Improvement of Reproductive Function in Mares by Oral Supplementation of L-Arginine

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Improvement of Reproductive Function in Mares by Oral Supplementation of L-Arginine
Kelley, Dale E
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[Gainesville, Fla.]
University of Florida
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Doctorate ( Ph.D.)
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University of Florida
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Animal Molecular and Cellular Biology
Committee Chair:
Mortensen, Christopher J
Committee Members:
Ealy, Alan D
Hansen, Peter J
Leblanc, Michelle M
Risco, Carlos A
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Amino acids ( jstor )
Blood flow ( jstor )
Horses ( jstor )
Ovulation ( jstor )
Oxides ( jstor )
Parturition ( jstor )
Perfusion ( jstor )
Plasmas ( jstor )
Pregnancy ( jstor )
Uterine artery ( jstor )
Animal Molecular and Cellular Biology -- Dissertations, Academic -- UF
l-arginine -- mare -- reproduction
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Animal Molecular and Cellular Biology thesis, Ph.D.


Increased blood flow to the ovulatory follicle has been associated with increased pregnancy rates in mares (Silva et al., 2006). L-Arginine is the nitrogen donor for nitric oxide a potent vasodilator which has been shown to increase blood flow in a variety of tissues (Bode-Bogeret al., 2007). The purpose of this dissertation was to determine the effect of orally supplemented L-arginine on reproductive blood flow and performance in mares. Mares supplemented with 100 g, 0.025% or 0.0125% L-arginine by body weight had increased (P < 0.05) plasma L-arginine concentrations. Experiment 2 was to investigate the uterine artery blood flow changes with oral L-Arginine (100g) supplementation 3 wk prior to foaling. Mares supplemented with L-arginine had a shorter gestation length (337 ± 1.7 d) compared to control (345 ± 2.1 d; P = 0.05) and greater blood flow prepartum in the uterine arteries (P = 0.05). Experiment 3 was to determine the effect of supplementing 100 g of L-arginine on follicular dynamics and ovarian and uterine artery blood flow during foal heat. There were no differences in follicular dynamics, ovarian or uterine resistance indices between groups; however, vascular perfusion of the F1 follicular wall was greater in L-arginine supplementedmares (37.3 ± 2.6%) than controls (25.4 ± 2.7 %; P < 0.05). L-Arginine supplemented mares had smaller uterine body and horns and accumulated less uterine fluid than controls (P < 0.05). The last experiment was to determine if supplemental L-arginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle and embryo recovery rates in mares. L-Arginine supplementation did not affect embryo recovery rate (arginine-treated: 54%, control: 48%). Mares treated with L-arginine had a larger follicle for the 10 d preceding ovulation than control mares (30.4 ± 1.2 and 26.3 ± 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 d prior to ovulation. Treatment of mares with L-arginine increased follicular blood and improved uterine conditions in the post partum. This raises the potential use of L-arginine to improve reproductive efficiency. ( en )
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Thesis (Ph.D.)--University of Florida, 2013.
Adviser: Mortensen, Christopher J.
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by Dale E Kelley.

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2 2013 Dale Edward K elley


3 ACKNOWLEDGMENTS I would like to thank my family, especially my mother and father, for supporting me over the years, my advisor Dr. Christopher Mortensen and committee, Dr. Alan Ealy, Dr. Peter Hansen, and Dr Michelle LeBlanc for their guidance. I would also like to thank Chris Cooper, Joss Cooper and the staff at the Equine Sciences Center for their valuable assistance over the cour se of my research


4 TABLE OF CONTENTS page ACKNO WLEDGMENTS ................................ ................................ ................................ .. 3 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 LITERATURE REVIEW ................................ ................................ .......................... 12 Introduction ................................ ................................ ................................ ............. 12 Cardiovascular syste m and blood flow regulation ................................ ................... 12 Cardiovascular system and reproductive system ................................ .................... 14 Introduction ................................ ................................ ................................ ....... 14 The ovary ................................ ................................ ................................ ......... 15 The uterus ................................ ................................ ................................ ........ 22 Pregnancy ................................ ................................ ................................ ........ 22 Doppler ultrasonography ................................ ................................ ......................... 23 Introduction ................................ ................................ ................................ ....... 23 Fluid mechanics ................................ ................................ ............................... 24 Principles of Doppler ultrasongraphy ................................ ................................ 28 Doppler ultrasonography measurements ................................ ......................... 30 Doppler ultrasonography and large animal r eproduction ................................ .. 32 L Arginine ................................ ................................ ................................ ............... 36 Introduction ................................ ................................ ................................ ....... 36 Endocrine effects ................................ ................................ .............................. 36 Nitric Oxide ................................ ................................ ................................ ....... 37 Polyamines ................................ ................................ ................................ ....... 39 Agmatine ................................ ................................ ................................ .......... 41 L Arginine and reproduction ................................ ................................ ............. 42 L Arginine and pregnancy ................................ ................................ ................ 42 Synopsis and Objectives ................................ ................................ ......................... 44 2 ORALLY SUPPLEMENTED L ARGININE IMPAIRS AMINO ACID ABSORPTION DEPENDING ON DOSE ................................ ................................ 45 Introduction ................................ ................................ ................................ ............. 45 Materials and Methods ................................ ................................ ............................ 46 Amino Acid Analysis ................................ ................................ ......................... 48 Statistical Analysis ................................ ................................ ............................ 48 Results ................................ ................................ ................................ .................... 49 Experiment 1 ................................ ................................ ................................ .... 49 Experiment 2 ................................ ................................ ................................ .... 50


5 Discussion ................................ ................................ ................................ .............. 51 3 SUPPLEMENTAL L ARGININE SHORTENS GESTATION LENGTH AND INCREASES MARE UTERINE BLOOD FLOW BEFORE AND AFTER PARTURITION ................................ ................................ ................................ ....... 62 Introduction ................................ ................................ ................................ ............. 62 Materials and Methods ................................ ................................ ............................ 63 Animals ................................ ................................ ................................ ............. 63 Experimental Design ................................ ................................ ........................ 63 Color Doppler ultrasonography ................................ ................................ ......... 64 Statistical Analyses ................................ ................................ .......................... 65 Results ................................ ................................ ................................ .................... 66 Gestation and foaling parameters ................................ ................................ .... 66 Uterine artery characteristics ................................ ................................ ............ 67 Uterine artery blood flow characteristics ................................ ........................... 68 Discusion ................................ ................................ ................................ ................ 69 4 ORAL L ARGININ E SUPPLEMENTATION IMPACTS SEVERAL REPRODUCTIVE PARAMETERS DURING THE POSTPARTUM PERIOD IN MARES ................................ ................................ ................................ ................... 80 Introduction ................................ ................................ ................................ ............. 80 Materials and Methods ................................ ................................ ............................ 81 Animals and experimental design ................................ ................................ ..... 81 Amino Acid Analysis ................................ ................................ ......................... 82 Ultrasonongraphy ................................ ................................ ............................. 83 Statistical analysis ................................ ................................ ............................ 85 Results ................................ ................................ ................................ .................... 85 Pla sma L arginine concentrations ................................ ................................ .... 85 Ovarian follicular dynamics, blood flow and follicular perfusion ........................ 85 Uterine involution and flui d clearance ................................ ............................... 86 Uterine blood flow ................................ ................................ ............................. 87 Discussion ................................ ................................ ................................ .............. 87 5 INFLUENCE O F L ARGININE SUPPLEMENTATION ON REPRODUCTIVE BLOOD FLOW AND EMBRYO RECOVERY RATES IN MARES ........................... 99 Introduction ................................ ................................ ................................ ............. 99 Materials and Meth ods ................................ ................................ ............................ 99 Animals ................................ ................................ ................................ ............. 99 Doppler Ultrasonography ................................ ................................ ................ 101 Embryo Collections ................................ ................................ ........................ 101 Statisical analysis ................................ ................................ ........................... 102 Results ................................ ................................ ................................ .................. 103 Discussion ................................ ................................ ................................ ............ 104 6 CONCLUSIONS ................................ ................................ ................................ ... 114 LIST OF REFERENCES ................................ ................................ ............................. 120


6 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 150


7 LIST OF TABLES Table page 5 1 Number of recover ed embryos classified by grade for each group or based on mare age. ................................ ................................ ................................ ... 108


8 LIST OF FIGURES Figure page 2 1 P lasma arginine concentrations for mares fed 0.025% of body weigh t L arginine ................................ ................................ ................................ .............. 56 2 2 Plasma citrul line concentrations and plasma ornithine concentrations for mares fed 0.025% of body weight L arginine .. ................................ .................... 57 2 3 P lasma lysine concentrations, methionine, histidine concentration s, glutamic acid conc entrations, proline co ncentrations and isoleucine concentrations for mares fed 0.025% of body weight L arginine .. ................................ .................... 58 2 4 P lasma arginine concentrations for mares fed 0.01 25% of body weight L arginine or 0.0087% of body w eight urea ................................ .......................... 59 2 5 P lasma citrulline concentrations and ornithine concentrations for mares fed 0.0125% of body weight L arginine or 0.0087% of body w eight urea ................ 60 2 6 P lasma lysine concentrations, met hionine, histidine concentrations, glutamic acid concentrations, pro line concentrations and isoleucine concentrations for mares fed 0 .0125% of body weight L ar ginine or 0.0087% urea ....................... 61 3 1 D iameter of the uterine arteries of L arginine treated and control mares in the 21 d before and 7 d after parturition. ................................ ................................ .. 77 3 2 P ulsatility index and R esistance index values in the uterine arteries leading to the gravid horn of pregnancy of L arginine treated and control mares in the 21 d before and 7 d after parturition. ................................ ................................ .. 78 3 3 P ulsatility index and R esistance index values in the uterine arteries leading to the nongravid horn of pregnancy of L arginine treated and control mares in the 21 d before and 7 d after part urition ................................ ............................. 79 4 1 P lasma arginine concentrations for 12 h following the consumption of 100 g L arginine. ................................ ................................ ................................ .......... 91 4 2 N umber of follicles for the 10 d preceding ovulation for L arginine treated and control mares.. ................................ ................................ ................................ .... 92 4 3 D iameter of the dominant and largest subordinate follicles for L arginine treated and control mares. ................................ ................................ .................. 93 4 4 R esistance index values in the ovarian arteries for the 10 d prior to ovulation.. ................................ ................................ ................................ ........... 94


9 4 5 P ercent of vascular perfusion of the ci rcumference of the ovulatory follicle as indicated by color power Doppler. ................................ ................................ ...... 95 4 6 D epth of the uterine body, diameter of the formerly gravid uterine horn and diameter of the formerly nong ravid horn for 30 d postpartum. ........................... 96 4 7 M aximal fluid accumulation in the postpartum uterus.. ................................ ....... 97 4 8 Re sistance index of the uterine artery on the side of formerly gravid and nongravid uterus. ................................ ................................ ................................ 98 5 1 D iame ter of the retro spectively identified dominant and largest subordinate follicles for L arginine supplemented and control mares separated by age. ..... 109 5 2 P ercent perfusion to the retrospe ctively identified dominant follicle for the 4 days prior to ovulation. ................................ ................................ ..................... 110 5 3 Diameter and p ercent perfusion of the Corpus luteum for the 6 days following ovulation. ................................ ................................ ................................ .......... 111 5 4 Resistance index to the ovarian artery ipsilateral and c ontralateral to ovulat ion for the 10 days prior to ovulation. ................................ ...................... 112 5 5 Resistance index of the ovarian artery ipsilateral and contralateral to the Corpus luteum for the 6 days following ovulation. ................................ ............ 113 6 1 Possible mechanisms by which L arginine supplementation could influence reproduction.. ................................ ................................ ................................ .... 119


10 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 IMPROVEMENT OF REPRODUCTIVE FUNCTION IN MARES BY ORAL SUPPLEMENTATION OF L ARGININE By Dale Edward Kelley May 2013 Chair: Christopher J. Morte nsen Major: Animal Molecular and Cellular Biology Increased blood flow to the ovulatory follicle has been associated with increased pregnancy rates in mares (Silva et al., 2006). L Arginine is the nitrogen donor for nitric oxide a potent vasodilator which has been shown to increase blood flow in a variety of tissues (Bode Bogeret al., 2007). The purpose of this dissertation was to determine the effect of orally supplemented L arginine on reproductive blood flow and performance in mares. Mares supplemented with 100 g, 0.025% or 0.0125% L arginine by body weight had increased (P < 0.05) plasma L arginine con centrations. Experiment 2 was to investigate the uterine arte ry blood flow changes with oral L Arginine (100g) supplementation 3 wk prior to foaling Mar es supplemented with L arginine had a greater blood flow prepartum in the uterine arteries determine the effect of supplementing 100 g of L arginine on follicul ar dynamics and ovarian and uterine artery blood flow during foal heat. There were no differences in follicular dynamics, ovarian or uterine re sistance indices between groups; however, v ascular perfusion of the F1 follicular wall was greater in L arginine supplemented


11 mares (37.3 2.6%) than controls (25.4 2.7 %; P < 0.05). L Arginine supplemented mares had smaller uterine body and horns and accumulated less uterine fluid than controls (P < 0.05). The last experiment was to determine if supplemental L ar ginine would increase ovarian arterial blood flow, vascular perfusion of the preovulatory follicle and embryo recovery rates in mares. L Arginine supplementation did not affect embryo recovery rate (arginine treated: 54%, control: 48%). Mares treated with L arginine had a larger follicle for the 10 d preceding ovulation than control mares (30.4 1.2 and 26.3 1.3 mm, respectively; P < 0.05) and vascular perfusion of the dominant follicle tended (P = 0.10) to be greater for the 4 d prior to ovulation. Trea tment of mares with L arginine increased follicular blood and improved uterine conditions in the post partum. This raises the potential use of L arginine to improve reproductive efficiency.


12 CHAPTER 1 L ITERATURE REVIEW Introduction Mares are distinctive among domestic livestock in their ability to return to estrus and potentially conceive shortly after giving birth. This first postpartum estrous period parturition (McCue a nd Hughes, 1990). Aditionally the first post partum estrous cycle is considered less fertile than other cycles. This may be in part to the changes occurring in the post partum mare uterus (McKinnon and Voss, 2011). Given the time constrants from foaling to conceiving, increasing uterine involution and decreasing uterine fluid accumulation could result in mares becoming preganant sooner. These are two major constrants that play a role in the discision to breed the mare during the first post partum estrus (Kn ottenbelt et al., 2003) Another challenge in equine reproduction is embryo recoveries from mares. Other species, due to anatomic reasons, are able to be effectively superovulated, while the mare at best can ovulated 4 follicles ( Meyers Brown et al., 2011 ). I ncreased blood flow to the dominant follicle is associated with increased pregnancy rates in mares (Silva et al., 2006) T hus it is concevievable that increasing blood flow may benefit fertility and possibly improve embryo recovery rates. Not only could increasing blood flow aid in embryo recovery but it could also enhance fertility in mares during the first post partum estrous cycle, mares bred via live cover or artificial insemination. Cardiovascular system and blood flow regulation Tissue and organs h ave the ability, to varying degrees, to regulate their own blood flow (Klubunde, 2012). Factors produced in the area surrounding blood vessels


13 act to either relax or contract the endothelial smooth muscle, altering resistance and thus blood flow. These fac tors can act directly on the smooth muscle or on sympathetic nerves. In highly metabolic tissues, substances are released that promote vasodilation. This is coined the Metabolic Theory of Blood Flow Regulation (Mosher et al., 1964) Some metabolic regulato rs of blood flow are adenosine (Berne et al., 1983) inorganic phosphate (Dobson et al., 1971) carbon dioxide (Ke ty and Schmidt, 1948) hydrogen ions (Enson et al., 1964) potassium ions (Branston et al., 1977) oxygen (Regg et al., 1944) and osmolarity ( Scott et al., 1977) in the tissue interstitium. Adenosine is a vasodilator in most organs, except the kidney where it is vasoconstrictive (Agmon et al., 1993) Hydrolysis of ATP leads to an increase of both adenosine and inorganic phosphate, which act as vasodilators in areas of skeletal muscle (Dobson et al., 1971) When blood flow is not sufficient to remove carbon dioxide from tissue, CO 2 increases and diffuses to the vascular smooth muscle of blood vessels where it also promotes vasodilation (Barer and Shaw, 1971) Additionally, increases in CO 2 cause increases in H + via the bicarbonate buffer system. H + causes local vasodilation particularly in cerebral circulation (Kety and Schmidt, 1948) Potassium released by contracting cardiac and skeletal muscle during membrane depolarization is normally restored to muscle cells via the Na + /K + ATPase pump. During times of rapid contractions K + can accumulate in the extracellular space around blood vessels. This causes hyperpolarization of the vascular smooth muscl e which results in smooth muscle relaxation, resulting in increased blood flow to contracting muscles (Duling, 1975) Low oxygen in tissue or hypoxia my induce vasodilation either directly via inadequate O 2 to maintain smooth muscle contraction or indirect ly via adenosine, lactic acid or H +


14 metabolite production ( Klunbunde, 2012 ) Additionally, non specific hyperosmolarity can induce vasodilation by local infusion of hyperosmolaric solutions into tissue (Sasa ki et al., 1986 ). The vascular endothelium plays an important role in smooth muscle regulation via the release of paracrine factors that can either induce vasoconstriction or vasodilation. Nitric oxide, under normal physiologic conditions, appears to be the most important regulator of blood flow (Klubun de, 2012). Prosctacyclin (PGI 2 ) induces vasodilation and prevents platelet aggregation (Rubin et al., 1982). Endothelin 1 is a potent vasoconstrictor (Yanagisawa et al., 1988) Other mechanisms include myogenic responses of smooth muscle caused by an incre ased luminal pressure which expands the diameter of the vessel resulting in vasoconstriction. Conversely when luminal pressure drops the smooth muscle relaxes. This behavior has been observed in various capillary systems but the functional significance var ies with organ (Meininger and Davis, 1992) It is difficult to study the mygenic response since pressure changes usually trigger metabolic mechanisms, which dominate myogenic responses (Klubunde, 2012). Mechanical compressive forces external to the vascula ture can affect vascular resistance and blood flow. If the pressure outside the blood vessel is greater than the pressure inside the blood vessel, the vessel will collapse, reducing blood flow. If the pressure difference is severe enough, blood flow throug h a vessel can be completely occluded (Klubunde, 2012). Cardiovascular system and reproductive system Introduction The growth of new blood vessels via angiogenesis and vasculogenesis occurs mainly during embryonic development; however, the female reproduc tive tract is unique


