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Development of an Elisa to Measure Crisp-3 Concentration in Equine Seminal Plasma

Permanent Link: http://ufdc.ufl.edu/UFE0041342/00001

Material Information

Title: Development of an Elisa to Measure Crisp-3 Concentration in Equine Seminal Plasma
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Connor, Meghan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 3, breeding, crisp, elisa, endometritis, equine, induced, plasma, proteins, seminal
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Breeding-induced endometritis is an acute inflammatory response mounted by the uterus of mares to clear excess spermatozoa, seminal plasma and bacterial contaminants after breeding. There is a population of mares in which this system fails. Such mares may suffer from prolonged uterine inflammation and are diagnosed with persistent breeding-induced endometritis. Three main types of protein are found in equine seminal plasma, including cysteine-rich secretory proteins (CRISP). Of the CRISP molecules secreted in equine seminal plasma, CRISP-3 is found in the greatest amount. CRISP-3 has been shown to have a protective effect on spermatozoa, protecting them from neutrophil binding and phagocytosis. Currently, no assay exists to quantify CRISP-3 in equine seminal plasma. One objective of this study was to develop an ELISA to measure the concentration of CRISP-3 in equine seminal plasma. An ELISA was the chosen type of assay, due to its high sensitivity and reliability. In addition to development and validation of an ELISA to measure CRISP-3 concentration, we aimed to determine if a correlation exists between the amount of CRISP-3 in seminal plasma and equine fertility. We hypothesized that due to the high amount of CRISP-3 secreted by the accessory sex glands of the stallion that there would be a correlation between CRISP-3 concentration in seminal plasma and stallion fertility. An indirect ELISA was successfully developed and validated using monoclonal mouse anti equine CRISP-3 antibodies. This assay was used to measure the amount of CRISP-3 found in 67 stallion seminal plasma samples. The average amount of CRISP-3 found in all seminal plasma samples (n=67) was 7.44mg/ml plus or minus 5.98. The minimum amount of measured CRISP-3was 0.49mg/ml and the maximum amount of measured CRISP-3 was 25.63. The r value was equal to -0.1433, indicating a poor correlation between CRISP-3 values and fertility. Possible explanations include a highly subjective assignment of stallion fertility and a lack of data concerning the mares which were bred by these stallions. Further study of equine CRISP-3 must be done to determine its exact role in fertility.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meghan Connor.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Verstegen, John P. L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041342:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041342/00001

Material Information

Title: Development of an Elisa to Measure Crisp-3 Concentration in Equine Seminal Plasma
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Connor, Meghan
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: 3, breeding, crisp, elisa, endometritis, equine, induced, plasma, proteins, seminal
Veterinary Medicine -- Dissertations, Academic -- UF
Genre: Veterinary Medical Sciences thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Breeding-induced endometritis is an acute inflammatory response mounted by the uterus of mares to clear excess spermatozoa, seminal plasma and bacterial contaminants after breeding. There is a population of mares in which this system fails. Such mares may suffer from prolonged uterine inflammation and are diagnosed with persistent breeding-induced endometritis. Three main types of protein are found in equine seminal plasma, including cysteine-rich secretory proteins (CRISP). Of the CRISP molecules secreted in equine seminal plasma, CRISP-3 is found in the greatest amount. CRISP-3 has been shown to have a protective effect on spermatozoa, protecting them from neutrophil binding and phagocytosis. Currently, no assay exists to quantify CRISP-3 in equine seminal plasma. One objective of this study was to develop an ELISA to measure the concentration of CRISP-3 in equine seminal plasma. An ELISA was the chosen type of assay, due to its high sensitivity and reliability. In addition to development and validation of an ELISA to measure CRISP-3 concentration, we aimed to determine if a correlation exists between the amount of CRISP-3 in seminal plasma and equine fertility. We hypothesized that due to the high amount of CRISP-3 secreted by the accessory sex glands of the stallion that there would be a correlation between CRISP-3 concentration in seminal plasma and stallion fertility. An indirect ELISA was successfully developed and validated using monoclonal mouse anti equine CRISP-3 antibodies. This assay was used to measure the amount of CRISP-3 found in 67 stallion seminal plasma samples. The average amount of CRISP-3 found in all seminal plasma samples (n=67) was 7.44mg/ml plus or minus 5.98. The minimum amount of measured CRISP-3was 0.49mg/ml and the maximum amount of measured CRISP-3 was 25.63. The r value was equal to -0.1433, indicating a poor correlation between CRISP-3 values and fertility. Possible explanations include a highly subjective assignment of stallion fertility and a lack of data concerning the mares which were bred by these stallions. Further study of equine CRISP-3 must be done to determine its exact role in fertility.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Meghan Connor.
Thesis: Thesis (M.S.)--University of Florida, 2009.
Local: Adviser: Verstegen, John P. L.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041342:00001


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1 DEVELOPMENT OF AN ELISA TO MEASURE CRISP -3 CONCENTRATION IN EQUINE SEMINAL PLASMA By MEGHAN C. CONNOR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2009

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2 2009 Meghan C. Connor

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3 To my m om: thank you for always believing in me

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4 ACKNOWLEDGMENTS I am extremely grateful to the Grayson Jockey Club Foundation for their funding of this research. I would like to thank the members of my supervisory committee: Dr. Mats Troedsson, Dr. John Verstegen and Dr. Bill Buhi. Dr. Troedsson, thank you so much for your constant guidance and unwavering support throughout this project. Your high level of care and concern was evident throughout my work and I am truly appreciative. It has been an incredible honor to work with you. Dr. Verstegen, thank you for bringing me into your lab and for teaching me so much about scientific technique. Thank you for being patient as we went through the long process of trial and error in developing this assay. I would like to extend a special thank s to Linda Green for helping me with all aspects of my project. Thank you so much for everything Im not sure where I would be without your help. Thank you to everyone who helped me get this project started. Andria Doty, thank you for all of your moral support and for providing the purified CRISP 3. Scherwin Henry, thank you for your tireless efforts in producing our monoclonal antibodies. Dr. Macpherson, thank you for sharing your seminal plasma samples with me. I am so grateful to the members of the Small Animal Reproduction Lab: Jim Burr ow and Tameka Phillips. Thank you for sharing the lab with me and for providing lots of laughs. My family has been a constant source of support throughout this project. I would like to thank my mother for her never ending support of my educational pursuit s Thank you for always being so interested in my work. I would also like to thank my boyfriend

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5 Damian for always being there for me and for helping me in any way possible Thank you for going to the lab with me on so many Sunday nights.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 ABSTRACT ........................................................................................................................ 10 CHAPTER 1 INTRODUCTION ........................................................................................................ 12 2 LITERATURE REVIEW .............................................................................................. 14 Introduction ................................................................................................................. 14 Breeding -induced Endometritis .................................................................................. 14 Sperm Transport and Elimination ............................................................................... 17 Seminal Plasma Proteins and Cysteine Rich Secretory Proteins ............................. 19 ELISA Development ................................................................................................... 22 3 ELISA DEVELO PMENT AND VALIDATION ............................................................. 28 Introduction ................................................................................................................. 28 Materials and Methods ............................................................................................... 29 Monoclonal Antibody Selection ........................................................................... 29 Antibody Titration ................................................................................................. 29 Standard Curve Development ............................................................................. 31 Competitive Assay Validation .............................................................................. 32 Results ........................................................................................................................ 33 Monoclonal Antibody Sele ction, Affinity and Specificity ..................................... 33 Antibody Titration ................................................................................................. 33 Standard Curve Development ............................................................................. 34 Competitive Assay Validation .............................................................................. 34 Disc ussion ................................................................................................................... 34 4 MEASUREMENT OF CRISP 3 .................................................................................. 42 Introduction ................................................................................................................. 42 Materials and Methods ............................................................................................... 43 Samples ................................................................................................................ 43 Animals ................................................................................................................. 43 Assay .................................................................................................................... 43

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7 Results ........................................................................................................................ 44 Discussion ................................................................................................................... 45 5 CONCLUSIONS .......................................................................................................... 55 LIST OF REFERENCES ................................................................................................... 58 BIOGRAPHICAL SKETCH ................................................................................................ 62

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8 LIST OF TABLES Table page 4 -1 CRISP -3 concentration in stallions with known fertility. ........................................ 51 4 -2 CRISP -3 concentrations in seminal plasma of stallions with unknown fertility. ... 52 4 -3 Fertility data provided for 40 stallions. ................................................................... 53

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9 LIST OF FIGURES Figure page 2 -1 Schematic diagram of an indirect ELISA. .............................................................. 26 2 -2 Schematic diagram of an indirect sandwich ELISA. ............................................. 27 3 -1 Western blot depicting recognition of CRISP 3 in seminal plasma by HL2175.. ................................................................................................................. 37 3 -2 HL2175 titration curves at diluti ons of 1:160,000 and 1:200,000 ..................... 38 3 -3 HL2175 titration curve shown at dilutions of 1:40,000; 1:80,000 and 1:120,000. ............................................................................................................... 39 3 -4 Titrat ion curves of HL2175 at 1:80,000 dilution showing no significant differences between regular and pre-incubation. .................................................. 40 3 -5 Comparison of the standard curve and curve generated using equine seminal plasma sample.. ..................................................................................................... 41 4 -1 Standard curve for the ELISA measuring CRISP -3 concentration in seminal plasma.. .................................................................................................................. 47 4 -2 Bar graph of CRISP -3 concentrations for stallions with known fertility. ............... 48 4 -3 Bar graph of CRISP -3 concentrations of stallions with unknown fertility. ............ 49 4 -4 Scatter plot and regression lines for fertility data in relation to CRISP 3 concentration. ......................................................................................................... 50

