Surface Architectural Anatomy of The Penile and Preputial Epithelium of Bulls by Lew G. Strickland A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama August 9, 2010 Keywords: Trichomoniasis, Penile epithelium, Preputial epithelium Copyright 2010 by Lew G. Strickland Approved by Dwight F. Wolfe, Chair, Professor of Clinical Sciences Robert L. Carson, Professor of Clinical Sciences Misty Edmondson, Assistant Professor of Clinical Sciences Herris Maxwell, Associate Professor of Clinical Sciences Soren P. Rodning, Assistant Professor, Extension Veterinarian ii Abstract It has been a long held assumption that older bulls develop deeper folds or crypts in the epithelium covering the penis and prepuce than are found in their younger counterparts. These deeper crypts reportedly facilitate the carrier state of bovine venereal disease by providing a protected environment suitable for long term maintenance of infection. No published reports have been found to support this assumption. This study was conducted to characterize the surface architectural anatomy of the epithelium and epithelial crypts in younger and older bulls. This survey included Angus bulls sampled between December 2008 and March 2009. Bulls were placed into two groups of six according to age. Group 1 consisted of bulls 2 years of age, and group 2 consisted of bulls ? 5 years of age. Penile epithelium was collected from three anatomical locations (Proximal, Middle, and Distal) and tissue samples were prepared for examination by light microscopy and scanning electron microscopy. Three parameters were examined; 1) area of the epithelium per unit of linear measurement, 2) area encompassed by the folds, and 3) total number of folds per unit of linear measurement. Findings were then compared within and between the age groups. Results indicated that there are no significant differences in the area of the epithelium, area encompassed by the epithelial folds or total number of epithelial folds per unit of linear measurement between the two age groups. iii Acknowledgments I would like to thank God Almighty my creator and sustainer for all that He has done for me. Without Him nothing would be possible. To His Son Jesus Christ my Savoir, thank you for providing a way to spend eternity with Him. To my beautiful wife Ellen, who has endured and supported me through my entire veterinary education and all endeavors that I have pursued. To my children, Jesse, Kayla, Andrew, and Anna for all of the all pleasure that they bring me. I pray that one day they can have families that are as much of a blessing to them as they have been to me. I also want to thank my daddy, J.G. Strickland (1924-2008), for all of the life?s lessons that he taught me. I hope that I can be the example in my daily life that he was in his. I thank both Dr. Dwight Wolfe and Dr. Robert Carson, my mentors and friends, for the tremendous influence they have had on my entire veterinary career, as well as their support for this research project. Also, I thank Dr. Herris Maxwell for his guidance on this project and for his assistance in my pursuit of board certification with The American College of Theriogenologists. To Dr. Soren Rodning and Dr. Misty Edmondson for their commitment to the writing of this project, I give thanks. I offer my additional thanks to Dr. Joe Newton for his help with light microscopy, Dr. Jim Wright for his help with statistics, and Ms. Karen Wolfe for her help with electron microscopy. Finally, I thank the food animal theriogenology students for their willingness to collect and process samples. iv Table of Contents Abstract........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iii List of Tables ................................................................................................................................. vi List of Illustrations........................................................................................................................ vii Chapter 1. Introduction ...................................................................................................................1 Chapter 2. Literature Review ........................................................................................................3 Penile Anatomy of the Bull ................................................................................................3 Venereal Disease ................................................................................................................6 Therapy ...............................................................................................................................8 Prevalence ...........................................................................................................................9 Economic Impact .............................................................................................................10 Chapter 3. Material & Methods ..................................................................................................11 Study of Penile and Preputial Epithelium in Beef Bulls ..................................................11 Animals ............................................................................................................................12 Sampling for Tritrichomonas foetus ..................................................................................12 Polymerase Chain Reaction (PCR) Verification................................................................13 Sampling for Campylobacter fetus subsp. venerealis........................................................13 Biopsy Technique ..............................................................................................................14 Light Microscopy...............................................................................................................14 vi Scanning Electron Microscopy..........................................................................................15 Areas Measured .................................................................................................................16 Area of Epithelium.............................................................................................................16 Area of