Arginine Supplementation during Estrus Has No Effect on Uterine Blood Flow or Fluid Clearance in Non-Pregnant Mares

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Arginine Supplementation during Estrus Has No Effect on Uterine Blood Flow or Fluid Clearance in Non-Pregnant Mares
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english
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Jacobs, Robert D
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Master's ( M.S.)
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University of Florida
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Animal Sciences
Committee Chair:
Warren, Lori
Committee Members:
Mortensen, Christopher J.
Fields, Michael J

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arginine -- blood -- equine -- flow -- fluid
Animal Sciences -- Dissertations, Academic -- UF
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Animal Sciences thesis, M.S.
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Abstract:
Uterine fluid accumulation has been identified as a factor that can negatively impact the reproductive performance of mares by reducing pregnancy and successful fertilization as well increasing early embryonic death rates. L-arginine is an amino acid responsible for various functions in the body and has been identified as an essential amino acid in equine nutrition. L-arginine supplementation has been shown to positively impact reproductive performance in pigs and mice, and alter uterine involution in mares. Acting as a nitric oxide donor, L-arginine has been demonstrated to increase blood flow. An increase in blood flow has been associated with hastened uterine involution and fluid clearance. The objectives of this study were to determine the effect of L-arginine supplementation on uterine arterial blood flow and fluid clearance in non-pregnant mares. It was hypothesized that mares supplemented with L-arginine would have hastened uterine fluid clearance when compared to control. Twelve non-pregnant light horse mares were used in a 3x3 Latin Square design study. The three treatments included dietary supplementation with L-arginine, isonitrogenous amounts of urea, or no supplementation coupled with administration of oxytocin. Treatments were initiated 10 d post ovulation, and continued until all fluid was absent from the uterus, resulting in a total length of supplementation of approximately 12 days. Mares were examined by transrectal ultrasonography and infused with 880 mL of sterile saline combined with 120 mL of semen extender upon discovery of a 33 mm follicle. Mares were examined by transrectal Doppler ultrasonography at 12-h intervals following infusion and uterine blood flow and fluid presence were recorded until all fluid was absent from the uterus. Mean fluid clearance across treatments was 53.8 plus/minus 4.9 h. Short-term arginine supplementation had no effect on rate or latency of fluid clearance or blood flow measured as pulsatility or resistance indices. Although previous research has demonstrated L-arginine supplementation improved uterine blood flow and hastened uterine fluid clearance in early postpartum mares, results of this study indicate L-arginine supplementation has limited effects on uterine hemodynamics and fluid clearance in nonpregnant mares.
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by Robert D Jacobs.
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Thesis (M.S.)--University of Florida, 2012.
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Adviser: Warren, Lori.
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RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2014-05-31

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1 ARGININE SUPPLEMENTATION DURING ESTRUS HAS NO EFFECT ON UTERINE BLOOD FLOW OR FLUID CLEARANCE IN NON PREGNANT MARES By ROBERT D. JACOBS A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2012

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2 2012 Robert Jacobs

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3 To Grandma Shirley

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4 ACKNOWLEDGMENTS I would first like to thank my advisors Dr. Lori Warren and Dr. Christopher Mortensen for their help and guidance in the development and completion of this project as well as their dedication to my education and assistance in completing this research h project is a testament to his devotion to seeing his students succeed. I would also like to thank the other member of my committee Dr. Michael Fields for his support and advice and h is work in laying down the groundwork to help develop my passion for reproductive physiology. I appreciate the time that Justin Callaham spent teaching me to ultrasound and palpate mares and his continued insight and assistance into this project. I would also like to thank Chris Cooper for providing me with any assistance that I needed at the Equine Sciences Center. Additionally, I would like to thank Joss Cooper for assisting me on a daily basis with my examinations and data collection as well as making s ure that everything ran smoothly. I would also like to thank the staff of the Equine Sciences Center, including Richard Hayes, Adel Dawson, and all other volunteers that helped with this project. Without the assistance that I received from the staff of the ESC, I would not have been able to successfully complete this project. I would also like to these long months. I would like to thank Dale Kelley for all of his assistance, guidance and teaching throughout this project and his assistance in data collection and examinations. I would also like to thank Jan Kivipelto for all of her assistance in not only

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5 the lab work, but all around willingness to help in any way necessary. In addition, I would like to thank the undergraduates who assisted me in this project. I would also like to thank my fiance, Alana Goldstein for everything that you do for me. In addition, all of the help that you gave me at the ESC with data collection is truly appreciated. Finally, I would like to thank my parents, without whom I would never have made it to this point. My parents have spent my entire life inspiring me and pushing me to achieve everything that I have. They have shown an unyielding devotion to my schooling and making sure that I achieve all of my goals.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 11 CHAPTER 1 INT RODUCTION ................................ ................................ ................................ .... 13 2 REVIEW OF LITERATURE ................................ ................................ .................... 16 Overview of Equine Reproductive Physiology ................................ ........................ 16 Reproductiv e Anatomy ................................ ................................ ..................... 16 Oviduct ................................ ................................ ................................ ............. 17 Uterus ................................ ................................ ................................ ............... 18 Cer v ix ................................ ................................ ................................ ............... 19 Uterine Vasculature and Blood Flow ................................ ................................ ....... 20 Reproductive Vasculature ................................ ................................ ................ 20 Arteries and Hemodynamics ................................ ................................ ............ 22 Blood Flow Indices ................................ ................................ ........................... 23 Arginine ................................ ................................ ................................ ................... 25 Arginine Requirement ................................ ................................ ....................... 25 Arg inine Properties and General Functions ................................ ...................... 25 Arginine Synthesis ................................ ................................ ............................ 26 Relationship between Arginine and Citrulline ................................ ................... 28 Effects o f Arginine and Nitric Oxide on Reproduction and Blood Flow ............. 29 Uterine Fluid ................................ ................................ ................................ ........... 31 Fluid Accumulation ................................ ................................ ........................... 31 Methods of Fluid Removal ................................ ................................ ................ 33 3 METHODS AND MATERIALS ................................ ................................ ................ 36 Animals ................................ ................................ ................................ ................... 36 Experimental Design ................................ ................................ ............................... 36 Dietary Treatments ................................ ................................ ................................ 37 Uterine Infusion ................................ ................................ ................................ ....... 38 Doppler Ultrasonography ................................ ................................ ........................ 39 Statistical Analysis ................................ ................................ ................................ .. 39

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7 4 RESULTS ................................ ................................ ................................ ............... 42 Feeding and Supplementation ................................ ................................ ................ 42 Latency to Total Fluid Cl earance ................................ ................................ ............ 42 Rate of Fluid Clearance ................................ ................................ .......................... 42 Latency to Follicle Development and Ovulation ................................ ...................... 42 Blood Flow ................................ ................................ ................................ .............. 43 5 DISCUSSION ................................ ................................ ................................ ......... 51 6 CONCLUSIONS ................................ ................................ ................................ ..... 55 LITERATURE CITED ................................ ................................ ................................ .... 57 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 61

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8 LIST OF TABLES Table page 2 1 Nitric o xide s ynthase p rofiles and their a ctions ................................ ................... 29 3 1 Nutrient composition of basal diet feeds and L arginine and urea supplements ................................ ................................ ................................ ....... 41 4 1 F ollicular and ovulatory dynamics in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration ......................... 50

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9 LIST OF FIGURES Figure page 2 1 L Arginine s ynthesis as adapted from Wu and Morris, 1998 .............................. 2 8 2 2 Citulline/ n itric o xide cycle as adapted from Wu and Morris, 1998 ....................... 29 4 1 Mean ( SEM) time (h) to total fluid clearance from the uterus in mares supplemented with L arginine, urea, or no supplement combined w ith oxytocin administration. ................................ ................................ ...................... 44 4 2 Mean ( SEM) diameter (mm) of fluid present wit hin the uterus of mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. ................................ ................................ ...................... 45 4 3 Mean ( SEM) resistance index of the nonovulatory uterine artery following uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. ................................ .......... 46 4 4 Mean ( SEM) resistance index of the ovulatory uterine artery follo wing uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. ................................ .......... 47 4 5 Mean ( SEM) pulsatility index of the nonovulatory uterine artery following uterine infusion i n mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. ................................ .......... 48 4 6 M ean ( SEM) pulsatility index of the ovulatory uterine artery following uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytocin admini stration. ................................ ........... 49

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10 LIST OF ABBREVIATION S ARG Arginine ASS Arginosuccinate Synthase ASL Arginosuccinate Lyase ATP Adenosine triphosphate BW Body Weight DCA Deep Circumflex Artery EDV End Dias tolic Velocity EIA External Iliac Artery eNOS Endothelial Nitric Oxide Synthase FSH Follicle Stimulating Hormone iNOS Inducible Nitric Oxide Synthase IU International Units NO Nitric Oxide NOS Nitric Oxide Synthase nNOS Neuronal Nitric Oxide Synthase OXY Oxytocin PI Pulsatility Index PSV Peak Systolic Velocity RI Resistance Index SE Standard Error TAMV Timed Average Mean Velocity UREA Urea VEGF Vascular Endothelial Growth Factor Vegfr2 Vascular Endothelial Growth Factor Receptor 2