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10 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science DEVELOP MENT OF AN ELISA TO MEASURE CRISP -3 CONCENTRATION IN EQUINE SEMINAL PLASMA By Meghan C. Connor December 2009 Chair: John Verstegen Major: Veterinary Medical Sciences Breeding -induced endometritis is an acute inflammatory response mounted by the uterus of mares to clear excess spermatozoa, seminal plasma and bacterial contaminants after breeding. There is a population of mares in which this system fails. Such mares may suffer from prolonged uterine inflammation and are diagnosed with persistent breeding -induced endometritis. Three main types of protein are found in equine seminal plasma, including cysteine-rich secretory proteins (CRISP). Of the CRISP molecules secreted in equine seminal plasma, CRISP 3 is found in the greatest amount. CRISP -3 has been s hown to have a protective effect on spermatozoa, protecting them from neutrophil binding and phagocytosis. Currently, no assay exists to quantify CRISP -3 in equine seminal plasma. One objective of this study was to develop an ELISA to measure the concentration of CRISP 3 in equine seminal plasma. An ELISA was the chosen type of assay, due to its high sensitivity and reliability. In addition to development and validation of an ELISA to measure CRISP 3 concentration, we aimed to determine if a correlation exi sts between the amount of CRISP -3 in seminal plasma and equine fertility. We hypothesized that due to the high amount of CRISP 3 secreted by the accessory sex glands of the stallion that there would be a correlation

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11 between CRISP 3 concentration in seminal plasma and stallion fertility. An indirect ELISA was successfully developed and validated using monoclonal mous e anti equine CRISP -3 antibodies. This assay was used to measure the amount of CRISP -3 found in 67 stallion seminal plasma samples. The average amount of CRISP 3 found in all seminal plasma samples (n=67) was 7.44mg/ml 5.98. The minimum amount of measured CRISP 3was 0.49mg/ml and the maximum amount of measured CRISP -3 was 25.63. The r value was equal to -0.1433, indicating a poor correlation bet ween CRISP -3 values and fertility. Possible explanations include a highly subjective assignment of stallion fertility and a lack of data concerning the mares which were bred by these stallions. Further study of equine CRISP 3 must be done to determine its exact role in fertility.

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12 CHAPTER 1 INTRODUCTION Breeding -induced endometritis is a natural immune response developed by the mare to clear the uterus of excess spermatozoa, seminal plasma and bacterial contaminants after breeding (Troedsson, 1999). Most normal mares are able to effectively clear the uterus within 36 hours of breeding (Kat ila, 1995; Troedsson, 1995). However, a population of mares exists that are unable to effectively clear the uterus of contaminants and remain in a state of prolonged inflammation (Troedsson et al., 1998). Such persistent endometritis is a common and costly cause of subfertility in horses. It has been demonstrated that seminal plasma suppresses the post breeding i nflammatory response in mares ( Alghamdi et al., 2004). There are a great variety of seminal plasma proteins found in the stallion ejaculate. A pr otein that is expressed in high quantity in the stallion ejaculate is Cys teine-Rich Secretory Protein 3 (CRISP -3) (Schambony et al., 1998). It was recently demonstrated that CRISP 3 is able to provide a protective effect toward spermatozoa by preventing polymorphonuclear neutrophil (PMN) binding and phagocytosis (Andria Doty, PhD Thesis). However, there is no existing assay to quantify CRISP -3 in stallion seminal plasma. Enzyme linked immunosorbent assay (ELISA) is a technique widely used in diagnostics a nd research to quantify specific molecules. ELISAs are highly sensitive and incredibly effective in measuring hormone, protein and antibody levels in serum and plasma (Crowther, 2001). Another advantage of ELISA is their flexibility. There are three differ ent types of ELISAs, and the investigator can develop a specific assay depending on reagent, antigen and antibody availability (Crowther, 2001).

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13 The overall objectives of this study were to develop and validate an ELISA to measure the concentration of CR ISP-3 in equine seminal plasma and to determine if CRISP -3 concentration is correlated with stallion fertility. The hypothesis was that stallion fertility would be correlated to CRISP -3 concentration. CRISP -3 comprises such a significant portion of the equine ejaculate, suggestive of a major biological function.

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14 CHAPTER 2 LITERATURE REVIEW Introduction The objectives of this work are to investigate the roles and functions of CRISP 3 in equine seminal plasma using a specific quantitative assay. CRISP 3 i s a protein found in equine seminal plasma that has been shown to play a role in stallion fertility as well as in suppressing the binding between polymorphonuclear neutrophils (PMNs) and spermatozoa. Breeding -induced Endometritis Immediately after breed ing, mares develop an acute form of endometritis. I t has been suggested that this inflammatory response to breeding serves the purpose to remove excess sperm, seminal plasma and bacterial contaminants from the uterus (Troedsson, 1999). Indeed, t he external genitalia of the stallion harbor different bacterial species and all physical barriers to the uterus are passed by the penis during natural mating (Troedsson et al., 1993). Thus, it was previously believed that bacteria introduced into the uter us during breeding were causing the uterine inflammatory response. However, a study published in 1993 by Kotilainen and others showed insignificant bacterial contamination of the ut erus six hours after breeding. In fact, n eutrophil concentrations were fou nd to be influenced by the amount of spermatozoa present (Kotilainen et al., 1993). The study suggested that spermatozoa were the cause of the post -breeding uterine inflammatory response. This was later confirmed in a study that demonstrated a role for spermatozoa in the mechanism of breedinginduced endometritis (Troedsson et al., 1995b).

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15 The inflammatory response o f the uterus causes an influx PMNs into the uterine lumen (Troedsson et al., 2001) PMNs may be found in the uterus as soon as 0.5 hours after breeding and reach a peak level between six to twelve hours post -breeding (Katila, 1996). However, sperm transport to the oviduct is not complete until four hours after breeding (Scott et al., 1995). The influx of PMNs is caused by the activation of comple ment ( Troedsson et al., 1995b; Troedsson, 1998). Once neutrophils are activated in the uterus, they begin to phagocytose both bacteria and spermatozoa (Troedsson, 2001). Large clusters of sperm bound to neutrophils may be seen as a result of neutrophil ext racellular traps, or NETs (Alghamdi et al., 2008). However, receptor ligand binding is probably also involved in binding between spermatozoa and PMNs, but the exact nature of this binding is unknown. PMNs may remain in the uterus for up to forty eight hour s after breeding, however most normal mares are able to achieve uterine clearanc e in thirty -six hours (Katila, 1995; Troedsson 1995 ) through increased uterine contractility. The release of prostaglandins after breeding induces contraction of smooth muscle layers, specifically of the myometrium (Troedsson et al., 1995 b ). Uterine contractions then aid in the removal of fluid and other contaminants. Once all excess fluids, spermatozoa and other contaminants are removed via myometrial activity and PMN phagocy tosis, the uterus generally return s to its normal state. Other species that display a sperm triggered inflammatory response include rabbits, humans, and pigs (Cohen, 1984; Rozeboom et al., 2001; Tyler, 1977). There is however, a population of mares in which these systems fail. Such mares are said to be susceptible to persistent breeding induced endometritis (Troedsson et al., 1998) In 1998, Zent and Troedsson completed a field study of Thoroughbred mares.

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16 They found that 15% o f mares enrolled in their study were unable to clear their uteri after breeding and remained in a state of prolonged uterine inflammation. The delay in uterine clearance is likely caused by poor myometrial function and decreased uterine contractility ( LeBl anc et al., 1994; Troedsson et al., 1991; Troedsson et al., 1993). A study performed in 2008 by Alghamdi and others showed that mares prone to persistent breeding induced endometritis had elevated levels of nitric oxide in the uterine lumen as a result of increased expression of inducible nitric oxide synthaoase (iNOS) in the endometrium Mares susceptible to persistent breedinginduced endometritis exhibit below baseline myoelectrical activity in the hours following the onset of inflammation. The ability o f nitric oxide to induce smooth muscle relaxation may explain this effect. P ersistent endometritis is one of the most common and costly causes of subfertility in horses. For this reason, identification of mares susceptible to breedinginduced endometriti s is of major clinical relevance. However, early detection may be difficult, as uterine fluid accumulation is not diagnosed until after breeding (Troedsson, 2008). Some mares may exhibit clinical signs such as vaginal discharge observed withn a few hours a fter breeding. However, i t is important to note that this may be a manifestation of the normal uterine clearance of excess seminal plasma and other contaminants. Generally, most normal mares have cleared the uterus from fluid within 6 hours after breeding A diagnostic technique has been proposed based on intrauterine inoculation of charcoal particles (LeBlanc et al., 1989). When inoculated, i t is possible to measure the amount of particles cleared within a forty eight hour t ime period (LeBlanc et al., 198 9; LeBlanc et al., 1994). Through this measurement, a veterinarian may be able to recognize a mare susceptible to persistent endometritis as compared to a