15 as it has continual remodeling of the vascular system both cyclically and during pregnancy (Augustin et al., 2001). Folliculogenesis, growth of the corpus luteum, uterine changes and pregnancy are linked with the growth of blood vessel s (Augustin et al., 2001). Studying the reproductive tract has increased our understanding of angiogenic processes. The first angiogenic cytokine basic fibroblast growth factor or fibroblast growth factor 2 (bFGF or FGF2) identified was in the ovary (Jakob et al., 1977). What is considered to be the most important angiogenic cytokine, vascular endothelial growth factor (VEGF), was characterized in the ovary (Ravindranath et al., 1992) as well as the first angio regressive factor, angiopoietin 2 (ANP 2), was a lso identified in the ovary (Maisonpierre et al., 1997). The ovary Pre antral follicles do not have a network of blood vessels associated with them, yet as the follicle reaches the antral stage, a capillary plexus forms in the theca externa and theca inter na, but not in granulosal cells layer (Augustin et al., 2001). It has been speculated that the vascular development of the follicle may contribute to selection of the dominant follicles and conversely contribute to atresia of the non dominant follicles (Au gustin et al., 2001). In women, increased vascularization appears to play a role in the selection and maturation of follicles from both spontaneous and stimulated IVF cycles (Weiner et al., 1993; Balakier and Stronell, 1994; Bassil et al., 1997). It is tho ught, follicles secrete substance to regulate angiogenesis as evident in experiments with extracts from pre ovulatory follicles which stimulate endothelial cell proliferation in culture (Makris et al., 1984; Koos et al., 1986; Rone et al., 1993 ; Kuo et al. 2011 ). Some of the factors identified in human follicles are stromal cell derived factor 1 (SDF1) and vascular endothelial growth factor (VEGF), which increase in the follicular fluid as


16 the follicle diameter increases (Nishigaki et al., 2011). SDF 1 enh ances the production of VEGF in a positive feedback loop in human umbilical vein endothelial cells (Salcedo et al., 1999). The corpus luteum has three phases of vascular changes: vascular growth, vascular maturation and vascular regression (Augustin et al ., 2001). During the first third of luteal growth there is an intense increase in corpus luteum size which requires the growth of blood vessels to maintain nutrient supply to the growing tissue (Augustin et al., 2001). Shortly after ovulation there is an i nvasion of endothelial cells into the corpus luteum (Gaede et al., 1985; Meyer and McGaechie 1988) with sprouting angiogenesis resulting in vessels invading the fibrin rich ovulatory cavity (Augustin et al., 2001). In rats this occurs as early as 16 h pos t ovulation (Matsushima et al., 1996). Up to 40% of the microvessles of an early corpus luteum have been found to have proliferating endothelial cells, with as much as 50% of the early corpus luteum composed of endothelial cells (Augustin et al., 2001). In hibition of FGF 2 signaling in bovine luteal cell culture between Days 0 3 reduced angiogenesis by 64% and between inhibition of FGF2 signaling between Days 3 6 by 81% (Woad et al., 2012). Additionally, FGF 2 infusion into the early bovine corpus luteum sti mulated progesterone secretion (Miyamoto et al., 1992) suggesting that FGF 2 plays a critical role in early luteal development. Additionally, d espite the view of ovulation as an inflammation reaction (Espy, 1980), there are few inflammatory cells in the co rpus luteum, suggesting the angiogenesis is a noninflammatory event (Vinatier et al., 1995). Mature corpus lutea has a dense capillary network with each cell in contact with at least two capillaries (Augustin et al., 2001). Blood vessels recruit pericytes which


17 ensheath the capillaries while larger vessels recruit smooth muscle cells (Goede et al., 1998). Roughly, 60% of the blood vessels in a mature corpus lutea aquire a mature phenotype. Analysis of luteal blood flow using microspheres found blood flow i s primarily regulated by systemic blood pressure, suggesting the development or function of smooth muscle along the vasculature is limited (Wiltbank et al., 1990; Wiltbank et al., 1994). Regression of the corpus lutea is characterized by a rapid phase of t issue disintegration and involution (within days) followed by a slow phase of remodeling and connective tissue growth (within weeks; Augustin et al., 2001). This is a unique process among adult tissues. Luteolysis results in a rapid functional loss and red uction in size; vacuolization, hyalination and cellular condensation that can be seen in all luteal cell types (Augustin et al., 2001). The massive apoptosis during regression primarily reflects luteal cell and not endothelial cells populations (Zheng et a l., 1994). During the slow period, smooth muscle proliferates resulting in an occlusion of blood vessels which shuts down vascular blood flow in the regressing corpus luteum (Augustin et al., 2001). During luteolysis, macrophages are recruited which play a n important role in regressing the corpus luteum (Augustin et al., 2001). VEGF is a major regul atory molecule of angiogenesis in the CL VEGF can be alternatively spliced into 4 isoforms (121, 165, 189 and 206 amino acids in length). VEGF 121 and VEGF 165 are the isoforms primarily found in the bovine corpus luteum (Berisha et al., 2000) The biological activity of VEGF is mediated by the receptors Flt 1(VEGFR 1) and Flk 1 (VEGFR 2; Miyamoto et al., 2009). VEGF and VEGFR 2 mRNA


18 expression is highest in the early corpus luteum and decreases significantly during the mid and late luteal phases in cattle (Berisha et al., 2000). Generally, VEGF stimulates progesterone secretion in the bovine ovary (Kobayashi et al., 2001). Regulation of angiogenesis is a complex series of interaction involving VEGF and other molecules. One important molec ule involved in angiogenesis is angiopoietin 1 (ANG 1) which is necessary to main tain and stabilize vessels. ANG2 acts as an antagonist to ANG1 resulting in active remodeling dest abilizing vessel structure; when VEGF is high the result is an increase in the vascular network, when VEGF is low regression occurs (Miyamoto et al., 2009). Other factors regulate vasoconstriction and vasodilation and appear to play a role in reproductiv e physiology. Endothelin 1 (EDN 1) is a potent vasoconstrictor and acts via the endothelin receptor A (ETR A ;a G coupled protein receptor); however the second recep tor, endothelin receptor B (ETR B), induces vasodilation. Angi o tensin II (ANG II) acts a vasoc onstrictor and is converted from angi tensin I (ANG I) by angiotensin converting enzyme (ACE) localized in the corpus luteum. Studies have su ggested a role of EDN 1 and A NGII in luteolysis (Miyamoto et al., 2009). EDN1 has been shown to inhibit progesterone s ecretion in cattle (Girsh et al., 1996A; Miyamoto et al., 1997), sheep (Hinckley and Milvae, 2001), and humans (Apa et al., 1998). Administration of an ETR A antagonist interrupted PGF induced luteolysis in sheep (Hinckley and Milvae, 2001). Angiotensin II decrease d progesterone secretion in bovine luteal cells (Stirling et al., 1990). Both EDN1 and ACE are upregulated during spontaneous and induced luteolysis (Girsh et al., 1996B; Le vy et al., 2000; Milvae, 2000; Wright et al., 2001; Berisha et al., 2002).


19 Nitric oxide (NO) is a potent vasodilator produced by n itric oxide synthase f rom L arginine (Miyamoto, 2009) and has been implicated in steroidogenesis, ovulation and luteolysis. E stradiol has been found to induce endothelial nitric oxide synthase, and estrogen induced vasodilation is mediated at least in part by NO (Van Buren et al., 1992; McNeill et al., 1999). The action of NO on estrogen production is unclear; some research sugg ests NO increases estrogen synthesis (Jablonka Sherriff and Olson, 1998) while other research suggests a negative effect (Mitsube et al., 1999). In eNOS knockout mice, higher estradiol concentrations (Jablonke Shariff and Olson, 1998) and chronic NO inhibi tion produced prolonged estrus (Dunnam et al., 1999). These authors concluded NO acts to inhibit estradiol production. eNOS knockout mice have a 63% decrease in ovulation rate, a delay in meiotic maturation delay, smaller litters and a higher stillborn rat e (Jablonka Shariff and Olson, 1998). A role of NO has been proposed in the ovulation process, thought to be mediated by effects on vasodilation or prostaglandin production (Dixit and Parvizi, 2001). This effect may be mediated in part by effects on prosta glandin production. Blocking intraovarian NO production decreased PGF and PGE 2 production, while giving a NO donor increased both (Faletti at al., 1999). NO appears to increase PG synthesis via COX II pathway activation to enhance the inflammatory process (Salvemini, 1997; Faletti et al., 1999). NO in rat ovaries increase d PGF production and diminished progesterone production (Motta et al., 1999). In the cow, NO may mediate luteolysis by inhibiting progesterone secretion (Skarzynski et al., 2000; Skarzynski et al., 2003). Injection of NO into the corpus luteum increased blood flow similar to that seen with PGF reduced progesterone secretion and reduced luteal volume (Skarzynski et al., 2003).


20 Administration of a nitric oxide synthase inhibitor suppressed the increase in luteal blood flow and delayed the decrease in pro gesterone secretion (Jaroszewski and Hansel, 2000).PGF injection stimulates endothelial nitric oxide syntase in the early and mid cycle corpus luteum in cattle, sheep and rabbits (Boiti et al., 2003; Vonnahme et al., 2006; Shirsuna et al., 2008). In hors es, Ferreira Dias et al. (2011) found that NO stimulates progesterone and PGE 2 secretion during early CL development Work in other species later during the CL lifespan suggests a different ro le for NO in particular, NO produced during the last 2 days of C L development increased PGF 2 production and decreased progesterone production (Motta et al., 19 99). In cattle, PGF has been found to increase endothelial nitric oxide synthase expression and increase luteal blood flow during luteolysis (Shirasuna et al., 2008), suggesting the incr ease in blood flow during this period is NO mediated. In the uterus, neurons containing NO have been identified in rats and mice (Papka and McNeill, 1992; Grozdanovic et al., 1994; Papka et al., 1995). In nonpregnant human uterus it has been suggested tha t NO plays a role in vasodilation, platelet aggregation (Norman and Cameron, 1996) and a role in controlling uterine contractions (Longo et al., 1999) NO is a smooth muscle relaxant; however, NO has been reported to affect cyclooxygenase activity to incre ase prostaglandin synthesis and alter uterine contractility in rat uterine cultures (Franchi et al., 1994). NO decreased uterine contractions during times of increased myometrial motility, but increased myometrial contractions when motility was low. Using pre term mice induced to undergo labor, Cella et al. (2010) found that treatment with low concentrations of a nitric oxide


21 donor (SNAP) decreased prostaglandin production whereas a higher amount of SNAP increased prostaglandin production. There also appear s to be crosstalk between NO and oth er molecules, such as VEGF stimulated nitric oxide release from cultured umbilical endothelial cells and upregulates nitric oxide synthase (Van der Zee et al., 1997; Hood et al., 1998). Simila r results are observed with TGF 2 (Inoue et al., 1995; Wu et al., 1996; Tiefenbaucher and Chilian, 1997). Angiogenesis is attenuated when nitric oxide is blocked (Cooke and Losordo, 2002). Nitric oxide is a survival factor, inhibits apoptosis in endothelial cells (Dimmeler et al ., 1999; Rossig et al., 1999), increases endothelial cell proliferation (Ziche et al., 1997; Dulak et al., 2000), increases endothelial cell migration (Ziche et al., 1994; Murohara et al., 1999), and stimulates endothelial cell podokinesis (Noiri et al., 1 998).Matsunaga et al. (2002) suggested nitric oxide may suppress angiostatin, an endogenous antagonist of angiogenesis. It has been reported that nitric oxide induces VEGF expression by enhancing HIF1 binding activity and accumulation of HIF1 protein in tu mor cells (Kimura et al., 2000). Nitric oxide is an inorganic free radical gas that acts as a biological messenger (Dixit and Parvizi, 2001) and can potentially influence the reproduction via the hypothalamus, ovary and uterus (Rosselli et al., 1998; Dix it and Parvizi, 2001). NO is implicated in stimulating gonadotrophin secretion (Dixit and Parvizi, 2001) via a calcium dependent, cGMP independent pathway (Pinilla et al., 1998). NO positive neurons are in close association to GnRH containing neurons of th e hypothalamus (Bhat et al., 1995). Microinjection of a nitric oxide synthase inhibitor into the third cerebral ventricle blocked LH pulses but had no effect on FSH release (Rettori et al., 1993). Knauf et al.


22 (2001) found that nitric oxide in the median eminence of rats was highest at proestrus and had a positive correlation with GnRH release. The proestrus GnRH surge was blocked in rats treated with a nitric oxide synthase inhibitor. In females, NO has been implicated in modulating the pre ovulatory LH s urge (Aguan et al., 1996; Bonavera et al., 1996). Sodium nitroprusside, a NO donor, has been found to stimulate GnRH release in vitro from rat medial basal hypothalamic fragments (Rettori, et al., 1993), via a cGMP dependent mechanism (Moretto et al., 19 93). The uterus The uterine blood supply is primarily from the aorta via the uterine and iliac arteries with contributions from the uterine branch of the vaginal artery and uterine branch of the ovarian artery (Ginther, 2007). In women, arcuate arteries within the endometrium give rise to the radial arteries that supply the endometrium, and after passing through the myometrial endometrial junction split to form smaller arteries that supply the basalis and spiral arteries which continue to the endometrial surface (Rogers and Gargett, 2001). The endometrial vasculature is indistinguishable at the ultrastructural level from other tissues (Rodger and Gargett, 2001). In women, most blood vessels show little change within the menstrual cycle; the exception is th e spiral arteries which, in women, grow toward the uterine lumen as the endometrial layer thickens (Ramsey, 1994) and breakdown during the secretory phase (Rodgers and Gargett, 2001). Pregnancy There is great diversity of placental structure among euther ian mammals ( Carter and Mess, 2007 ). In all species, the placenta is involved in fetal nutrition, respiration and endocrinology, with different placental types having specialized adaptations to meet


23 these demands (Charnock Jones and Smith, 2001). Mares hav e an epitheliochoral placenta which is a simple apposition of unchanged fetal and maternal epithelia with interdigitated microvilli. Between Days 35 38 post conception, the allantois has vasculaized 90% of the trophectoderm (Steven 1982) and by Day 40 the trophectoderm/ chorion establishes a mircovillous attachment to the endometrium (Wilscher and Allen, 2011). By Day 60, fetal villi are longer and group together with adjacent villi grouping to form microcotelydons and continued branching of the villi l eading to the formation of a mature microcotelydon (Wilscher and Allen, 2011). There are no extravasations of maternal blood at the microvillar junction (Wooding and Burton, 2008). The blood vessel pattern in the fetal horse villus consists of a central a rteriole passing directly to the villus tip which then divides into a perpherial capillary network draining back down to the base of the villus. The maternal arteries run to the villi tip and then drain down to the villi base via capillaries before returni ng to collecting veins. The maternal villi are inserted between the fetal villi. This allows for the classic countercurrent flow between fetal and maternal capillaries, provided they are in close proximity for a long enough distance for exchange to occur ( Wooding and Burton, 2008). At parturition the interdigitated microvilli separate, with the fetal and maternal villi disengaging, possibly due to a decrease in blood pressure or blood volume (Stevens et al., 1979). Doppler ultrasonography Introduction Hea rt rate, stroke volume and peripheral resistance are the primary factors that control the relationship between blood pressure and flow rate (Waite and Fine, 2007). In


24 order to understand the potential applications and limitations of Doppler ultrasonography it is necessary to have a basic understanding of fluid mechanics, particularly how fluid mechanics applies to biological systems. Fluid mechanics A fluid is classified by the effect of a deforming force on the material and can be either a liquid or gas. When a deforming force acts on a fluid, it deforms and remains deformed even after the removal of the force (Zamir, 2000). Fluid behavior depends on its mechanical properties. These properti es include density, specific gravity, surface tension, viscosity vapor pressure and compressibility. Density and specific gravity are measures of the heaviness of a fluid (Waite and Fine, 2007) and can influence fluid behavior. The viscosity of a fluid is its internal resistance t o deformation. V iscous properties redu ce sudden change s in velocity within a fluid field upon application of a force Thus, when a fluid moves through a tube the velocity of the fluid adjacent to the tube wall remains zero with the maximum velocity at the center of the tube. This phenomenon is is the reason a pump is needed to maintain flow of a fluid through a tube. Two classifications of flow exist; laminar and turbulent (Zamir, 2000). In laminar flow, when viscous forc es dominate, all elements of the fluid move in the same direction. In turbulent flow, when inertial forces dominate, there is a mean flow direction, as well as velocity components in other directions, and thus the fluid is engaged in random flow to a degre e (Zamir, 2000). In term of the cardiovascular system, during laminar flow all components (i.e. red blood cells) are moving in a similar direction; while in turbulent flow, components are moving in various random directions, though flow still progress thro ugh the tube. In practical terms, turbulent flow cannot be accurately


25 measured using Doppler ultrasonography, since the velocity profile is based on sound wave frequency shifts reflected from red blood cells, which are not all moving in the same direction. Efforts have been made to mathematically describe flow through a tube with the viscosity, flow, pressure and resistance (Klabunde, 2012), where Q is the volume of flow through a tube, r the tube radius, P the pressure difference between the length of the tube, n the viscosity of the fluid and L the length of biological systems at times. The equation assumes steady, laminar, incompressible, and viscous flow of a Newtonian fluid in a rigid, cylindrical tube of constant cross section (Waite and Fine, 2007). Flow in blood vessels can be laminar or turbulent depending on the circums laminar or turbulent and in the body can range from 1 in small arteries to 4000 in the largest arteries (Ku, 1997). Typically, blood flow is laminar except under intense exercise and in l arge arteries of large an imals (Waite and Fine, 2007). Blood, strictly speaking is not a Newtonian fluid; however, it can be modeled as such in vessels greater than 1 mm when it flows with shearing strain greater than 100 s 1 (Waite and Fine, 2007). Blood flow is not a steady flow, but rather pulsatile flow due to the rhythmic contractions of the heart. The flow pattern becomes more complicated w ith arterial walls consisting of s mooth muscle and elastic tissue Pulsatile flow combined with an elastic


26 tube introduces the idea of waves traveling through the system. As pressure increases with each pump of the heart the radius of the vessel increases and between pumps the pressure decreases allowing the vessel to relax. This results in the formation of waves m oving through the system. Flow, no matter how small the elasticity of the tube, propagates down the tube in waves and when obstacles appear, waves can be reflected altering the local flow in a vessel (Zamir, 2000). Parameters that describe the elastic beh avior of arteries are distensibility and p ), while compliance is the absolute change p ) (Yim, 2008). These are generally expresses in changes of the cross sectional area of the arterial lumen during a cardiac cycle, which is allowed since vessel length changes very little within a cardiac cycle (Yim, 2008). In other word, a s compliance increases, the artery becomes more distensible or more flaccid, and as compliance decreases the artery becomes less distensible or firmer (Waite et al., 1990). Lumped parameter models based on electrical circuits have been devised to allow sim ulations of the cardiovascular system (Thiriet, 2008). These use electrical analogues, compliance (vessel storage ability), inertance (a measure of the pressure gradient of a fluid required to cause a change of flow rate with time), and resistance (impedan ce components with inertial and viscous forces) based on the similarities of AC electricity and blood flow (Thiriet, 2008). These analogies lead to the vascular impedance concept, which are complex quantities introduced into unsteady electrical analogues ( Thiriet, 2008). Resistance and its close relation impedance both measure