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11 Abstract of Thesis Pr esented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science ARGININE SUPPLEMENTATION DURING ESTRUS HAS NO EFFECT ON UTERINE BLOOD FLOW OR FLUID CLEARANCE IN NON PREGNANT MARES By Robert D. Jacobs May 2012 Chair: Lori K. Warren Major: Animal Sciences Uterine fluid accumulation has been identified as a factor that can negatively impact the reproductive performance of mares by reducing pregnancy and successful f ertilization as well increasing early embryonic death rates. L arginine is an amino acid responsible for various functions in the body and has been identified as an essential amino acid in equine nutrition. L arginine supplementation has been shown to posi tively impact reproductive performance in pigs and mice, and alter uterine involution in mares. Acting as a nitric oxide donor, L arginine has been demonstrated to increase blood flow. An increase in blood flow has been associated with hastened uterine inv olution and fluid clearance. The objectives of this study were to determine the effect of L arginine supplementation on uterine arterial blood flow and fluid clearance in non pregnant mares. It was hypothesized that mares supplemented with L arginine would have hastened uterine fluid clearance when compared to control. Twelve non pregnant light horse mares were used in a 3x3 Latin Square design study. The three treatments included dietary supplementation with L arginine, isonitrogenous amounts of urea, or n o supplementation coupled with administration of oxytocin. Treatments were initiated 10 d

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12 post ovulation, and continued until all fluid was absent from the uterus, resulting in a total length of supplementation of approximately 12 days. Mares were examine d by transrectal ultrasonography and infused with 880 mL of sterile saline combined with 120 mL of semen extender upon discovery of a 33 mm follicle. Mares were examined by transrectal Doppler ultrasonography at 12 h intervals following infusion and uterin e blood flow and fluid presence were recorded until all fluid was absent from the uterus. Mean fluid clearance across treatments was 53.8 4.9 h. Short term arginine supplementation had no effect on rate or latency of fluid clearance or blood flow measure d as pulsatility or resistance indices. Although previous research has demonstrated L arginine supplementation improved uterine blood flow and hastened uterine fluid clearance in early postpartum mares, results of this study indicate L arginine supplementa tion has limited effects on uterine hemodynamics and fluid clearance in nonpregnant mares.

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13 CHAPTER 1 INTRODUCTION Uterine fluid accumulation has been shown to negatively impact the reproductive performance of the mare, including reduced pregnancy rates as well as increased early embryonic loss (McKinnon and Voss, 2005). The accumulation of fluid in the uterus of the mare is also associated with endometritis and has a negative correlation to the reproductive efficiency of the mare (McKinnon and Voss, 2005). In the equine industry there are economic incentives that influence breeders to maintain a 12 month breeding schedule (McKinnon and Voss, 2005). Mares are unique in that they have a fertile foal heat occurring approximately 10 d post foaling (Blanchard e t al., 2002). Breeding on foal heat allows the breeder to maximize breeding efficiency and thus maintain an economic advantage. Many breed registries mandate a universal birthday of January 1 for foals. Breeding on foal heat can allow for the foal to be bo rn at or near that date and maintain a competitive advantage. Factors such as uterine fluid accumulation can negatively impact the success achieved when breeding during this foal heat period (McKinnon and Voss, 2005). Delayed uterine involution as well as prolonged endometritis are often responsible for the decline in breeding efficiency during foal heat (McKinnon and Voss, 2005). Ultrasonography is an extremely valuable tool for estimating the quality and quantity of intrauterine fluid, as well as other k ey reproductive attributes (McKinnon and Voss, 2005). Blood flow measurements obtained via Doppler ultrasonography can also help determine the reproductive efficiency of the mare (Ginther, 1992). An increase in blood flow has been shown to increase reprodu ctive efficiency in pigs (Mateo et al., 2007) and rats (Greene et al., 2012). Increases in blood flow have also been associated

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14 with increased rates of uterine involution following parturition (Ginther,1992). Uterine blood flow is measured using indices su ch as the pulsatility index (PI) and the resistance index (RI; Ginther, 1992). A decrease in RI is associated with an increase in blood flow and there is a strong correlation between RI and PI (Ginther, 1992). Arginine is one of the most versatile amino a cids and serves as a precursor for a multitude of molecules (NRC, 2007). Arginine is recognized as an essential amino acid for horses, but the requirements are not yet known (NRC, 2007). However, as in other species, arginine is presumably synthesized in t he horse, but in insufficient quantities and must be supplied in some part by the diet (NRC, 2007). Arginine supplementation has been investigated for its potential reproductive benefits in rats and pigs and has shown favorable results including increased litter size, number of offspring born alive, and increased placental attachments. (Mateo et al., 2007; Li et al., 2010; Greene et al., 2012). Arginine supplementation has also been associated with an increase in uterine arterial blood flow and hastened ut erine involution in postpartum mares (Mortensen et al., 2011). Amit et al., (1998) stated that arginine is believed to influence blood flow by impacting the production of nitric oxide (NO). The conversion of arginine to citrulline involves the production of NO through the citrulline/NO cycle (Wu and Morris, 1998). Nitric oxide generated from arginine by the vascular endothelium has been shown to be an important regulator of vascular tone and subsequent blood flow (Amit et al., 1998). Arginine has also bee n shown to influence important angiogenic and vasculogenic factors such as vascular endothelial growth factor (VEGF; Greene et al., 2012).

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15 We hypothesized that mares supplemented with L arginine would have increased uterine blood flow and consequently has tened uterine fluid clearance when compared to a control Increased blood flow is associated with increased lymphatic drainage, which is associated with uterine fluid clearance. The objectives of the current study were to investigate the effects of L argin ine supplementation on uterine fluid removal, uterine arterial blood flow and hemodynamics. In addition, this study investigated the effects of knowledge, this is the first s tudy to investigate the effects of L arginine supplementation on the rate of uterine fluid clearance.

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16 CHAPTER 2 REVIEW OF LITERATURE Overview of Equine Reproductive Physiology Reproductive Anatomy The main reproductive organs of the mare include the ovar ies, oviducts, uterus, and cervix. The ovary of the mare consists of three layers including the cortex, medulla, and the hilum (Berne et al., 1998). The ovary of the mare is unlike that of other farm animals. The inner zone is considered the cortex and con sists of germinal epithelium, which contains the oocytes (Berne et al., 1998, Senger, 1997). The outer layer, known as the medulla consists of heterogenous cells (Senger, 1997). The hilum is located on the convex side of the ovary and is the area of attach ment to the abdominal cavity as well as a passage for blood vessels and nerves (Senger, 1997). The follicles on the ovary are one of four types. The most immature and undeveloped follicles, primordial follicles contain immature oocytes surrounded by flat, squamous granuosa cells (Senger, 1997). Primordial follicles can lie dormant for up to 50 years in humans and 30 years in horses and have little to no biological activity (Berne et al., 1998). Primary follicles develop from primordial follicles and are cha racterized by a single layer of cuboidal cells surrounding the oocyte. At this time, primary follicles develop receptors to follicle stimulating hormone (FSH), but are still considered gonadotropin independent (Hillier, 2001). Primary follicles can then de velop into secondary follicles, which have multiple layers of granulosa cells that undergo cytodifferentiation into the theca externa and theca interna (Hillier, 2001). Tertiary follicles, also known as antral follicles are characterized by the development of a fluid filled antrum (Senger, 1997).

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17 Folliculogenesis occurs in a wavelike pattern that results in the selection of a dominant follicle (Senger, 1997). The mare is unique in that ovulation occurs at one point in the ovary known as the ovulation fossa (Senger, 1997). Mares are considered a monotocous species, meaning that they generally ovulate a single oocyte, resulting in a single embryo (Senger, 1997). Oviduct The oviduct is the passageway for oocytes from the ovary to the uterus, as well as a pathw ay for spermatozoa from the uterus to the oocyte (Pauerstein et al., 1974). The oviduct is supported in the abdomen of the mare by the mesosalpinx region of the broad ligament (Pauer s tein et al., 1974). The oviduct does not directly connect to the ovary, r ather it surrounds the ovary with a structure known as the infundibulum (Senger, 1997). The infundibulum has many ciliated projectiles that function to move the ovulated oocyte into the oviduct and towards the uterus (Pauerstein et al., 1974). Following th e infundibulum, is a region of the oviduct called the ampulla. The ampulla is a ciliated structure that functions to move the oocyte toward the site of fertilization (Pauerstein et al., 1974). Fertilization occurs at a site termed the ampullary isthmus jun ction (Senger, 1997). The distinction between the ampulla and isthmus can be seen histologically, as the isthmus is surrounded by smooth muscle cells and characterized by a smaller luminal area, while the cells lining the ampulla are ciliated to assist in transport of the oocyte towards the site of fertilization (Senger, 1997). This smooth muscle acts in a peristaltic function to push sperm cells toward the oocyte and the site of fertilization (Senger, 1997). The oviduct joins the uterus at the uterotubual junction (Pauerstein et al., 1974).

