

caused by PUFAs may lead to improved fertility through enhanced luteal function,

reduced embryonic loss and increased pregnancy rates.

The obj ective of this investigation was to examine the effects of n-6 and n-3

PUFAs on phorbol 13, 14-dibutyrate (PDBu)-induced PGF2a biosynthesis in BEND cells.










We hypothesized that PUFAs may be more effective in inhibiting PGF2a prOduction

when compared to saturated fatty acids.

Materials and Methods

Materials

Polystyrene tissue culture dishes (100 x 20 mm) were purchased from Corning

(Corning Glass Works, Corning, NY). The Ham F-12 medium, antibiotic/antimycotic

(ABAM), phorbol 13, 14-dibutyrate (PDBu), horse serum, D-valine, insulin, fatty acid-

free bovine serum albumin (BSA), stearic acid (ST, Cis:o), aprotinin, leupeptin, and

pepstatin were from Sigma Chemical Co. (St. Louis, MO). The Minimum Essential

Medium (MEM) and fetal bovine serum (FBS) were from US Biologicals (Swampscott,

MA) and Atlanta Biologicals (Norcross, GA), respectively. Linoleic acid (C18:2n-6),

LNA (C18:3n-3), eicosapentaenoic acid (EPA, C20:5n-3), PGHS-2 and PGES antibodies,

and PGF2a Standard were from Cayman Chemicals (Ann Arbor, MI). The PPARS and

secondary anti-rabbit IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz,

CA). The enhanced chemiluminescence (ECL) kit was purchased from Perkin Elmer

(Boston, MA). Hanks Balanced Salt Solution (HBSS) and TriZol reagent were from

GIBCO BRL (Carlsbad, CA). Isotopically-labelled PGF2a (5, 6, 8, 9, 11, 12, 14, 15[n-

3H] PGF2a; 208 Ci/mmol) was from Amershan Biosciences (Piscataway, NJ). The anti-

PGF2a antibody was purchased from Oxford Biomedicals (Oxford, MI). BioTrans nylon

membrane and [ot-32P]deoxycitidine triphosphate (SA 3000 Ci/nmol) were from MP

Biomedicals (Atlanta, GA). The PGHS-2 cDNA probe was cloned from an ovarian

follicular cDNA library (Liu et al., 1999), the PGES cDNA probe was cloned from









endometrial cDNA (Guzeloglu et al., 2004), whereas the PPARS probe was generated

from bovine endometrial RNA (Balaguer et al., 2005).

Cell Culture and Treatment

Bovine endometrial (BEND; American Type Culture Collection # CRL-2398;

Manassas, VA) cells were plated on 100-mm tissue culture plates in complete culture

medium (40% Ham F-12, 40% MEM containing 10 ml ABAM/L, 0.0343g D-Valine/L,

200 U insulin/L, 10% fetal bovine serum and 10% horse serum) and grown to confluence

at 370C in a humidified atmosphere containing 95% 02 and 5% CO2. CellS then were

rinsed twice with Hanks balanced salt solution (HBSS), and cultured in fresh serum-free

medium containing appropriate treatments for an additional 24 h.

To examine the effects of PDBu on PGF2a prOduction, subconfluent BEND cells

were incubated with medium alone (control, n=2) or medium supplemented with 100

ng/ml PDBu (n=2) for 6 h. After incubation, aliquots (1 ml) of cell-conditioned media

were collected and stored at -200C for subsequent analysis of PGF2a USing RIA. The

entire experiment was repeated five times.

To investigate the effects of supplemental PUFAs on uterine PGF2a Synthesis,

BEND cells were treated with PDBu alone (100 ng/ml) or with a combination of PDBu

and stearic (ST; 100 CLM), LA (100 CLM), LNA (100 CLM) or EPA (100 CLM).

Subconfluent cells were incubated with serum-free medium alone (PDBu) or with

appropriate treatments (listed above) completed with BSA (1:3 ratio) for a period of 24

h. Cells then were rinsed twice with HBSS, and challenged with phorbol ester 12, 13-

dibutyrate (PDBu, 100 ng/ml) for an additional 6 h. Control plates were cultured in the

absence of PDBu. Samples of conditioned media (1 ml/plate) were collected after PDBu









challenge and stored at -200C until analyzed for PGF2a COncentration. The remaining cell

monolayer was rinsed with HBSS, lysed in TriZol reagent, and stored at -800C for

subsequent mRNA analysis. For analysis of protein abundance, cells were collected and

lysed as described later in the section for Western blot analysis. These experiments were

repeated two times.

PGF2a Radioimmunoassay

The concentration of PGF2a in Cell-COnditioned media was measured as described

by Danet-Desnoyers et al. (1994) and modified by Binelli et al. (2000). One hundred and

fifty microliters of Tris buffer (50 mM Tris-HC1, 1 g/1 sodium azide, pH 7.5), 50 Cll of

conditioned media or standards (range 10 1000 pg/tube), 100 Cll of Tris buffer/serum-

free medium, 100 C1l of anti-PGFza antibody (1: 30,000 in Tris buffer) and 100 C1l of 3H-

PGF2a (18,000 dpm) were added sequentially to 12x75 mm disposable glass tubes,

vortexed and incubated for 24 h at 4oC. After incubation, 500 Cll of dextran-coated

charcoal solution (25 mg dextran and 250 mg charcoal into 100 ml RIA buffer) was

added, and the mixture was incubated for an additional 4 min at 4oC. Assay tubes were

then centrifuged (3200 rpm for 15 min at 4oC), decanted and counted in a beta counter.

Assay sensitivity was 0.5 ng/ml and intra- and inter-assay coefficients of variation were

8.2 and 15.8%, respectively. Final PGF2a COncentrations were expressed as picograms

per milliliter.