27 physical properties that tend to inhibit blood flow (Bateman, 2004). Simply put, vascular resistance specifies the opposition to mean flow generated by a mean pressure and thus by def inition is a single number (Adamson, 1999). Vascular impedance is a frequency dependent complex quantity that specifies the opposition to pulsatile flow generated by a pulsatile pressure (Adamsom, 1999; Thiriet, 2008). Blood flow can be shown as the sum of the steady flow and pulse flow components (Bateman, 2004). This is important since it can be shown that a change in vascular resistance alters the steady flow but not the pulse flow component and conversely impedance can alter the pulse flow but not the s teady flow component (Bateman, 2004). Impedance is often lumped flow depends on the viscosity of the fluid, vessel length, and the fourth power of the radius, while imped ance depends fundamentally on the compliance or visoelastic properties of the vessel wall that allow it to expand and contract (Bateman, 2004). The terms vascular resistance and impedance tend to be used interchangeably in the literature thus the two wil l be defined, here as; resistance is the opposition to the steady flow component and impedance is the opposition to the pulse flow component of blood flow (Adamson, 1999). There are three main determinates of blood flow pulsatility: 1) arterial blood press ure pulsatility, 2) resistance and 3) impedance. Arterial blood pressure pulsatility depends on upstream factors such as heart rate, stroke volume, aortic compliance, and total peripheral vascular resistance. Resistance expresses the total opposition to st eady flow and is usually due to resistance at the arteriole level. Impedance (pulse pressure divided by pulse flow) expresses opposition to pulsatile flow at the heart rate frequency measured at the entrance to the vessel of interest


28 (Adamson, 1999). For e xample, when a blood vessel constricts the decrease in diameter increases resistance but at the same time it becomes less compliant (or less elastic), impedance increased. Principles of Doppler ultrasongraphy The use of modern Doppler ultrasound is the r esult of two discoveries, the Doppler Effect, first published in 1841 by Christian Andreas Doppler and the Doppler Effect is the observed changes in the frequency of tran smitted waves relative to a source and observer when mo tion exists between the two. The first prototype pulse wave Doppler device was developed in the 1950 s (Satomura et al., 1956) This breakthrough allowed the discrimination of blood vessels from other s tructures. The next real breakthrough was the development of real time 2 dimensional Doppler technology. This integrated a filtering technique developed by Angelsen and Kristofferson (1979), and an autocorrelation technique developed by Namekawa (1982). Th e autocorrelation technique allows the mean Doppler phase shift to be processed in real time and the color mapping to be displayed while the scan occurs. The first use of the prototype was in 1983. Since then Doppler technology has continued to advance wit h the development of 3 dimensional and 4 dimensional Doppler ultrasonography (Maulik, 2005). There are three basic types of Doppler machines: 1) continuous wave, 2) pulse wave, and 3) duplex. Continuous wave Doppler constantly transmits a signal. The trans ducer contains crystals dedicated to either sending or receiving signals. There is no range discrimination meaning any tissue movement in the path of the ultrasound beam is detected by the machine. The second type is the pulse wave system. Pulse


29 wave Doppl er uses the same transducer crystals to both send and receive information. The time between sending and receiving dictates the distance of the target for the transducer to be evaluated (range gating). The duplex Doppler system combines B mode utrasonograph y with pulse wave Doppler technology. This allows one to move the sampling location to known locations on the screen. This represents the majority of machines used today (Maulik, 2005). Several variations exist on the above ideas. Multi gated Doppler uses pulse wave Doppler but with multiple sampling gates sequentially sampled. Color flow mapping is based on this principle, using multiple scan lines with burst of short ultrasound pulses. The general process involves a transducer and receiver. The informati on from the transducer is converted to digital format and stored for integration with color flow mapping. Backscattered echoes are converted to digital data. The digital signals are filtered to remove noise, and the data analyzed to determine phase shifts. The data is then color coded and overlaid with the B mode image. This can be done using power flow (Doppler) or color power flow (Doppler) analysis. Power Doppler mode displays both the direction and magnitude of flow in a range of colors from red to blue The direction/color representation can be changed on machines. Color power Doppler displays the magnitude in a range or red colors but does not provide information on direction. Color power Doppler mode is more sensitive to low blood flow than power Dopp ler mode. These modes can be used in combination with spectral gating to aid in locating small blood vessels. Data from this can examine the percent area of a structure receiving blood flow (Maulik, 2005).


30 Doppler ultrasonography measurements Data from spe ctral measurements provide a graph of the flow rate per unit time in the vessel examined. If the angle of insonation is know and the diameter of the vessel measured, the volume of flow can be calculated using, where V is the flow volume, TAMV is the time d average mean velocity and D is the vessel diameter. Additionally, ratios can be calculated based on the wave form. The most common are resistance index (RI) where PSV is the peak systolic velocity and EDV is the end diastolic velocity; pulsatility ind ex (PI), and systolic/diastolic (S/D). These ratios area used due to their relative independence of Doppler angle: velocity is calculated from, where F d is the frequency shift, f is the frequency transmitted in Hertz, is the velocity (i.e. PSV, EDV or TAMV) in m/s, is the insonation angle and c is the speed of sound (1500 m/s). If the Doppler angle is 90, cos(90)=0, velocity cannot be calculated. The relative independence is due to the cosine canceling in both the denominator and


31 numerator in the vascular indices equations thus removing the effect of angle from the equation. There is some confusion in the interpretation of these. The dogma is the higher the value the more resistance to flow and thus lower flow. In r eality, PI, RI and S/D are a measure of the amount of pulse in the system and the relationship does not always hold true depending on if there is an increase in resistance (opposition to steady flow), an increase in impedance (opposition to pulse flow) or a combination of the two. In fact, the actual PI for an artery can vary based on mechanical factors such as: distensiblity, vessel diameter, resistance and distensiblity of vessels downstream of the measurement point (Waite et al., 1990). The determinatio n of the combined effect of resistance and compliance on velocity waveforms is complex, and a simple relationship between waveform shape, vascular index, resistance and compliance is not available (Waite et al., 1990). Theoretical models indicate certain r elationships between these factors (Waite, 1987). A change in resistance strongly influences the mean velocity, but can also affect the shape of the waveform and peak to trough value (Waite, 1987). A change in compliance primarily affects the shape of the waveform and peak to trough value (Waite, 1987). Adamson et al. (1990) noted that indices such as S/D and pulsatility index were not reliable indicators of umbilical blood flow. This was also found to be the case in previous work examining cerebral blood f low (Handen et al., 1983; Batton et al., 1983). Sauders et al. (1998) noted that pharmacologically induced vascular resistance failed to induce any changes in RI while pulse flow decreased. Saunders et al. (1998) concluded that multiple hemodynamic factors affect Doppler waveform shape, the Doppler Resistance index will be an accurate measure of vascular resistance when resistance is the sole


32 variable; however, in situations in which pressure pulsatility and fundamental impedance are altered, Doppler Resist ance index and other pulsatility indices fail to measure vascular resistance changes. In other studies, vascular indices have been reported to be linearly related to vascular resistance (Norris et al., 1984; Spencer et al., 1991); however these studies do not take into account the effect of compliance on the vascular index index. Bude and Rubin (1999) developed a model to study the relation between Resistance index and volume of flow in relation to changes in compliance. Bube and Rubin (1999) found that w hen compliance was zero, Resistance index values were independent of the actual resistance. Bude and Rubin (1999) demonstrated that compliance is necessary for resistance to affect Resistance index values and that the lower the compliance the lower the Res istance index value was for the same amount of resistance. Bude and resistance alone but also by vascular compliance and suggest the name be changes to Impedance index Dop pler ultrasonography and large animal reproduction Using cattle, W aite et al. (1990) examined change in uterine arterial blood flow and the relationship to estrogen and progesterone concentrations using Doppler transducers implanted on the uterine arteries Uterine blood flow increased 3 to 4 days prior to estrus, peaking at estrus and then declined for the next 2 days (Waite et al., 1990). The authors divided the estrous cycle into three periods to analyze uterine arterial blood flow; a period of high flow with estrogen to progesterone ratio at its highest, a transition period and period of low blood flow. The period of high blood flow was found concluded to have a high but constant compliance and a large increase in


33 blood flow, resulting in a decrease of P I. During the transition period mean blood flow remained constant although PI decreased (Days 4 to 7; Waite et al., 1990). It was concluded that during the transition period, compliance of the vessels decreased while mean flow remained constant resulting i n the decreasing PI values. During the remainder of the luteal phase (low blood flow, Days 7 to 14), compliance remained low while blood flow decreased, resulting in an increase in PI (Waite et al., 1990). Bollwein et al. (2000) using transrectal Doppler u ltrasonography found similar results in cattle validating the use of transrectal Doppler ultrasonography in evaluating uterine blood flow. In post partum dairy cattle, the largest reduction in uterine blood flow volume occured during the first 7 days post partum and continues to decrease until about 18 days (Krueger et al., 2009). Blood flow volume then remained constant for the next 60 days. During this time PI was found to increase for the first 28 days and then began to decrease for the next 60 days (Bo llwein et al., 2002). In cows, RI in the uterine artery ipsilateral to the conceptus was consistently lower than the contraltateral uterine artery (Bollwein et al., 2002). Additionally during the first 8 mo of pregnancy RI continually decreased, while the TAMV, diameter and volume of blood flow increased during the same period (Bollwein et al., 2002). In mares, uterine blood flow volume has been found to increase from Day 11 (Bollwein et al., 2003) to Day 14 (Ousey et al., 2012) until term. By 2 weeks, blo od flow becomes significantly increased in pregnant versus nonpregnant mares while RI is decreased (Bollwein et al., 2003), and increases with gestational age (Bailey et al., 2012). Between 18 and 26 weeks, diastolic notching disappears in pregnant mares


34 ( Bollwein et al., 2004). Younger mares had higher uterine blood flow which increased from 0.28 ml/min/kg at Day 14 to 27.7 ml/min/kg, and was higher than older mares at both Day 14 (0.24 ml/min/kg) and term (23.8 ml/min/kg; Ousey et al., 2012). This data su ggested older mares have poor placental microvillus development which may have contributed to the reduced birth weights observed in older mares (Ousey et al., 2012). Acosta et al. (2005) found in cattle that blood flow prior to deviation was the same betwe en the largest and second largest follicle; however, after deviation blood flow increased to the dominant follicle. Additionally, blood flow to the dominant follicle increased during the period of time prior to ovulation (Acosta et al., 2003). Mare which b ecame pregnant after hCG induced ovulation had a greater percentage of perfusion to the pre ovulatory follicle and lower RI and PI to the ovulatory ovary than mares which did not become pregnant (Silva et al., 2006). In heifers undergoing TAI, those that become pregnant had greater percent of the follicle with perfusion 26 hours post GnRH, than those that did not become pregnant (Siddiqui et al., 2009). In mares, blood flow to the preovulatory follicle increased as ovulation approached; however, within the hours preceding ovulation there is a decrease in blood flow to the future ovulatory follicle (Gastal et al., 2006, 2007; Palmer et al., 2006; Ginther et al., 2007b, 2009). During ovulation, blood flow concentrates at the base of the structure and is assoc iated with the granulosal layer, but the middle and apical aspects have a lack of blood flow (Gastal and Gastal, 2011). These results ask whether Doppler ultrasonography may be a useful tool to predict the success of breeding on a normal cycle, but also wh ether blood flow evaluation may aid in predicting the success of a superovulation protocol.


35 During CL development there is an increase in blood flow that coincides with the increase in progesterone (Acosta et al., 2003). Around the time of sponateous lute al regression (Days 16 17) there is an increase in luteal perfusion which precedes the decrease in progesterone by one day (Shirasuna et al., 2004; Miyamoto et al., 2005, 2006). During induced luteolysis, with PGF an increase in CL perfusion occurs 30 m in post injection and remained increased for 2 h. One suggested application of Doppler ultrasonography is the differentiation of follicular and luteal cysts in cattle, since the use of color Doppler can facilitate s wall (Matsui and Miyamoto, 2009). It has been suggested in cattle, evaluating CL blood flow during pregnancy may be a diagnostic tool. CL perfusion increases post ovulation ; however in cycling cows decreases around luteolysis on Days 19 21. It has been proposed this period of decreased luteal perfusion may be used as an early indicator of pregnancy (Matsui and Miyamoto, 2009). CL blood flow appears to be closely related to progesterone (Glock and Brumsted, 1995; Bourne et al., 1996) and thus color Dopple r may provide a useful assessment of luteal function (Matsui and Miyamoto, 2009). Matsui and Miyamoto (2009) have also proposed the use of luteal blood flow as a prognostic indicator for fetal loss in addition to pregnancy recognition. Utt et al. (2009) at tempted to use luteal blood flow as an early indicator of pregnancy in beef cattle; however, they concluded that luteal blood flow alone was a poor indicator for early pregnancy diagnosis due to low specificity and low sensitivity. In mares, an early vasc ular indicator of the future embryonic position was noted (Silva and Ginther, 2006). The Doppler ultrasound exam demonstrated a colored spot in


36 the uterus 0.5 0.1 d after fixation and 2.5 0.2 d prior to detection of the embryo (Silva and Ginther, 2006) L Arginine Introduction Since the discovery of L arginine as a mammalian protein 1885 (Hedin, 1895), roles in several metabolic pathways have been found. This led to the idea of functional amino acids, which regulate key metabolic pathways to benefit s urvival, growth, development, reproduction and health of animals and humans (Wu et al. 2010). Arginine is classified as a functional amino acid (Wu et al., 2010) and can be supplied by the diet and/or synthesized in mammals from glutamine via pyrroline 5 c arboxylate synthase and proline oxidase (Appleton, 2002). Supplemental arginine is readily absorbed by the intestines and roughly half is rapidly converted to ornithine (Appleton, 2002). Ornithine can be metabolized to glutamate and proline (Appleton, 200 2). L Arginine can be converted to citrulline and nitric oxide via nitric oxide synthase, can be a precursor for ornithine (which acts as a precusor for polyamines and L proline), agmatine, creatine or used in protein synthase (Raghavan and Dikshit, 2004). Due to arginine acting as a precursor for a variety of molecules, the potential exists for numerous biological effects. Endocrine effects Endocrine mechanisms may contribute to the effects seen by L arginine supplementation. Administration of certain ami no acids can increase plasma concentrations of pituitary hormones (Knopf et al., 1965). Intravenous doses of L arginine stimulate growth hormone, pancreatic insulin, glucagon and pituitary prolactin secretion (B ger and Bode


37 Arginine infused intravenously (183 mg/kg body weight) increased plasma growth hormone con centrations in women 20 fold (Merimee et al., 1969) and in men (30 g) 8.6 fold (Alba Roth et al., 1988). Isidor et al. (1981) found administering an oral combination of lysine and arginine resulted in a greater growth hormone release than either alone. An arginine metabolite, ornithine, has been found to increase growth hormone at high doses which caused patients side effects (Bucc et al., 1990). The exact mechanism of arginine increasing growth hormone is unclear; however, evidence suggests nitric oxide ma y play a role (Rettori et al., 1994; Fisker et al., 1999) via a Ca 2+ cGMP independent pathway. In culture a nitric oxide donor increase pituitary growth hormone secretion which was not altered by Ca 2+ channel blockers (Pinilla et al., 1991). Nitric Oxide Nitric oxide is formed from L arginine a reaction catalyzed by nitric oxide syntase (Bode B ger et al., 2007). There are two mechanisms for loss of NO bioactivity: reduced synthesis and increased oxidative inactivation by reactive oxygen intermediates (Bode dysfunctional endothelium an d the limitation of reactive oxygen species increases the availability of NO, evident by treatment with antioxidants (Gokce et al., 1999) and cholesterol lowering therapy (Yamamoto et al., 1998). Other methods to increase NO availability include the use of agonists to stimulate nitric oxide release from the endothelium and providing additional substrate, such as L arginine, to endothelial cells (Bode B ger et al., 2007).


38 The exact mechanism by which L arginine improves endothelial function remains elusive (Bode not appear to limiting; intracellular L arginine concentrations are i n the millimolar range Menton constant (k m ) is in the micromolar range (Bredt al., 2007). One possible mechanism is arginase may alter cellular concentrations of L arginine, decreasing the amount available for nitric oxide synthase (Wei et al., 2000). A second explanation is decreased arginine transport into the cytoplasm via y+ transports which also transport other cationic amino acids. Thus, supplementation increases arginine available to compete for the transporter (Buga et al., 1996). A third possibility is L arginine is compartmentalized in the cytoplasm leading to low amounts in the vicinity of nitric oxide synthase (McDonald et al., 1997) Finally, endogenous analogs of L arginine, asymmetrical dimethylarginine (ADMA) and monomethylarginine (L NMMA) exist which competitively inhibit nitric oxide synthase (Vallance et al., 1992). Increased plasma ADMA and a reduced arginine/ADMA ration are correlated with a decrease in endothelium dependent flow mediated vasodilation (B ger et al., 1998; Lundman eat al., 2001; Sydow et al., 2003). Data suggests that ADMA during a pathophysiology may alter the K m without impeding arginine transport into the cell al conditions there is enough arginine to create nitric oxide; however, under certain pathologies supplementing arginine has a beneficial effect. This is supported by research which found that normal patients supplemented with L arginine had no affect


39 on v asodilation, while those patients with chronic heart failure had an increase in endothelial dependent vasodilation (Bode B ger et al., 2007). Polyamines Polyamines are low molecular weight aliphatic compounds composed of carbon chains of variable lengths with two or more amino groups (Lefvre et al., 2011). Polyamines are synthesized from arginine, proline and methionine eith er from the diet or intestinal flora (Lefvre et al., 2011). Polyamines are synthesized from L arginine and L proline through ornithine and L methionine intermediates via S adenosylmethionine (Lefvre et al., 2011). Ornithine decarboxylase (ODC1) is the ra te limiting step of polyamine synthesis and catalyzes the decarboxylation of L ornithine to putrescine (Lefvre et al., 2011). Putrescine combined with decarboxylated S adenosylmethionine is converted to spermidine and spermine by either spermidine synthas e or spermine synthase (Lefvre et al., 2011). Polyamines play a role in spermatogenesis and are thought to be important for oogenesis, embryogenesis, implantation, placentation, parturition, lactation and post natal development (Lefvre et al., 2011).This is suggested by the induction of ODC1 activity by FSH, LH, eCG and hCG (Johnson and Sashida, 1977; Nureddin, 1978; White and Ojeda, 1981). ODC1 localizes to the theca interna of preantral and antral follicles in a similar pattern to the LH receptor (Iceks on et al., 1974; Persson et al., 1986). Specific polyamines are essential for follicular development; however, excessive amounts can be detrimental (Lefvre et al., 2011).Transgenic expression of SAT1 (an enzyme in the catabolic pathway of polyamine syntha sis) causes an infertile phenotype characterized by follicular arrest at the secondary stage (Pietil eta l., 1997). Additionally, LH was more effective at inducing ODC1 than FSH it is hypothesized that polyamines play a role in ovulation (Lefvre et al., 2011).