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18 Uterus The uterus of the mare is considered a bicornuate uterus with a single body and less developed horns than that of the sow (Senger, 1997). The uterus lies horizontal in the mare and is suspended by the broad ligam ent (Ginther, 1992). The arrangement of the uterus in regards to the attachment to the broad ligament allows for palpation of the uterus through the rectum (Blanchard et al., 2002). As the mare ages and parity increases, the uterus is suspended lower in th e abdomen as the broad ligament stretches and extends to accommodate gestation (Blanchard et al., 2002). The uterus is characterized by three distinct layers. The serous layer is continuous with the broad ligament and is the outermost layer of the uterus. The myometrium consists mainly of smooth muscle, and its main function is to push the foal into the birth canal during parturition. In addition, this smooth muscle is responsible for contractions required to clear the uterine lumen of fluid and during ute rine involution following parturition (Ginther, 1992). The myometrium is significantly thick and responsible for the changes in tone that are seen throughout the estrous cycle of the mare. During behavioral estrus, under the influence of estrogen, more ton e can be expected in the uterus than during anestrus (Hayes and Ginther, 1986). The innermost layer, known as the endometrium is a complex mucosal membrane containing a rich blood supply that houses and supports the developing fetus during pregnancy (Ginth er, 1992). The endometrium of the uterus is glandular and secretory in nature (Ginther, 1992). The uterus of the mare is characterized by a large uterine body anterior to the cervix with two smaller uterine horns that terminate at the oviduct (Blanchard et al., 2002). In a nonpregnant state, the uterine lumen is nearly indistinguishable and defined by dominant endometrial folds, which can be palpated through the rectum. The

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19 v iewed on an ultrasound (Blanchard et al., 2002). Uterine endometrial biopsies can be used to grade the uterus of the mare based on inflammation and fibrosis of the uterus (McKinnon and Voss, 2005). These biopsies are useful in helping to diagnose reasons for infertility in mares and create an accurate Grading is done on a scale from Grade I, which is a normal endometrium to a Grade III, which is indicative of severe inflamm ation or diffuse fibrosis (Blanchard et al., 2002). A grade of IIB results in a 10 50% less chance of conceiving or carrying a foal to term, while a grade of III indicates less than a 10% chance of carrying a foal to full term (Blanchard et al., 2002). Inc reased inflammation is also a factor in reduction in uterine fluid clearance or an increase in uterine fluid accumulation (Blanchard et al., 2002). Cer v ix The cervix of the mare is an adaptable organ that is lined with secretory epithelial cells. These ep ithelial cells secrete a thin mucus for lubrication during estrus and a thicker mucus during diestrus that is less permeable to foreign contaminants and bacteria (Senger, 1997). During estrus under the influence of estrogen the cervix can expand to accommo passage of the foal (Senger, 1997). During diestrus as well as pregnancy under the influence of progesterone, the cervix is tightly closed (McKinnon and Voss, 2005). The cervix of the mar e is characterized by longitudinal folds that extend from the endometrial folds of the uterus (Blanchard et al., 2002). The cervix of the mare is unique from the cervix of other farm animals in that its lumen can greatly expand and contract because of a th ick layer of muscular fibers as well as an absence of transverse cervical rings

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20 (Blanchard et al., 2002). These unique characteristics allow the uterus to be more easily accessed through the cervix than that of the cow, which allows for ease of artificial insemination as well as other breeding practices (Blanchard et al., 2002). Although the cervix can easily be palpated through the rectum due to its thick walled nature, it is more readily palpated under the influence of progesterone, which causes the cervi x to maintain a more rigid state (Hayes and Ginther, 1986). In contrast, under the influence of estrogen during estrus, the cervix is flaccid and difficult to palpate (Hayes and Ginther, 1986). Maintaining the integrity of the cervix, and especially the ex ternal os of the cervix, is important in regards to fluid clearance as well as fluid accumulation in the uterus of the mare (McKinnon and Voss, 2005). Cervical injuries that can occur during foaling or breeding can compromise the integrity of the uterus as well as impact the overall reproductive efficiency of the mare (McKinnon and Voss, 2005). Uterine Vasculature and Blood Flow Reproductive Vasculature The reproductive tract of the mare is suspended in the abdomen by the broad ligament (Ginther, 2007). Ar teries supplying blood to the uterus and ovaries pass through the broad ligament and connect to the ovaries and the uterus after branching off of the aorta (Ginther, 2007). The broad ligament is broken into three unique areas that are distinctive in their function and can be useful in locating individual blood vessels (Ginther, 2007). The mesometrium attaches to the uterus, while the mesovarium attaches to the ovaries, and the mesosalpinx projects from the mesovarium and supports the oviduct (Ginther, 2007)

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21 The aorta passes along the spinal column dorsal to the reproductive tract in the mare (Bollwein et al., 2002). The ovarian artery branches off of the aorta and runs dorsally along the abdominal wall until it enters through the mesovarium and connects to the ovary (Ginther, 2007). The location and anatomy of the uterine artery is unique in the mare from that of the cow in that it branches off of the aorta at a different origin (Ginther, 2007). The uterine artery extends from the external iliac artery in h orses, which is in contrast to cows and heifers where it branches from the internal iliac artery (Ginther, 2007). The uterine artery forms both a caudal and a smaller cranial branch. The uterine artery branches course along the antimesometrial border and g ives origin to branches that run over the individual horns (Ginther and Pierson, 1984). The uterus receives blood from the uterine branch of the ovarian artery, the uterine artery, and the uterine branch of the vaginal artery, and all three sources are int erconnected to varying degrees, in addition to a variation between mares (Ginther, 2007). In older, multiparous animals, the uterine artery follows a convoluted path as a result of uterine involution and stretching of the broad ligament during gestation (G inther and Pierson, 1984). The uterine artery is looser than the other arteries in support of physiological changes seen during gestation and uterine involution (Ginther, 2007). In addition, the diameter of the uterine artery changes substantially during g estation (Ginther, 2007). Ultrasound has been identified as a key tool in identifying arteries as well as measuring blood flow through the arteries (McKinnon and Voss, 2005). The uterine artery is identified on the screen of an ultrasound by determining w here the external iliac artery (EIA) branches from the aorta (Ginther and Pierson, 1984). The branching of the EIA is followed closely by the branching of the uterine artery from the EIA. The

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22 branching of the deep circumflex artery (DCA) follows closely af ter, but may originate directly from the aorta before the branching of the EIA. The uterine artery will always pass above and close to the DCA regardless of its origin (Ginther, 2007). In open mares, the artery may be barely detectable because of its small size and diameter, which ranges from 2 6 mm in mares between the ages of 6 and 13 (Ginther, 2007). Arteries and Hemodynamics Blood flow is characterized by both systemic and pulmonary circulation (Maton et al., 1993). Pulmonary circulation is defined as the portion of the cardiovascular system which pumps deoxygenated blood from the heart to the lungs via the pulmonary artery and subsequently returns oxygenated blood to the heart via the pulmonary vein (Maton et al., 1993). Systemic circulation is the fra ction of the cardiovascular system, which transports oxygenated blood away from the heart through arteries, and returns deoxygenated blood to the heart through veins (Maton et al., 1993). Doppler ultrasonography has been identified as a noninvasive method of assessing uterine blood flow in women and more recently in horses (Bollwein et al., 1998). The relationship between blood flow and other reproductive characteristics including ovulation, pregnancy, and pregnancy loss is an emerging area of research acr oss species (Bollwein et al., 1998). Research in humans has shown that low uterine blood flow and perfusion is a cause of infertility (Bollwein et al., 1998). Additionally, the quality of the uterine blood flow is important in obtaining a successful embry o transfer or artificial insemination (Bollwein et al., 1998). Age has been shown to cause a difference in blood flow of the uterine artery. Additionally, parity can influence the blood flow in the uterine artery. Older multiparous mares have been shown t o have significantly less blood flow than younger maiden

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23 mares (Bollwein et al., 1998). The period of the estrous cycle is also a significant factor in regards to blood flow, with significantly lower blood flow on day 0 10 as compared to days 15 and 20 (Bo llwein et al., 1998 ). Increased blood flow in ovarian and uterine arteries has been shown to hasten uterine involution (Mortensen et al., 2011). Mares with increased blood flow to reproductive tissues showed hastened uterine involution when measured as u terine body diameter as well as gravid and non gravid horn diameter (Mortensen et al., 2011). Additionally, mares supplemented with L arginine displayed a decrease in days with measurable uterine fluid ( 3.4 1.5 d) as compared to control mares (7.11 3.1 d ) ( Mortensen et al., 2011 ). Blood Flow Indices Blood flow can be measured by obtaining waveform velocities and reflected through a Doppler index (Ginther, 2007). These indices are ratios of velocity measurements, which make them independent of Doppler an gles (Alcazar, 2004). Doppler angle, also known as insonation angle is important in measuring blood flow through the use of Doppler ultrasound (Dav and Milner, 2000). Insonation angle is the angle between the Doppler ultrasound beam and the direction of b lood flow in a vessel (Dav and Milner, 2000). The Doppler instrument identifies only the blood flow velocity component being directed straight towards the transducer along the Doppler ultrasound beam (Dav and Milner, 2000). The relationship is equal to V cos A, where V is the true blood flow velocity in the vessel and A is the Doppler angle (Dav and Milner, 2000). An error in the measurement of the Doppler angle causes an error in the estimation of the true blood flow velocity that increases as the error in Doppler angle increases (Dav