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17 normal animal LeBlanc et al., (1994) also suggested that scintigraphy can be used to measure clearance of radio-colloids from the uterus, hence identifying mares with delayed uterine clearance. To prevent or reduce the occurrence of breedinginduced endometritis it is important to decrease the amount of uterine exposure to semen and bacteria (Troedsson, 2006). Similarly, breeding the mare only once, reducing bacterial and sperm concentration, may be helpful in reducing post -breeding inflammation. C areful monitoring of follicular development and hormonal treatment will then be necessary (Troedsson, 2006). Mares that are diagnosed with persistent breed ing induced endometritis, can be treated either with drugs that increase myometrial activity, such as oxytocin or by performing a post breeding uterine lavage to assist uterine clearance (LeBlanc, 1994 ; Troedsson et al., 1995a ). Sperm Transport and E limination In the horse, semen is deposited in the uterus during both natural mating and artificial insemination. After entering the uterus, spermatozoa must be transported to the oviduct for fertilization This transport may be completed in just two hours, but the highest numbers of sperm are found in the oviduct four hours after insemination (Bader, 1982). During transport, the physical barrier that sperm must pass through is the utero tubal junction (Troedsson et al., 1995). The transport of sperm to the oviduct may be affected by a variety of factors that include the fertility of both the mare and stallion. The motility of spermatozoa is important, as the sperm actively participates in its transport through the female reproductive tract. Uterine contractility also affects sperm transport and is stimulated by mating (Drobnis and Overstreet, 1992). Insemination, physical or

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18 visual contact with a stallion, and teasing all trigger the release of oxytocin from the pi tuitary gland of the mare (Madill, 2000). The release of oxytocin stimulates uterine contractions. Oxytocin and prostaglandin in semen may also stimulate uterine contractions (Katila, 2001). After breeding, the uterus mounts an inflammatory response to sem en and bacterial contaminants. Increased inflammation causes a release of prostaglandins from the mares uterus. The release of prostaglandins induces contraction of sm ooth muscle including the myometrium (Troedsson et al., 1995). These contractions assist spermatozoa in moving through the ut erus into the oviduct. Scott and others (1995) found greater numbers of spermatozoa in the caudal isthmus of the oviduct in fertile mares than in sub -fertile mares. Motile sperm were not recovered bilaterally from the oviducts of sub -fertile mares, but were recovered from both oviducts of fertile mares. Higher numbers of motile sperm were found in the oviducts of fertile mares. Scott and others (1995) also investigated the transport of sperm from fertile and sub-fertile stallions. They found that fertile stallions had a greater total number of spermatozoa and of these spermatozoa a higher percentage were found to be morphologically normal. This study indicates that the transport patterns of sperm may differ between fertil e and sub-fertile mares and stallions. Abnormal transport of spermatozoa can be a direct cause of reproductive failure (Scott et al., 1995). During insemination, a large number of spermatozoa in 20-80mL volumes is deposited into the uterus. However, less than one percent of inseminated spermatozoa reach the oviduct for fertilization (Bader and Krause, 1980). Any remaining sperm and seminal fluids must be effectively eliminated from the female reproductive tract. Semen eliminated from the uterus moves through the cervix into the vagina (Bader and Krause,

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19 1980). While uterine contractions are essential in eliminating sperm from the female reproductive tract, it is important for the cervix to remain relaxed and open to allow for the movement of sperm into and out of the uterus (Katila, 2001). Seminal Plasma P roteins and Cysteine Rich Secretory P roteins Seminal plasma is the fluid portion of semen that consists of secretions from the epididymis and accessory sex glands of the male genital tract (Topfer -Peterson et al., 2005; Troedsson et al., 2005). The biochemical components of these secretions include proteins, enzymes and electrolytes (Kareskoski and Katila, 2008). In the stallion, most seminal plasma proteins are synthesized in the ampulla and seminal vesicles (Schambony et al., 1998). In horses, the bulbourethr al glands and prostate gland also secrete seminal plasma proteins, but in minimal amounts (Schambony et al., 1998). Seventy percent of equine seminal plasma proteins have molecular weights between 1130 kDa (Topfer -Peterson et al., 2005). It has been shown that some of these proteins are correlated with stallion fertility (Brandon et al., 1999). Seminal plasma has been shown to suppress complement activation and decrease phagocytosis (Dahms and Troedsson, 2002; Troedsson et al., 2000; Troedsson et al., 200 2). A study performed by Kotilainen and others in 1993 showed that neutrophil concentrations were lower when mares were inseminated with raw semen and extended fresh semen than with spermatozoa only. The ability of seminal plasma to decrease inflammation h as also been observed in pigs (Rozeboom et al., 2001). The main protein components of equine seminal plasma can be catergorized into fibronectin type II modules (Fn2 type proteins), cysteine-rich secretory proteins (CRISP), and spermadhesins (Kareskoski and Katila, 2008). The most abundant

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20 proteins in equine seminal plasma are SP 1 and SP 2, which are Fn2 type molecules (Ekhlasi -Hundrieser et al., 2005). These proteins account for 7080% of the protein comp onent of equine seminal plasma. Fn -2 type protei ns are thought to have a regulatory role in capacitation due to their ability to modulate sperm plasma membrane properties (Topfer -Peteron et al., 2005). Equine spermadhesin is localized to the sperm surface and is able to bind to the zona pellucida and ca rbohydrates. This may point to a role in fertilization (Topfer -Peterson et al., 2005). A member of the CRISP family that is found in high quantity in equine seminal plasma is CRISP -3 (Schambony et al., 1998). CRISP -3 has a molecular weight of approximately 28 kDa and is expressed by many mammals (Schambony et al., 1998). The HSP -7 protein found in equine seminal plasma is homologous to spermadhesin AWN found in porcine seminal plasma. This protein is synthesized in the testis, ductus epididymis and seminal vesicles of the stallion genital tract (Hoshiba and Sinowatz, 1998; Topfer Peterson et al., 2005). The functions of seminal plasma proteins have been widely investigated and include effects on fertilization as well as sperm transport and elimination. In 2005, Troedsson and others demonstrated that seminal plasma proteins exhibit a protective effect toward viable spermatozoa, protecting them from PMN phagocytosis. The specific seminal plasma protein responsible for this interaction has now been identified as equine CRISP 3 (Andria Doty, PhD Thesis). Determination of this function supports the finding that seminal plasma is involved in the inflammatory response of the equine uterus after breeding by decreasing phagocytosis of spermatozoa by PMNs ( Dahms and Troedsson 2002; Troedsson et al., 2000; Troedsson et al., 2002 ). It is unclear however, if subfertile stallions are deficient in CRISP -3.

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21 Cysteine rich secretory proteins are of particular interest in the horse as they are secreted in high amounts in equine seminal plasma (Schambony et al., 1998). CRISPs are characterized by the presence of sixteen conserved cysteine residues (Cohen at al., 2008). Members of the CRISP family have molecular weights of 20-30 kDa and are expressed primarily in mammals (Cohen at al., 2006). The CRISP family appears to be highly conserved, as mammalian CRISPs share between 5080% sequence identity (Schambony et al., 1998). Four CRISP family members have been identified, CRISP -1 being the first. CRISP -1 is expressed in the epididym is of the rat, mouse, horse and human. CRISP 1 is thought to be involved in sperm oocyte fusion (Cohen et al., 2000; Udby et al., 2002). CRISP -2 is expressed only in the testis and has been identified in the human, rat, mouse, horse and guinea pig (Schambony et al., 1998; Udby et al., 2002). It is hypothesized that CRISP 2 facilitates cell to cell interactions between developing spermatocytes and Sertoli cells in the testis (Madea t al., 1999). CRISP -3 is the most widely expressed member of the CRISP family It was first identified in the salivary glands of mice and humans (Schambony et al., 1998). CRISP -3 is also expressed in the genital tract and immune cells of many mammals (Hamann et al., 2007). CRISP -4 is the most recently described member of the CRISP family and is expressed only in the epididymis (Jalkanen et al., 2005; Nolan et al., 2006). Additionally, a protein with 45% homology to mammalian CRISPs has been identified in the venom of lizards (Mocha Morales et al., 1990; Morrissette et al., 1995). I n the horse, CRISP 3 comprises a major portion of seminal plasma proteins found in the ejaculate (Schambony et al., 1998). CRISP 3 is expressed mainly in the seminal vesicles and ampulla of the stallion genital tract (Schambony et al., 1998). It

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22 has been s hown that CRISP -3 is associated with fertility in Hanoverian stallions. Indeed, Hanoverian stallions homozygous for the CRISP 3 gene had a 7% higher pregnancy rate per cycle than stallions heterozygous for the CRISP 3 gene (Hamann et al., 2007). ELISA D ev elopment Enzyme linked immunosorbent assays (ELISAs) are highly sensitive, precise and versatile assays used in diagnostics and research. ELISA allows for the semi quantitative or quantitative evaluation of antigens present in solutions of either blood, se rum or plasma or after extraction for different tissues or supports (Crowther 2001). ELISAs have slowly supplanted radioimmunoassay as they do not require the use of radioactive tracers (John Verstegen, personal communication). In an ELISA, these tracers are replaced by enzymes used for the detection of the antigen. ELISAs are incredibly flexible, in that more than one system can be used to measure the same unknown. This is useful, as the investigator can adapt their assay to the availability of reagents ( Crowther 2001). There are three main types of ELISAs; direct, indirect and sandwich (Crowther 2001). The direct ELISA is the simplest form of the assay. In a direct ELISA, antigen is diluted in a buffer and a set volume is placed into each well of the sol id phase. The solid phase of an ELISA is usually a 96well flat bottom plate. It is important that the buffer contains no interfering proteins, as the antigen will passively bind to the solid phase of the assay (Crowther 2001). After incubation, the plate is washed and any unbound antigen is removed from the system. Next, a blocking buffer such as bovine serum albumin (BSA) is added to the solid phase. This step coats any areas of the well that were not bound by antigen and decreases non-specific binding. A fter incubation, the plate is washed and any excess blocking buffer is removed. Next, the detecting