40 Treatment of rats with an ODC1 inhibitor during proestrus decreased plasma LH and prolactin concentrations, reduced polyamine concentrations in the pituitary, decreased the number of oocytes ovulated and decreased plasma progesterone concentration ( Nicholson et al., 1988; Nicholson and Wynne Jones, 1989). There is a paucity of information on the role of polyamines in mammalian oogenesis (Lefvre et al., 2011). In Xenopus laevis meiotic maturation can be induced with progesterone or hCG, and this matu ration is preceded by an increase in ODC1 (Younglai et al., 1980; Sunkara et al., 1981). Using an ODC1 inhibitor, oocyte maturation can be blocked in Xenopus laevis (Sunkara et al., 1981). Additionally, in vitro oocytes treated with polyamines have a highe r rate of development to blastocysts and more trophoblast outgrowth (Muzikova and Clark, 1995). ODC1 expression increases in the two cell mouse embryo and during the blastocyst stage (Domashenko et al., 1997). Null mutations in ODC1 are not lethal until ga strulation (Pendeville et al., 2001). In vitro, null ODC1 mutants do not survive past the late morula/ early blastocyst due to apoptotic loss of the inner cell mass (Pendeville et al., 2001). Polyamine biosynthesis is a feature of both invasive and nonin vasive placental types, suggesting a conserved role of polyamines during implantation and placentation (Lefvre et al., 2011). In ruminants and pigs the principle site of polyamines appears to be the uterus (Lefvre et al., 2011). In pigs, polyamine synthe sis in the uterus peak on Day 12 of pregnancy when estrogen from the conceptus signals the prescence of embryos (Rodriguez Sallaberry et al., 2001). ODC1 and polyamine concentrations in the luminal epithelium remain elevated on Days 20 to 40 of pregnancy i n sows (Wu et al., 1998) and decrease thereafter (Wu et al., 2005).In porcine allantoic fluid, ornithine


41 and polyamine concentrations increase between Days 40 to 60 gestation (Wu et al., 1998). Ruminants follow a similar pattern to those observed in pregna nt swine (Kwon et al., 2003; Gao et al., 2009). Polyamines appear to play an important role in placental development. Polyaine deprivation is associated with abnormal development of extraembryonic structures, yolk sac and placenta (Lpez Garca et al., 20 08), a reduction in the number of blood islands and lowered expression of genes coding for embryonic globins (Lpez Garca et al., 2009). ODC1 localizes to the labyrinthine zone of the placenta and development of the placenta and spongiotrophoblast layer a re abnormal in ODC1 knockouts (Lpez Garca et al., 2009). Depending on the level of ODC1 during post implantation the detrimental effects can vary. At low levels of ODC1 inhibition the effects are similar to intrauterine growth restriction, with reduced p lacental dramatic effects can be seen (Lefvre et al., 2011). Agmatine Agmatine is formed from L arginine via arginine decarboxylase. Recent studies have found agmatine p resent in plasma and tissue, widely and unevenly distributed throughout the body (Feng et al., 1997). When administered intravenously, agmatine, caused a decrease in blood pressure which was reversed by L NAME a nitric oxide inhibitor (Haulica et al., 1999 ). Agmatine acts as an agonist to imidazoline and 2 adrenergic receptors (Reis et al., 1998; Yang et al., 1999; Blantz et al., 2000). Treatment of individuals with either Imidazolin receptor 2 adrenergic receptors agonists results in a decrease in blood pressure (Yu an Frishman, 1996; Link et al., 1996), suggesting a role of agmatine in vascular regulation. Later it was shown that polyamines synthesized from agmatine induced relaxation in rabbit thoracic aorta via a


42 nitric oxide synthase independent but Ca 2+ agonistitic mechanism (Myung et al. 200 0). Interestingly, agmatine has been reported to inhibit inducible nitric oxide synthase (Blantz et al., 2000; Raasch et al., 2001) but activate endothelial nitric oxide synthase (Gao et al., 1995; Morrissey et al., 1997) suggesting not only actions via im idazoline 2 adrenergic receptors but also potentially through NO. L Arginine and reproduction Battaglia et al. (1999) examined the effect of orally administered L arginine in women who respond poorly to ovarian hyperstimulation in an IVF program. Wome n treated with L arginine had an increase in the number of oocytes collected and embryos transferred (Battaglia et al., 1999). Plasma and follicular fluid concentrations of arginine, citrulline, NO 2 /NO 3 and IGF 1 were increased with L arginine treatement (Battaglia et al., 1999). Both ovarian and uterine vascular indices (RI and PI) were lower in women receiving L arginine (Battaglia et al., 1999). The authors concluded L arginine supplementation may improve ovarian response, endometrial receptivity and p regnancy rates in women who respond poorly to an IVF program. The same group followed up with a randomized blind trial using normally responding patients using IVF (Battaglia et al., 2002). This study found patients treated with L arginine had an increase in vascular indices on the day of oocyte retrieval, lower pregnancy rate per cycle and lower pregnancy rate per embryo transferred (Battaglia et al., 2002). This suggests a potential role of L arginine in aiding with subfertile patients, but a potential ad verse effect on reproduction in healthy patients. L Arginine and pregnancy The use of L arginine during pregnancy has generated the most research. In pregnant pigs, up to 60% or the arginine in catabolized in the small intestines by the


43 enzyme arginase ( Bergen and Wu, 2009). Due to this catabolism, it is thought increased arginine intake could improve pregnancy out come in pigs (Wu et al., 2010). Several experiments support this idea. First, increasing maternal plasma arginine by 69% increased endothelial NO synthesis (Wu and Meininger, 2002) and increased utero placental blood flow (Neri et al., 1995). In women diagnosed with asymmetrical fetal growth restriction, L arginine treatment resulted in increased birth weights compared to women undergoing standa rd therapy, but still lower than normal pregnancies (Xiao and Li, 2005). In pregnant rats exposed to hypoxia during gestation, L arginine treatment prevented the decrease in fetal weight that hypoxia induces (Vosatka et al., 1998). Supplementation of under nourished ewes with L arginine increased birth weight 21% compared to non supplement ewes (Lassala et al., 2010). In gilts fed 1.0% of their diet L arginine (fry matter basis) beginning on Day 30 gestation, the number of piglets born increased from 9.4 to 11.4 pigs/litter and the birth weight increased by 24% (Mateo et al., 2007). In mice, arginine supplementation between D ays 12 and 18 increased litter size, number of live pups born and birth weight (Greene et al., 2012). Additionally, L arginine treatment resulted both in an earlier rise Vegfr2 transcription and increased amounts of transcript. Vegfr2 is a cell surface receptor for VEGF and Vegfr2 is the primary receptor by which VEGF promotes angiogenesis (Otrock et al., 2007). In sows, arginine supplemen tation increases chorioallantoic expression of eNOS, but no difference was observed between control in VEGFA or PlGF1 transcript abundance (Wu et al., 2012). When the diet is supplemented with 0.8% L arginine in gilts from Days 0 to 25 gestation, arginine increased the vascularity of the uterus but reduced uterine weight, number of fetuses, CL number, fetal weight, progesterone concentrations and


44 estrone concentrations (Li et al., 2010). The data thus far suggests that the stage of gestation and dose of ar ginine fed can have impact fetal survival possibly via impacts on placental angiogenesis (Wu et al., 2009). The mechanism by which arginine work is largely unknown; however, it is postulated this may involve changes in CL development and function, progeste rone production, NO signaling, cellular redox status (Wu et al., 2010) and angiogenesis (Otrock et al., 2007). Synopsis and Objectives Based on a review of the literature, blood flow to the reproductive tract plays a critical role in fertility. Doppler u ltrasonography offers a minimally invasive method to evaluate changes in blood flow to the reproductive tract. L Arginine has been shown to have beneficial effects on reproduction in other species which are thought to be mediated by its potential cardiovas cular effects. The objectives of this dissertation are to: 1. Determine whether orally supplement L arginine can increase plasma arginine concentrations in mares, 2. Determine if L arginine supplementation can improve embryo recovery rates in mares, 3. Determine i f L arginine supplementation can alter uterine blood flow during late pregnancy 4. Determine if L arginine supplementation alters follicular dynamics or ovarian blood flow.


45 CHAPTER 2 ORALLY SUPPLEMENTED L ARGININE IMPAIRS AMINO ACID ABSORPTION DEPENDING ON DOSE Introduction L Arginine supplementation improves vascular performance in humans (Boger et al., 2001) and reproductive performance in swine (Mateo et al., 2002) L Arginine can affect these and other systems via several mechanisms. Many of the effects of arginine are thought to be mediated by the actions of nitric oxide (NO), which appears to play a role in blood pressure maintenance, inflammation, apoptosis and oxidative damage (Appleton, 2002) Polyamines are synthesized from arginine, proline and methionine either from the diet or intestinal flora and are thought to be important for oogenesis, embryogenesis, implantation, placentation, parturition, lactation and post natal development (Appleton, 2002) Arginine also alters adrenal and pituitary function, stimulating catecholamines, insulin, glucagon, prolactin and growth hormone (Appleton, 2002) Thus, it is not surprising that L arginine supplementation can have effects on reproductive, cardiovascular, pulmonary, liver, renal, gastrointestinal and immune physiology (Wu et a ., 2000) Many of the effects of arginine are thought to be mediated by the actions of NO, which appears to play a role in blood pressure maintenance, inflammation, apoptosis and oxi dative damage (Appleton, 2002) The effects of L arginine supplementation observed in other species have generated an interest in using arginine as a n utraceutica l for horses. Studies have found L arginine supplementation improves endothelial cell function, decr eases gastric ulcers, enhances wound healing and improve insulin s ensitivity in diabetic humans (Gad, 2010) L Arginine infused intravenously (183 mg/kg body weight) increased plasma growth hormone co ncentrations in women 20 fold (Merimee et al., 1969) and in men (30


46 g) 8.6 fold (Alba Roth et al., 1988) ,suggesting L arginine supplementation may be of benefit to performance horses, such as racehorses. Additionally, L arginine supplementation may increase blood fl ow to the reproductive tract (Mortensen et al. 2011) and improve the uteri ne environment post foaling (Kelley et al., in press) increasing reproductive efficiency. Current knowledge on dose and absorption of orally supplemented L arginine must be inferred from other species. Supplementation in swi ne ranges from 0.4% to 1.0% of the diet (on a dry matter basis) (Mateo et al., 2007; Wu et al., 2007; Li et al., 2010) and in human studies doses u p to 30 g are well tolerated (Gad, 2010) There is a paucity of literature on the effect of increased supplem entation of one amino acid on the absorption of other amino acids. Lysine is the limiting amino acid in horses (NRC, 2007) and thus must be considered in diet formulation. Considering, arginine, lysine, cysteine, and histidine can all utilize cationic amino acid tr ansport system with varying affinities [ (Palacin et al., 1998) ] it is possi ble increasing L arginine in the diet may adversely effect the absorption on other amino acids, including lysine. The purpose of this study was to observe the effect of two oral L arginine doses on plasma arginine concentrations and determine whether they alter the absorption of other amino acids in mares. Materials and Methods Studies were approved by the University of Florida Institute of Food and Agricultural Sciences (IFAS) Animal Research Committee and were conducted at the IFAS Equine Science Center (Ocala, FL). Experiment 1 used a mix of Thoroughbred and Quarter Horse mares, blocked based on age and breed, then randomly assigned into an L arginine group (n=6) or Control group (n=6). The mean (SEM) age of the L


47 Arginine group was 10.2 4.2 yr and c ontrol was 9.2 6.8 yr (NS). Both groups were fed 0.25% of body weight a commercial grain (minimum guarantees: 16% CP, 3.5% Arginine mares had their grain supplemented with L arginine (CAS number 74 79 3; Ajinomoto AminoScience LLC, Raleigh, NC, USA) at 0.025% of body weight (mean (SEM) L arginine supplemented: 134.3 3.6 g) once at the beginning of the trial. with the L arginine mares receiving a mean (SEM) of 1.34 0.04 kg grain and control mares receiving 1.36 0.04 kg (NS) grain. Mares were fed and housed individually in stalls for the duration of the study with free access to water. Blood samples (10 ml) were taken via jugular catheters, prior to feeding (0 h) and at 0.5, 1, 2, 3, 4 and 5 h post feeding and placed in heparinized tubes. Samples were then centrifuged at 2,400 x g for 15 min and plasma was stored at 80 C until analyzed. Experiment 2, utilized Thoroughbred (n=3) and Quarter Horse (n=3) mares in a cro ss over design. On the first day of the experiment mares were randomly assigned to either L arginine (n=2), urea (isonitrogenous; n=2) or control (no supplement; n=2) group. Mares were given a 6 d washout period, rotated to another group and the trial was rerun, and repeated until each mare received each treatment. Groups were fed 0.25% of a commercial grain (minimum guarantees; 16% CP, 3.5% crude fat, 0.9% Ca, with the mares rec eiving a mean ( SEM) of 1.40 0.04 kg of grain per feeding. Mares had their grain supplemented with L arginine (CAS number 74 79 3; Ajinomoto AminoScience LLC, Raleigh, NC, USA) at 0.0125% of body weight (study mean: 69.9 2.2 g) and urea at 0.0087% of body weight (study mean: 48.9 1.5 g) once on the day


48 of the study. Mares were fed and housed individually in stalls for the duration of the study (12h). Blood samples were taken via jugular catheters, prior to feeding (0 h) and at 1, 2, 4, 6, 8, 10 and 12 h post feeding on the day of the study, in heparinized tubes. Samples were then centrifuged at 2,400 x g for 15 min and plasma was stored at 80 C until analyzed. Amino Acid Analysis Plasma samples from both experiments were deproteinized using 35% (w/v) sulfosalicylic acid. The acid soluble fraction was separated by centrifugation (4C, PA, USA) and then mixed 1:1 with 0.02 N HCL (Le Bocher et al., 1997) Plasm a samples were then analyzed for amino acid composition using an Amino Acid Analyzer (L 8900,Hitachi High Technologies, Pleasanton, CA, USA) as previously described (Ma et al., 2010) Statistical Analysis Data was analyzed using the SAS MIXED procedure wit h a random statement to account for variability of mares within group and a repeated measures statement to account for sequential measurements taken each hour (SAS version 9.2: SAS Institute, Cary, NC, USA). Dietary treatment, hour, and hour x dietary trea tment were included in the model as fixed effects and a compound symmetry covariate structure was used. Data is represented as least squared means ( SEM). A probability of P < 0.05 was ndicated a trend towards significance.


49 Results Experiment 1 Mares fed 0.025% of body weight L arginine had an increase in mean plasma respectively; P < 0.05). L Arginine m ares had elevated ( P < 0.05) plasma arginine concentrations by 2 h post feeding (Fig. 1) and remained higher than control through the remaining 5 h blood drawing period. There were no significant differences in plasma citrulline concentrations (Fig. 2A) b etween groups. Ornithine concentrations (Fig. 2B) were increased in arginine treated mares compared to control mares (112 10 respectively; P < 0.05) with concentrations being higher ( P < 0.05) at 2 h post feeding and remaining elevated through the remainder of the experiment. Mean plasma lysine concentrations (Fig. 3A) were lower in L arginine mares (91 P < 0.05) with L arginine mares having lower ( P < 0.05) plasma lysine concentrations at 0.5, 1, 2, 3, 4 and 5 h post feeding. Mean plasma methionine concentrations (Fig. 3B) were lower in L arginine mares (20.6 P < 0.05). Methionine concentrations were lower ( P < 0.05) at 1 h in L arginine mares than control mares, which peaked at that time (Fig. 3B). Mean plasma valine concentrations were lower in L arg P < 0.05). There were no differences in the mean plasma concentrations of histidine, glutamic acid, proline and isoleucine (Fig 3B F). L Arginine mares had lower ( P < 0.05) concentrations of each of the respective amino acids one hour post feeding compared to control. In the


50 control mares, amino acid concentrations peaked in each of these amino acids at 1 hour post feeding. The same effect seen in Fig. 3B F was also observed with threonine, p henylalanine, leucine, valine, alalinine and taurine (data not shown). None of these amino acids were significantly different between groups; however, each amino acid had a group*time interaction with L arginine mares having lower ( P < 0.05) the respectiv e amino acid concentration 1 h post feeding, which reached peak concentrations in control mares at that time. No differences were observed between L arginine mares and control mares in either the mean or at any time point between ammonia (mean: 36 3 Experiment 2 Mares fed 0.0125% of their body weight L arginine had an increased ( P < 0.05) mean plasma arginine concentrations compare Arginine mares had elevated plasma arginine concentration (Fig. 4) at 1, 2, 4, 6 and 8 h compared to urea or control mares which were not significantly different by 10 h po st feeding. No significant differences were found between groups in citrulline concentrations (Fig. 5A). Mean plasma ornithine concentrations were higher ( P < 0.05) in L arginine mares. Plasma ornithine concentrations (Fig. 5B) were elevated ( P < 0.05) in L arginine mares at 1, 2, 4, 6, and 8 h compared to urea and control mares and by 10 h were not significantly different.