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24 and Milner, 2000). Use of indices including pulsatility and resistance indices allows for accurate blood flow measurements without the acquisition of an insonation angle. Waveform velocities are depicted on ultrasound dis plays as velocity displays (Ginther, 2007). The changes in the Doppler shift frequencies and the signal amplitudes are displayed as a waveform, representing a cardiac cycle depicting the pulsatile nature of arterial blood flow (Ginther, 2007). The maximum point in the traced outline of a spectrum represents the peak systolic velocity (PSV), while the low point before the next systolic increase represents the end diastolic velocity (EDV) (Ginther, 2007). An average of the maximum values over a single cardia c cycle is known as the timed average maximum velocity (TAMV) (Ginther, 2007). The resistance index (RI) is a routinely used Doppler index (Ginther, 2007). The RI relates the extent of resistance in the tissues and the extent of vascular perfusion (Ginthe r, 2007). A higher RI will result in less blood flow through the individual vessel. Conversely, a lower RI is indicative of increased blood flow. Resistance index is calculated as follows: Resistance Index (RI)= Peak Systolic Velocity (PSV) End Diastolic Ve locity (EDV) Peak Systolic Velocity (PSV) Another widely used index in determining blood flow is the pulsatility index (PI). Pulsatility index measures the difference in PSV and ESV as it relates to the timed average mean ve locity (TAMV) (Ginther, 2007). An increase in PI is indicative of decreasing perfusion of the distal tissues (Ginther, 2007). Pulsatility index and RI are highly correlated (r > 0.9) and only one is usually necessary when measuring blood flow (Ginther, 200 7). Pulsatility index is calculated as follows:

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25 Pulsatility Index (PI)= Peak Systolic Velocity (PSV) End Diastolic Velocity (EDV) Timed Averaged Maximum Velocity (TAMV) Arginine Arginine Requirement Arginine is an amino ac id that is found in many different proteins in the body. Proteins are a major component of almost all tissues in the body, as well as, enzymes, hormones, and other substances (NRC, 2007). Horses like other nonruminants do not have a protein requirement, ra ther they have a requirement for individual amino acids (NRC, 2007). However, with the exception of lysine, the requirements for individual amino acids have not been determined in the horse (NRC, 2007). Arginine is considered one of the ten essential amin o acids to the horse in addition to histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine (Efron and Barbul, 2000). Essential amino acids cannot be synthesized by the body in sufficient quantities and must be supplied by the diet (NRC, 2007). Arginine is not considered a limiting amino acid, which is an amino acid that if deficient will impact the effectiveness of other amino acids (Efron and Barbul, 2000). Arginine Properties and General Functions The amino ac id side chain of arginine consists of a 3 carbon aliphatic straight chain, while the distal end is capped by a guanidium group. Arginine imparts basic qualities and allows for the formation of multiple hydrogen bonds (Tapiero et al., 2002). Arginine is on e of the twenty most common natural amino acids, but is considered an essential amino acid and needs to be supplied by the diet (Tapiero et al., 2002). The common form seen in the body is L arginine (Tapiero et al., 2002). L arginine has many functions in the body and has many health benefits including functioning as a precursor

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26 for nitric oxide, as a component in collagen and enzymes, as well as the synthesis of important proteins including creatine and insulin (Evoy et al., 1998). L arginine has also been suggested to play a role as a minor antioxidant (Evoy et al., 1998). L arginine is a major component of seminal fluid and is important for maintaining a healthy ejaculate volume. As a result, it has been deemed as an important factor in sperm motility (Ke ller and Polakoski, 1975). Research into the role of arginine and reproductive efficiency of stallions has not been conducted. L arginine can also function to remove excess ammonia from the body via the urea cycle and help to maintain whole body nitrogen b alance (Visek, 1986). Arginine supplementation is gaining support in the equine world because of its reputation as a potent vasodilator (Clarkson et al., 1996). Vasodilation is not only important in delivering oxygen rich blood to muscles, but also in the removal of lactic acid which can have a negative effect on the performance of the horse (Wu and Meininger, 2000). The performance horse industry is also interested in arginine (Sewell and Harris, 1995). Creatine is a nitrogenous organic acid that aids in the production of energy rich ATP molecules (Sewell and Harris, 1995). It is important to remember that the daily requirements of L arginine in the horse are unknown, so supplem entation may simply be meeting the requirements necessary for normal function. Arginine Synthesis In the body, arginine can be synthesized from L glutamine, proline, citrulline, and ornithine (Wu and Morris, 1998) (Figure 2 1). Many of the enzymes required to synthesize L arginine are found in c ells through out the body; however certain enzymes are restricted to the liver and intestinal mucosa (Wu and Morris, 1998). Endogenous

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27 arginine synthesis varies by species as well as age, nutritional status and devel opmental stage (Wu and Morris, 1998). At birth, the intestine is the major site of arginine synthesis, but as the animal ages, the intestine becomes a major site of citrulline production as intestinal arginase production increases (Wu and Morris, 1998). De spite recent interest in arginine, very little is known about the regulation of intestinal citrulline and arginine synthesis (Wu and Morris, 1998). Approximately 60% of net endogenous arginine synthesis occurs in the kidney (Wu and Morris, 1998). Citrulli ne is extracted from the blood and converted to arginine by the enzymes arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) (Wu and Morris, 1998). A correlation between renal citrulline uptake and renal arginine output has been demonstrated in a dult humans and rats (Wu and Morris, 1998). This correlation means that in vivo arginine synthesis in the kidney is closely associated to citrulline production in other organs. The liver is another major site of arginine synthesis (Wu and Morris, 1998). A rginine synthesis occurs in hepatic cells via the hepatic urea cycle (Wu and Morris, 1998). Arginine synthesis by the liver is only accomplished when urea cycle intermediates such as ornithine are produced in sufficient quantities (Wu and Morris, 1998).

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28 Figure 2 1. L Arginine Synthesis as adapted from Wu and Morris, 1998 Relationship b etween Arginine and Citrulline A strong relationship exists between the amount of citrulline in the system and the amount of arginine that is produced (Wu and Morris, 1998) Citrulline is co produced with nitric oxide (NO) as a product of the breakdown of arginine by nitric oxide synthase (NOS; Wu and Morris, 1998). In addition, citrulline can be recycled back to arginine via the citrulline/NO pathway (Figure 2 2). The enzym es ASS and ASL are necessary for this recycling and are present in most cell types. Figure 2 2 also depicts the products of the catabolism of L arginine. Arginine is a major precursor for NO (Wu and Morris, 1998). A byproduct of the catabolism of L argini ne is citrulline, which can then be converted back to L arginine through the citrulline/NO cycle (Wu and Morris, 1998).

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29 Figure 2 2. Citulline/Nitric Oxide cycle as adapted from Wu and Morris, 1998 Effects of Arginine and Nitric Oxide on Reproduction an d Blood Flow Arginine is a major precursor for NO and is considered a NO donor molecule (Wu and Morris, 1998). Nitric oxide is produced from L arginine by a family of enzymes known as nitric oxide synthases (Wu and Morris, 1998; Table 2 1). Nitric oxide is an important cellular signaling molecule that has many functions including acting as a signal to control vascular tone and angiogenesis (Wu and Morris, 1998). Table 2 1. Nitric Oxide Synthase Profiles and their Actions Name Location Function Neuronal N OS (nNOS) Nervous tissue Skeletal muscle Cell communication Inducible NOS (iNOS) Immune system Cardiovascular system Immune defense Endothelial NOS (eNOS) Endothelium Vasodilation Nitric oxide generated from L arginine by the vascular endothelium is th ought to play a major role in the regulation of vascular tone, which affects blood pressure, blood perfusion, and blood flow (Amit et al., 1998). The endothelium of blood vessels uses NO as a signaling molecule, triggering relaxation of smooth muscle surro unding blood

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30 vessels, resulting in dilation of blood vessels and increased blood flow (Amit et al., 1998). Research has shown that an increase in a NO donor such as L arginine increases endothelial production of NO (Amit et al., 1998). The effect of L arg inine supplementation on enhancement of reproductive function is a current topic that is under review by many people. Gilts supplemented with 1.0% L arginine from d 30 of gestation until parturition had a 22% increase in number of pigs born alive, as well as 24% increase in litter birth weight (Mateo et al., 2007). Although NO was not measured, the authors speculated that these increases could be due to the angiogenic effects of NO on the placenta of pigs during gestation (Mateo et al., 2007). Supplementat ion of pregnant mice with 2% L arginine resulted in an increase in weight gain during the latter one third of gestation, total litter size, number of pups born alive, litter birth weight, and litter weight of pups born (Greene et al., 2012). Additionally, the transcriptional activity of Vegfr2, a receptor for vascular endothelial growth factor (VEGF), a potent endothelial growth factor was shown to increase in fetoplacental tissues after L arginine supplementation (Greene et al., 2012). L arginine supplemen ted to pregnant gilts at 0.8% of the diet for 25 days enhanced the vascularity of the chorionic and allantoic membranes compared to control groups (Li et al., 2010). Enhancement of vascularity in the placenta has demonstrated improvements in number of anim als born alive as well as provides an area to examine in order to fully understand the effects of L arginine supplementation during pregnancy. The role of NO on uterine blood flow has been investigated in vivo in the porcine model ( Barszczewska et al., 200 5). A bolus of NO donor was injected into the uterine artery of pigs during the estrous period. Results from these studies, including blood