RNA Isolation and Analysis

Total RNA was isolated using TriZol reagent, following the manufacturer's

instructions. Ten Cpg of total RNA was fractioned in a 1.5 % agarose formaldehyde gel

using the MOPS buffer (Fisher Scientifie, Pittsburgh, PA) (Ing et al., 1996) and









transferred to a Biotrans nylon membrane by downward capillary transfer. The RNA was

cross-linked to the nylon membrane by exposure to a UV light source for 90 sec and

baked for 1 h. Membranes were prehybridized for 2 h at 420C in ultrasensitive

hybridization buffer (ULTRAhyb; Ambion, Austin, TX) followed by an overnight

incubation at 420C in the same ULTRAhyb solution containing the 32P-labeled bovine

prostaglandin H synthase 2 (PGHS-2), prostaglandin E synthase (PGES), and peroxisome

proliferator-activated receptor 6 (PPARS) cDNA probes. Filters were sequentially

washed in 2X SSC (lX= 0.15 M sodium chloride, 0.015 M sodium citrate)-0.1% SDS

and in 0.1x SSC-0.1% SDS two times each at 420C and then exposed to X-ray film to

detect radiolabeled bands. Equal loading of total RNA for each experimental sample was

verified by comparison to 18S rRNA ethidium bromide staining.

Western Blot Analysis of PGHS-2, PGES and PPAR8

For Western blot analyses, whole cell lysates were prepared as described by

Binnelli et al. (2000). Five hundred microliters of ice-cold whole cell extract buffer (50

mM Tris, pH 8.0, 300 mM NaC1, 20 mM NaF, 1 mM Na3VO4, 1 mM Na4P207, 1 mM

EDTA, 1 mM EGTA, 1 mM dithiothreitol, 0.5 mM PMSF, 10% v/v glycerol, 0.5% v/v

NP-40, and 10 Clg/ml each of Aprotinin, Leupeptin, and Pepstatin), were added to each

dish and cells were collected in 1.5 ml microcentrifuge tubes and incubated for 30 min at

40C. Cell lysates then were centrifuged at 40C for 10 min (13000 rpm) to remove cell

debris. Protein concentration in the supernatant was determined by the Lowry method

(Lowry et al., 1951). Protein (25 Gig) from each dish was resolved on a 7.5% SDS-

PAGE, and electrophoretically transferred to a nitrocellulose membrane. The Membrane

was blocked for 2 h in 5% (w/v) nonfat dried milk in Tris-buffered saline (TBS)









containing 0.1% Tween-20 (TBST, pH 7.4), rinsed with TBST and hybridized with

antibodies against either PGHS-2, PGES, or PPARS diluted (1:500) in 5% nonfat dried

milk in TBS. The secondary antibody was anti-rabbit IgG (1:3000 in 5% nonfat dried

milk in TBS). Target proteins were detected by enhanced chemiluminescence.

Statistical Analyses

Data were analyzed by Least-Squares analysis of variance (ANOVA) using the

General Linear Model (GLM) procedure of the Statistical Analysis System (SAS, 2001).

For PGF2a TOSponse, the mathematical model included fixed effects of treatment (n = 5),

experiment (n = 5), treatment by experiment interaction, and random effect of dish (n =

2/treatment) nested within treatment by experiment interaction. The variance of dish

nested within treatment by experiment interaction was used as the error term for all

effects. For Northern and western blot data, hybridization volumes obtained from

densitometric analysis were subj ected to ANOVA using the GLM procedure. For

Northern blot analysis, the mathematical model included independent fixed effects of

treatment (n = 5), experiment (n = 2), treatment by experiment interaction, and random

residual error. Results are presented as ratios of densitometric values for the target

mRNA over those for 18S rRNA ethidium bromide staining, and are presented as LS

means + SEM. For Western blot analysis, the statistical model included only the main

effect of treatment. Treatment effects were further analyzed using preplanned orthogonal

contrasts. These contrasts were constructed to compare PDBu vs. fatty acids responses;

SFA vs. PUFA; n-6 vs. n-3 PUFA; and n-3 LNA vs. longer chain n-3 EPA.









Results

To test the hypothesis that PUFAs may be more effective in inhibiting endometrial

PGF2a Secretion, we examined endometrial PGF2a TOSponse to PDBu in the presence of

n-6 (LA) and n-3 (LNA and EPA) PUFAs in comparison to saturated fatty acid (ST).

In vitro systems designed to evaluate endometrial PGF2a Secretion involved

stimulation of cells with PDBu, an activator of protein kinase C which induces PGHS-2

gene expression and secretion of PGF2a (Binelli et al., 2000). Treatment of BEND cells

with PDBu resulted in a 25-fold induction (P < 0.0001) of PGF2a Secretion (Figure 3-1).

The PDBu-induced PGF2a Secretion coincided with increased abundance of PGHS-2

mRNA (P < 0.0001; Figure 3-2) and protein (P = 0.0003; Figure 3-3). Stimulation of

BEND cells with PDBu also resulted in induction (P < 0.0001) of PGES gene expression

(Figure 3-4), but no differences were observed for PGES protein abundance (Figure 3-5).

Co-incubation of BEND cells with fatty acids decreased (P < 0.0001) PGF2a

response to PDBu (Figure 3-6). Analysis of individual fatty acid effects revealed that

EPA greatly reduced PGF2a induction by PDBu. The other fatty acids had minimal

effects on PDBu-induced prostaglandin production.

To determine the molecular mechanism by which fatty acids altered PGF2a

production in BEND cells, we examined PGHS-2, PGES and PPARS mRNA and protein

responses to PDBu in the presence of various fatty acids. On average, long-chain fatty

acids had no detectable effects on PGHS-2, PGES and PPARS mRNA responses to PDBu

(Figures 3-7, 3-9 and 3-11). The long-chain fatty acids stimulated (P = 0.0001) PGES

protein expression, but failed to alter PGHS-2 or PPARS protein response to PDBu

(Figures 3-8, 3-10 and 3-12).









Discussion

Polyunsaturated fatty acids have been associated with improvement of reproductive

efficiency in cattle, and modulation of prostaglandin synthesis has being suggested as a

potential mechanism. In the present study, we examined the effects of selected long-

chain fatty acids on PDBu-induced PGF2a Synthesis in BEND cells. On average, the

long-chain fatty acids tested in this study decreased PGF2a TOSponse to PDBu in BEND

cells. EPA decreased PGF2a prOduction to a greater extent than did the other fatty acids.