51 Mean plasma lysine (Fig. 6A), methionine (Fig. 6B), histi dine (Fig. 6C), glutamic acid (Fig. 6D), proline (Fig. 6E) and isoleucine (Fig.6F) concentrations were not different ( P were greater ( P < 0.05) at 0 h in L arginine mares than control but were not different ( P No differences between groups were observed in threoni ne, phenylalanine, leucine, valine, alalinine and taurine concentrations (data not shown). Mean plasma ammonia concentrations were not different between groups; however, ammonia concentrations tended to be higher ( P l/L) at 6 h compared to L Mean plasma urea concentrations were not different ( P however, at 2 h, plasma urea concentrations were higher ( P < 0.05) with urea treatment P P < L) and tended ( P plasma urea concentrations tended ( P P arginin e P urea concentrations. Discussion L Arginine has received much attention due to the wide effects it has on a variety of physiological systems. Arginine could have poss ible beneficial effects in reproduction, insulin r esistance and gastric ulcers (Wu et al., 2007) each of which is of interest to the


52 equine industry. However, knowledge on the proper dosage and absorption is currently unknown. This study demonstrates the L arginine supplemented at both 0.025 and 0.0125% of body weight increased plasma arginine concentrations. Arginine supplementation has the potential to affect other amino acids absorption by competition for the same amino acid transporters, as lysine, cy steine and hi stidine (Palacin et al., 1998) It is possible that the dose fed in Experiment 1 was enough to saturate the amino acid transporters while in Experiment 2 this did not occur. In swine, feeding arginine > 2.5% of dry matter intake reduced growth and can cause death due to amino acid imbalanc e (Edmonds et al., 1987) but supplementation of up to 0.8 % of the diet had no effect on lysine or histidine absorption (Li et al., 2010) The explanation for the difference from these results and our study in horses is unclear. Woodward et al. (Woodward et al., 2012) reported no differences in L max or K M between porcine and equine jejunum and proximal colon, but that ponies had a greater diffusion of protein in brush border membrane ve sicles from both sections than hogs. The question remains as to whether these transporters have varying affinity for L arginine between species. Although our study did not observe diarrhea in any mares, pigs supplemented with 1.2% of the diet with L argin ine had an in crease incidence of diarrhea (Haydon et al., 1990) suggesting a gastrointestinal toxicity (Austric et al., 1981) This may be caused directly by arginine or mediated by nitric oxide production Tucker et al., 1991) Nitric oxide plays a role in water and electrolyte balance (Wu et al., 2000) altering blood flow to the gastrointestinal tract (Bochroder et al., 1994) alters intestinal motility (Van Weyenberg et al., 2006; Woodward et al., 2010; Woodward et al., 2012) and the


53 immune system (Akisu et al., 2002) The role of nitric oxide in gastrointestinal function is complex, but depending on the concentration may alt er secretion and absorption (Grimble, 2007) Although this study does not address the intestinal effects of arginine supplementation it raises the question as whether arginine or nitric oxide can inhibit amino acid absorption at high concentrations. Both experiments 1 and 2 had a large increase in plasma ornithine concentrations. This is consistent with previous work in other species, and it has been reported that up to 70% of ingested L arginine in converted to ornithine as it passes thr ough the intestinal membrane (Wu et al., 2000) Pregnant gilts supplemented at 1% of the diet with L arginine HCl beginning on Day 30 gestation had in crease plasma concentrations of proline, ornithine, and arginine and a lower plasma glutamine concentration on Days 70 and 110 of gestation. Wu et al. (2007) found that in sheep and swine, arginine administration either i.v. or orally increased serum conce ntrations of ornithine, urea and proline, while glutamine and ammonia decreased in a dose dependent manner. Wu et al. (2007) did not note changes in any other amino acids. The amount fed in Experiment 1 of our study corresponded to 1% of total diet and wou ld be comparable to the study by Mateo et al.(2007) Unexpectedly in Experiment 2, plasma concentrations of some amino acids begin to increase between 4 h and around 8 h post feeding. The timing of this increase suggests that these amino acids may be abso rbed as feedstuff passing through the hindgut. Originally, research suggested that amino acids were not absorbed via the hindgut (Bochoder et al., 1994) however, more recent work suggests that lysine is absorbed from the equine hindgut (Woodward et al., 201 2) The horse hindgut contains


54 mRNA for several amino acid transporters (Woodward et al., 2010) Both LAT 3 and b 0,+ AT amino acid transport systems were found to have significant amounts of mRNA in the hindgut. The authors concluded that the equine hindgu t may contribute to the absorption of cationic and neutral amino acids (Woodward et al., 2010) Interestingly, lysine, arginine and histidine are all cationic amino acids; however, only lysine appears to have incre asing plasma concentration at 8 h post feeding. Based on our study, it appears that the hindgut may contribute to the absorption of lysine, methionine and isoleucine. This raises questions on the specificity of the amino acid transport system in the hindgu t and the contribution of the hindgut to amino acid absorption. For Experiment 1, we chose to draw blood for only 5 h, based on work performed in swine (Wu et al., 2007) Wu et al. (2007) found that in both pregnant and nonpregnant gilts supplemented with L arginine HCl, arginine peaked around 1 h post feeding and after 5 h could find no difference in plasma L arginine concentrations between supplemented and non supplemented gilts. Wu et al. (2007) gave L arginine HCl at 0.76% (dry matter basis) of fed. Thi s is at a dose roughly between what we fed in Experiments 1 and 2. Our data from both experiments demonstrates that when supplementing L arginine HCl and evaluating plasma amino acid concentrations in horses, blood should be taken for at least 12 h. It is possible the differences in timing relate t o delivery method. Wu et al. (2007) administered L agrinine via oral gavage while our study mixed L arginine into the feed ration. The fiber length in our study may have been long and thus resulted in a decreased rate of passage through the digestive system (Van Weyenberg et al., 2006) thus increasing the time for amino acids to be


55 absorbed. This may in part explain the differences in the absorption profile between what has been observed in hogs and the present st udy. The results from our two experiments demonstrate that L arginine is absorbed via the gastrointestinal tract and depending on the amount fed, alters the absorption of other amino acids in non pregnant, non lactating, cycling mares. Although further re search is needed to determine the long term effects of supplementing a diet high in one amino acid, the possibility exist to create a deficit in other amino acids.


56 Figure 2 1. Least squares mean ( SEM) plasma arginine concentrations for mar es fed 0.025% of body weight L arginine for 5 h post feeding. An asterisk (*) denotes a significant (P < 0.05) difference between groups on a given hour.


57 Figure 2 2. Least squares means ( SEM) Panel A: Plasma citrull ine concentrations and Panel B: plasma ornithine concentrations for mares fed 0.025% of body weight L arginine for 5 h post feeding. An asterisk (*) denotes a significant ( P < 0.05) difference while a pound sign 0.10) bet P 0.10) between groups on a given hour.


58 Figure 2 3. Least squares mean ( SEM) Panel A: plasma lysine concentrations, Panel B: plasma methionine, Panel C: plasma histidine concentration s, Panel D: Plasma glutamic acid concentrations, Panel E: plasma proline concentrations and Panle F: plasma isoleucine concentrations for mares fed 0.025% of body weight L arginine for 5 h post feeding. An asterisk (*) denotes a significant (P < 0.05) dif 0.10) between groups on a given hour. Concentration mol/L Concentration mol/L Time (h)


59 Figure 2 4. Least squares mean ( SEM) plasma arginine concentrations for mares fed 0.0125% of body weight L arginine or 0.0087% of body weight urea fo r 12 h between urea and c ontrol on a given hour.


60 Figure 2 5 Least squares mean ( SEM) Panel A: plasma citrulline concentrations and Panel B: plasma ornithine concentrations for mares fed 0.0125% of body weight L arginine or 0.0087% of body weight urea for 12 h post feedi ng. An arginine and urea, a arginine and control control on a given hour.


61 Fig ure 2 6 Least squares mean ( SEM) Panel A: plasma lysine concentrations, Panel B: plasma methionine, Panel C: plasma histidine concentrations, Panel D: Plasma glutamic acid concentrations, Panel E: plasma proline concentrations and Pan le F: plasma isoleucine concentrations for mares fed 0.0125% of body weight L arginine or 0.0087% urea for 12 h post feeding. a significant ( P < 0.05) difference between L arginine and control treatments. Concentration mol/L Concentration mol/L Time (h)


62 CHAPTER 3 SUPPLEMENTAL L ARGININE SHORTENS GESTATION LENGTH AND INCREASES MARE UTERINE BLOOD FLOW BEFORE AND AFTER PARTURITION I ntroduction L Arginine (Arg) is one of ten essential AA for horses (NRC, 2007). Arg supplementation has been shown to improve reproduction, cardiovascul ar, pulmonary, renal, gastrointestinal, liver and immune functions in various species (Wu et al., 2009). In addition, Arg metabolism results in production of nitric oxide (NO), polyamines, proline, glutamate, creatine and agmatine (Wu and Morris, 1998). Ou r primary interest is the production of NO from Arg metabolism, due to the crucial role of NO in reproduction (McCann et al., 1999) and its vasodilation properties. Previous work has established the safety of supplementing Arg in diets of pregnant sheep, p igs and rats (Wu et al., 2007). Supplementing diets with Arg has been shown to enhance reproductive performance of pigs (Mateo et al., 2007) and reduced embryonic mortality in rats (Zeng et al., 2008). In the study of Mateo et al. (2007), Arg supplemented in the diets of gilts from Day 30 of gestation until term led to a greater number of live piglets increases could have been greater utero placental blood flow and great er placental angiogensis. In another study, Takasaki et al. (2010) reported women with a thin endometrium supplemented orally with L arginine (24g/d) had greater blood flow to the uterine radial arteries and improved endometrial thickening. Both Bollwein e t al. (2003) and Mortensen et al. (2010) have documented changes in uterine blood flow during early pregnancy in mares. The biological changes leading up to parturition in the mare have been documented; however, uterine blood flow characteristics during la te pregnancy and immediately following parturition have


63 not. Therefore, our goals were twofold; document the uterine blood flow changes leading up to, and the immediate days following, parturition; and evaluate any potential uterine blood flow increases to mares by supplemental feeding of Arg beginning in late pregnancy. Materials and Methods Animals Sixteen pregnant light horse mares were utilized for this trial. Animal use was approved by the Institute of Food and Agricultural Sciences Animal Care and Us e Committee at the University of Florida. Mares were maintained on pasture until signs of impending parturition were detected. Those mares nearing parturition were moved to a 1 acre dry lot for overnight monitoring until foaling. Once first signs of labor were detected, mares were moved into an approximate 6 m by 3 m foaling stall. The following foaling parameters were recorded: time from rupture of the chorioallantois to termination of stage II (foal delivery), time from foal delivery to shedding of the pl acental tissues, placental weight, and time from foal delivery to first nursing. Mares and foals were allowed back on pasture 24 h after foaling. Experimental Design Mares were blocked by age (range 5 19 yr), breed (Thoroughbred (n = 8) and American Quart er Horse (n = 8)) and expected foaling date (EFD); then randomly assigned to one of two dietary treatments: L arginine supplementation (n = 8) or non supplemented control (n = 8). The basal diet consisted of ad libitum access to Coastal bermudagrass hay an d 3.8 0.3 kg of a grain mix concentrate formulated for gestating Feed and Supply, Ocala, FL, USA) fed individually in stalls at 0700 and 1500 h.


64 Beginning 21 days prior to EFD Arg supplemented mares received 100 g of L Arginine (Ajinomoto AminoScience LLC, Raleigh, NC, USA), which was hand mixed into the 0700 h concentrate feeding. This amount of Arg represented approximately 1% of the total diet, and was based on previous rep orts of the efficacy and safety of 1% Arg supplemented to pregnant pigs (Mateo et al., 2007). An isonitrogenous vehicle was not fed to control mares to permit the evaluation of Arg supplementation on a standard pregnant mare diet. Additionally, the use of an isonitrogenous vehicle, such as L Alanine (Ala), has been shown to lead to false positive results. The study of Lewis and Langkamp Henken (2000) showed that the observed positive response of Arg was due to comparisons to Ala supplemented animals, and w as not evident when compared to animals fed a standard diet. Color Doppler ultrasonography Transrectal examinations of blood flow to the reproductive tract of all mares began 21 d prior to the EFD between 0800 and 1100 h by an operator blind to treatment. Pregnant mares were evaluated every other day until parturition. Twenty four hours following parturition ultrasound exams were continued daily until 7 d post foaling. A digital color Doppler ultrasound with a 10 5 MHz broadband 52 mm linear probe (Microm axx Sonosite, Bothell, WA, USA) was used for all examinations. All exams were digitally recorded (Sony DVDIRECT San Diego, CA, USA) and subsequent videos were reviewed for analysis. Spectral Doppler measurements of both uterine arteries were evaluated and calculated by the algorithm package in the Micromaxx ultrasound. The sample cursor gate was set at 5 mm and at a magnification depth of 7.7 cm. The measurements were: resistance index (RI) [(peak systolic velocity (PSV) end diastolic velocity (EDV) )/PSV;


65 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 of both uterine arteries taken near the branching of the extern al iliac artery or deep circumflex artery or both. Similar to the methods described by both Siddiqui et al. (2009) and Silva et al. (2006) the angle cursor in relation to the direction of blood flow in the uterine arteries (insonation angle) was unknown; t hus the RI and PI provide relative, rather than actual, velocity measurements. Ginther (2007) stated that the indices (RI and PI) are ratios of velocity measurements and therefore are independent of the Doppler angle. A reciprocal relationship exists betwe en these indices and blood flow, whereby an increase in either PI or RI indicates a decrease in blood flow through that examined vessel. The setting for the range of flow velocity was adjusted to clearly visualize the spectral graph, and a Doppler spectrum with at least two uniform cardiac cycles was generated, with one of the cycles used for spectral measurements. This was done a second time, and the mean of the two measurements was used for statistical analysis. Finally, diam eter of both uterine arteries was recorded during each exam for the duration of the trial. Following parturition, mares underwent ultrasonic evaluation as described above and the uterine arteries were determined to be either ipsilateral or contralateral to the previously gravid horn. A retrospective analysis was then conducted on the blood flow measurements prior to parturition and defined as the gravid uterine artery (GUA) or non gravid uterine artery (NGUA). Statistical Analyses Differences in blood flow velocities (RI and PI) were compared within and between the GUA and NGUA using the SAS MIXED procedure to determine the main


66 effects of treatment and day and their interactions with a repeated measures statement; and LSMEANS to evaluate differences between treatment groups over time (SAS version 9.2; SAS Institute, Cary, NC, USA). Dietary treatment, day and the day x treatment interaction were included in the model as fixed effects. Data collected prior to parturition was analyzed separately from the 7 d following parturition. Data f or days prior to parturition were normalized to day of parturition (Day 0). Means of both indices and arterial diam. between the day prior to parturition and the days immediately following parturition were compared by ANOVA followed by a post hoc Tukey tes t. Comparisons of gestation length between age, parity, EFD, sex of resulting foal, as well as all foaling parameters, between treated and control mares were evaluated by Students t test. Values are reported as mean SEM. A probability of P ed a significant difference and a probability between P significance. R esults Gestation and foaling parameters All mares completed the study without complication and no feed refusals were noted in response to Arg supplementation. The mean age for control and Arg mares was 11.4 1.3 and 11.5 1.7 yr, respectively. The EFD for all mares ranged from January 14 to May 14, 2010. No deleterious physical effects of feeding Arg were observed in treated mares. Parturitio n proceeded without complication in all mares and all foals were born clinically healthy with no noted complications or physiological abnormalities. Mares fed Arg beginning 21 d prior to EFD had a shorter gestation length (337.9 1.7 d) than control mares (345 2.1 d; P


67 foaling parameters observed. Combined means for: time from rupture of the chorionallantois to foal delivery was 16.5 1.5 min; time from rupture of chorionallantois to placenta s hed was 34.8 12.2 min; time from foal delivery to first nursing 95.1 9.5 min; placental weight was 6.98 .31 kg. When separated for gender, gestation length for fillies born from Arg supplemented mares was shorter (n = 4; 336.4 2.2 d) compared to co ntrol (n = 5; 342.8 2.2 d; P born from Arg supplemented mares to have a shorter gestation length (n = 3; 340.3 2.7 d) when compared to control (n = 4; 347.3 3.3 d; P = 0.09). When data was combined for both groups, fillies tended to h ave a shorter gestation length compared to colts ( P for this project as control (n = 7) had a mean gestation length of 342.9 2.0 d, and Arg supplemented mares (n = 6) was 342.3 2.3 d; no significant difference. Finally, no acute effects were observed on initiation of parturition in treatment mares, with rupture of the chorionallantois ranging from 2059 to 0620 h. Time of chorionallantois rupture in control mares ranged from 20 02 to 0412 h. Finally, no differences were found in gestation length between EFD, breed, parity or age of mares. Uterine artery characteristics Differences in diam. of the gravid compared to non gravid uterine arteries were found ( P ach was evaluated separately. A day effect was observed in both GUA and NGUA following parturition with decreasing uterine artery diameter ( P up parturition (Figure 3 1 A ); however, a treatment effect was observed in the GUA following parturition, with Arg supplemented mares having smaller uterine artery diam. ( P


68 with treated mares having sm aller uterine artery diam. ( P significance in the days following parturition ( P = 0.10; Figure 3 1 B). Across treatments, a decrease in diam. of the GUA was found when comparing the day prior to parturition (1.21 .05 cm) to D ay 7 post parturition (0.67 .06 cm; P was found in the NGUA (1.07 .07 cm and 0.59 .02 cm, respectively; P Interestingly, control mares NGUA had a larger diam. the day prior to parturition ( P 0.05) but no differe nces were evident the day following parturition. Uterine artery blood flow characteristics Differences in blood flow measurements were found between the GUA (Figure 3 2) and NGUA (Figure 3 3) prior to and the days following parturition ( P therefo re, each were evaluated separately. In the days leading up to parturition, a day effect was observed in both the RI and PI as index values decreased in the GUA ( P 0.01). This was not observed in the NGUA. Prior to parturition, no overall treatment effect was found in the GUA; however, control mares had higher PI values on Days 7 and 3 (Figure 3 2 A; P 17 (Figure 3 2 B; P 0.0001) and RI (P < 0.0001) in the NGUA leading up to parturition. Control mares had higher NGUA PI values on Days 21, 19, 13 ( P Day 3 (Figure 3 3 A; P = 0.07). Additionally, control mares had higher RI values in the NGU A on Days 21, 19, 13 ( P 17 and 1 (Figure 3 3 B; P When compared to the measurements to the day prior to parturition, blood flow significantly decreased across both groups in both uterine arter ies the day after parturition. Across treatments, uterine blood flow significantly ( P