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23 antibody is diluted in blocking buffer and is added to the solid phase. In a direct ELISA, the detecting antibody is conjugated to an enzyme. After incubati on, the plate is washed to remove any unbound antibody. The final step of a direct ELISA is the addition of a substrate that is able to recognize the enzyme bound to the detecting antibody (Crowther 2001). During substrate incubation, a reaction occurs that allows color development through enzyme catalysis. The optical density of the liquid in each well is measured using a spectrophotometer that has been set at an appropriate wavelength (Crowther 2001). There is one main difference between a direct and an indirect ELISA. In an indirect ELISA, the detecting antibody is not bound to an enzyme. After the solid phase is coated with antigen, washed, blocked and washed again, the detecting antibody is added and allowed to incubate. After incubation, the plate is washed and an enzymelabeled, antispecies antibody is added to the solid phase as shown in figure 2 -1. This antibody is allowed to incubate, the plate is washed and then substrate is added to begin the colorimetric reaction (Crowther 2001). The developmen t of an indirect ELISA is generally associated with a higher sensitivity of the system, allowing for the detection of lower concentrations of the target hormone or protein. A disadvantage of indirect ELISAs is that they are generally more expensive and tim e consuming to develop. The sandwich ELISA is divided into two forms: direct sandwich and indirect sandwich. A schematic diagram of an indirect sandwich ELISA is shown in Figure 22. Sandwich ELISAs begin with the addition of antibody directly to the soli d phase. This antibody is diluted in a buffer with low protein content, similar to the antigen in a direct or indirect ELISA. This allows the antibody to passively bind to the plate during the

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24 incubation period. This antibody is referred to as the capture antibody (Crowther 2001). After incubation of the capture antibody the plate is washed. Next, antigen diluted in blocking buffer is added. The antigen will bind to the capture antibody. After incubation, the plate is washed. Now, an antibody antigen complex is attached directly to the solid phase (Crowther 2001). A detecting antibody diluted in blocking buffer is now added to the solid phase. In a direct sandwich ELISA this antibody is labeled with enzyme. In an indirect ELISA this antibody is unlabeled and an addition of antispecies, enzymeconjugated antibody will be necessary for detection. The detecting antibody is allowed to incubate and the plate is washed. The final step of a sandwich ELISA is addition of substrate to produce color development. After the colorimetric reaction is complete, the plate can then be placed in a spectrophotometer and optical density readings taken (Crowther 2001). Each stage in an ELISA is able to be adapted to the specific needs of the investigator. However, there are some general practices that are common to most assays. The solid phase most commonly used in an ELISA is a 96 well flat bottomed microtiter plate. Most plates are manufactured from either polyvinyl chloride or polystyrene (Crowther 2001). The coating step of th e assay, which involves passive binding of an antigen o r antibody to the plate also have a few key features. It is important for the antigen or antibody to be diluted in buffer with low protein content, such as phosphate buffered saline (PBS). This helps ensure that only the antigen or antibody binds to the plate (Crowther 2001). The coating concentration is variable, but usually ranges from 1-10 ug/mL at a volume of 50uL (Crowther 2001). The incubation time used in coating is highly variable, as is coating temperature. The washing step of

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25 most assays is similar, as plate washing machines are readily available for purchase. The washing solution is generally PBS with a small amount of added detergent (Crowther 2001). Enzymes that are commonly conjugated to an tibodies are Horseradish Peroxidase (HRP) and Alkaline Phosphatase (ALP) (Crowther 2001). The final step of an ELISA, the measurement of color change is usually done by taking optical density readings using a spectrophotometer. However, some assays call fo r reading by eye (Crowther 2001). It is important to remember that these are simply general procedures followed in most assays. The key feature of an ELISA is its flexibility; therefore the investigator may modify these processes based on the needs of thei r assay.

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26 Figure 21. Schematic diagram of an indirect ELISA. Red dots represent antigen, yellow Y represents detecting antibody, blue Y represents enzyme-conjugated antibody. The bottom portion of the diagram shows a microtiter plate with wells demonstrating the occurrence of a colorimetric reaction. (http://www.microvet.arizona.edu/courses/mic419/ToolBox/elisa3.jpg )

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27 Figure 22. Schematic diagram of an indirect sandwich ELISA. The Y on top of the microtiter plate represents the capture antibody bound to the blue oval that represents the target antigen. The gray Y on top of the blue oval represents the detection antibody. The orange Y bound to the detection antibody represents th e enzyme linked antibody. (http://www.newenglandbiolabs.de/en/index.php?option=com_content&task=v iew&id=14&Itemid=20)

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28 CHAPTER 3 ELISA DEVELOPMEN T AND VALIDATION Introduction Endometritis induced by breeding is an inflammatory response mounted by the uterus to remove excess spermatozoa, seminal plasma and bacterial contaminants (Troedsson, 1999). It has been demonstrated that this response is activ ated by spermatozoa (Troedsson et al., 1995b). Seminal plasma has been demonstrated as having a role in suppressing the inflammatory response (Rozeboom et al., 2001). Furthermore, a protective effect of seminal plasma on spermatozoa to prevent PMN binding and phagocytosis has been described (Alghamdi et al., 2004; Alghamdi et al., 2008). Through further investigation of seminal plasma proteins it has been determined that CRISP -3, a protein found in equine seminal plasma is involved this protective effect in the horse (Andria Doty, PhD Thesis). CRISP 3 is a major protein component of the equine ejaculate and has been shown to have an effect on fertility in the horse (Hamann et al., 2007; Schambony et al., 1998). However, it is not known if concentrations of C RISP -3 in seminal plasma differs among stallions with high and low fertility. Enzyme linked immunosorbent assay (ELISA) is a highly precise assay used in diagnostics and research. ELISAs are able to measure protein, hormone and antibody concentrations in fluids such as serum, plasma and seminal plasma. There are three main types of ELISAs: direct, indirect and sandwich. The specific steps of each type of assay differ, but all involve binding between antigens and antibodies (Crowther 2001). A colorimetic r eaction is the final step in an ELISA. This reaction takes place between an enzyme conjugated antibody and substrate (Crowther 2001). The measurement of the target substance is done through analysis of optical density values.

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29 The objective of this chapter is to develop and validate an ELISA to measure CRISP -3 concentration in equine seminal plasma. There are no assays available to measure the concentration of CRISP -3 in equine seminal plasma. As CRISP 3 comprises a significant portion of the equine ejacula te, it is of interest to determine its concentration. Materials and Methods Monoclonal Antibody S election Mouse anti equine monoclonal antibodies were produced by the Hybridoma Core Lab at the Interdisciplinary Center for Biotechnology Research at the Uni versity of Florida campus in Gainesville, FL, from CRISP 3 purified from equine seminal plasma (Andria Doty, PhD Thesis). Three antibody lines were generated and made available for use according to established Hybridoma Core Lab protocol. The antibody line s were tested for specificity to purified CRISP -3 by Western blotting. Antibody affinity toward equine CRISP 3 was measured using the kinetics experiment program on the Octet QK machine (ForteBio, Menlo Park, CA). Once an antibody line was chosen, the anti body was diluted in PBS/glycerol at a 1:200 dilution to serve as a stock solution for the foll owing evaluation. The glycerol treated antibody was stored at 20 C. The antibody line was selected based on titer, specificity and affinity for CRISP -3 as descri bed by Crowther (2001) and demonstrated in affinity studies (Linda Green, personal communication). Antibody T itration Upon selection of the monoclonal antibody line, titration of the antibody to determine optimal concentration was completed. A Nunc Maxisor p 96well flat bottomed microtiter plate (Fisher Scientific, Waltham, MA) was coated with purified CRISP -3.

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30 Purified CRISP 3 from equine seminal plasma was obtained at a concentrati on of 3.6 mg/ml (Andria Doty, PhD Thesis) Aliquots of 100ul volume were ad ded to polypropylene cryogenic vials (Corning, Fisher Scientific, Waltham, MA) and were stored at 80 C until needed. CRISP -3 was diluted in PBS (Fisher Scientific, Waltham, MA) to a final concentration of 1 ug/ml. This concentration was chosen after plat e coating with decreasing CRISP -3 concentrations (10ug/ml; 5ug/ml; 2.5ug/ml and 1ug/ml) showed no significant difference in final binding of the antibody based on optical density values. Fifty microliters of this dilution were added to 92 wells of the 96w ell plate. Two wells were coated with 10ug/ml mouse IgG (Sigma Aldrich, St. Louis, MO) to serve as a positive control and two wells were uncoated to serve as a negative control. The purified CRISP 3 was allowed to incubate overnight at 4C. After incubatio n was complete, the plate was washed three times using the WellWash plate washer (Fisher Scientific, Waltham, MA). The wash solution consisted of 0.05% PBS -Tween20. A blocking solution of 1% BSA (Fisher Scientific, Waltham, MA) was added at a volume of 20 0ul to each well of the plate to block plates and prevent non -specific binding. The blocking solution was allowed to incubate for one hour at room temperature on a plate shaker. After incubation was complete, the plate was washed three times using the Well Wash plate washer and the previously described washing solution. The antibody was diluted in 1% BSA to concentrations of 1:10,000; 1:20,000; 1:40,000; 1:80,000; 1:120,000; 1:160,000 and 1:200,000, then added to duplicate wells of the plate at a volume of 5 0ul and allowed to incubate for one hour at room temperature. Throughout the incubation, plates were kept on a shaker. After incubation was completed, the plate was again washed three times as previously described. Next, a 1:1000 dilution of rabbit