However, both saturated and unsaturated fatty acids had no detectable effects on PGHS-

2, PGES and PPARS mRNA responses to PDBu.

Reports on the effects of feeding n-6 PUFA on prostaglandin synthesis in vivo have

been inconsistent. Recent studies showed that in vitro supplementation of LA to maternal

intercotyledonary endometrial cells isolated from late pregnant ewes caused a significant

reduction of 2-series prostaglandins (Cheng et at., 2003, 2004). Similarly, cyclic dairy

cows fed a diet high in LA had reduced endometrial prostaglandin production (Cheng et

al., 2001). On the other hand, Cheng and others (2005) showed that late pregnant ewes

fed a diet high in LA had increased endometrial and placental prostaglandin production.

In the present study, incubation of BEND cells with EPA caused a reduction of PGF2a

secretion in response to PDBu stimulation. These findings are in agreement with reports

indicating that n-3 PUFAs inhibit prostaglandin secretion in vitro (Achard et al., 1997;

Levine and Worth, 1984; Mattos et al., 2001, 2003; Caldari-Torres et al., 2006) and in

vivo (Staples et al., 1998; Abayasekara and Wathes, 1999; Mattos et al., 2000, 2002,

2004).









The mechanisms by which EPA alters PGF2a TOSponse to PDBu are not well

understood. It has been proposed that PUFAs may reduce PGF2a prOduction through

modulation of one or more steps of the PG biosynthetic pathway. These include

decreasing availability of the precursor AA, competing for PGHS-2 activity and directly

inhibiting synthesis and/or activity of PGHS-2 (Mattos et al., 2000; Cheng et al., 2001).

In the present study, no changes were observed in PGHS-2 mRNA response to PDBu.

This is consistent with a previous study (Mattos et al., 2003) and suggests that EPA may

be competing with AA for the available binding sites on PGHS-2, thus shifting to

production of 3-series prostaglandins (PGF3u and PGE3). This hypothesis does not rule

out the possibility that EPA may also affect the PGHS-2 enzyme activity through

posttranslational modifications.

The observation that the long-chain fatty acids tested in this study had no effects on

PPARS gene or protein expression indicates that these fatty acids likely affect PGF2a

production through mechanisms which do not involve PPARS induction. However,

whether or not long-chain fatty acids alter PPARS activity warrants further investigation.

Summary

Phorbol-ester stimulated PGF2a prOduction and up-regulated PGHS-2 gene and

protein expression within 6 h in cultured BEND cells. Priming of BEND cells with ST,

LNA, and EPA reduced PGF2a TOSponse to PDBu by 17%, 14%, and 66%, respectively.

Both saturated and unsaturated fatty acids had not detectable effects on PGHS-2, PGES

or PPARS mRNA response to PDBu, suggesting that long-chain fatty acids tested in this

study likely affect PGF2a prOduction through PGHS-2-, PGES-, and PPARS-independent

mechanisms. Whether and how these fatty acids affect the activities of various enzymes







57


and transcription factors involved in the PGF2a biosynthetic cascade warrants further

information.





8000
6000
E


1


Control PDBu
Treatments
Figure 3-1. Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin Fza (PGF2ac)
secretion in bovine endometrial (BEND) cells. Treatment by experiment
interaction was significant (P < 0.05). Data represents least square means
SEM of five independent experiments (treatment effect, P < 0.0001).





Co nt rol


(A)


Control


PDBu


PDBu


PGHS-2

rRNA


(B)


4.4 kb

19S


1 2 3 4


0.12 C


0.08 E


0.04 C


D.00


Treatments


Figure 3-2. Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide
synthase (PGHS-2) mRNA abundance in bovine endometrial (BEND) cells.
Ten micrograms of total cellular RNA isolated from control and treated
BEND cells were subj ected to Northern blot analysis (A), and resulting
densitometric values were analyzed by the GLM procedure of SAS (B). The
top panel shows a representative Northern blot, whereas the bottom panel
represents means + SEM calculated over two experiments (n = 4 for each
treatment, P < 0.0001). There was no treatment by experiment interaction (P
= 0.06).





1 2 3 4


(A)


Control


PDBu


PGHS-2
(77 kD a)

(B)
150



S125


PDBu


C control


50 '


Treatments

Figure 3-3. Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin endoperoxide
synthase (PGHS-2) protein levels in bovine endometrial (BEND) cells.
Twenty five micrograms of total cellular protein extracted from control and
treated cells were subj ected to Western blot analysis (A), and resulting
densitometric values were analyzed by the GLM procedure of SAS (B). The
top panel shows a representative Western blot whereas the bottom panel
represents means + SEM calculated over two experiments (n = 4 for each
treatment, P = 0.0003).





(A)


Control


PDBu


PAGES


rRNA


1.3 kb


18S


PDBu


1 2 3 4


(B)








n~r
EB
vln
UI~
N
I~
E
r


Control


0.3 C


0.2 C


0.0 '


Treatments



Figure 3-4. Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase
(PGES) mRNA abundance in bovine endometrial (BEND) cells. Ten
micrograms of total cellular RNA isolated from control and treated BEND
cells were subj ected to Northern blot analysis (A), and resulting densitometric
values were analyzed by the GLM procedure of SAS (B). The top panel
shows a representative Northern blot, whereas the bottom panel represents
means + SEM calculated over two experiments (n = 4 for each treatment, P <
0.0001). Treatment by experiment interaction was significant (P = 0.009).














PAGES
(1 6 k Da)
1 2 3 4


(A)


Control


PDBu


(B)


120 t


PDBu


too C


C control


80 '


Treatments

Figure 3-5. Effect of phorbol 12, 13 dibutyrate (PDBu) on prostaglandin E synthase
(PGES) protein levels in bovine endometrial (BEND) cells. Twenty five
micrograms of total cellular protein extracted from control and treated cells
were subj ected to Western blot analysis (A), and resulting densitometric
values were analyzed by the GLM procedure of SAS (B). The top panel
shows a representative Western blot whereas the bottom panel represents
means + SEM calculated over two experiments (n = 4 for each treatment, P
0. 3).