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31 pressure measurements, indicate that NO is an important factor in the regulation of blood flow through the porcine r eproductive tract during estrus regardless of the period of the estrous cycle. (Barszczewska et al., 2005). Sheep pregnant with multiple fetuses administered L arginine parenterally, had a 23% decrease in the number of lambs born deceased and a 59% increas e in the number of lambs born alive compared to control (Lassala et al., 2011). This enhancement of pregnancy outcome was attributed to the increased circulating levels of arginine, ornithine, cysteine, and proline (Lassala et al., 2011). Research into th e reproductive benefits of arginine in horses has been limited to a few studies. Supplementation of 1.0% L arginine to pregnant mares beginning 21 d prior to expected foaling date has been shown to increase blood flow in the uterine and ovarian arteries du ring the early postpartum period (Mortensen et al., 2011). Additionally, postpartum mares displayed a significant reduction in the amount of fluid present in the uterus as well as an increase in uterine arterial blood flow to the formerly gravid uterine ho rn when fed L arginine as compared to control mares (Kelley et al., 2011). Uterine Fluid Fluid Accumulation Endometritis has long been associated with a decrease in the fertility of mares, and free fluid accumulation is a significant indicator of endometr itis (McKinnon and Voss, 2005). Approximately 15% of Thoroughbred mares develop endometritis, resulting in accumulation of uterine fluid due to impaired uterine clearance mechanisms (Drost et al., 2002) Reproductively normal mares respond to an infection i n the uterus by activation of the immune system as well as increased uterine luminal contractions to

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32 evacuate the contents of the uterus (McKinnon and Voss, 2005). Mares that are unable to do this are generally less reproductively fit and are susceptible t o a decrease in fertility (Blanchard et al., 2002). Ultrasonographic examination is a useful tool in estimating the quantity of fluid in the uterine lumen (McKinnon and Voss, 2005). Ultrasound is a beneficial tool in deciding whether or not to breed a mar e given the condition of the uterus. Uterine fluid can easily be identified on the display of an ultrasound and is distinguishable as a dark area inside the lumen of the uterus (McKinnon and Voss, 2005). Uterine fluid can be categorized by appearance on ul trasound with grades ranging from I IV, with grade I being white (hyperechoic) to grade IV being black (anechoic) on the display (McKinnon and Voss, 2005). A small amount of anechoic fluid in the lumen of the uterus was once thought to have no impact on r eproductive performance and was normal (Blanchard et al., 2002). However, controlled research studies as well as case study observations have determined that even small amounts of uterine fluid c an adversely affect fertility (Blanchard et al., 2002). Fluid is most commonly seen during diestrus and immediately following ovulation, however any fluid seen in the uterine lumen should be considered abnormal (Blanchard et al., 2002). Accumulation of fluid is indicative of endometritis, and can identify mares that have impaired ability to mechanically evacuate the uterus (Blanchard et al., 2002). The degree of fluid echogenicity is a function of the concentration of inflammatory cells and debris (McKinnon and Voss, 2005). Hyperechoic fluid is characterized by a hig her concentration of debris and immune cells, normally resulting from an infection.

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33 Some mares also generate anechoic fluid that is sterile in nature (Blanchard et al., 2002). This fluid can reduce fertility and decrease the chances of successful fertiliza tion (McKinnon and Voss, 2005). It has been hypothesized that these mares have deficient lymphatic drainage of endometrial edema that causes it to collect in the uterine lumen (Blanchard et al., 2002). This anechoic fluid can become hyperechoic quickly if not treated and lead to increased endometritis and decreased fertilityn (Blanchard et al., 2002). Uterine fluid accumulation has been determined to be associated with increased early embryonic death as well as reduced day 50 pregnancies (McKinnon and Voss, 2005). Additionally, fewer mares become pregnant when uterine fluid is present during the first postpartum ovulatory period also known as foal heat (McKinnon and Voss, 2005). Methods of Fluid Removal Prudent management is the preferred way of maintaining a reproductively sound mare with a better chance of conception and maintenance of pregnancy to term (Blanchard et al., 2002). The use of ultrasound is important in identifying mares that may be predisposed to accumulating uterine fluid (Blanchard et al., 2002). Ultrasonography is also useful in determining the quality of the fluid present, which aids in determining the preferred course of corrective action (Blanchard et al., 2002). Additionally, the use of uterine endometrial biopsies as described previous ly is a key management technique that can help identify mares that may be predisposed to endometritis and fluid accumulation (McKinnon and Voss, 2005). Uterine lavage is a technique that is routinely used to remove fluid, especially hyperechoic fluid from the uterus (McCue and Hughes, 1990). Uterine lavage is usually

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34 performed before breeding or 4 8 hours after breeding (McCue and Hughes, 1990). Uterine lavage functions by expanding the uterine lumen through the use of sterile fluid and subsequently flushi ng the infused fluid out of the uterus, along with to the hyperechoic fluid containing neutrophils, antigens, and debris (McCue and Hughes, 1990). Research has shown that uterine lavage performed after breeding does not decrease pregnancy rates or interfer e with sperm in the oviduct (McCue and Hughes, 1990). Uterine lavage is normally conducted in conjunction with an infusion of antibiotics post infusion (McCue and Hughes, 1990). The most routine route of uterine fluid removal is through the use of ecbolic s (Blanchard et al., 1991). Ecbolics such as oxytocin or prostaglandins are administered to stimulate uterine contractility and expulsion of uterine contents (Blanchard et al., 1991). Administration of oxytocin has been shown to be safe for 2 3 days follow ing ovulation, but administration of prostaglandins post ovulation has been shown to reduce the function of the corpus luteum and negatively affect the ensuing estrous cycle (Blanchard et al., 2002). In addition, the use of prostaglandins has been associat ed with increased embryonic death in subsequent ovulations (Blanchard et al., 2002). Ecbolics are also used to promote uterine clearance without the use of uterine lavage (Blanchard et al., 2002). Mares that are susceptible to uterine fluid accumulation ma y have an inability to clear the uterine lumen of fluid without the administration of ecbolics (Blanchard et al., 2002). Current industry standards are the administration of 20 IU oxytocin intramuscularly once or twice a day (Blanchard et al., 2002). Howev er, case study reports from Florida have shown that former industry standards of multiple

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35 injections of oxytocin 4 6 hours apart can cause uterine spasms that are unproductive in eliminating uterine fluid (Blanchard et al., 2002). Naturally, oxytocin is s ynthesized in the hypothalamus and secreted by the posterior pituitary gland (Gimpl and Fahrenholz, 2001). Smooth muscle cells in the uterus contain receptors that bind to oxytocin (Gimpl and Fahrenholz, 2001). Receptor number is increased late in pregnanc y and in some animals oxytocin injections are given to facilitate parturition. However this is not normally the case in horses, as induction of early parturition can lead to foal death (Blanchard et al., 2002). Oxytocin binds to a G protein coupled recepto r and causes contraction through second messengers (Gimpl and Fahrenholz, 2001). The half life of oxytocin is relatively short in the blood, averaging about three minutes (Gimpl and Fahrenholz, 2001). The study presented in this thesis was undertaken to de termine if an increase in blood flow attributed to L arginine supplementation would accelerate uterine fluid clearance. Previous research has demonstrated that L arginine supplementation hastened uterine involution (Mortensen et al., 2011) and increased ut erine blood flow (Kelley et al., 2011). The increase in blood flow may permit more rapid clearance of fluid from the uterus. The objectives of the current study were as follows: Determine the effect of L arginine supplementation on uterine artery blood flo w Determine the effect of L arginine supplementation on uterine fluid clearance Determine the effect of L arginine supplementation on follicular and ovulatory dynamics.