PAGE 31

31 anti mo use whole molecule IgG antibody conjugated to alkaline phosphatase was prepared (Sigma Aldrich, St. Louis, MO) and added to each well at a volume of 50ul. The secondary antibody was allowed to incubate for one hour at room temperature while on a plate shak er. Upon completion of secondary antibody incubation, the plate was washed and 100ul of pnitrophenyl phosphate (P -NPP) substrate was added to each well. The P NPP substrate was prepared at a concentration of 1mg/ml (SigmaAldrich, St. Louis, MO). The subs trate was allowed to incubate for one hour at room temperature while on a plate shaker. After incubation was complete, the plate was read using a VMax spectrophotometer (Molecular Devices, Sunnyvale, CA) set at a wavelength of 405nm to measure optical dens ity values. A minimum of three repetitions were completed for each antibody dilution. The optimum antibody concentration was determined as being the concentration allowing an optical density binding of approximately 1.8 (Crowther, 2001). Standard Curve D ev elopment After the optimum antibody concentration was determined, a standard curve was developed using known values of purified CRISP -3 and the selected monoclonal antibody at the optimal dilution. A Nunc Maxisorp 96well, flat bottomed microtiter plate wa s coated with purified CRISP -3 at a concentration of 1ug/ml and allowed to incubate at 4 C overnight. Upon completion of incubation, the plate was washed and blocked using 1% BSA as previously described. Next, a dilution curve of purified CRISP 3 was prepa red, as was the chosen antibody diluted to the chosen concentration. The dilution of CRISP 3 used as standard was from 40ug/ml up to a final dilution of 0.0025 ug/ml (2.5ng/ml) using a 1 by 2 dilution in 14 points. The CRISP -3 dilutions were added to dupli cate wells of the plate at a volume of 50ul. The CRISP -3 addition was immediately

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32 followed by an addition of 50ul of monoclonal antibody at the determined optimum concentration. These additions were allowed to incubate for one hour, shaking at room temperature. The plate was washed three times as previously described after incubation was completed and a 1:1000 dilution of rabbit anti mouse IgG secondary antibody was added at a volume of 50ul to each well. The plate was allowed to incubate at room temperatur e, shaking for one hour. Upon completion of incubation, the plate was washed and P -NPP substrate was added as previously described. The substrate was allowed to incubate for one hour, shaking at room temperature. Finally, optical density measurements were taken using the VMax spectrophotometer set at a wavelength of 405 nm. In an effort to determine if the sensitivity of the assay could be increased, preincubation of the purified CRISP 3 and monoclonal antibody was tested using the described ELISA protocol. Pre -incubation was simply mixing 50ul of each CRISP 3 dilution with 50ul of diluted monoclonal antibody in a polypropylene tube for one hour prior to addition to the plate. After one hour, the antibody -CRISP -3 mixture was added to the plate and the prev iously described protocol was followed to completion. Competitive Assay V alidation Preliminary assays were completed using seminal plasma samples collected from two stallions housed at the Large Animal Hospital of the Veterinary Medical Center at the Univ ersity of Floridas College of Veterinary Medicine. The samples were obtained from the equine theriogenology service and were stored in polypropylene cryogenic tubes at -80 C until use. The indirect ELISA protocol described above was followed to test the c ompetitive ELISA and demonstrate the identity of response of the standard curves generated from the purified CRISP -3 and the fresh CRISP 3 in the seminal

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33 plasma samples. A standard curve was developed as described above and the unknown sample behavior in t he assay was evaluated adding 50ul of a 1 by 2 dilution curve of seminal plasma from 1:1 initially to 1:8. Samples were diluted in 1% BSA. Upon completion of initial testing, it was determined that the samples contained high amounts of CRISP 3 and further dilution was necessary to obtain CRISP -3 measurements. The samples dilution curve was finally established as a 1 by 2 dilution in 8 points from 1:2400 to 1:307200 in 1% BSA. The results obtained from assays using these diluted seminal plasma samples were used to confirm parallelism of the curve and to calculate the coefficients of variation of the assay (inter and intra) by measuring the same samples at least 5 times within the same assay and at least 5 times in different assays. Results Monoclonal Antibody Selection, Affinity and S pecificity Affinity measurements of the three monoclonal antibody lines, HL2175, HL2199 AND HL2212 obtained using the Octet QK showed that HL2175 exhibited the highest affinity for purified CRISP 3. Western blots using sem inal plasma and HL2175 shows four bands, one for each of the 2 glycosylated forms of CRISP 3 and one for each of the non-glycosylated forms of CRISP -3 as seen in Figure 31. Based on these results, HL2175 was chosen as the monoclonal antibody line to be us ed in development of the ELISA to measure CRISP -3 concentrations. Antibody Titration Dilution of HL2175 to various concentrations allowed us to determine the optimum concentration for use in the ELISA. Dilutions at 1:10,000 and 1:20,000 were found to have low sensitivity and reached a plateau earlier than further dilutions. Similarly, dilutions at 1:160,000 and 1:200,000 had low sensitivity and were of poor repeatability,

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34 as seen in Figure 3 -2. The dilutions of antibody at 1:40,000; 1:80,000 and 1:120,000 were the most sensitive and had the greatest detection ranges and best repeatability, as seen in Figure 3 3. Analysis of percent binding at each antibody concentration showed that dilution of HL2175 to 1:80,000 had high sensitivity and also had the greates t detection range. Therefore, HL2175 diluted to a concentration of 1:80,000 was the antibody concentration chosen to develop our ELISA. Standard Curve Development Using the antibody concentration previously described resulted in a standard curve with reliable measurements from 40ug/ml CRISP -3 to 0.0025ug/ml CRISP -3. Analysis of preincubation of CRISP -3 and antibody did not provide any significant increase in assay sensitivity. There was no marked difference in standard curves or unknown sample curves betw een regular and pre-incubation, as seen in Figure 3-4. Competitive Assay V alidation As previously described, analysis of unknown samples showed a need for dilution of the sample. Very high dilutions were necessary to obtain reliable measurements of CRISP -3. The intraassay coefficient of variation at 20% binding and 80% binding was 6% and 16%, respectively, while the inter assay coefficient of variation at the same percentage of binding were 4% and 1%, respectively. Parallelism between the standard curve and unknown sample curve is seen in Figure 35. Discussion The objective of this part of our work was to develop an assay which would allow us to later quantify CRISP -3 concentration in stallion seminal plasma. This assay would also be used to characterize CRISP 3 biological action and potential roles in stallion fertility and the development of breeding-induced endometritis in the mare.

PAGE 35

35 Monoclonal antibodies were produced by the Hybridoma Core Lab at the University of Florida following standard protocols described for monoclonal antibody production. Three monoclonal antibody lines were obtained and characterized for affinity and binding to CRISP 3. The chosen monoclonal antibody line, HL2175, is characterized by a high affinity and binding. It is available in large supply and is a proven detecting antibody, specifically recognizing the four classical bands of CRISP 3 shown in Western blot. The parallelism of the curves generated using purified CRISP -3 and the diluted seminal plasma tend to demonstrate an d c onfirm the specificity of th is antibody. The other two antibody lines were characterized by a lesser affinity for the purified CRISP -3 and a lower concentration demonstrated by dilution curves. It may however be of interest to use these other antibody line s in development of a sandwich ELISA. The use of a sandwich ELISA may serve to increase assay specificity. Additionally, the amount of CRISP 3 needed for a sandwich ELISA is considerably less than that used in our indirect assay. Purifying CRISP 3 from equine seminal plasma is a difficult task and there may be differences found in the protein between purification runs. As the dilution of HL2175 used in this assay is so high, the volume of antibody that was necessary for each dilution was very small. It was for this reason that we chose to dilute the purified antibody 1:200 in a 50:50 mixture of PBS:glycerol. This dilution also prevented the antibody from being subjected to repeated periods of freezing and thawing, as glycerol does not freeze at 20 C. This helped protect the antibody from denaturation and from a loss of function. The chosen dilution of 1:80,000 was able to detect in the range of 15 -85% binding. The intra assay coefficient of variation at 20% binding and 80% binding was 6% and 16%, respectively, while the inter assay

PAGE 36

36 coefficient of variation at the same percentage of binding were 4% and 1%, respectively. We can expect values between 2070% binding to be reliable in the measurement of CRISP 3. Preliminary testing of two readily available seminal plasma samples indicated a need for great dilution of unknown samples. The high dilution factor may account for variability in the assay, especially at the highest dilution levels. While the average intra assay coefficient of variations for the assay was in the acceptable range (11%), it is however important to note the individual intra assay coefficients of variation were significantly higher at the highest dilution (17%). On the other hand, the coefficients of variation for samples at the lowest dilution were only 7.5%. The high sensitivity of our assay in which high concentrations of CRISP 3 are detected in the seminal plasma can to some extent be considered a problem. Pre -incubation of the CRISP -3 addition and monoclonal antibody could have increased the sensitivity of the assay. However, as seen in Figure 35, there was no clear difference between curves. Due to these results and the extra effort involved in preparing samples for pre-incubation, the protocol of regular incubation in the plate was foll owed as previously described. The ELISA described here is the first quantitative assay described for the evaluation of CRISP 3 concentration in equine seminal plasma. Up to now, no other similar assays have been developed in any species in which CRISP pro teins have been described. This assay will be of significant interest in the analysis and characterization of the roles of CRISP 3 both in the evaluation of male fertility and in the study of breeding-induced endometritis in mares.