10000 C









(V 6000
L.



4000


200d


PDBu ST LA LNA EPA

Treatments


Figure 3-6. Effect of fatty acids on prostaglandin Fza (PGF2a) TOSponse to phorbol 12, 13
dibutyrate (PDBu) in bovine endometrial (BEND) cells. When treatment
effects were detected (P < 0.05), means were separated using orthogonal
contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P < 0.0001.
Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P = 0.04. Contrast 3: (LA) vs.
(LNA) + (EPA), P < 0.0001. Contrast 4: (LNA) vs. (EPA), P < 0.001. When
main treatment effect was significant (P < 0.05), then differences between
treatments are represented by different letters. There was no treatment by
experiment interaction (P = 0.14).











(A)


PDBu ST LA LNA EPA


4.4 kb

18S


PGHS-2
rRNA


1234567891


(B)


0.15


z

ZV)
P~OO
r
E
B
hi
c~P
IN
~3Q
L
O


0.05


0.00


PDBu ST LA LNA EPA


Treatme nts


Figure 3-7. Effect of fatty acids on prostaglandin endoperoxide synthase (PGHS-2)
mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cells. Ten micrograms of total cellular RNA isolated from control
and treated BEND cells were subj ected to Northern blot analysis (A), and
resulting densitometric values were analyzed by the GLM procedure of SAS
(B). The top panel shows a representative Northern blot, whereas the bottom
panel represents means & SEM calculated over two experiments (n = 4 for
each treatment). When treatment effects were detected (P < 0.05), means
were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) +
(LA) + (LNA) + (EPA), P = 0.1. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA),
P = 0.06. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.06. Contrast 4: (LNA)
vs. (EPA), P = 0.02. When main treatment effect was significant (P < 0.05),
then differences between treatments are represented by different letters. There
was no treatment by experiment interaction (P = 0.38).











(A)


PDBu ST LA LNA EPA


12345678910


PGHS-2
(77 kD a)


(B)


150


135



120


105


PDBu ST LA LNA EPA


Treatments

Figure 3-8. Effect of fatty acids on prostaglandin endoperoxide synthase (PGHS-2)
protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial
(BEND) cells. Twenty five micrograms of total cellular protein extracted
from control and treated cells were subjected to Western blot analysis (A), and
resulting densitometric values were analyzed by the GLM procedure of SAS
(B). The top panel shows a representative Western blot whereas the bottom
panel represents means & SEM calculated over two experiments (n = 4 for
each treatment). When treatment effects were detected (P < 0.05), means
were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) +
(LA) + (LNA) + (EPA), P = 0.1. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA),
P = 0.01. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.2. Contrast 4: (LNA)
vs. (EPA), P = 0.5.










(A)


PDBu ST


LA LNA


EPA


PGES

rRNA


1.3 kb

18S


12345678910


(B)


0.40


0.32


0.24


0.16


0.08


0.00


Zo


PDBu ST LA LNA EPA
Treatments


Figure 3-9. Effect of fatty acids on prostaglandin E synthase (PGES) mRNA response to
phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells. Ten
micrograms of total cellular RNA isolated from control and treated BEND
cells were subj ected to Northern blot analysis (A), and resulting densitometric
values were analyzed by the GLM procedure of SAS (B). The top panel
shows a representative Northern blot, whereas the bottom panel represents
means & SEM calculated over two experiments (n = 4 for each treatment).
When treatment effects were detected (P < 0.05), means were separated using
orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) + (LNA) + (EPA),
P = 0.09. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P < 0.0001. Contrast
3: (LA) vs. (LNA) + (EPA), P = 0.04. Contrast 4: (LNA) vs. (EPA), P =
0.003. When main treatment effect was significant (P < 0.05), then
differences between treatments are represented by different letters. Treatment
by experiment interaction was significant (P = 0.0003).






67



(A)
PDBu ST LA LNA EPA

PGES
(16 kDa)

(B) 1 2 3 4 5 6 7 8 9 10


b bc
150 C b





-) a









75
PDBu ST LA LNA EPA
Treatments

Figure 3-10. Effect of fatty acids on prostaglandin E synthase (PGES) protein response
to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND) cells.
Twenty five micrograms of total cellular protein extracted from control and
treated cells were subj ected to Western blot analysis (A), and resulting
densitometric values were analyzed by the GLM procedure of SAS (B). The
top panel shows a representative Western blot whereas the bottom panel
represents means & SEM calculated over two experiments (n = 4 for each
treatment). When treatment effects were detected (P < 0.05), means were
separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) + (LA) +
(LNA) + (EPA), P = 0.0001. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA), P:
0.02. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.05. Contrast 4: (LNA) vs.
(EPA), P = 0.5. When main treatment effect was significant (P < 0.05), then
differences between treatments are represented by different letters.












(A)


PDBu ST


LA LNA EPA


PPARE

rRNA


3.5 kb

18S


1 2 3 4 5 6 7 8 9 10


(B)


Q



cl


E
cf


PDBu ST LA LNlA EPA


Treatments

Figure 3-11. Effect of fatty acids on peroxisome proliferator-activated receptor 6
(PPARS) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine
endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated
from control and treated BEND cells were subj ected to Northern blot analysis
(A), and resulting densitometric values were analyzed by the GLM procedure
of SAS (B). The top panel shows a representative Northern blot, whereas the
bottom panel represents means & SEM calculated over two experiments (n = 4
for each treatment). When treatment effects were detected (P < 0.05), means
were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (ST) +
(LA) + (LNA) + (EPA), P = 0.2. Contrast 2: (ST) vs. (LA) + (LNA) + (EPA),
P = 0.9. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.5. Contrast 4: (LNA) vs.
(EPA), P = 0.2.