69 the day before to the day after parturition. Postpartum, a day ( P ( P atment by day effect observed in the GUA PI ( P supplemented mares had smaller PI values on Days 3, 4, 6 ( P P P GUA postpartum ( P 0.05), with no significant differences between groups. D iscusion The results of this study have shown that uterine arterial blood flow can be measured during late pregnancy in mares and the immediate post partum period. Our results ind icated that supplemental feeding of Arg beginning 21 d prior to EFD shortened gestation length. An increase in uterine artery blood flow through the NGUA was observed in treated mares prior to parturition, with no differences in the GUA. The day immediatel y following parturition for both groups, indices significantly increased and uterine artery diameter decreased in both the GUA and NGUA as mares transitioned back to the non pregnant state. Additionally, a treatment effect was observed with Arg supplemente d mares having greater blood flow (lower indices) in the GUA. As previously mentioned, supplemental Arg in the diets of pregnant animals has no apparent deleterious effects on pregnancy, fetus or resulting neonate (Wu et al., 2007). This also appears to be true with short term feeding of Arg during late pregnancy in mares. The most surprising finding in the current study was the significantly shorter gestation length in Arg supplemented mares compared to un supplemented controls. Many factors influence ges tation length in mares, including season, breed, gender of fetus, parity, and age of mare. While we did not find any differences in season, breed,


70 age or parity of the mares in this study, we did find differences in foal gender with mares carrying fillies having a shorter gestation length in comparison to mares carrying colts, regardless of treatment. However, when foal gender and gestation length were examined by treatment, Arg supplemented mares had shorter gestation length in both fillies and colts. To e valuate any potential mare differences, records from the previous foaling season were evaluated for gestation length. Of the thirteen mares that foaled in the previous year, no differences to gestation length were found when mares were separated based on t his study, control or Arg supplemented mares. Therefore, it appears other factors were responsible for the shorter gestation length observed in Arg treated mares. These findings are in contrast to the study of Mateo et al. (2007), who did not find any diff erences with gestation length in gilts fed Arg from Day 30 to term. There are differences in characteristics of pregnancy between pigs and horses, most noticeably a litter bearing versus a typical singleton bearing species, respectively. In the mare the se quence of prepartum endocrine events are generally similar to those seen in other farm animals; however there are differences (Fowden et al., 2008). Across mammalian species, a fetal rise in glucocorticoids and a fall in maternal progesterone levels are the endocrine changes most consistently observed leading up to parturition. However, the prepartum rise in fetal cortisol concentrations occurs much closer to term in the mare than in other species (Fowden and Silver, 1995). Fowden et al. (2008) stated th at the basal fetal cortisol levels remain low until 4 5 d before term and rise exponentially only in the final 24 36 h before birth. This rise in cortisol is essential for fetal maturation, and foals born prior to this increase typically do not


71 survive (Ou sey, 2006). Conversely in swine, a rise in cortisol happens earlier and is observed beginning the final 2 wk of gestation (Heo et al., 2003). How NO might influence fetal foal, or fetal pig, corticoid synthesis is currently unknown. Riquelme et al. (2002) established that NO is important in regulating adrenal blood flow and corticomedullary function in the llama fetus. In their study, treatment with an NO synthase inhibitor (L NAME) decreased fetal adrenal blood flow and prevented increases of fetal plasma cortisol under hypoxic conditions. While inhibition of NO has shown to effect fetal adrenal function, to our knowledge increased NO production has not. However, increased NO production through Arg supplementation could possibly enhance fetal adrenal funct ion or efficiency and deserves further investigation. Other endocrine differences exist between porcine and equine pregnancy. In pigs, estrogen levels steadily increase 3 weeks prior to parturition, while progestins steadily decrease over the last 2 weeks of gestation (Bladwin and Stabenfeldt, 1975; Ellendorff et al., 1979). In contrast, mare estrogen concentrations decrease over the final 30 d of gestation, while total progestagen concentrations rise during the last 20 30 d of gestation and decline only i n the last 24 48 h before parturition (Fowden et al., 2008). Of particular interest is the decrease in estrogen, a potent vasodilator, in mares during the final weeks of gestation. Compared to other species, it may be possible that lower estrogen levels du ring late pregnancy in the mare may lead to decreased uterine blood flow. Estrogen has been shown to increase uterine blood flow by increased uterine vessel vasodilation (Griess and Anderson, 1970). In the pregnant ewe, estrogen induced vasodilation leadin g to increased uterine blood flow has been associated with increased levels of NO cyclic


72 monophosphate (cGMP; Rossenfeld et al., 1996). Similar studies in other species have found direct links between estrogen, NO and enhanced blo od flow (Chen et al., 2004; Khan et al., 2010). Zhang et al. (2010) stated that enhanced endothelial NO production contributes significantly to the vasculature effects of estrogen. Our results of a shorter gestation length may be due to the observed great er blood flow in the NGUA in Arg supplemented mares, leading to greater NO production, which may under normal conditions be lower due to naturally declining estrogen concentrations. The fetal foal continues to develop during the last month of gestation dur ing the final maturation process leading up to parturition. An increase in uterine blood flow could have hypothetically partitioned more nutrients, oxygen and other substrates to the utero placental unit that hastened this development period, leading to ea rlier foaling. Another contributing factor to shorter gestation length may be due to mare prostaglandin metabolites slowly increasing over approximately the last 30 d until term (Fowden et al., 2008), whereas in pigs no rises in prostaglandins are detecte d until term (Silver et al., 1979). This is interesting because NO has been positively correlated in the mare endometrium with both prostaglandin E synthase (PGES) and prostaglandin F synthase (PGFS), enzymes responsible for conversion of important reprod uctive prostaglandins (PGE 2 and PGF respectively; Roberto da Costa et al., 2008). In the mare, PGE 2 promotes cervical ripening and relaxation while PGF stimulates uterine contractions (Rigby et al., 1998; Fowden et al., 2008). NO, like PGE 2 has also been shown to be a critical compone nt in cervical ripening, and interestingly has been shown to inhibit spontaneous uterine contractions (Chwalisz et al., 1998). Therefore, it is possible that Arg treatment late in gestation may enhance prostaglandin synthesis and


73 cervical ripening, thus p laying a role in hastening the onset of labor and deserves further study. As stated in the results, no noticeable differences were observed in labor, or neonatal outcomes between our Arg supplemented or control mares. The second most interesting data from this study is the increase in uterine arterial blood flow in the NGUA prepartum, and the increase in uterine arterial blood flow in the GUA postpartum. Color Doppler ultrasonography is becoming a commonly used diagnostic tool to evaluate blood flow to re productive tissues in livestock animals. Doppler indices (RI and PI) as ratios are useful in quantitative assessment of blood flow velocity measurements for reproductively important tortuous vessels such as the uterine arteries, as stated by Ginther (2007) The studies of Mortensen et al. (2010) and Bollwein et al. (2003) both observed differences in uterine arterial blood flow leading to the gravid or non gravid uterine horns during early equine pregnancy. Results from both studies showed greater blood flo w through the uterine artery leading to the horn (gravid) of established pregnancy. Our results indicated that this also appears to hold true during late pregnancy in mares, with greater blood flow to the GUA compared to the NGUA, regardless of group. Work in other species has shown that during pregnancy uterine blood flow increases from 10 to 100 fold above nonpregnant levels (Osol and Mandala, 2008). As expected, uterine arterial blood flow decreased in the days following parturition, as the foal and pla centa were shed. These decreases have previously been documented in sheep and dogs (Assali et al., 1958) and more recently in postpartum dairy cows (Krueger et al., 2009). This appears to be the first study in mares documenting differences pre and post par tum in uterine arterial blood flow and diameter.


74 The differences in blood flow in the uterine arteries between Arg treated and control pre partum mares could be attributed to the known vasodilative properties of NO (Rosselli et al., 1998; Valdes et al., 2 009). Nitric oxide is produced by the conversion of Arg to L citrulline by nitric oxide synthase (NOS) enzymes and is a potent vasodilator that has been shown to regulate blood flow to reproductive tissues in ewes (Gibson et al., 2004) and sows (Barszczews ka et al., 2005). It is interesting that this was more evident in the NGUA prepartum, and may possibly indicate that during late pregnancy in mares blood flow through the GUA is at maximal flow and is not manipulated with a NO donor such as Arg; or convers ely, the indices are so low that significant changes in blood flow are not detectable with the Doppler ultrasound utilized in this trial. These results are supported by the study of Neri et al. (1996) who only observed increases in blood flow (lower PI) in the non placental side uterine artery in Arg treated women. Another significant observation was the significant treatment effect with Arg supplemented mares having greater uterine arterial blood flow the days following parturition in the GUA. Why this w as not observed in the NGUA post foaling, when prior to foaling there were significant differences is curious. Osol and Madala (2008) stated that the available evidence points to local rather than systemic factors playing a primary role in gestational uter ine vascular remodeling and increases in blood flow. Additionally, the authors stated that a reduction in downstream resistance would be an effective stimulus for increasing the velocity of blood flow in upstream arteries. In women, enhanced myometrial vas cularity (EMV) is common in the early postpartum period, is located at the former placental site and is associated with lower pulsatility indices and a higher maximal flow velocity in the uterine arteries (Van Schoubroeck et al., 2004).


75 Therefore, it is pl ausible from our results that as the uterus recover from the trauma of parturition and undergoes involution, greater vascular development in the previously gravid horn benefits more from an NO donor such as Arg compared to the non gravid horn. How greater blood flow to the uterus might benefit the mare remains to be elucidated; however, greater blood flow to the reproductive tract has been associated with higher fertility in mares (Silva et al., 2006) and cows (Siddiqui et al., 2009). Finally, uterine arte ry diam. did not differ prior to parturition in the GUA, but was observed to be smaller in the NGUA in Arg supplemented mares. Conversely, following parturition Arg treated mares had smaller GUA diam. with increased flow. This is interesting, as outward hy pertrophic growth of uterine arteries has been correlated with increased blood flow (Osol and Madala, 2008). However, based on these results, Arg supplemented mares had smaller uterine artery diam. with increased blood flow. It is possible that these diffe rences may reflect physiological differences between the two groups, rather than a treatment effect. Yet, it is also possible that Arg supplementation, and presumably through NO action, may have altered the vascular remodeling further downstream than our p oint of examination of the uterine arteries. As stated above, greater vasculature downstream increases blood flow upstream, and based on our results appeared to not influence uterine artery diameter. Further work should be attempted to try and delineate th ese differences. In summary, supplementing pregnant mares with Arg 21 d prior to EFD appeared to be safe and reduced gestation length. Additionally, supplemented mares were observed to have greater uterine arterial blood flow prior to, and the days immedi ately following parturition. The sharp reduction in uterine arterial blood flow and diam.


76 following parturition could be predicted as the mares transitioned from the pregnant to non pregnant state. Since most equine breed registries recognize an annual Jan uary 1 birth date, and due to the long equine gestation length (range 329 to 353 d in this study), each day a mare remains open following parturition potentially reduces the value Therefore, any reduction in gestation length, or ensuring a mare foals near her EFD, is extremely valuable to horse breeders. Previous attempts at reducing gestation length through pharmacological manipulation potentially leads to dystocia or still born foals (Jeffcot and Rossdale, 1977; Ousey 2006). Therefore, if results from the current study can be confirmed, this would appear to be an important finding in safely reducing gestation length in mares by diet manipulation.


77 Figu re 3 1. Mean (SEM) diameter of the uterine arteries of L arginine treated and control mares in the 21 d before and 7 d after parturition with Day 0 as day of parturition. A) Gravid uterine artery for Days 21 to 1 (T: NS, D: NS, T*D: NS) and Days 1 to 7 artery for Days 21 to an asterisk (*) indicates significance ( A B # #


78 Figure 3 2. Mean (SEM) PI and RI values in the uterine arteries leading to the gravid horn of pregnancy of L arginine treated and control mares in the 21 d before and 7 d after parturition with Day 0 as day of parturition. A) Gravid PI for Days 21 to 21 to T*D: P = 0. B A


79 Fig ure 3 3 Mean (SEM) PI and RI values in the uterine arteries leading to the nongravid horn of pregnancy of L arginine treated and control mares in the 21 d before and 7 d after parturitio n with Day 0 as day of parturition. A) Non gravid PI for Days 21 to 1 (T: P (T: NS, D: P gravid RI for Days 21 to 1 (T: P 0.0001, D: ND, T*D: P P .01, T*D: NS). T = treatment, D = day, NS = not significant, an asterisk (*) indicates significance ( P significance ( P > 0.05 to P B A


80 CHAPTER 4 ORAL L ARGININE SUPPLEMENTATION IMPACTS SEVERAL R EPRODUCTIVE PARAMETERS DURING THE POSTPARTUM PERIOD IN MARES Introduction L Arginine is one of ten essential amino acids in horses (NRC, 2007 and a biologically active regulator of several systems including the reproductive, cardiovascular, pulmonary, rena l, and immune systems (Wu et al., 2009). L arginine can behave both as a receptor ligand (Joshi et al., 2007) and as a substrate for biosynthesis of nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine (Wu and Morris, 1998). L arginine activates nitric oxide synthase (Joshi et al., 2007; Morrissey et al., 1997) which catalyzes the conversion of L arginine to NO and L citrulline. Nitric oxide is a vasodilator that inhibits vasoconstrictor signals (Thiriet, 2008) and acts downstream of V EGF signaling to promote angiogenesis (Murohora et al., 1998). In gilts, oral supplementation of L arginine starting on Day 30 of gestation has been shown to increase litter size and birth weights (Mateo et al., 2007). Recently, it has been shown that su pplementing L arginine (1% of diet) in pre and postpartum mares increased uterine blood flow (Mortensen et al., 2011). Given that blood flow surrounding the dominant follicle is associated with increased pregnancy rates in mares (Silva et al., 2006); fert ility may be improved in mares by feeding L arginine. Here we further examined the impact of L arginine supplementation on the reproductive characteristics of the postpartum mare. Mares are distinctive among domestic livestock in their ability to return to estrus and potentially conceive shortly after ovulating from 8 to 13 d after giving birth parturition (McCue and Hughes, 1990). Scant literature exists on follicula r development and follicle selection during the first immediate


81 postpartum estrous cycle in mares. Shortly after parturition, mares the diameter of largest follicle ranges from 13 to 16 mm (Ginther et al., 1994) and reaches a diameter of 36.4 mm by Day 8 ( Gndz et al., 2008). The objectives of this study were to observe baseline follicular dynamics and ovarian blood flow during the first postpartum estrous cycle in mares and evaluate the influence of supplemental L arginine on ovarian follicular dynamics, ovarian and uterine blood flow and uterine involution. Materials and Methods Animals and experimental design Studies were approved by the Institute of Food and Agricultural Sciences Animal Care (IFAS) and Use Committee at the University of Florida and wer e conducted at the Experiment 1, six multiparous Quarter horse mares were randomly assign to either a control or L arginine group (n=3) during the last 3 weeks of pregnancy to evaluate plasma availability of arginine in response to a meal supplemented with L arginine. Both groups were fed 2.4 kg of a commercial mixed concentrate ration formulated for gestating and lactating mares (minimum guarantees: 16% crude protein, 3.5% cru de fat, stalls L arginine supplemented mares received 100 g of L arginine ( CAS number 74 79 3; Ajinomoto AminoScience LLC, Raleigh, NC, USA) that was hand mixed into the gra in ration. Mares in both groups were fed Coastal Bermuda grass (3.5 kg) hay 4 and 8 h after receiving grain. Control mares were not fed an isonitrogenous (diets equal in nitrogen) vehicle to ensure that the comparison would be between L arginine and a stan dard pregnant mare diet. Blood samples were obtainied via jugular catheters into heparinized tubes, prior to feeding (0 h) and at 1, 2, 3, 4, 6, 8, 10 and 12 h post feeding.


82 Samples were immediately centrifuged at 11,000 g for 15 min and plasma was stored at 80 C until analysis. In Experiment 2, sixteen mares were blocked by age (range 5 19 yr), breed (Thoroughbred (n=8) and Quarter Horse (n=8)), and expected foaling date and assigned randomly to receive L arginine or no supplementation (n=8/group). The mean (SEM) age of mares was 11.5 1.7 yr for L arginine and 11.4 1.3 yr for controls. The basal diet consisted of ad libitum Coastal Bermuda grass hay and 3.8 0.3 kg of a commercial mixed concentrate ration formulated for gestating and lactating mares Feed and Supply, Ocala, FL, USA). Mares were fed individually in stalls at 0700 and 1500 h. L arginine supplemented mares r eceived 100 g of L arginine ( CAS number 74 79 3; Ajinomoto AminoScience LLC, Raleigh, NC, USA) that was hand mixed into the grain ration immediately before each morning feeding. Treatments began 21 d before expected foaling dates and continued for 30 d postpartum. Amino Acid Analysis Plasma samples from Experiment 1 were deproteinized using 35% (w/v) sulfosalicylic acid. The acid soluble fraction was separated by centrifugation (4C, PA, USA) and then mixed 1:1 with 0.02 N HCl (Boucher et al., 1997). Plasma samples were then analyzed for amino acid composition using an amino acid analyzer (L 8900, Hitachi High Technologies, Pleasanton, CA) as previously described by Ma et al. (2010).