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37 Figure 31. Western blot depicting recognition of CRISP -3 in seminal plasma by HL2175. Lane of interest is the third from the left, or first lane showing the 4 bands typical of CRISP -3. This Western blot was run on 7/29/08 by Scherwin Henry at the Hybridoma Lab of the Interdisciplinary Center for Biotechnology Research on the University of Florida campus.

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38 Figure 32. HL2175 titration curves at dilutions of 1:160,000 and 1:200,000. Curves are shown for regular incubation and pre-incubation. 0.5 0 0.5 1 1.5 2 2.5 5000 2500 1250 625 312 156 78 39 19 O p t i c a l d e n s i t y CRISP 3 concentration (ng/ml)HL2175 titration 1:160 REGULAR INCUBATION 1:200 REGULAR INCUBATION 1:160 PRE INCUBATION 1:200 PRE INCUBATION

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39 Figure 33. HL2175 titration curve shown at dilutions of 1:40,000; 1:80,000 and 1:120,000. 20 0 20 40 60 80 100 120 5000 2500 1250 625 312 156 78 39 19 P e r c e n t B i n d i n g CRISP 3 concentration (ng/ml)HL2175 Titration 1:40 REGULAR INCUBATION 1:80 REGULAR INCUBATION 1:120 REGULAR INCUBATION

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40 Figure 34. Titration curves of HL2175 at 1:80,000 dilution showing no significant differences between regular and pre-incubation. 20 0 20 40 60 80 100 120 5000 2500 1250 625 312 156 78 39 19 P e r c e n t B i n d i n g CRISP 3 concentration (ng/ml)Pre incubation analysis 1:80 REGULAR INCUBATION 1:80 PRE INCUBATION

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41 Figure 35. Comparison of the stan dard curve and curve generated using equine seminal plasma sample. Parallelism of the curves demonstrates validity of the competitive ELISA and shows low variability of the assay. 0 10 20 30 40 50 60 70 80 90 100 P e r c e n t B i n d i n g CRISP 3 concentration (ng/ml)Parallelism of standard and unknown curve std curve 1 rep 1

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42 CHAPTER 4 MEASUREMENT OF CRISP 3 Introduction CRISP -3 is a major c omponent of equine seminal plasma and is thought to be associated with fertility in horses (Schambony et al., 1998). It was recently demonstrated that equine CRISP 3 exhibits a protective effect toward spermatozoa in the equine uterus by preventing PMN bin ding and phagocytosis (Andria Doty, PhD Thesis). In mares, breeding-induced endometritis is a natural process in which the uterus is able to clear itself of excess spermatozoa, seminal plasma and bacterial contaminants (Troedsson, 1999). Studies have demonstrated that the uterine inflammatory response is triggered by spermatozoa (Troedsson et al., 1995b). The protective ability of CRISP 3 toward spermatozoa may be important when mares are bred in the presence of a uterine inflammatory environment. It may al so protect spermatozoa from being phagocytosed and eliminated from the uterus during the first four hours after breeding. While PMNs enter the uterus within 0.5 hours after breeding, sperm transport to the oviduct is not completed until four hours after br eeding. Normal concentrations for CRISP -3 in equine seminal plasma have not yet been established. The objectives of this chapter are to analyze CRISP 3 concentration in the seminal plasma of a population of stallions and to establish a normal value of CRISP -3 concentration in equine seminal plasma. Analysis of a possible correlation between CRISP -3 con centration and stallion fertility was also performed.

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43 Materials and Methods Samples A total of 67 seminal plasma samples from various stallions were provided by Dr. Margo MacPherson, Associate Professor of Theriogenology in the department of Large Animal Clinical Sciences of the College of Veterinary Medicine at the University of Florida. The samples were collected during normal semen collection in 2000. The seminal plasma samples were aliquoted into cryogenic polypropylene tubes and stored at 80 C until analysis. All samples were analyzed a minimum of three times during consecutive assays. Stock solutions of the seminal plasma samples were made at a dilution of 1:200 in 1% BSA. Animals Seminal plasma from 67 stallions was used. Fertility data was available for 40 animals, as seen in Table 4-3. For use in this assay, fertility was defined as the percentage of first cycle pregnancy. The use of these animals was in agreement with the University of Floridas regulations on animal use. Assay An indirect ELIS A was used with protocol as described in the previous chapter to measure the concentration of CRISP -3 in equine seminal plasma samples. In brief, each plate was coated with 1ug/ml CRISP -3 diluted in PBS. Coating incubation was overnight at 4 C. After compl etion of coating, plates were washed three times using WellWash and were blocked with a volume of 200ul 1%BSA. After incubation at room temperature, shaking for one hour the plate was washed three times with WellWash. Every other plate included a standard curve ranging from 10ug/ml to 25ng/ml purified CRISP -3, a total binding measurement as well as a non-specific binding measurement.

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44 Seminal plasma samples were further diluted for analysis, from 1:2400 to 1:307200 in a 1 by 2 dilution curve in 8 points. Eac h dilution was duplicated within the plate and at least 3 repetitions were completed for each sample. Each dilution from the seminal plasma samples was added to the plate at a 50ul volume in the appropriate wells. HL2175 was diluted from glycerol stock sol ution to a dilution of 1:80,000 and 50ul of antibody was added to appropriate wells of the plate. The plate was incubated for one hour, shaking at room temperature. The plate was washed three times using WellWash and 50ul secondary antibody was added at a 1:1000 dilution to each well. The secondary antibody incubated for one hour, shaking at room temperature. The plate was washed three times using WellWash and 100ul P -NPP substrate was added to each well. After incubating for one hour, shaking at room temperature the plate was read by VMax set at 405nm wavelength. Optical density measurements for each dilution duplicate were averaged using Microsoft Excel (Microsoft Corporation, Redmond, WA). Each average was converted into percent binding using total bind ing and non-specific binding measurements. The percent binding of unknown samples as compared to percent binding of the standard curve was used to calculate the concentration of CRISP -3 in each unknown sample. SigmaPlot 11 (Sigmaplot Software Inc., San Jo se, CA) was used to perform these calculations and for statistical analysis. Results The average amount of CRISP 3 found in all seminal plasma samples (n=67) was 7.44mg/ml 5.98. The standard error around the mean was 0.73. The confidence intervals arou nd the mean were 5.98 for the lower 95% and 8.90 for the upper 95%. The minimum amount of measured CRISP 3was 0.49mg/ml and the maximum amount of

PAGE 45

45 measured CRISP 3 was 25.63. The median amount of CRISP 3 in the seminal plasma samples was 5.78mg/ml. By use of the ShapiroWilk normality test, it was found that the data were not normally distributed. Due to the abnormal distribution of data, the Spearman-Rank correlation was used to determine the correlation between the concentration of CRISP 3 and fertility. The r value was equal to 0.1433, indicating a poor correlation between CRISP -3 values and fertility. Discussion The objectives in this section of our study were to evaluate CRISP -3 seminal plasma concentration and to attempt to determine the possible existence of a correlation between CRISP 3 seminal plasma concentration and fertility. Using the previously developed CRISP 3 ELISA we were able to confirm a high concentration of CRISP 3 in equine seminal plasma. An average of approximately 7.5mg CRISP -3 per mL of seminal plasma was detected. Even the lowest amount of detected CRISP 3 can be considered high, as the lowest concentration was 0.49mg/ml, or 490ng/ml. The fact that each sample was measured a minimum of three times and that for the majority o f samples the standard deviation of the mean was relatively low confirms the validity of the assay. With the exception of a few samples, the coefficient of variance was always under 20%, which is generally accepted for most immunoassays. The fact that CR ISP 3 is present in such h igh concentration is indicative of a significant biological activity. However, analysis of the data disputes the hypothesis that the concentration of CRISP -3 in seminal plasma would be correlated with stallion fertility. We have n ot been able to establish any significant correlation between the

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46 seminal plasma concentration of CRISP 3 and the fertilty data provided for 40 o f the samples, as seen in Figure 44 The basis for assessing stallion fertility was subjective. Fertility data provided for 40 of the tested stallions was limited to the number of mares bred, pregnancy rate percentage per season, percent of mares pregnant at first cycle and the amount of cycles needed to become pregnant. This data was on ly recorded for one breeding season. There was great variation between stallions in the number of mares bred, with a maximum of 109 mares bred and a minimum of 2 mares bred. The variation in this number has a great effect on pregnancy rate and must be cons idered when defining fertility in this population of stallions. No specific fertility data was provided for the mares bred by these stallions, with the exception of pregnancy rates and cycles needed to become pregnant. It would be useful to know if any o f the mares bred were susceptible to breedinginduced endometritis or if any mares that were bred in consecutive cycles were suffering from persistent endometritis. As CRISP -3 is able to protect spermatozoa from being bound and phagocytosed by PMNs, its e ffectiveness is most likely highest in mares suffering from prolonged uterine inflammation due to breeding -induced endometritis. While CRISP -3 was not found to be correlated with stallion fertility in this study, it remains an important component of equi ne seminal plasma. Further investigations are necessary to determine the extent of CRISP 3s effect on the fertility of both the mare and stallion.