(A)



PPARE
(53.2 kDa)


PDBu ST


LA LNA


EPA


1 2 3 4 5 6 7 8 9 10


r
O
m
cn

Kr
ur~
r~
,,
Ir
n
,ra
u


n


100





80


PDBu ST LA LNA EPA


Treatments


Figure 3-12. Effect of fatty acids on peroxisome proliferator-activated receptor 6
(PPARS) protein response to phorbol 12, 13 dibutyrate (PDBu) in bovine
endometrial (BEND) cells. Twenty five micrograms of total cellular protein
extracted from control and treated cells were subj ected to Western blot
analysis (A), and resulting densitometric values were analyzed by the GLM
procedure of SAS (B). The top panel shows a representative Western blot
whereas the bottom panel represents means & SEM calculated over two
experiments (n = 4 for each treatment). When treatment effects were detected
(P < 0.05), means were separated using orthogonal contrasts. Contrast 1:
(PDBu) vs. (ST) + (LA) + (LNA) + (EPA), P = 0.6. Contrast 2: (ST) vs. (LA)
+ (LNA) + (EPA), P = 0.8. Contrast 3: (LA) vs. (LNA) + (EPA), P = 0.2.
Contrast 4: (LNA) vs. (EPA), P = 0.9.















CHAPTER 4
EFFECTS OF CONJUGATED LINOLEIC ACIDS ON PROSTAGLANDIN Fza
PRODUCTION BY BOVINTE ENDOMETRIAL CELLS

Introduction

Conjugated linoleic acid (CLA) refers to a group of geometrical and positional

isomers of LA resulting from incomplete biohydrogenation in the rumen (Chin et al.,

1992; Ma et al., 1999; Griinari et al., 2000). The number of double bonds remains the

same as in the parent LA, but one of the double bonds is shifted to a new position by

microbial isomerases. A broad number of cis- and trans-t~t~rt~t~rt~t~rt~ CLA isomers have been

identified in food; however, the most commonly occurring CLA isomer is the cis-9,

ttrttrttrttrttrttrtrans-11-octadecadienoic acid with minor but significant proportions of trans-trt~r~rt~r~rt~t~rt 10, cis-12-

18:2 (Parodi, 1977, 1997; Chin et al., 1992; McGuire et al., 1998).

After ruminal synthesis of cis-9, trans-11 CLA, it may be absorbed in the small

intestine or further biohydrogenated to trans-11 -octadecenoic acid (trans vaccenic acid)

by rumen microorganisms. The trans-1rt~r~rt~r~rt~t~rt 1 18:1 can be reduced to stearic acid (18:0) or

transported to peripheral tissues as MUJFA. In the peripheral tissues (i.e. mammary

gland, muscle), this trans-isomer may be converted back into cis-9, trans-11 CLA by the

action of A9 desaturase (Holman and Mahfouz, 1981; Pollard et al., 1980). This appears

to be a maj or source of cis-9, trans-11 CLA in the cow' s milk (Corl et al., 1998; Griinari

and Bauman, 1999; Santora et al., 2000). The trans-10, cis-12 CLA detected in ruminal

tissues (Griinari and Bauman, 1999; Dhiman et al., 1999) seem to originate solely from









ruminal synthesis, since mammalian tissues do not have the Al2 desaturase necessary for

conversion of trans-trt~r~rt~r~rt~t~rt 10-octadecenoic acid (elaidic acid) back to trans-trt~r~rt~r~rt~t~rt 10, cis-12 CLA.

A large number of beneficial effects have been attributed to CLA ranging from

enhancing feed efficiency and growth (Chin et al., 1994; Bee, 2000), decreasing body fat

in mice (DeLany et al., 1999) and pigs (Dugan et al., 1997; Ostrowska et al., 1999), and

reducing milk fat synthesis in lactating dairy cows (Griinari et al., 1998; Romo et al.,

2000; Baumgard et al., 2001). Although it is likely that some biological effects of CLA

may be induced and/or enhanced synergistically by these isomers, there is evidence

suggesting that the effects of CLA are due to separate actions of these biologically active

isomers (Pariza et al., 2000).

The anticarcinogenic effect of CLA has been linked to its ability to modulate

eicosanoid production (Sugano et al., 1998). Recent studies detected an inhibitory effect

of CLA on eicosanoid synthesis in various animal and cell systems (Liu and Belury,

1998; Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al, 2003). Feeding

pregnant rats with dietary CLA resulted in inhibition of uterine PGF2a independent of

PUFA content (Harris et al., 2001). In addition, Cheng et al. (2003) reported that

treatment of endometrial cells isolated from late pregnant ewes with CLA suppressed

PGF2a in a dose dependent manner, while low doses of CLA stimulated PGE2 generation.

Cheng and coworkers (2003) showed that treatment of intercotyledonary endometrial

cells with CLA resulted in a dose-dependent inhibition of PGF2a prOduction. Similarly,

CLA has also been shown to inhibit PGF2a Synthesis in rat placental and uterine tissues

(Harris et al., 2001). Prostaglandins of the 2-series are involved in several reproductive

processes such as ovulation (Espey, 1980), follicular growth and development (Wallach









et al., 1975), and CL function (Abayasekara et al., 1995; Poyser, 1995; Wathes and

Lamming, 1995). Hence, CLA isomers may regulate reproductive processes through

modulation of PGF2a biosynthesis.

The objective of this investigation was to examine the effects of cis-9, tranzs-11 and

trans-trt~r~rt~r~rt~t~rt10, cis-12 CLA isomers on PBDu-induced PGF2a prOduction in bovine endometrial

(BEND) cells. We hypothesized that both CLA isomers would be equally effective in

inhibiting PDBu-induced PGF2a prOduction in BEND cells. Additionally, if these effects

are specific to CLA, LA should have minimal effect on PGF2a prOduction.