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36 CHAPTER 3 METHODS AND MATERIAL S Animals Twelve non pregnant light horse mares (mean SE, 540.5 56.5 kg) were used for this study The experimental protocol was reviewed and approved by the Institute of Food and Agricultural Sciences (IFAS) Animal Care and Use Committee at the University of Florida. Mares were maintained on two pastures and housed at the IFAS pasture throughout the supplementation period. While on pasture, mares had free choice access to water and salt blocks. Mares were confined in 3.6 x 3.6 m box stalls at 0730 and 1500 h for approximately 30 min to facilitate feeding. Mares scheduled to be examined or weighed were removed from their stalls and placed in small paddocks devoid of vegetation, but with free choice access to water and Coas tal bermudagrass hay for approximately 1 h. During ultrasound examinations, mares were moved into a covered barn equipped with fans to facilitate air circulation and placed in individual stocks. Experimental Design The study was designed as a 3x3 Latin Sq uare, evaluating 3 dietary treatments over 3 consecutive periods, permitting all mares to undergo all treatments. Each period consisted of one estrous cycle. To accommodate frequent and timely ultrasound examination, mares were initially blocked by age (10 .5 5.8 y; range 3 22 y ) and breed (Thoroughbred (n=5), and stock type (n=7)), then randomly assigned to one of four estrous synchronization groups (3 mares per group) Synchronization of mares was accomplished using a single dose of Lutalyse (dinoprost tromethamine; Pfizer Inc.,

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37 New York City, NY) injected IM followed by daily oral administration of 0.044mg/kg BW Regumate (altrenogest; Intervet Inc., Summit, NJ) for 2 wk, and finally a second dose of Lutalyse injected IM on d 15. Synchronization of mar e g roups was staggered at 3 to 5 d interval s, and each group had equal representation of the three dietary treatments. Dietary Treatments Mares in each synchronization group were randomly assigned to one of three dietary treatments in a 3X3 Latin Square de sign: arginine supplementation (ARG), urea supplementation (UREA), or no supplementation coupled with oxytocin administration (OXY). Arginine was supplied as L arginine (Ajinomoto AminoScience LLC, Ral eigh, NC) at a rate of 200 mg/kg BW/d. This amount is b ased on the rate of supplementation used by Mateo et al. (2007) in pregnant gilts, and represents approximately 1% of estimated DM intake for a non pregnant mare. A feed grade source of urea was used for the UREA treatment, which served as an isonitrogenou s control. Urea was fed at a rate of 114 mg/kg BW/d. No dietary supplementation was provided to mares on the OXY treatment. Supplementation rates were determined from body weights obtained on d 7 post ovulation in each period. While on the ARG treatment, a pproximately 75% of daily L arginine intake originated from the ARG supplement. The basal diet consisted of ad libitum access to mixed bahiagrass pasture and Coastal bermudagrass hay and 0.5% BW/d of a grain mix concentrate formulated for gestating and la ctating mares ( The basal diet was designed to meet or slightly exceed the nutrient requirements of horses at maintenance (NRC, 2007). ARG and UREA supplements were hand mixed into the concentrate portion of the ration and fed once daily at 1500 h. Dietary treatment began 10 d post ovulation and continued until all fluid was absent in the uterus as determined

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38 by ultrasound examination (approximately 12 d total of supplementation). Nutrient composition of basal fee ds and supplements is presented in Table 3 1. Uterine Infusion Mares were initially evaluated by transrectal ultrasonography to map follicular development beginning 13 d after ovulation. Examinations were conducted once daily until a 33 mm follicle was ob served. Once a follicle of this size was observed mares received a uterine infusion consisting of 880 mL of sterile saline (0.9% NaCl) mixed with 120 mL of semen extend er (E Z Mixin OF, Animal Reproductive Systems, Chino, CA). This fluid mixture was det ermined based on preliminary studies measuring rate of fluid clearance over a range of fluid volumes. The fluid mixture was prepared on the day of infusion and stored at 4 C until the time of infusion. Sterile tubing was passed through the cervix of the ma re and into the uterine body. Tubing was secured through the use of an air filled bladder, and fluid was gradually infused at a rate of approximately 300 mL/min. Completion of the infusion was considered time=0 h. Mares assigned to the OXY treatment receiv ed 20 IU oxytocin (AgriLabs, St. Joseph, MO) administered IM immediately after fluid infusion. Mares were examined via ultrasound at 12 h intervals to record uterine blood flow and fluid disappearance. When all fluid was cleared from the uterus, dietary tr eatment was terminated, and mares continued to undergo once daily ultrasound evaluation When no discernible fluid was detected for 48 h, all mares received 20 IU oxytocin administered IM to ensure total fluid clearance before starting the next treatment p eriod. When the next ovulation was confirmed by presence of a corpus luteum, treatments were switched and mares began a new dietary treatment 10 d post ovulation. The procedures described above were repeated until all mares had completed all treatments.

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39 D oppler Ultrasonography All uterine and ovarian examinations were conducted transrectally using a digital color Doppler ultrasound with a 10 5 MHz broadband 52 mm linear probe (Micromaxx, Sonosite, Bothell, WA). Examinations were conducted by a single oper ator that was not blinded to treatment. Prior to discovery of a 33 mm follicle, only follicle size and presence of a corpus luteum were evaluated on both the left and right ovary. Once a 33 mm follicle was discovered and the mare had received the uterine i nfusion, follicle size of only the dominant follicle, amount and location of uterine fluid, and blood flow of the uterine artery were recorded at each exam. The amount of uterine fluid was estimated by measurement of the largest pocket of fluid. Location o f the fluid was noted as either uterine body or left or right uterine horn. Spectral Doppler measurements of both uterine arteries were calculated by the algorithm package in the Micromaxx ultrasound unit. The sample cursor gate was set at 5 mm and at an initial magnification depth of 7.7 cm. The measurements taken included resistance index (RI) [(peak systolic velocity (PSV) end diastolic velocity (EDV))/PSV] and pulsatility index (PI) [(PSV EDV)/ time averaged maximum velocity (TAMV)] (Ginther, 2007). Uterine arteries were identified as described by Bollwein et al. (1998), with measurements taken near the branching of the external iliac artery or deep circumflex artery or both. Statistical Analysis Blood flow and rate of fluid clearance data were analy zed using the PROC MIXED procedure of SAS (Version 9.2, SAS Institute Inc., Cary, NC.) with repeated measures. Latency to corpus luteum development, latency to ovulation, and latency to total fluid clearance were evaluated using the PROC GLM procedure of SAS (Version 9.2, SAS Institute Inc., Cary, NC.). The Kolmogorov Smirnov for multiple comparisons was used

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40 to test for normal distribution for follicular characteristics. The LS MEANS statement of PROC MIXED was used to compare the treatment means. All dat a are expressed as the mean SE. Differences were considered significant at P and trends for significance were acknowledged at P <0.10. One mare completed her first treatment cycle (UREA), but was removed from the study when she failed to produce a 33 mm follicle in the subsequent ovulatory cycle. Data collected from this mare while on the UREA treatment was retained in the statistical analyses, but no data was generated or included for this mare on the ARG and OXY treatments.

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41 Table 3 1. Nutrient composition of basal diet feeds and L arginine and urea supplements Nutrient a Grain Bermudagrass Hay L arginine Supplement Urea Supplement DE Mcal/kg 3.47 1.87 --Crude fat, % 4.2 2.3 --Crude protein, % 16.8 9.4 --NDF, % 20.8 75.4 --L arginine, % 1.26 0.01 99.0 -Urea, % -b --99.7 a Values presented on a 100% DM basis. b Not determined.

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42 CHAPTER 4 RESULTS Feeding and Supplementation Mares consumed both the ARG and UREA supplements with little to no initial refusal. In addition, mares maintained BW during the study (data not shown). Total days of supple mentation (15 1.8 d) did not differ between treatments. Latency to Total Fluid Clearance Uterine fluid was absent in all mares prior to uterine infusion, indicating all mares began each treatment period with similar uterine fluid status. On average, total fluid clearance occurred within 53.8 4.9 h (Figure 4 1). Dietary treatment had no effect on average latency to total fluid clearance ( P =0.7075) (Figure 4 1). Rate of Fluid Clearance Fluid clearance was influenced by time ( P <0.0001) and time*treatment ( P =0.0088) and tended to be affected by treatment ( P =0.0925; Figure 4 2). Mares undergoing the OXY treatment cleared fluid faster ( P =0.0388) than those receiving ARG. There was also a trend to clear fluid faster ( P =0.0100) when receiving OXY than UREA. Rate of fluid clearance was not different between ARG and UREA. Fluid clearance from the uterus was steady from 0 through 48 h across all treatments, with the amount of fluid remaining at each 12 h interval significantly less than the previous measurement ( P <0 .0001). Latency to Follicle Development and Ovulation On average mares developed a 33 mm follicle within 8.3 1.2 d of treatment initiation. The interval between the development of a 33 mm follicle from one estrus to

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43 the next averaged 21.7 2.1 d across treatments. Dietary treatment had no effect on follicle development (Table 4 1). On average, mares ovulated within 12.4 1.9 d of treatment initiation. The interval between ovulations during the study period averaged 22.0 1.4 d across treatments. Dieta ry treatment had no effect on ovulatory dynamics (Table 4 1). Blood Flow Across treatments, mean resistance index was 0.68 0.03 and 0.70 0.03 for the ovulatory and nonovulatory uterine artery, respectively. Resistance indices in the ovulatory and nono vulatory uterine arteries were unaffected by dietary treatment (Figure 4 3 and Figure 4 4). The pulsatility index of the ovulatory uterine artery was affected by time ( P <0.0001), where the PI was higher ( P <0.0001) immediately after infusion (0 h) compared to all subsequent measurements, regardless of treatment. Similarly, the PI of the nonovulatory uterine artery showed a trend for a time effect ( P =0.0830), where the PI was higher immediately after infusion (0 h) compared to all subsequent time points ( P <0 .05). The PI of the ovulatory and nonovulatory uterine arteries were not affected by treatment or the time*treatment interaction (Figure 4 5 and Figure 4 6).