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47 Figure 41. Standard curve for the ELISA measuring CRISP 3 concentration in seminal plasma. The Y axis represents percent binding and the X axis is a logarithmic representation of the unknown sample dilution curve. Standard CurveX Data 1e+0 1e+1 1e+2 1e+3 1e+4 1e+5 Y Data -20 0 20 40 60 80 100

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48 Figure 42. Bar graph of CRISP 3 concentrations for stallions with known fertility. 0 5 10 15 20 25 30 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 CRISP 3 concentration mg/ml StallionCRISP 3 concentrations of stallions with known fertilty Stallion

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49 Figure 43. Bar graph of CRISP 3 concentrati ons of stallions with unknown fertility. 0 5 10 15 20 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 CRISP 3 concentration mg/ml StallionCRISP 3 concentrations of stallions with unknown fertility

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50 Figure 44. Scatter plot and regression lines for fertility data in relation to CRISP -3 concentration. FCPR stands for first cycle pregnancy rate and TPR stands for total pregnancy rate. The highest regression line is for TPR v. CRISP -3 and the lower regression line corresponds to FCPR v. CRISP 3. The regression lines are nearly straight, indicating poor correlation between both first cycle pregnancy rate and total pregnancy rate with CRISP -3 concentration in eq uine seminal plasma.

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51 Table 4 1. CRISP 3 concentration in stallions with known fertility. Horse CRISP 3 mg/ml All Gone 11.53 Amazin Appraisin 0.49 Bandit 4.26 Big Boy 2.69 Byars 1.52 Carnewstie 6.06 Cliff 25.09 Colonel T 9.83 Doc's Lonesome Lena 5.1 Doctor Dynamite 8.38 Dundee Colonel 3.41 Ed's Cody 5.78 Eskimo 2.91 Festive 8.66 Gold Alert 8.49 Hancock 5.84 Heaven's Wish 3.35 Herat 4.960 Jolie's Appeal 10.88 Kissin Kris 4.45 Lost Soldier 13.04 Lucky 15.68 Lucky North 15.79 Magic 5.7 Max 3.4 Monty 13.97 Mr. Diamante 0.894 Native Regent 23.27 Nu Bar 12.84 Nu Bar Rey 3.99 PJ 2.42 Precocity 1.76 Premiership 25.63 Private Talk 3.269 Skip A Stake 3.41 Starman 3.2 Tizon 3.12 Too Light To Quit 2.49 VIP 3.680 Wharf 12.18

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52 Table 4 2. CRISP 3 concentrations in seminal plasma of stallions with unknown fertility. Horse CRISP 3 mg/ml Cosmo 3.21 Robinhood 4.09 Sport 6.26 Straight 12.38 Tank 8.36 Trebb WW 3.7 Tribb 9.68 Water Magic 6.83 Jamestown 15.54 Maria 2.34 Palcluey 8.63 Commando 14.75 Darkside 6.88 Incantarie Z 6.41 Jairus 23.5 Draft Maria 3.87 WhataMagicC 6.23 Three Wishes 9.74 Answer Lively 4.23 Little Bit Lively 3.25 Paint 15.18 Precicionist 0.57 Presialist 2.33 Sonny 7.08 Super May 6.75 Baraco 2.09 Chaseman 1.26

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53 Table 4 3. Fertility data provided for 40 stallions. Horse # of mares Preg./Season 1st Cycle Preg. Cycles/Preg. Doc's Lonesome Lena 10 90 73 Colonel T 13 91 Paint Pal Cluey Doctor Dynamite 40 90 70 Carnewstie 40 92 72 Bandit 25 96 71 1.4 Big Boy 12 83 70 1.6 Nu Bar 18 94 82 1.2 Monty 12 92 82 1.5 Lucky 24 92 96 1.2 PJ 11 82 89 1.6 Hancock 19 89 94 1.2 Max 18 94 77 1.4 Nu Bar Rey 3 100 67 1.3 Starman 4 100 100 1 Dundee Colonel 29 100 83 1.2 Ed's Cody 6 100 83 1.2 Skip A Stake 5 80 75 1.5 All Gone 60 85 53 2.1 Byars 22 95 80 1.5 Eskimo 21 65 31 3.8 Festive 5 100 40 1.6 Gold Alert 19 89 41 2.3 Heaven's Wish 8 100 88 1.1 Kissin Kris 109 84 66 1.8 Lost Soldier 82 85 47 2.1 Lucky North 36 67 75 2.2 Native Regent 34 91 68 1.7 Precocity 41 95 56 1.8 Premiership 53 75 63 2.3 Too Light To Quit 29 97 79 1.3 Amazin Appraisin 8 75 83 2.2 Magic 2/1 17 82 57 2 Mr. Diamante 3 100 100 1 Private Talk 16 94 80 1.3 Jolie's Appeal 3 100 100 1 Sonny Wharf 2 0 0 0 Cliff 10 70 Royal Appearance 21 52 43 3.6 Contucci 78 81 68 1.8 Parabol 28 79 63 2.1 Cor Noir 24 88 80 1.6 Riverman 94 71 86 1.9 Tizon 2 0 0 0 Hawthorne 3 67 0 3.5

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54 Table 4 3. Continued VIP 9 56 33 3.4 Herat 3 0 0 0

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55 CHAPTER 5 CONCLUSIONS The overall objectives of this study were to develop and validate an ELISA to measure CRISP -3 concentration in stallion seminal plasma and to determine if CRISP 3 concentration is correlated with stallion fertility. The general hypothesis was that stallion fertility would be correlated to CRISP -3 concentration. This hypothesis was based on the fact that CRISP 3 comprises a huge amount of the equine ejaculate, suggesting a major biological function. Furthermore, we have recently identified an important role of CRISP -3 in breeding -induced endometritis. An indirect ELISA was successfully developed and validated. Three monoclonal antibodies were produced by the Hybridoma Core Lab at the Interdisciplinary Center for Biotechnology Research at the University of Fl orida. The antibodies were tested for affinity and binding to CRISP 3 purified from equine seminal plasma. One of these antibodies, HL2175, was found to have the highest affinity for purified CRISP 3 as well as exhibiting a high level of binding to the pro tein. This antibody was diluted in several titration assays to determine the optimum concentration for binding to CRISP 3. The chosen concentration was 1:80,000. This dilution provided the highest sensitivity and standard curves using this dilution were reliable and repeatable. Initial testing of two readily available seminal plasma samples indicated the need for a high dilution in order to obtain reliable CRISP -3 measurements. T his was due to the high levels of CRISP 3 expressed in equine seminal plasma. I nter and intra assay coefficients of variation were determined using the seminal plasma samples. The coefficients of variation were in the acceptable range for an assay, the inter assay coefficient of variance was 3% and the intraassay coefficient of var iance was 10%.

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56 A total of 67 equine seminal plasma samples were assayed to determine the concentration of CRISP 3. Fertility data was available for forty of the stallions. Fertility data included pregnancy rate, number of mares bred, number of cycles need ed to achieve pregnancy and percent of mares pregnant at first cycle. The average amount of CRISP -3 found in all seminal plasma samples (n=67) was 7.44mg/ml 5.98. The minimum amount of measured CRISP 3was 0.49mg/ml and the maximum amount of measured CRIS P3 was 25.63. The median amount of CRISP 3 in the seminal plasma samples was 5.78mg/ml. By use of the Shapiro Wilk normality test, it was found that the data were not normally distributed. Due to the abnormal distribution of data, the Spearman-Rank correl ation was used to determine the correlation between the concentration of CRISP 3 and fertility. The r value was equal to 0.1433, indicating a poor correlation between CRISP 3 values and fertility. The poor correlation between CRISP -3 concentration and s tallion fertility was in direct dispute of our hypothesis that these two factors would be correlated. It is evident that CRISP -3 has a major role in equine breedinginduced endometritis, however it is unclear how this role may affect male fertility. We mus t also take into account the subjective nature of assigning fertility. In this study, fertility data was provided but it was incredibly broad. Many factors affect pregnancy rate and cycles needed to achieve pregnancy. One such factor is the number of mares each stallion bred. Also, no data was provided concerning the fertility of the mares bred by these stallions. Although we did not observe a correlation between CRISP -3 concentration and stalli on fertility our success in developing an ELISA to measure C RISP-3 concentration must not be overlooked. This is the first assay developed to quantify the amount of

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57 CRISP -3 in equine seminal plasma. Through further study of the biological roles and processes involving CRISP 3 we may be able to determine if CRISP 3 has an effect on stallion fertility.

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58 LIST OF REFERENCES Alghamdi, A.S., Foster, D.N., Troedsson, M.H.T., 2004. Equine seminal plasma reduces sperm binding to polymorphonuclear neutrophils (PMNs) and improves the fertility of fresh semen inseminated into inflamed uteri. Reproduction 127, 593-600. Alghamdi, A.S. Foster, D.N., 2005 a Seminal Dnase frees spermatozoa entangled in neutrophil extracellular traps. Biol. Reprod. 73, 1174-1181. Alghamdi, A.S., Foster, D.N., Carlson, C.S., Troedsson, M.H., 2005b. Nitric oxide levels and nitric oxide synthase expression in uterine samples from mares susceptible and resistant to breeding-induced endometritis. Am. J. Reprod. Immunol. 53(5), 230-237. Alghamdi A.S., Lovaas B.J., Bird S.L., Lamb G.C., Rendhal A.K ., Taube P.C., Foster D.N., 2008 Species -specific interaction of seminal plasma on sperm neutrophil binding. Anim. Repro. Sci. 114(4), 331344. Bader, H., 1982. An investigation of sperm migration into the oviducts of the mare. J Reprod Fertil (Suppl). 32, 59-64. Bader, H., Krause, A., 1980. Investigations about the tr ansport, distribution and the fate of spermatozoa in the genital tract of the mare. Proc 9th Int Congr Anim Reprod AI. 5, 197-205. Brandon, C.L., Heussner, G.L., Caudle, A.B., Fayrer -Hosken, R.A., 1999. Two dimensional polyacrylamide electrophoresis of equine seminal plasma proteins and their correlation with fertility. Theriogenology 52, 863 873. Cohen, D.J., Ellerman, D.A., Cuasnicu, P.S., 2000. Mammalian sperm egg fusion: evidence that epididymal protein DE plays a role in mouse gamete fusion. Biol. Repr od. 63, 462468. Cohen, D.J., Busso, D., Da Ros, V., Ellerman, D.A., Maldera, J.A., Goldweic, N., Cuasnicu, P.S., 2008. Participation of cysteine -rich secretory proteins (CRISP) in mammalian sperm egg interaction. Int. J. Dev. Biol. 52, 737742. Crowther, J.R., 2001. The ELISA Guidebook. Humana Press, New Jersey. Dahms, B.J., Troedsson, M.H.T., 2002. The effect of seminal plasma components on opsonization and PMN phagocytosis of equine spermatozoa. Theriogenology. 58, 457-460. Drobnis, E.Z., Overstreet, J .W., 1992. Natural history of mammalian spermatozoa in the female reproductive tract. Oxford Rev Reprod Biology 1 45.