Materials and Methods

Materials

Polystyrene tissue culture dishes (100 x 20 mm) were purchased from Corning

(Corning Glass Works, Corning, NY). The Ham F-12 medium, antibiotic/antimycotic

(ABAM), phorbol 13, 14-dibutyrate (PDBu), horse serum, D-valine, insulin, fatty acid-

free bovine serum albumin (BSA), stearic acid (ST, Cis:o), aprotinin, leupeptin, and

pepstatin were from Sigma Chemical Co. (St. Louis, MO). The Minimum Essential

Medium (MEM) and fetal bovine serum (FBS) were from US Biologicals (Swampscott,

MA) and Atlanta Biologicals (Norcross, GA), respectively. Linoleic acid (LA, C18:2n-6),

cis-9, trans-11 conjugated linoleic acid (CLA, C18:2), trans-10, cis-12 CLA (C18:2),

PGHS-2 and PGES antibodies, and PGF2a Standard were from Cayman Chemicals (Ann

Arbor, MI). The PPARS and secondary anti-rabbit IgG antibodies were from Santa Cruz

Biotechnology (Santa Cruz, CA). The enhanced chemiluminescence (ECL) kit was

purchased from Perkin Elmer (Boston, MA). Hanks Balanced Salt Solution (HBSS) and

TriZol reagent were from GIBCO BRL (Carlsbad, CA). Isotopically-labelled PGF2ae (5,










6, 8, 9, 11, 12, 14, 15[n-3H] PGF2a; 208 Ci/mmol) was from Amershan Biosciences

(Piscataway, NJ). The anti-PGFza antibody was purchased from Oxford Biomedicals

(Oxford, MI). BioTrans nylon membrane and [ot-32P]deoxycitidine triphosphate (SA

3000 Ci/nmol) were from MP Biomedicals (Atlanta, GA). The PGHS-2 cDNA probe

was cloned from an ovarian follicular cDNA library (Liu et al., 1999), the PGES cDNA

probe was cloned from endometrial cDNA (Guzeloglu et al., 2004), whereas the PPARS

probe was generated from bovine endometrial RNA (Balaguer et al., 2005).

Cell Culture and Treatment

Bovine endometrial (BEND) cells were plated and cultured as described in chapter

3. To investigate the effects of supplemental CLA acids on uterine PGF2a Synthesis,

BEND cells were treated with PDBu alone (100 ng/ml) or PDBu in combination with 100

CLM linoleic acid (LA), cis-9, trans-11 CLA or trans-10,rtrt~t~r~rtrt~t~ cis-12 CLA. Fatty acids were

completed with BSA (1:3 ratio) for 2 h, and then treatments were applied to cells for a 24

h period. After treatment, cells were rinsed with HB SS and challenged with PDBu for

another 6 h. The remaining cell monolayer was rinsed with HB SS, lysed in TriZol

reagent, and stored at -800C for subsequent mRNA analysis. This experiment was

repeated two times. Radioimmunoassay of PGFza and Northern and Western blot

analyses were performed as described in chapter 3.

Statistical Analyses

Data were analyzed by Least-Squares analysis of variance (ANOVA) using the

General Linear Model (GLM) procedure of the Statistical Analysis System (SAS, 2001).

For PGF2a TOSponse, the mathematical model included fixed effects of treatment (n = 4),

experiment (n = 4), treatment by experiment interaction, and random effect of dish (n =









2/trt) nested within treatment by experiment interaction. The variance of dish nested

within treatment by experiment was used as the error term for all upstream effects. For

Northern and western blot data, hybridization volumes obtained from densitometric

analysis were subj ected to ANOVA using the GLM procedure. For Northern blot

analysis, the statistical model included independent fixed effects of treatment (n = 4),

experiment (n = 2), treatment by experiment interaction, and random residual error.

Results are presented as ratios of densitometric values for target genes over those for 18S

rRNA ethidium bromide staining, and are presented as LS means + SEM. For Western

blot analysis, the mathematical model included only the main effect of treatment.

Treatment effects were further analyzed using preplanned orthogonal contrasts. These

contrasts were constructed to compare PDBu vs. fatty acids responses; LA vs. CLA

isomers; and cis-9, trans-1rt~r~rt~r~rt~t~rt 1 CLA vs. trans-10,rtrt~t~r~rtrt~t~ cis-12 CLA.

Results

In the present study we used bovine endometrial (BEND) cells as a model to study

CLA regulation of PGF2a prOduction. BEND cells are a line of spontaneously replicating

endometrial cells originating from d 14 cycling cows (Staggs et al., 1998). Priming of

BEND cells with cis-9, trans-11 or trans-10, cis-12 CLA isomers resulted in a 1.3-fold

inhibition (P < 0.0001) of PGF2a TOSponse to PDBu (Figure 4-1). Conversely, both CLA

isomers stimulated (P < 0.05) PGHS-2 mRNA response to PDBu (Figure 4-2). Co-

incubation with C18 fatty acids resulted in a significant reduction (P < 0.003) in PGES

mRNA response to PDBu (Figure 4-3). In contrast with PGES gene expression, PPARS

mRNA levels were increased (P < 0.0008) by all the polyunsaturated fatty acids tested in










this study (Figure 4-6). Average PPARS protein levels did not differ among treatments

(Figure 4-7).

Discussion

Prostaglandins of the 2-series affect numerous processes in reproduction, including

ovulation (Espey, 1980), follicular development (Wallach et al., 1975), corpus luteum

function (Poyser, 1995; Wathes and Lamming, 1995; Abayasekara and Wathes, 1999)

and parturition (Challis et al., 1997). The anticarcinogenic effect of CLA has been

attributed partially to its inhibitory effect on eicosanoid synthesis (Banni et al., 1999;

Gregory and Kelly, 2001).

In the present study, incubation of BEND cells with either cis-9, trans-11 or trans-

10, cis-12 CLA isomers resulted in a 1.3-fold inhibition (P < 0.0001) of PDBu-induced

PGF2a Secretion. This is in agreement with several studies that have detected inhibitory

effect of CLA on eicosanoid synthesis in vivo and in vitro (Liu and Belury, 1998;

Kavanaugh et al., 1999; Uruquhart et al., 2002; Eder et al., 2003). In addition, Cheng et

al. (2003) reported that treatment of endometrial cells isolated from late pregnant ewes

with CLA suppressed PGF2a prOduction. However, CLA inhibition of endometrial

PGF2a TOSponse to PDBu in BEND cells was not mediated through repression of PGHS-2

gene expression. On the contrary, priming of BEND cells with CLA isomers resulted in

increased PGHS-2 mRNA steady-state levels, though no changes were observed at the

protein level.