83 Ultrasonongraphy In Experiment 2 ovaries and ovarian arteries were examined via transrectal ultrasonography daily from the day after foaling until the first postpartum ovulation. The uterus and uterine arteries were examined daily from foaling until 30 d post foaling. Ultrsonographic e xams (900 1100 h) were performed using a Micromaxx Sonosite digital color Dopplar ultrasound with a 5 10 MHz broadband 52 mm linear array (Bothell, WA, USA). Exams were recorded (Sony DVDIRECT, San Diego, CA, USA) and subsequent videos were reviewed for a nalysis. The length of foal heat was defined as the number of days from foaling to the first post partum ovulation. Follicles were grouped by diameter (6 to 10, 11 to 15, 16 to 20 and > 20 mm) each day without regard to day to day identity as previously d escribed by Kelley et al (2011). Data were normalized to the 10 d preceding ovulation with the day of ovulation considered Day 0. Following ovulation, retrospective analysis determined the largest (ovulatory) follicle (F1), largest subordinate follicle (F2 ) and the second largest subordinate follicle (F3). The diameter of the F1 and F2 at the time of deviation was ascertained as described elsewhere (Kelley et al., 2011). Deviation was defined as occurring on the day (exam) prior to the examination with the greatest change in differences in diameter between the two largest follicles. For uterine measurements the screen depth was increased to visualize the entire uterine section and an outer cross sectional measurement for the uterine body was taken at the point of maximum height using electronic calipers. The diameter of each uterine horn was calculated by averaging the height and width measurements and was taken at approximately the midpoint of each horn. Maximum intrauterine fluid


84 accumulation was measure d within the uterine body via ultrasonography using electronic calipers. Spectral Doppler measurements of both ovarian arteries and uterine arteries were evaluated as described by Ginther (2007) and calculated by an algorithm package in the Micromaxx ul trasound. Retrospective analysis identified the ovarian arteries as either ipsilateral or contralateral to the ovulatory follicle. The sample cursor gate was set at 5 mm at a magnification depth of 7.7 cm. Resistance index (RI) [(peak systolic velocity (PS V) end diastolic velocity (EDV))/PSV] was recorded without concern for the Doppler angle. The RI is a measure of spectral waveform variation and used to estimate downstream impedance of the vasculature. Generally, a decrease in RI indicates an increase i n blood flow. The setting for the range of flow velocity was adjusted to visualize the spectral graph, and Doppler spectrum with at least two uniform cardiac cycles generated, one of which was used for measurements. This was repeated with the mean of the t wo RI measurements taken were used for statistical analysis. Follicular perfusion was defined as the blood flow area surrounding the wall of the follicle using color Doppler ultrasonography (Ginther and Utt, 2004). Follicular perfusion was evaluated for t he retrospectively identified dominant follicle (F1) using the color power Doppler (CPD) mode, which is more sensitive to low or weak blood flow than the color Doppler mode. Measurements for CPD perfusion were taken from DVD recordings using a radial grid for the 5 d preceding ovulation. Briefly, a radial grid was constructed by dividing a circle into 32 even slices, each slice covering 11.25 of the circle. The center of the radial grid was aligned to the center of the F1 and the slices containing perfusio n, indicated by the color, were counted. The numbers of slices


85 containing perfusion were then used to determine the percent [Percent perfusion = (slices with perfusion/total number of slices) x 100] of the F1 that contained perfusion. Statistical analysis Data were analyzed using the SAS MIXED procedure with a random statement to account for variability of mares within group and a repeated measures statement with treatment, da y, and day x dietary treatment were included in the model as fixed effects. Data is represented as least squared means SEM. A probability of P < 0.05 was considered significant and a probability between P towards significance. Results Plasma L arginine concentrations In Experiment 1, there were no significant differences in plasma arginine concentrations at 0 h between groups. Beginning 1 h post feeding and lasting unti l 8 h post feeding L arginine mares had higher( P < 0.05) plasma arginine concentrations than control mares (Fig. 4 1). Plasma arginine concentrations were not statistically different between groups at 10 and 12 h post feeding. Ovarian follicular dynamics, blood flow and follicular perfusion All mares in Experiment 2 foaled without complications. Gestational length of L arginine treated mares was shorter than control mares and no differences were found between groups in placental weight. Mean length of foa l heat for L arginine treated and control mares was 12.6 1.9 d and 16.5 1.8 d ( P = 0.15), respectively. All mares ovulated a single follicle. No differences were observed between groups in the mean number of follicles within categories of 6 10, 11 15, 16 20, or > 20 mm (see Fig. 4 2 A


86 D). Similarly, there was no difference in diameter of F1 or F2 (Fig. 4 3). The mean time from deviation to ovulation in L arginine treated and control mares was 8.0 0.6 and 6.6 0.6 d ( P = 0.10), respectively. L Argini ne treated mares had a mean F1 and F2 diameter at deviation of 22.9 1.2 mm and 20.5 1.4 mm, respectively, which was not significantly different than controls (23.0 1.2 mm and 20.9 1.4 mm, respectively). There was no effect of treatment effect on RI for either the ovarian artery ipsilateral or contralateral to ovulation (Fig. 4 4 A and B). The mean percent perfusion for the F1 was greater in the L arginine treated group (37.3 2.6%), than, control (25.4 2.7 %; P < 0.05). L Arginine treated mares h ad increased perfusion ( P < 0.05) on Days 2 and 3 from ovulation and there was trend towards increased perfusion ( P = 0.07) on the day of ovulation (Fig. 4 5). Uterine involution and fluid clearance L Arginine treated mares had a smaller mean diameter in the formerly gravid and nongravid uterine horns as well as the uterine body ( P < 0.05; Fig. 4 6). Diameter of the formerly gravid horn in L arginine treated mares was smaller on Days 1, 2, 5, 6, 7, 8, 10, 16 and 22 ( P < 0.05) relative to controls. Diamete r of the formerly nongravid horn was smaller on Days 2, 10, 14, and 24 ( P < 0.05) in L arginine treated mares. The depth of the uterine body was decreased in L arginine treated mares on Days 3, 4, 6, 7, 10, 14, and 16 ( P < 0.05). Fluid accumulation was le ss than controls (Fig. 4 7), on Days 3 and 4 ( P < 0.05). There was a trend (0.10 P > 0.05) towards less fluid on Day 4 postpartum. L arginine treated mares had detectable fluid present for a mean of 3.41.5 d after foaling compared to control mares, 7.13.1d ( P < 0.05).


87 Uterine blood flow No difference within groups in RI was obser ved for the uterine artery of either the formerly gravid or formerly nongravid side of the uterus ( P < 0.05; Fig. 8 A and B). RI increased in the uterine artery supplying the formerly gravid horn in L arginine treated mares from Day 1 to Days 12, 13, 15 a nd 29 and from Day 1 to Days 4, 8, 12, 15, 17, 18, 21 and 23 ( P < 0.05) for uterine artery to the formerly nongravid uterine horn,. In control mares RI increased from Day 1 to Day 3 through 30 ( P < 0.05) in the formerly gravid horn and from Day 1 to Days 1 3, 15, 25 and 27 ( P < 0.05) in the formerly non gravid horn. There were no differences ( P > 0.05) in the RI between the uterine artery on the formerly gravid side and the non gravid side within groups or between groups for the 30 d following parturition. Discussion This study demonstrated that supplementation of mar es with 100 g/d of L arginine increased plasma arginine concentrations, increased perfusion of the ovulatory follicle, and reduced uterine diameter and uterine fluid accumulation postpartum. L A rginine had no effect on foal heat length, follicular development or RI for either the ovarian or uterine arteries. Results lead to the possibility that supplementation of mares that foal later in the breeding season with L arginine may be beneficial to hasten uterine fluid clearance without altering follicular development or duration of foal heat in an effort to retain an annual foaling interval. Mares treated with L arginine had a decrease in uterine size and fluid accumulation. L arginine can potential ly affect many biological pathways that impact growth hormone, polyamine and nitric oxide synthesis. L Arginine treatment increases growth hormone production in humans (Merimee et al., 1969). In rats, growth hormone


88 increased uterine proliferation and cell growth (Kennedy and Dorktorcik, 1988; Gunin, 1997), while in sheep growth hormone increases the weight of the myometrium and endometrium (Jenkinson et al., 1999). L Arginine is a precursor for both polyamines and nitric oxide (Wu and Morris, 1998). Polyam ines can exert many actions but their role in reproduction remains unclear. Spermine, a polyamine, has been found to increase PGF synthesis and inhibit PGE 2 production (Igarashi et al., 1981; Maruta et al., 1985). The role of other polyamines and their e ffect on prostaglandins is an area in need of further investigation. Nitric oxide is a smooth muscle relaxant (Thiriet, 2008) and has been found to be elevated in mares with a predisposition to breeding induced endometritis (Alghamdi et al., 2005) although they did not establish whether the increase in nitric oxide was a cause or result of breeding induced endometritis. Mares susceptible to post breeding endometritis have been reported to have elevated concentrations of PGF 2 Nitric oxide has been reported to affect cyclooxygenase activity to increase prostaglandin synthesis and effect uterine contractility in rat uterine cultures (Franchi et al., 1994). Interesti ngly, nitric oxide decreased uterine contractions during times of increased myometrial motility, but increased myometrial contractions when motility was low. Using pre term mice induced to undergo labor, Cella et al. (2010) found that treatment with low co ncentrations of a nitric oxide donor (SNAP) decreased prostaglandin production whereas a higher amount of SNAP increased prostaglandin production. This result raises the possibility that treatment with L arginine may increase prostaglandin production in th e uterus and impact uterine contractions. Although this study did not evaluate uterine motility, prostaglandin or local nitric oxide synthesis, it is


89 possible that L arginine treatment increases nitric oxide concentrations in the uterus enough to increase prostaglandin production. Further work is needed to test this hypothesis. L Arginine increased perfusion of the ovulatory follicle. Silva et al. (2006) reported mares that became pregnant had greater vascular perfusion of the ovulatory follicle compared to those that did not become pregnant. Additionally, we found lower perfusion (25 37%) during the immediate postpartum estrous cycle compared to previous reports in mares from non periparturient cycles (approximately 50% in non pregnant and 75% in pregnant mares; Silva et al., 2006 Although our study did not evaluate fertility, further research is needed to determine whether increasing blood flow to the dominant follicle increases fertility. There was no difference in RI between groups for any arterial meas urements. The RI was selected as the blood flow measurement as it cancels out errors caused by not knowing the Doppler angle and is perceived to be more accurate than pulsatility index (Dickey, 1997). Our data are interpreted to mean that there was no diff erence in the amount of blood flow to the ovaries or uterus between groups. Interestingly, the amount of perfusion to the ovulatory follicle was increased in L arginine mares, as evaluated by color power Doppler ultrasonography. This observation raises the possibility that L arginine increased perfusion of the ovulatory follicle may be caused by a local angiogenic effect at the level of the ovary rather than the vasodilatory actions of L arginine. There were no differences in the number of follicles, the di ameter of F1 or F2, and deviation parameters between groups. Our interpretation is that L arginine


90 supplementation has no effect of follicular growth and development. In comparing foal heat to non periparturient cycles, mares from both groups during foal h eat appear to have fewer follicles 6 10 and 11 15 mm than previously reported by Pierson and Ginther (1987) which reported that non periparturient ponies had between six to ten follicles, compare to 2 to 6, between 6 10 mm. Additionally, Pierson and Ginthe r (1987) reported four to seven follicles in the 11 15 mm range compared to the value of 2 to 4 obtained here. Furthermore, we found that the number of follicles that were 16 20 and >20 mm remained relatively steady throughout foal heat while Pierson and Ginther (1987) observed an increase in the number of these follicles over time, followed by a decline. Deviation diameters of the F1 and F2 follicles during foal heat were similar to previous reports in mares with a single dominant follicle. Jacob et al. (2009) reported in diameters of 22.7 and 21.7 mm for F1 and F2 in single ovulating mares. Supplementation of mares with L arginine increased vascular perfusion to the retrospectively identified dominant follicle without altering follicular growth and deve lopment or ovarian artery RI. Mares supplemented with L arginine had a reduction in uterine size and fluid accumulation but no change in uterine artery RI. The combination of reducing uterine fluid accumulation, while not altering follicular development, r aises the possible use of L arginine supplementation as a breeding management tool during the postpartum period to increase reproductive success.


91 1. Figure 4 1. Least squares mean ( SEM) plasma arginine concentrations for 12 h following the co nsumpt ion of 100 g L arginine An asterisk (*) denotes a difference ( P < 0.05) between groups. Concentration ( mol/L) Hour


92 Figure 4 2. Least squares mean ( SEM) number of follicles for the 10 d preceding ovulation for L arginine treated (solid line) and control (dotted line) mare s. Panel A: diameter between 6 and 10 mm. Panel B: diameter between 11 and 15 mm. Panel C: diameter between 16 and 20 mm. Panel D: diameter greater than 20 mm. An asterisk (*) denotes a significant difference (P < 0.05) between groups, while a pound sign ( #) indicates a trend towards significance Days Prior to Ovulation Days Prior to Ovulation Number of Follicles Number of Follicles


93 Figure 4 3. Least squares mean ( SEM) diameter of the dominant (F1) and largest subordinate (F2) follicles for L arginine treated and control mares. There were no differences between treatments. Days Prior to Ovulation Diameter (mm)


94 Figure 4 4. Least squares mean ( SEM) resistance index (RI) values in the ovarian arteries for the 10 d prior to ovulation. Panel A: RI of the ovarian artery ipsilateral to the ovary with the ovulatory follicle. Panel B: RI of t he ovarian artery contralateral to the ovary with the ovualtory follicle. An asterisk (*) denotes a significant difference between groups (P < 0.05), while a pound Days Prior to Ovulation Resistance Index Resistance Index A B


95 Figure 4 5. Least squares mean ( SEM) percent of vascular perfusion of the circumference of the ovulatory follicle as indicated by color power Doppler. An asterisk (*) denotes a significant difference between groups (P < 0.05), while Percent of Follicle Circumference Days Prior to Ovulation


96 Figure 4 6. Least squares mean ( SEM) Panel A: depth of the uterine body, Panel B: diameter of t he formerly gravid uterine horn and Panel C: diameter of the formerly nongravid horn for 30 d postpartum. An asterisk (*) denotes a difference (P < 0.05) between groups. Days postpartum Depth (mm) D iameter (mm) D iameter (mm) A B C


97 Figure 4 7. Least squares mean ( SEM) maximal fluid accumulation in the postpar tum uter us. An asterisk denotes a significant difference (P < 0.05) while a pound Days postpartum Maximal Uterine Fluid Accumulation (mm)


98 Figure 4 8. Least squares mean ( SEM) resistance index of the uterine artery on the side of forme rly gravid and nongravid uterus. Panel A: Uterine artery on the side of the formerly gravid uterine horn. Treatment mares had a significant increase in RI between Day 1 and Days 12, 13, 15 and 29 in the uterine artery on the side of the formerly gravid hor n while RI was increased in control mares between Day 1 and Day 3 through 30. Panel B: Uterine artery on the side of the formerly nongravid uterine horn for 30 d postpartum. Treatment mares had in increase in RI between Day 1 and Days 4, 8, 12, 15, 17, 18, 21 and 23 (P < 0.05). Control mares had an increase in the uterine artery on the nongravid side of the uterus between Day 1 and Days 13, 15, 25 and 27 (P < 0.05). An asterisk(*) denotes a significant difference (P < 0.05)between groups on a given day. Days postpartum Resistance Index Resistance Index


99 CHAPTER 5 I NFLUENCE OF L ARGININE SUPPLEMENTATION ON REPRODUCTIVE BLOOD FLOW AND EMBRYO RECOVERY RATES IN MARES Introduction Proper vascularization plays an important role in the selection, growth and maturation of follicles (Weiner et al., 1993) and incr eased blood flow to the dominant follicle is associated with increased pregnancy rates in mares (Silva et al., 2006). One way to improve blood flow to the reproductive tract is through administration of L arginine (Wu et al., 2009). This amino acid acts a s a substrate for biosynthesis of nitric oxide (NO), polyamines, proline, glutamate, creatine, and agmatine (Wu and Morris, 1998). Nitric oxide is a vasodilator that inhibits vasoconstrictor inputs (Thiriet, 2008), acts downstream of vascular endothelial g rowth factor (VEGF) signaling to promote angiogenesis (Murohora et al., 1998) and is thought to modulate pre ovulatory ovarian blood flow (Ben Shlomo et al., 1994; Anteby et al., 1996). Supplementation of women that have a history of poor response to ovari an hyperstimulation with L arginine increased ovarian blood flow, oocytes retrieved and embryos transferred (Battaglia et al., 1999). In mares, oral supplementation with L arginine increased uterine arterial blood flow and hastened the involution period i n postpartum mares (Mortensen et al., 2011). The objective of this experiment was to determine if oral supplementation with L arginine would increase ovarian blood flow and embryo recovery in mares. Materials and Methods Animals Animal use was approved b y the University of Florida IFAS Animal Research Committee. Mares were blocked into a control or treatment group based on age and breed. The control consisted of five Thoroughbreds (mean age: 14.3 1.7 yr) and three

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100 American Quarter Horse (mean age: 12.3 5.2 yr) mares aged 2 to 19 yr (control mean age: 12.0 2.4 yr). The treatment group contained five Thoroughbred (mean age: 15.8 1.3 yr) and four American Quarter Horse (mean age: 5.3 1.6 yr) mares aged 2 to 21 yr (treatment mean age: 12.6 2.3 yr). Mares had apparently normal reproductive activity as determined by ultrasound examination of the cervix, uterus and ovaries. All mares had uterine biopsies obtained before the study and were examined by a Diplomate of the American College of Theriogenolog y using the Kenney system (1978). All mares had either a biopsy score of II or IIa and there was no difference between groups in biopsy score as determined by chi square test. Lo Data were collected from up to 6 cycles; however if uterine bacterial culture was positive mares were treated and not bred. Each cycle was was bred during heat induced with prostaglandin.. Mares were housed on pasture and supplemented with of a commercial mixed concentrate ration formulated for gestating and lactating mares (minimum guarantees : 16% crude protein, 3.5% crude fat, 0.9% Ca, 0.55% P; Ocala daily. All mares had free access to water and trace mineral salt. Treatment mares were fed 100 g of L arginine (Aj inomoto, Raleigh, NC, USA) once daily in the morning beginning 17 d before the start of the study and remained on supplementation once daily until study completion. Ovulation was determined by transrectal ultrasonography.