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59 Ekhlasi-Hundrieser, M., Schafer, B., Kirchhoff, C., Hess, O., Bellair, S., Muller, P., Topfer Peterson, E., 2005. Structural and molecula r characterization of equine sperm -binding fibronectinII module proteins. Mol. Reprod. Dev. 70, 45-57. Hamann, H., Jude, R., Sieme, H., Mertens, U., Topher -Petersen, E., Distl, O., Leeb, T., 2007. A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses. Anim. Genet. 38, 259264. Hoshiba, H., Sinowatz, F., 1998. Immunohistochemical localization of the sperm adhesin AWN -1 in the equine male genital tract. Anat. Histol. Embryol. 27, 351353. Jalkanen, J., Huhtaniemi, I., Poutanen, M., 2005. Mouse cysteine-rich secretory protein 4 (CRISP-4): a member of the Crisp family exclusively expressed in the epipidymis in an androgen -dependent manner. Biol. Reprod. 72, 1268-1274. Kareskoski, M., Katila, T., 2008 Components of stallion seminal plasma and the effects of seminal plasma on sperm longevity. Anim. Repro. Sci. 107, 249256. Katila, T., 1995 Uterine defence mechanisms in the mare. Anim. Repro. Sci. 42, 197204. Katila, T., 2001. Sperm -uterine interact ions: a review. Anim. Repro. Sci. 68, 267-272. Katila, T., 2005. Effect of the inseminate and the site of insemination on the uterus and pregnancy rates of mares. Anim. Repro. Sci. 89, 31-38. Kotilainen, T. Huhtinen, M., Katila, T., 1993. Sperm induced leukocytosis in the equine uterus. Theriogenology 41, 629636. LeBlanc, M.M., 1994. Oxytocin-the new wonder drug for treatment of endometritis? Equine Vet. Educ. 6, 3943. LeBlanc, M.M., Neuwirth, L., Asbury, A.C., 1994. Scintigraphic measurement of uteri ne clearance in normal mares and mares with recurrent endometritis. Equine Vet. J. 26, 109113. Madea, T., Nishida, J., Nakanishi, Y., 1999. Expression pattern, subcellular localization and structure-function relationship of rat Tpx 1, a spermatogenic cell adhesion molecule responsible for association with Sertoli cells. Dev. Growth Differ. 41, 715722. Madill, S., Troedsson, M.H.T, Alexander, S.L., Shand, N., Santschi, E.M., Irvine, C.H.G., 2000. Simultaneous recording of pituitary oxytocin secretion and m yometrial activity in oestrous mares exposed to various breeding stimuli. J. Reprod. Fertil. (Suppl). 56, 351361.

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60 Mochca Morales, J., Martin, B.M., Possani, L.D., 1990. Isolation and characterization of helothermine, a novel toxin from Heloderma horridum horridum. Toxicon 28, 299309. Morrissette, J., Kratzschmar, J., Haendler, B., El -Hayek, R., Mochca Morales, J., Martin, B.M., Patel, J.R., Moss, R.L., Schleuning, W.D., Coronado, R., Possani, L.D., 1995. Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors. Biophys. J. 68, 22802288. Nolan, M.A., Wu, L., Bang, H.J., Jelinsky, S.A., Roberts, K.P., Turner, T.T., Kopf, G.S., Johnston, D.S., 2006. Identification of rat cysteine-rich secretory protein 4 (Crisp 4) as the otholog to human CRISP1 and mouse Crisp4. Biol. Reprod. 74, 984 -991. Rozeboom, K.J., Troedsson, M.H., Rocha, G.R., Crabo, B.G., 2001. The chemotactic properties of porcine seminal components toward neutrophils in vitro. J. Anim. Sci. 79, 9961002. Schambony, A., Gentzel, M., Wolfes, H., Raida, M., Neumann, U., Topher -Petersen, E., 1998. Equine CRISP -3: primary structure and expression in the male genital tract. Biochim. Biophys. Acta 1387, 206216. Scott, M.A., Lie, I.K.M., Overstreet, J.W., 1995. Sperm transport to the oviducts: Abnormalities and their clinical implications. Proc Am Assoc Eq Pract. 41, 1-2. Topher -Petersen, E., Ekhlasi -Hundrieser, M., Kirchhoff, C., Leeb, T., Sieme, H., 2005. The role of stallion seminal proteins in fertilization. Anim. Repro. Sci. 89, 159170. Troedsson, M.H., Liu, I.K., 1991. Uterine clearance of nonantigenic markers (51Cr) in response to bacterial challenge in mares potentially susceptible and resistant to chronic uterine infections. J. Reprod. Fertil. (Suppl). 44, 283288. Troedsson, M.H.T., Liu, I.K.M., Thurmond, M., 1993. Function of uterine and blood derived polymorphonuclear neutrophils in mares susceptible and resistant to chronic uterine infection: phagocytosis and chemotaxis. Biol. Reprod. 49, 507 -514. Troedsson, M.H.T., Scott, M., Liu, I.K.M., 1995a. Comparative treatments of mares susceptible to chronic uterine infection. Am. J. Vet. Res. 56(4), 468-472. Troedsson, M.H.T., Steiger, B.N., Ibrahim, N.M., King, V.L., Foster, D.N., Crabo, B.G., 1995b. Mech anism of sperm -induced endometritis in the mare. Biol. Reprod. 52(Suppl. 1), 133. Troedsson, M.H.T., Liu, I.K.M., Crabo, B.G., 1998. Sperm transport and survival in the mare: a review. Theriogenology 50, 807818. Troedsson, M.H.T., 1999. Uterine clearanc e and resistance to persistent endometritis in the mare. Theriogenology 52,461471.

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61 Troedsson, M.H.T., Lee, C.S., Franklin, R., 2000. Postbreeding uterine inflammation: the role of seminal plasma. J Reprod. Fertil. (Suppl). 56, 341349. Troedsson, M.H. T., Alghamdi, A.S., Mattisen, J., 2002. Equine seminal plasma protects fertility of spermatozoa in an inflamed uterine environment. Theriogenology. 58, 453-456. Troedsson, M.H.T., Loset, K., Alghamdi, A.M., Dahms, B., Crabo, B.G., 2001. Interaction between equine semen and the endometrium: the inflammatory response to semen. Anim. Repro. Sci. 68 ,273278. Troedsson, M.H.T., Desvousges, A., Alghamdi, A.S., Dahms, B., Dow, C.A., Hayna, J., Valesco, R., Collahan, P.T., Macpherson, M.L., Pozor, M., Buhi, W.C. 2005. Components in seminal plasma regulating sperm transport and elimination. Anim. Repro. Sci. 89, 171186. Troedsson, M.H.T., 2006. Breeding-induced endometritis in mares. Vet. Clin. North Am. Equine Pract. 22, 705 -712. Troedsson, M.H.T., 2008. Probl ems after breeding. J. Equine Vet. Sci. 28, 635639. Udby, l., Cowland, J.B., Johnsen, A.H., Sorensen, O.E., Borregaard, N., Kjeldsen, L., 2002. An ELISA for SGP28/CRISP3, a cysteine rich secretory protein in human neutrophils, plasma, and exocrine secre tions. J. Immunol. Methods 263, 43-55. Zent, W.W., Troedsson, M.H.T., 1998. Post breeding uterine fluid accumulation in a normal population of Thoroughbred mares: a field study. Proc Am Assoc Eq Pract. 44, 64-65. ELISA. Decker, J., 1 Feb. 2006. Web. 20 Oc t. 2009. < http://www.microvet.arizona.edu/courses/mic419/ToolBox/elisa3.jp g > New England Biolabs PathScan ELISA Kits. New England Biolabs, 21 Nov. 2008. Web. 20 Oct. 2009. < http://www.newenglandbiolabs.de/en/index.php?option=com_content&task=view &id=14&Itemid=20 >

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62 BIOGRAPHICAL SKETCH Meghan Carey Connor was born in West Palm Beach, Florida. She received an International Baccalaureate diploma from Suncoast Community High School in 2002. In June 2002, she began undergraduate coursework at the University of Florida and graduated with a Bachelor of Science in animal s cie nces and a Bachelor of Arts in s panish in 2007. In 2007, she was given the opportunity to pursue a Master of Science with Dr. Mats Troedsson at the University of Floridas College of Veterinary Medicine. Upon completion of her degree, Meghan hopes to atten d veterinary school and specialize in equine theriogenology.