The mechanism of CLA inhibition of PGF2a has not been fully elucidated. CLA

may be competing with AA for incorporation into the membrane phospholipids as well as

for PGHS-2 activity (Mattos et al., 2000; Cheng et al., 2001). Banni and others (1999)









showed that CLA supplementation to rat mammary tissue increased accumulation of

CLA metabolites and decreased LA metabolites, such as AA. These isomers may also be

directly inhibiting the activity of PGHS-2 (Mattos et al., 2000; Cheng et al., 2001). CLA

metabolites have been shown to be powerful inhibitors of PGHS enzyme (Nugteren and

Christ-Hazelhof, 1987).

Alternatively, CLA may regulate the conversion of PGF2a to PGE2, aS reported by

Gross and Williams (1988) in bovine placental cells. However, in this study, incubation

of BEND cells with both CLA isomers resulted in reduced PGES mRNA expression,

with no changes in protein concentration. This finding provides no evidence that CLA

favors PGE2 Synthesis at the expense of PGFza prOduction. Whether CLA affects PGES

activity was not documented in this study.

CLA is a naturally occurring ligand of PPARs (Moya-Camarena et al., 1999).

Priming of BEND cells with cis-9, trans-11 and trans-1rt~r~rt~r~rt~t~rt 0, cis-12 CLA isomers induced

PPARS gene expression, but no changes were observed in PPARS protein concentration.

There was no correlation between PPARS mRNA levels and PGF2a COncentration in cell-

conditioned medium. Collectively, these findings indicate that CLA-induced attenuation

of PGF2a Secretion in BEND cells is not mediated through modulation of PGHS-2 or

PPARS gene expression. Whether and how CLA isomers affect PGHS-2 enzymatic

activity warrants further study.

Summary

Evidence is rapidly accumulating that CLA isomers modulate eicosanoid

biosynthesis in various cell systems. In the present study, priming of BEND cells with

cis-9, trans-11 or trans-10, cis-12 CLA isomers greatly decreased PGF2a TOSponse to









PDBu. Interestingly, co-incubation with both CLA isomers increased PGHS-2 mRNA

abundance in PDBu-stimulated BEND cells, suggesting that these fatty acids alter PGF2a

production through a mechanism that does not require repression of PGHS-2 gene

expression. Further studies are needed to test whether or not CLA isomers modulate the

activity of various enzymes and transcription factors involved in the prostaglandin

biosynthetic cascade.














6000




4500 bbb bbb b b




S3000




1500





PDBu LA CLA (c9,tl 1) CLA (t10,cl2)

Treatments


Figure 4-1. Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin Fzca (PGF2ac)
response to phorbol 12, 13 dibutyrate (PDBu) in bovine endometrial (BEND)
cells. When treatment effects were detected (P < 0.05), means were separated
using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) + (cis-9, trans-11) +
(trans-10,rt~t~r~rtrt~t~r cis-12), P < 0.0001. Contrast 2: (LA) vs. (cis-9, trans-11) + (trans-
10, cis-12), P < 0.0001. Contrast 3: (cis-9, trans-11t~t~rtrt~r~rt~t~ ) vs. (trans-10, cis-12), P
= 0.44. When main treatment effect was significant (P < 0.05), then
differences between treatments are represented by different letters. Treatment
by experiment interaction was significant (P < 0.0001).











(A)


PGHS-2

rRNA



(B)



0.20


PDBu LA c9,tl1


t10,c12


4.4 kb

18S


1 2 3 4 5 6 7 8


Q
z
Q
Zu,

Eo
cl
h(
rhg
IN

L
O


0.16



0.12


0.08


PDBu LA CLA(c9,tl1) CLA(t 0, cl2)

Treatments


Figure 4-2. Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin endoperoxide
synthase (PGHS-2) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. Ten micrograms of total cellular RNA
isolated from control and treated BEND cells were subj ected to Northern blot
analysis (A), and resulting densitometric values were analyzed by the GLM
procedure of SAS (B). The top panel shows a representative Northern blot,
whereas the bottom panel represents means & SEM calculated over two
experiments (n = 4 for each treatment). When treatment effects were detected
(P < 0.05), means were separated using orthogonal contrasts. Contrast 1:
(PDBu) vs. (LA) + (cis-9, trans-11l) +(trans-10, cis-12), P < 0.002. Contrast
2: (LA) vs. (cis-9, trans-1 1) + (trans-10, cis-12), P < 0.04. Contrast 3: (cis-9,
trans-trt~r~rt~r~rt~t~rt 11) vs. (trans-1rt~t~rtrt~t~rtrt~ 0, cis-12), P = 0.006. W hen main treatment effect was
significant (P < 0.05), then differences between treatments are represented by
different letters. There was no treatment by experiment interaction (P = 0.65).










(A)


PDBu


LA


c9,t 11


t10,cl12


PGES
rRNA



(B)


1.3 kb

18S


1 2 3 4 5 6 7 8


0.14


Lu


0.12


0.10


0.08


0.06


PDBu LA CLA(c9,tl1) CLA(tl0,cl2)


Treatme nts

Figure 4-3. Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin E synthase
(PGES) mRNA response to phorbol 12, 13 dibutyrate (PDBu) in bovine
endometrial (BEND) cells. Ten micrograms of total cellular RNA isolated
from control and treated BEND cells were subj ected to Northern blot analysis
(A), and resulting densitometric values were analyzed by the GLM procedure
of SAS (B). The top panel shows a representative Northern blot, whereas the
bottom panel represents means & SEM calculated over two experiments (n = 4
for each treatment). When treatment effects were detected (P < 0.05), means
were separated using orthogonal contrasts. Contrast 1: (PDBu) vs. (LA) +
(cis-9, trans-trt~r~rt~r~rt~t~rt 11) + (trans-t~t~rt~t~rt~t~rt~ 10, cis-12), P < 0.003. Contrast 2: (LA) vs. (cis-9,

10, cis-12), P = 0.6. When main treatment effect was significant (P < 0.05),
then differences between treatments are represented by different letters. There
was no treatment by experiment interaction (P = 0.10).