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101 Doppler Ultrasonography Ovarie s and ovarian arteries were examined via transrectal ultrasonography using Doppler ultrasonography with a 5 10 MHz broadband linear array (Micromax, Sonosite, Bothell, WA, USA) every other day during estrus until the largest follicle reached a diameter of 35 mm and then daily until ovulation. Profiles of the eight largest follicles (designated F1 through F8, respectively) were categorized using a non tracking method (without regard to day to day follicle identity). A retrospective analysis identified the d ominant ovulatory follicle (F1) and largest subordinate follicle (F2). Corpus luteum measurements were taken on Days 2, 4 and 6 post ovulation. Diameters (average of height and width) of the largest follicle and corpus luteum (CL) were measured and record ed. Vascular perfusion to the follicle and CL was determined with color power Doppler ultrasonography. Resistance index (RI) was measured for both ovarian and uterine arteries as described by Smith et al. (2012). All examinations were recorded digitally fo r future analysis. Images from the DVD recordings were used to determine the percent perfusion of the retrospectively identified dominant follicle and CL as previously described by Kelley et al. (2009) and Smith et al. (2012). Embryo Collections Mares wer e bred with semen from one of two fertile stallions. Semen was collected by artificial vagina and evaluated for total progressive motility and sperm concentration. Concentration was estimated using an Equine Densimeter (Animal Reproduction Systems, Chino, CA, USA). Each mare received a total dose of 500 x 10 6 fresh, motile spermatozoa extended to a volume of 15 ml using E Z Mixin OF (Animal Reproduction Systems). Mares were bred every other day until ovulation via artificial insemination, beginning when t hey displayed estrus to a teaser stallion and had a

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102 follicle with a diameter of 35 mm or greater. All ovulations were spontaneous (not induced). Non surgical embryo collection was performed on D 7 after ovulation. The uterus was infused four times with 500 ml flush medium ( Biolife TM Advantage, Agtech, Manhattan, KS, USA ) utilizing a silicone 34 French catheter ( Reproduction Resources, Walworth, WI, USA ) and the effluent was collected through Y junction tubing ( Reproduction Resources ) into a 75 Reproduction Resources ). Mares were Pfizer, New York, NY, USA), 10 mg i.m., at the conclusion of the flushing procedure. The contents of the filter were rinsed into collection dishes, which were searched using a stereomicroscope at 20 for presence of an embryo. Embryos were evaluated based on a 1 (excellent) through 4 (non viable) scale as previously described ( Vanderwall, 1996 ). Statisical analysis Mares were divided by age into young (< 16 yr; arginine: n= 4; control: n=4 ) and old and RI data (during the follicular phase) were normalized to ovulation (Day 0). Corpus luteum and RI data (during the luteal phase) were normalized to o vulation beginning an estrous cycle (Day 0). Continuous data were analyzed using the SAS MIXED procedure (Version 9.2; SAS Institute Inc., Cary, NC, USA). A random statement was used to account for variability of animals within treatment and cycle by anima ls within treatment. A repeated statement for day was used, with the subject being cycle by animals within treatment, using compound symmetry as the model best fitting the covariance matrix. A g groups within days when there was a significant interaction between group and day. The embryo recovery rates were compared using a Chi square test. Data are presented as least square

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103 means SEM. A significant difference was denoted by P < 0.05, whereas a tendency was denoted by 0.05 Results The mean interovulatory interval for mares supplemented with L arginine was 18.1 2.6 d and was not different than for control mares 20.7 2.3 d (NS); however there was a tendency (P = 0.08) for a longer interovulatory interval in you ng (22.6 2.6 d) when compared to older (16.3 2.3 d) mares. There was no difference between stallions on embryo recovery rate, thus datum from both stallions were combined for further analysis. There was no significant difference in embryo recovery rate s of arginine (13/24, 54%) and control mares (16/33, 48%); or between young (14/25, 56%) and old (15/32, 47%) mares. In addition, there was no difference between treatments or ages in the stage of embryonic development or embryo score (Table 5 1). There wa s no effect of cycle on diameter of F1, F2, or CL or vascular perfusion of F1 or CL and therefore data are combined across cycles for additional analysis. Arginine treated mares had a larger ( P < 0.05) diameter F1 follicle (30.4 1.2 mm) than control (26 .2 1.2 mm) mares during the 10 days preceding ovulation (Fig. 5 1A) and during this time, younger mares had a larger ( P < 0.05) F1 (30.2 1.2 mm )than older mares (F1: 26.3 1.3 mm; Fig. 5 1A). No difference between groups was observed between F2 foll icles (Fig. 5 1B); however, younger mares had a larger ( P < 0.05) F2 (21.8 1.1 mm ) than older mares (18.5 1.1 mm ) during the 10 d preceding ovulation. There was a tendency ( P = 0.10) for arginine treated (45.1 1.7 %) mares to have greater perfusi on to the F1 for the 4 d preceding ovulation compared to control (40.2 2.1 %;Fig. 5 2) and a tendency ( P = 0.05) for younger mares (45.3 1.8 %) to

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1 04 have increased perfusion of the F1 than older mares (39.9 2.0 %; Fig. 5 2). No differences were observ ed between groups or age in diameter (Fig. 5 3A) or vascular perfusion of the CL, and data from both ages were combined (Fig. 5 3 B). The mean CL perfusion was 47.4 5.2% in arginine and 42.5 4.0% in control mares. There was a tendency ( P = 0.08) for CL perfusion to increase from Day 4 to 6 post ovulation. As no difference in Resistance Index (RI) of the ovarian artery was found between the two age groups during the follicular or luteal phase, data were combined for analysis. No differences were observed in RI between groups during the follicular phase in the retrospectively identified ovulatory (Fig 5 4 A) or non ovulatory (Fig 5 4 B) ovarian artery for the 10 d preceding ovulation. The mean RI of the ovarian artery ipsilateral to ovulation was 0.95 0. 05 for arginine treated and 0.86 0.05 for control mares. The mean RI of the ovarian artery contralateral to ovulation was 0.88 0.02 for arginine treated and 0.87 0.01 for control mares. Resistance index was also normalized to ovulation and examined d uring the luteal phase (Days 2, 4 and 6 post ovulation; Fig. 5 5 ). There was no difference between RI in the ovarian artery ipsilateral to the CL, mean values were 0.90 0.02 and 0.87 0.02, for arginine treated and control mares, while values for the o vary contralateral to the CL were 0.89 0.03 and 0.90 0.03 for arginine treated and control mares. Discussion Oral supplementation of mares with L arginine increased the mean dominant follicle diameter during the 10 d preceding ovulation and tended to increase perfusion of the dominant follicle for the 4 d prior to ovulation, but had no effect on yr) had a significantly smaller dominant and largest subordinate folli cle than younger

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105 mares for the 10 d preceding ovulation. There also was a tendency for older mares to demonstrate less perfusion to the dominant follicle for the 4 d prior to ovulation and these mares tended to have a shorter interovulatory interval. No di fferences were found by age with regards to CL diameter, perfusion, embryo recovery rates or RI. Our findings demonstrate that L arginine can increase growth of the dominant follicle possibly by increasing blood flow. Our study found older mares have red uced follicular blood flow for 4 days prior to ovulation and smaller dominant and largest subordinate follicles for the 10 days prior to ovulation as compared to younger mares. These results are consistent with observations in women, in which increasing a ge was associated with a decrease in ovarian blood flow (Kurjak and Kupesic, 2002; Kupesic et al., 2003; Ng et al., 2004; Costello et al., 2006). This is in contrast to findings of Altermatt et al. (2012), who reported increased blood flow to the ovulator y follicle in mares > 20 yrs of age compared to younger mares in the 24 h prior to ovulation treated with FSH or LH. Additionally, Ginther et al. (2009) found no difference in follicular vascularity in young and old mares for the four days leading to ovula tion. Our study also demonstrated that older mares have reduced dominant and largest subordinate follicle diameters during the10 days preceding ovulation. The reduction in preovualtory follicle diameter has been found in older mares, as well as a reductio n in the number of follicles throughout the interovulatory interval (Ginther et al., 2008). In cattle, a reduction in follicle diameter has been associated with a smaller CL and reduced progesterone secretion and decreased fertility (Vasconcelos et al., 20 01). It is unclear if the reduction in blood flow with age is a cause or result of the smaller follicular diameter in older mares.

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106 Our study found a tendency for increased vascular perfusion of the F1 follicle with arginine treatment in younger mares. S ilva et al. (2006) found that increased blood flow to the pre ovulatory follicle was positively correlated with pregnancy rates in mares with hCG induced ovulations. Our study may lack enough statistical power to detect a difference in embryo recovery rat e that may be associated with alterations in would need 411 animals per group. Additionally, Altermatt et al. (2012) found a positive correlation of increasing blood flow from follicle which had oocytes collected and increasing rates of blastocyst development in culture. Our study found no difference in either development stage or embryo grade between age or treatment groups. This may be due to a lack of statistical power a nd further work is needed to establish a relationship between changing follicular blood flow and its effect on fertility. Additionally, our study did not find any differences in embryo recovery rates between young and old mares. As previously discussed, th is is likely due to a lack of statistical power. Recovery rates in mares range from 50% in mares to 90% (Fleury et al., 1989; Camillo et al. 2001). Embryo recovery rates in our study were low and this may be due in part to no rest cycles between flushes, u se of subfertile mares and environment factors, such as heat and humidity (Woods et al., 1985; Stout, 2006; Mortensen et al., 2009). Arginine supplementation in women that poorly respond to a follicular hyperstimuation protocol by either failing to achi eve an adequate number of mature follicles and/or an adequate serum estradiol concentration after hyperstimulation, increased the number of oocytes retrieved and embryos transferred (Battaglia et al., 1999). In contrast, women supplemented with arginine wh o responded normally to a

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107 hyperstimulation protocol and then underwent an IVF program had decreased embryo quality and pregnancy rates (Battaglia et al., 2002). Battaglia et al. (2002) hypothesized that a reduction in embryo quality was related to NO2 /NO3 concentrations in follicular fluid a adversely affect ATP production resulting in the formation of oxidizing molecules. Resistance Index was not different between groups in our study for arterial measurements during the luteal phase. This is in contra st to Takasaki et al. (2009) which found women treated with arginine starting after ovulation and through the luteal phase had a decreased RI compared to previous cycles. A decrease in RI is generally associated with improved blood flow through the examine d vessel (Ginther, 2007). Based on our findings, arginine supplementation seemed to have little effect on luteal blood flow. In conclusion, supplementation of mares with arginine resulted in a larger dominant follicle in the 10 d preceding ovulation and tended to increase ovarian blood flow, but had no effect on embryo recovery rates. Additionally, older mares had a smaller dominant follicle and tended to have reduced blood flow but no difference in embryo recovery rates. It is well established that olde r mares are less fertile than younger mares, and the number of animals used in this study yielded a low statistical power and thus it was unlikely to detect differences in embryo recovery between groups or ages. This research raises the question, whether follicular blood flow plays a factor in mare fertility and if developing methods to alter follicular blood flow will enhance fertility in mares.

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108 Table 5 1. Number of recovered embryos classified by grade (Vanderwall, 1996) for each group (control or argi 16 yr). There was no difference in embryo score between groups or ages. Item Group Age Control Arginine Young Old Uterine flushes performed 24 33 25 32 Embryos recovered 16 (54%) 13 (48%) 14 (56%) 15 ( 46%) Embryos classified by grade 1 10 (63%) 9 (69%) 11 (79%) 8 (53%) 2 4 (25%) 4 (31%) 2 (14%) 6 (40%) 3 1 (6%) 0 (0%) 1 (7%) 0 (0%) 4 1 (6%) 0 (0%) 0 (0%) 1 (7%)

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109 Figure 5 1. Least squares mean ( SEM) diameter (mm) of the (Panel A) retrospect ively identified dominant (F1) and (Panel B) largest subordinate (F2) follicles for L arginine supplemented and control mares separat ed by age (Young < 16 yr; between control old and arginine young mares; a B denotes a significant difference (P < 0.05) between control young and arginine young mares; and C denotes a significant difference (P < 0.05) between control old and arginine old mares on a given day. In Panel B, an D denotes a significant difference (P < 0.05) between control old and arginine young mares; a E denotes a significant difference (P < 0.05) betwe en control old and control young mares; a F denotes a significant difference (P < 0.05) between control young and arginine old mares; and a G denotes a significant difference (P < 0.05) between control young and arginine young mares on a given day.

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110 Figu re 5 2. Least squares mean ( SEM) percent perfusion to the retrospectively identified dominant (F1) follicle for the 4 days prior to ovulation. An A denotes a significant difference (P < 0.05) between control old and control young mares; a B denotes a si gnificant difference (P < 0.05) between arginine old and arginine young mares; and a C denotes a significant difference (P < 0.05) between control young and arginine old mares on a given day.

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111 Figure 5 3. Least squares mean ( SEM) Panel A: diameter a nd, Panel B: percent perfusion of the Corpus luteum for the 6 days following ovulation.

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112 Figure 5 4. Least squares mean ( SEM) Resistance index (RI) Panel A: to the ovarian artery ipsilateral to ovulation and Panel B: ovarian artery contralateral to o vulation for the 10 days prior to ovulation. An asterisk (*) denotes a significant difference (P < 0.05) between groups on a given day.

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113 Figure 5 5. Least squares mean ( SEM) Resistance index (RI) Panel A: of the ovarian artery ipsilateral to the Corpus luteum and Panel B: contralateral to the Corpus luteum for the 6 days following ovulation. In Panel A, and asterisk (*) denotes a significant difference (P < 0.05) between arginine treated mares and control on a given day.

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114 CHAPTER 6 CONCLUSIONS L Argin ine can potentially effect reproduction via several pathways (Fig. 6 1) which could impact follicular and fetal development. These data show that supplementing the post partum mare with L Arginine shortened gestation length and decreased uterine RI before parturition while decreased uterine body and horn sizes and accumulated less uterine fluid than controls, while not altering follicular development. Open mares treated with L arginine had a larger follicle for the 10 d precedin g ovulation than control mare s and vascular perfusion of the dominant follicle tended to be greater for the 4 d prior to ovulation. No differences were observed between groups in diameter or vascular perfusion of the corpus luteum. Resistance indices, normalized to ovulation, were not significantly different between groups during the follicular or luteal phase. Oral L arginine supplementation increased the size and tended to increase perfusion of the F1, but had no effect on luteal perfusion or embryo recovery rates in open mares. It is unclear whether L arginine supplementation altered ovarian or uterine blood flow. Changes in RI during late pregnancy suggested an increase in uterine blood flow with L arginine supplementation; however, there were no differences in uterine or ovarian R I neither during the post partum period nor in open mares were suggesting no difference in uterine blood flow with L arginine supplementation. Resistance Index takes into account both vascular resistance and vascular impedence. Vasodilators can alter both resistance and impedance of the vasculature (Zobel et al., 1980) thus complicating the interpretation of the index. If impedance remains unchanged while resistance increases, RI will increase while blood flow decreases; however, if resistance remains

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115 const ant while impedance increases the RI decreases while blood flow decreases (Bateman, 2004). Vasodilation and vasoconstriction act to alter both both resistance and impedance simultaneously. The result is, for example during vasodilation, the effect of the l ower resistance (to decrease RI) is counteracted by the effect of lower impedance (to increase RI) and thus the RI remains relatively constant while blood flow is in fact increasing. The same phenomon can happen during vasoconstriction, and has been observ ed by Adamson et al. (1990), who saw a significant reduction in blood flow while vascular indices remained unchanged. Follicular blood flow did increase as eviedent by the alteration in perfusion of follicles demonstrated by color power Doppler ultrasonog raphy. Interestingly, RI did not change between treatment and control while perfusion was increased. This could be due to two reasons. First, L arginine supplementation increased angiogensis in the tissue surrounding the follicle. This seems unlikely as an increase in angiogensis should result in a decrease in RI. This has been documented with ovarian neoplasms (Wu et al., 1994) where an incremental decrease in RI was found as cancer progressed. During these circulstances the RI decreases along with the res istance. Considering these factors it is possible that L arginine supplementation did effect vasodilation and blood flow; however, the current technique could not detect these changes due to the inverse relationship of resistance and impedance on RI. While L arginine supplementation increased follicular perfusion this did not translate into increased embryo recovery rates. This may be due in part to the low number of animals used resulted in a lack of statistical power to detect a difference. Silva et al. ( 2006) found mares that became pregnant had increased perfusion to the

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116 ovulatory follicle, lower RI in the corresponding ovarian artery and smaller diameter follicle than those which did not become pregnant. This raises the question was the decreased pregna ncy rates due to the decreased follicular blood flow, or whether impaired follicular development resulted in the decreased. Supplementation of L arginine increased blood flow but did not impact embryo recovery rates, suggesting that increasing blood flow t o the ovulatory follicle does not improve fertilization. This raise the question as to whether follicular blood flow posses a problem to fertility in mono ovulatory species. These potential vascular effects could be mediated by NO, a potent vasodilator (B oger and Bode Boger, 2001 ) which also plays a role in angiogenesis ( Hood et al., 1998 ) or by agmatine which can potentially adre nergic and imidizoline receptor ( Blantz et al., 2000 ) Adrenergic and imidizoline receptor agonists have been shown to induce vasodilation in arteries of dogs ( Link et al. 1996 ). Further work is needed to determine if agmatine can elicit these same effects. L Arginine supplementation did not alter follicular dynamics in post partum mares but increased the size of the dominant follicle during the follicular period. L A rginine supplementation could alter follicular growth via growth hormone, insulin or potentially IGF1 L Arginine has been show to increase growth hormone concentrations in humans ( Merimee et al., 1969 ) and swine ( Kim et al., 2004 ). Growth hormone has been shown to increase IGF1concentrations in cattle. In osteoblast cell s, L arginine directly stimulates IGF 1 prod uction (Chevalley et al., 1998). Although it remains unclear how L arginine may be acting on follicle formation, IGF1 has been show in vitro to increase follicle growth and enhance antrum formation (Gutierrez et al., 2000). Thus alterations in

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117 follicular dynamics may be due to the potential effects of L arginine on IGF1 production; h owever, further work is needed to investigate this possibility. Additionally, L arginine can effect insulin secretion. Insulin receptor knockout mice have reduced follicular development ( Kitamura et al., 2003 ). The exact mechanism remains to be elucidated but represents another possible pathway by L arginine could effect reproduction Considering the role of blood in supplying nutrients and signaling molecules while removing waste from tissues, it is plausible this may affect reproductive performance. The im portance of gonadotropins on reproductive performance is well established. Increasing blood flow to the ovulatory follicle not only could increase the delivery of these hormones to the ovarian tissue, but also increase the delivery of nutrients needed to s upport the growth of follicles. Thus the possibility exists that alteration in growth could be due to the increase delivery of signaling molecules and substrates needed for growth. Further work is needed to clarify this, such as examing follicular fluid. T he role of polyamines in reproduction is unclear; however, there is eveidence that suggests potential affects on reproduction. Polyamines have been idenetified in the reproductive tract of mice and treatment with an ornithine decarboxylase inhibitor decrea ses the number of oocytes ovulated. Research has demonstrated L arginine can stimulate the mTOR pathway although the exact mechanism is unclear ( Yao et al., 2008 ). This pathway plays a critical role in cell growth and proliferation ( Kim et al., 2002 ). I t is possible the increased amount of L arginine in the diet may stimulate this pathway in many cell types in the body include those involoved in reproduction.

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118 Mares supplemented orally with L arginine had an increase in follicular blood flow; however, th is did not translate into increased embryo recovery rates. Interestingly, mares during the post partum period fed L arginine had a decrease in uterine fluid and an increase in uterine involution rate. Although, the mechanism of these actions remains to be determined, it is clear L arginine can exert its effects thorugh a large number of pathways and the exact mechanisms remain to be determined. This research does demonatrate that in the post partum period, that L arginine has the potential to be a valuable management tool.

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119 Figure 6 1. Possible mechanisms by which L arginine supplementation could influence reproduction. Enzymes: 1 Arginine Decaboxylase, 2. Agmatinase, 3. Ornithine Decarboxylase, 4. Nitric Oxide Synthase, 5. Arginase. Abbreviations: VEGF Vascular Endothelial Growth Factor, mTOR Mammalian Target of Rapomycin, ISR1 Insulin Receptor Substrate 1, IGF1 Insulin Like Growth Factor. A dotted line and question mark (?) indictes the mechanism of action remains to be fully elucidated.

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150 BIOGRAPHICAL SKETCH Dale Kelley graduated from North Carolina State University with an AAS in agricu ltural business mana gement and a B S in animal science. After spending several years in the equine industry ; he graduated fro m Clemson University with a MS in animal and veterinary sciences. He then earned a PhD in animal molecular and cellular biology from the University of F lorida in the spring of 2013