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44 Figure 4 1. Mean ( SEM) time (h) to total fluid clearance from the uterus in mares supplement ed with L arginine, urea, or no supplement combined with oxytocin administration. Overall effect of treatment ( P =0.7075)

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45 Figure 4 2. Mean ( SEM) diameter (mm) of fluid present within the uterus of mares supplemented with L arginine, urea, or no suppl ement combined with oxytocin administration. Diameter was measured as the largest width of fluid observable via ultrasound examination. Overall effects of time ( P <0.0001), treatment ( P =0.0925), and time*treatment ( P =0.0088). A pound sign (#) indicates a ti me*treatment effect as OXY
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46 Figure 4 3. Mean ( SEM) resistance index of the nonovulatory uterine artery following uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytoc in administration. Overall effects of time ( P =0.9447), treatment ( P =0.3789), and time*treatment ( P =0.4070).

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47 Figure 4 4. Mean ( SEM) resistance index of the ovulatory uterine artery following uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. Overall effects of time ( P =0.6308), treatment ( P =0.7755), and time*treatment ( P =0.8952).

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48 Figure 4 5. Mean ( SEM) pulsatility index of the nonovulatory uterine artery following uterine infusion i n mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. Overall effects of time ( P =0.0830), treatment ( P =0.7827), and time*treatment ( P =0.1644).

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49 Figure 4 6. Mean ( SEM) pulsatility index of the ovulatory ute rine artery following uterine infusion in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration. Overall effects of time ( P <0.0001), treatment ( P =0.3054), and time*treatment ( P =0.3421). An asterisk (*) indicates a ll time points different from 0 h.

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50 Table 4 1. Follicular and ovulatory dynamics in mares supplemented with L arginine, urea, or no supplement combined with oxytocin administration Measurement Arginine Oxytocin Urea SEM P Value Days to develop a 33 mm fo llicle since treatment initiation 8.3 7.9 9.3 1.2 0.7180 Days to develop a 33 mm follicle since the previous 33 mm follicle 20.7 22.2 22.3 2.1 0.8603 Days to ovulation since treatment initiation 12.0 12.2 13 1.2 0.8196 Ovulatory interval, days 22.0 22. 2 21.8 1.4 0.9714

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51 CHAPTER 5 DISCUSSION The results of the current study demonstrated that uterine arterial blood flow could successfully be measured in nonpregnant mares using Doppler ultrasound. However, short term dietary supplementation with L arginin e had no effect on uterine arterial blood flow nor the rate and latency of uterine fluid clearance in non pregnant mares. The findings of this study contradict previous work in postpartum animals, which is likely related to the use of nonpregnant mares in the present study. For example, Mortensen et al. (2011) began feeding mares L arginine 21 d prior to expected foaling and documented increases in uterine arterial blood flow, accelerated uterine involution, and more rapid fluid clearance in the early postp artum period. Significant changes to the uterine environment during and immediately following pregnancy are well documented (Silva et al., 2011; Ousey et al., 2012). These changes occur in the uterus and the cervix in preparation for parturition, and serv e as a repair mechanism for uterine involution following parturition. A study investigating nitric oxide synthase (NOS) isoforms in pregnant rats demonstrated that progesterone played a significant role in regulating NOS expression in the uterus and cervix (Ali et al., 1997). Another study investigated uterine blood flow in pregnant ewes and found an increase in blood flow to ovaries containing a progesterone producing corpus luteum when compared to ovaries without a corpus luteum (Rosenfeld et al., 1974). The current study evaluated blood flow and fluid clearance during estrus with the presence of a dominant follicle on the ovary. The follicle produces estrogen, which is associated with low progesterone levels until a spike in luteinizing hormone signals fo r ovulation to occur. The absence of a progesterone producing corpus luteum in the nonpregnant mares

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52 used in the current study could be one contributing factor as to why no changes in uterine blood flow were observed in response to L arginine supplementati on. Changes in the vascular architecture are also common during pregnancy (Rosenfeld et al., 1974). In nonpregnant mares, the uterine artery ranges in diameter from 2 6 mm (Ginther, 2007). In pregnant mares, the uterine artery diameter increases significa ntly (Ousey et al., 2012). The change in uterine artery diameter is related to a decrease in resistance index (RI), which is associated with an increase in blood flow and decreased vascular resistance (Ousey et al., 2012). It may be that the uterus undergo es some type of priming event before pregnancy that allows it to make all necessary changes required to support gestation. The conceptus has also been identified as a key factor in modulating endometrial tissue remodeling and vascular development in mares (Silva et al., 2011). Silva et al. (2011) observed an increase in the area occupied by blood vessels in early pregnancy and preimplantation in mares, as well as an increase in the endometrial mRNA abundance of numerous angiogenic factors. The authors concl uded that the conceptus played a role in directing angiogenesis in the uterus. The use of non pregnant mares in the current study would have had no conceptus mediated angiogenesis. Placental development is a characteristic of uterine change during pregnan cy. The placenta of the mare is characterized as a diffuse, epitheliochorial placenta (Senger, three maternal uterine layers maintained (Senger, 1997). The placenta is supplied with nutrients through vasculature connections in microcotyledons consisting of highly vascularized chorionic villi, which extend into invaginations of the endometrium (Senger,

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53 1997). Placental development is a process that includes a large amount of angiogenesis and angiogenic factors that are absent in the nonpregnant mare. A study demonstrated that L arginine supplemented mice had increased transcriptional activity of vascular endothethial growth factor receptor 2, an important angiogenic compou nd, in the fetoplacental unit (Greene et al., 2012). Changes seen in these studies would not be displayed in the current study due to the use of non pregnant mares. The vascular remodeling occurring in the placenta downstream of the uterine artery can incr ease uterine artery blood flow upstream where it is being measured. The lack of placental vascular remodeling in the nonpregnant mares used in this study may be a factor in explaining a failure to influence blood flow with L arginine supplementation. One of the objectives of the current study was to determine the effect of L arginine supplementation on uterine fluid clearance. The authors hypothesized that mares supplemented with L arginine during the estrous period would display increased uterine arterial blood flow and subsequent hastened uterine fluid clearance. There are numerous factors that can influence the rate of uterine fluid clearance of mares including endometrial quality, cervical integrity, and general arrangement of the uterus in the abdomina l cavity. Endometrial biopsy scores are used to help to determine mares that are susceptible to endometritis. Mares susceptible to endometritis are also susceptible to accumulation of uterine fluid (McKinnon and Voss, 2005). The integrity of the cervix is a major factor in uterine fluid clearance and is influenced by the age and parity of the mare, as well as hormone status. As a mare ages, and parity increases, cervical integrity may decrease due to an increase in the number of parturitions as well as pos sible injury to the cervix during parturition. Period

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54 of the estrous cycle is also influential due to the varying levels of reproductive hormones. In this study, uterine infusion occurred during the estrus period of the estrous cycle when the cervix, under the influence of estrogen, was loose and open. This period of the estrous cycle was chosen to simulate post breeding uterine fluid accumulation, but it may be necessary to evaluate fluid clearance during the diestrus period, when the cervix, under the inf luence of progesterone, is closed tightly. A high degree of variability between horses was noted in this study; thus, a larger number of horses are likely required to ascertain the effects of L arginine supplementation on blood flow to reproductive tissue s in nonpregnant mares. Additionally, a larger n umber of mares may allow for improved understanding of uterine fluid clearance in nonpregnant mares. Finally, this study demonstrates the effectiveness of the ecbolic oxytocin on uterine fluid clearance. App roximately 33% of uterine fluid was absent 12 h post uterine infusion through the use of a single intramuscular administration of 20 IU oxytocin. Current industry standards recommend administration of 20 IU oxytocin intramuscularly every 12 hours for total fluid clearance. This study confirms that the use of oxytocin to aid in uterine fluid clearance is beneficial and successful.

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55 CHAPTER 6 CONCLUSIONS The current study demonstrated that supplementation of L arginine to nonpregnant mares had no significant impact on the rate of uterine fluid clearance or uterine arterial blood flow. Supplementing the diets of pregnant animals with L arginine has been shown to enhance reproductive function. In contrast to those studies, this study suggests that in order for the effects of L arginine on uterine blood flow to be observed, the uterus must be primed and the animal must be pregnant. The main objective of the current study was to investigate the effect of L arginine supplementation on uterine fluid clearance in ma res. Because all mares are different it is important to understand the different causes of uterine fluid accumulation. Some mares may be more susceptible to uterine fluid accumulation and less able to sufficiently evacuate the uterus of fluid. Additional studies should be attempted to mimic uterine fluid build up throughout the estrous cycle and to determine whether L arginine can hasten its removal. The current study demonstrates that the supplementation of L arginine to nonpregnant mares may not be an ap propriate model for the evaluation of uterine fluid clearance and uterine blood flow compared to the postpartum mare. Supplementation of L arginine to postpartum mares may be beneficial in improving rebreeding efficiency during the foal heat period as an a id in uterine involution. Any improvement in foal heat rebreeding efficiency would allow the producer to maintain a schedule of one foal/mare/year, resulting in a positive economic impact. The small number of horses investigated in this trial could have im pacted the results and future trials should use greater numbers of horses.