(A)


PDBu


LA ,tl


t10,cl2


PG3HS-2
(7 2 k Da)


(B)


150


1 2 3 4 5 6 7 8


125 C


100 E


75 E


PDBU


LA cuncsltil)


cuA(ti0, ci2)


Treatments


Figure 4-4. Effect of c9, t11 and tl0, cl2 CLA isomers on prostaglandin endoperoxide
synthase (PGHS-2) protein response to phorbol 12, 13 dibutyrate (PDBu) in
bovine endometrial (BEND) cells. Twenty five micrograms of total cellular
protein extracted from control and treated cells were subj ected to Western blot
analysis (A), and resulting densitometric values were analyzed by the GLM
procedure of SAS (B). The top panel shows a representative Western blot
whereas the bottom panel represents means & SEM calculated over two
experiments (n = 4 for each treatment). When treatment effects were detected
(P < 0.05), means were separated using orthogonal contrasts. Contrast 1:
(PDBu) vs. (LA) + (cis-9, trans-1 1) + (trans-10, cis-12), P < 0.2. Contrast 2:
(LA) vs. (cis-9, trans-1rt~r~rt~r~rt~t~rt 1) + (trans-10,rt~t~r~rtrt~t~r cis-12), P < 0.2. Contrast 3: (cis-9,
ttrttrttrttrttrttrtrans-11)vs. (trans-1rt~t~rtrt~t~rtrt~ 0, cis-12), P = 0.3.











(A)
PDBu LA d~i 10,cl2

PGES
(1 7 k Da)
1 23 4 5 6 7 8

(B)


150








60 -0
PDuL L,9t1)CAt0c2
Tretmnt

Fiue45 feto 9 1 n lc2 L smr npotgadnEsnhs







Figurextra ffcte fro control and treated L cels weresubjcte tro Wst erlnd n bloyth


analysis (A), and resulting densitometric values were analyzed by the GLM
procedure of SAS (B). The top panel shows a representative Western blot
whereas the bottom panel represents means & SEM calculated over two
experiments (n = 4 for each treatment). When treatment effects were detected
(P < 0.05), means were separated using orthogonal contrasts. Contrast 1:
(PDBu) vs. (LA) + (cis-9, trans-1 1) + (trans-10, cis-12), P < 0.6. Contrast 2:
(LA) vs. (cis-9, trans-1rt~r~rt~r~rt~t~rt 1) + (trans-10,rt~t~r~rtrt~t~r cis-12), P = 0.05. Contrast 3: (cis-9,
ttrttrttrttrttrttrtrans-11)vs. (trans-1rt~t~rtrt~t~rtrt~ 0, cis-12), P = 0.2.











(A)


PDBu


LA


c9,tl1


t10,c12


3.5 kb
18S


1 2 3 4 5 6 7 8


0.06


z
L
Zeo
P~r
Eo
c,


~ca
E
L
O


0.04





0.02


0.00


PDBu LA CLA(c9,tl1) CLA(tl0,cl2)


PPARS

rRNA



(B)


Treatments


Figure 4-6. Effect of c9, t11 and tl0, cl2 CLA isomers on peroxisome proliferator-
activated receptor 6 (PPARS) mRNA response to phorbol 12, 13 dibutyrate
(PDBu) in bovine endometrial (BEND) cells. Ten micrograms of total
cellular RNA isolated from control and treated BEND cells were subj ected to
Northern blot analysis (A), and resulting densitometric values were analyzed
by the GLM procedure of SAS (B). The top panel shows a representative
Northern blot, whereas the bottom panel represents means & SEM calculated
over two experiments (n = 4 for each treatment). When treatment effects were
detected (P < 0.05), means were separated using orthogonal contrasts.
Contrast 1: (PDBu) vs. (LA) + (cis-9, trans-1rt~r~rt~r~rt~t~rt 1) + (trans-10,rt~t~r~rtrt~t~r cis-12), P <
0.0008. Contrast 2: (LA) vs. (cis-9, trans-1rt~r~rt~r~rt~t~rt 1) + (trans-10,rt~t~r~rtrt~t~r cis-12), P < 0.02.
Contrast 3: (cis-9, trans-11) vs. (trans-10,rt~t~r~rtrt~t~r cis-12), P = 0.01. When main
treatment effect was significant (P < 0.05), then differences between
treatments are represented by different letters. There was no treatment by
experiment interaction (P = 0.08).












(A)


PDBu LA


d~til 10,cl2


PPAR8
(53.2 kDa)


12 34 5 6 7


(B)


125


r

loa
~cl
xr
lu~
r~
cl 75
n
n
,b


n r;R


PDBu LA CLA~c9,tl1) CLA(tl0, cl 2)


Treatments


Figure 4-7. Effect of c9, t11 and tl0, cl2 CLA isomers on peroxisome proliferator-
activated receptor 6 (PPARS) protein response to phorbol 12, 13 dibutyrate
(PDBu) in bovine endometrial (BEND) cells. Twenty five micrograms of
total cellular protein extracted from control and treated cells were subj ected to
Western blot analysis (A), and resulting densitometric values were analyzed
by the GLM procedure of SAS (B). The top panel shows a representative
Western blot whereas the bottom panel represents means & SEM calculated
over two experiments (n = 4 for each treatment). When treatment effects were
detected (P < 0.05), means were separated using orthogonal contrasts.
Contrast 1: (PDBu) vs. (LA) + (cis-9, trans-1rt~r~rt~r~rt~t~rt 1) + (trans-10,rt~t~r~rtrt~t~r cis-12), P < 0.4.
Contrast 2: (LA) vs. (cis-9, trans-trt~r~rt~r~rt~t~rt 11) + (trans-1rt~t~rtrt~t~rtrt~ 0, cis-12), P < 0.2. Contrast 3:
(cis-9, trans-1rt~r~rt~r~rt~t~rt 1) vs. (trans-10,rt~t~r~rtrt~t~r cis-12), P = 0.2.