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56 Finally, investigation on a molecular scale should be completed to better understand the effect that L arginine has on transcriptional regulation of nitric oxide synthase as well as other angiogenic factors in reproductive tissues.

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57 LITERATURE CITED Alcazar J. L 2004. Doppler assesement of t he early intrauterine pregnancy hemodynamics. Ultrasound Rev. obstet. Gynec 4:165 172 Ali, M., I. Buhimschi, K. Chwalisz, and R. E. Garfiel. 1997. Changes in expression of the nitric oxide synthase isoforms in rat uterus and cervix during pregnancy and parturition. Mol. Hum. Reprod 3(11):995 1003. Amit, A., I. Thaler, Y. Paz, and J. Tiskovitz Eldor. 1998. The effect of a nitric oxide donor on Doppler flow velocity waveforms in the uterine artery during the first trimester of pregnancy. Ultrasound Obstet Gynecol 11:94 98. Barszczewska, B., W. Markiewicz, T. Maslanka, M. Chrotowska, and J. J. Jaroszewski. 2005. Influence of nitric oxide on the blood flow in the porcine uterine artery: an in vivo study. Pol J Vet Sci 8:195 200. Berne, R. M., M. N. Levy B. M. Koeppen and B. A. Stanton. 1998. Physiology 4 th Ed. Mosby St. Louis, MO Pp 987 988 Blanchard, T. L., D. D. Varner, S. P. Brinsko, K. Quirk, J. N. Rugila, and L. Boehnke. 1991. Effects of ecbolic agents on measurements of uterine involution in the mare. Theriogenology 36(4):559 571. Blanchard, T. L., D. D. Varner, S. P. Brinsko, C. C. Love, S. L. Rigby, and J. Schumacher. 2002. Manua l of equine reproduction, 2 nd Ed. Elsevier Health Sciences, St. Louis, MO. Bollwein H R. Mayer and R. Stolla 1998. Transrectal Doppler so nography of the A. Uterina in c yclic mares Theriogenology 49: 1483 1488 Bollwein, H F. Weber B. Kolberg and R Stolla 2002. Uterin e and ovarian blood flow during the estrus cycle in mares. Theriogenology 57:2129 2138. Clarkson, P., M. R. Adams A. J. Powe A. E. Donald, R. McCredie, and J. Robinson. 1996. Oral L arginine improves en dothelium dependent dilation in hypercholesterolemic young adults. J Clin Invest 97:1989 1994. Dave, D. P. and T. E. Milner 2000. Doppler angle measurement in highly scattering media. Opt. Lett 25:1523 1525 Drost, M., P. G. A. Thomas, B. Seguin, and M. H. T. Troedsson. 2002. Dis eases of the reproductive system. Large Animal Internal Medicine Mosby. Maryland Heigths, MS. Pp 1304 1306. Efron, D. and A. Ba rbul 2000. Role of arginine in immunonutrition. J Gastro entero. 35: 20 23

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58 Evoy, D., M. D. Lieberman, T. J. Fahey, and J. M. Da ly. 1998. Immunonutrition: The role of arginine. J Nutr 14:611 617. Gimpl, G. and F. Fahrenholz. 2001. The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81(2):629 683. Ginther, O. J. and R. A. Pierson 1984 Ultrasonic anat o my and pathology of the equine uterus. Theriogenology 21:505 516 Ginther, O. J. 1992. Reproductive B iology of the M are : B asic and A pplied A spects 2nd Ed Equiservices Publishing, Cross Plains, WI. Ginther, O. J. 2007. Ultrasonic I maging and A nimal R epr oduction: Color Doppler Ul trasonography. Equiservices Publishing, Cross Plains, WI. Greene, J. M., C. W. Dunaway, S. D. Bowers, B. J. Rude, J. M. Feugang, and P. L. Ryan. 2012. Dietary L arginine supplementation during gestation in mice enhances reproducti ve performance and Vegfr2 transcription activity in the fetoplacental unit. J. Nutr. 142(3):456 460. Hayes, K. E. N. and O. J. Ginthe r. 1986. Role of progesterone and estrogen in d evelopment of uterine tone in mares. Theriogenology 25(4):581 590. Hillier, S. G. 2001. Gonadotropic Control of ovarian follicular g rowth and development Mol. And Cell. Endo 1 79: 39 46 Keller, D. W. and K. L. Polakoski. 1975. L arginine stimulation of human sperm motility in vitro. Biology of Reproduction 13:154 157. Kelley, D E., L. K. Warren, and C. J. Mortensen. 2011. L arginine supplementation reduces uterine fluid accumulation post foaling in the mare. J. Equine Vet. Sci. 31(5):315 316. Lassala, A., F. W. Bazer, T. A. Cudd, S. Datta, D. H. Keisler, M. C. Satterfield, T. E Spencer, and G. Wu. 2011. Parenteral administration of L arginine enhances fetal survival and growth in sheep carrying multiple fetuses. Journal of Nutrition 141(5):849 855. Li, X., F. W. Bazer, G. A. Johnson, R. C. Burghardt, D. W. Erikson, J. W. Frank T. E. Spencer, I. Shinzato, and G. Wu. 2010. Dietary supplementation with 0.8% L arginine between days 0 and 25 of gestation reduces litter size in gilts. J. Nutr. 140(6):1111 1116. Mateo, R. D., G. Wu, F. W. Bazer, J. C. Park, I. Shinzato, and S. W. Kim 2007. Dietary L arginine supplementation enhances the reproductive performance of gilts. J. Nutr. 137:652 656.

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59 Maton, A J. Hopkins C. W. McLaughlin S. Johnson M. Q. W arner, D. LaHart, and J. D. Wright 1993. Human Biology and Health. Prentice Health Englewood Cliffs, NJ McCue, P. M. and J. P. Hughes. 1990. The effect of postpartum uterine lavage on foal heat pregnancy rate. Theriogenology 33(5):1121 1129. McKinnon, A. O., J. L. Voss 2005. Equine Reproduction. 6 th ed. Blackwell Publishing, Ames I A Mortensen, C. J., D. E. Kelley and L. K. Warren 2011 Supplemental L arginine shortens gestation length and increases mare uterine blood flow before and after parturition. J. Equine Vet. Sci 31(9): 514 520 NRC ( National Research Council ) 2007. Nutri ent requirements of horses, 6 th rev. ed. National Academies Press Washington, DC. Ousey, J. C., M. Kolling, R. Newton, M. Wright, and W. R. Allen. 2012. Uterine haemodynamics in young and aged pregnant mares measured using Doppler ultrasonography. Equine Vet. J. 44(Suppl. 41):15 21. Pauerstein, C. J., B. J. Hodgson and M. A. Kramen 1974. The anatomy and physiology of the oviduct. Obstet Gynecol Annu 3(0): 137 201 Rosenfeld, C. R., F. H. Morriss, Jr., E. L. Makowski, G. Meschia, and F. C. Battaglia. 19 74. Circulatory changes in the reproductive tissues of ewes during pregnancy. Gynecologic and Obstetric Investigation 5:252 268. Senger, P. L. 1997 Pathways to Pregnancy and Parturition. Cu rrent Concepts,Inc., Pullman, WA. Sewell, D. A. and R. C. Harris. 1995. Effects of creatine supplementation in the Thoroughbred horse. Equine Vet. J. 27(18):239 242. Silva, L. A., C. Klein, A. D. Ealy, and D. C. Sharp. 2011. Conceptus mediated endometrial vascular changes during early pregnancy in mares: an anatomic, hi stomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study. Reproduction 142:593 603. Tapiero, H., G. Mathe, P. Courver, and K. D. Tew. 2002. I. Arginine. Biomed Pharmacother 56(9):439 445. Visek W. J. 1986. Arginine needs, physiological state and usual diets. A reevaluation. J. Nutr 116(1):36 46.

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60 Wu, G. and S. M. Morris. 1998. Arginine metabolism: nitric oxide and beyond. Biochem J. 336:1 17. Wu, G. and C. J. Meininger. 2000. Arginine nutrit ion and cardiovascular function. J. Nutr 130:2626 2629.

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61 BIOGRAPHICAL SKETCH Robert Jacobs is a native of Florida, and grew up in Plantation, Florida. From a young age, he was exposed to animals both at home and in his academic career. routinely ride and care for the thoroughbred that his grandfather had rescued from the track. Robert began his collegiate education at Purdue University where he hoped to go to vet school Robert made the decision to transfer to the University of Florida before his junior year and began his education at the University of Florida in August of 2007. Robert gained an interest in reproductive physiology in his Reproductive Physiology and Endo crinology course taught by Dr. Michael Fields, which sparked his desire to pursue this area of research as a career. Additionally, Robert was introduced to equine nutrition by Dr. Lori Warren whose passion for the topic inspired Robert to seek further know ledge on the topic. thesis student with plans to apply to vet school and pursue his interests in reproductive physiology and nutrition as a lear to Robert that he had a passion for research and he decided to switch to a thesis program. Dr. Christopher Mortensen and Dr. Lori Warren were supportive enough to develop a ogy. Robert plans on continuing his education at Virginia Tech University and obtaining his doctorate in equine reproductive physiology and he hopes to continue doing research as well as pursue his interests in teaching as a member of academia in the futu re.