Infoldings..............................................................................................................21 Total Number of Infoldings ...............................................................................................24 Statistical Analysis.............................................................................................................24 Examination for Crypts......................................................................................................25 Chapter 4. Results .........................................................................................................................29 Electronic Microscopy Evaluation.....................................................................................34 Chapter 5. Discussion ....................................................................................................................35 References ....................................................................................................................................38 Appendix A. Polymerase Chain Reaction Protocol......................................................................43 Appendix B. Individual Bull Epithelial Area and Mean...............................................................52 Appendix C. Individual Bull Infolding Area and Mean ...............................................................53 Appendix D. End Notes ................................................................................................................54 vi List of Tables Table 1. Age and Breed of Bulls....................................................................................................29 Table 2. Range of Epithelial Area..................................................................................................30 Table 3. Range of Epithelial Fold Area ........................................................................................32 vii List of Illustrations Illustration 1. Research Design Schematic ....................................................................................11 Illustration 2. Set Scale to Known Distance ..................................................................................17 Illustration 3. Convert to Grayscale...............................................................................................18 Illustration 4. Convert to Binary Image ........................................................................................18 Illustration 5. Using Tracing Tool .................................................................................................19 Illustration 6. Analyze Particles.....................................................................................................19 Illustration 7. Set Particle Size.......................................................................................................20 Illustration 8. Outline of Area.......................................................................................................20 Illustration 9. Set Scale to Known Distance .................................................................................21 Illustration 10. Using Arrow Tool ................................................................................................22 Illustration 11. Tracing the Outline of the Fold ............................................................................22 Illustration 12. Adjust Image to Threshold ...................................................................................23 Illustration 13. Convert to Binary Image ......................................................................................23 Illustration 14. Analyze Particles ..................................................................................................24 Illustration 15. Scanning Electron Image Proximal Prepuce 2 yr old ...........................................25 Illustration 16. Scanning Electron Image Middle Penis 6 yr old..................................................26 Illustration 17. Scanning Electron Image Distal Penis 5 yr old....................................................27 Illustration 18. Scanning Electron Image Intestinal Crypts..........................................................28 viii Illustration 19. Mean Epithelial Area and Standard Deviation.....................................................31 Illustration 20. Mean Area of Folds and Standard Deviation ......................................................32 Illustration 21. Total Number of Infoldings and Standard Deviation...........................................33 1 Chapter 1 Introduction The gross anatomy of the bovine penis and prepuce has been described in detail, and the intricate mechanisms leading to erection and ejaculation defined. (1) Compounded wrinkles and folds of the prepucial epithelium and creases on the surface of the glans penis are present and thought to allow for the mechanics of extension during erection (Personal communication Dr. David Bartlett). The surface architecture of the genital epithelium has been speculatively discussed for decades but qualititative study of the microscopic anatomy and the effects of ageing are lacking. Pervasive opinion suggests that that the epithelial microstructure of the penis and prepuce undergoes significant age-associated changes and that these changes are important in establishment of a state of persistent infection with Tritrichomonas foetus (T. foetus) or Campylobacter fetus subsp. venerealis (C. fetus). (2) Trichomoniasis and campylobacteriosis, the two most commonly recognized bovine venereal diseases, results in substantial economic loss in cow herds. Bulls may be asymptomatic carriers for either the protozoan Tritrichomonas foetus (T. foetus) or the bacterium Campylobacter fetus subsp. venerealis (C. fetus). Infection in females may result in early embryonic death, abortion, pyometra, fetal maceration, or infertility, all of which negatively influence the profitability of a cattle operation. Either venereal disease may persist without detection in endemic herds. (3-6) Herd infection with either organism significantly reduces a cattle producer?s profits. 2 Persistence of infection with these organisms is thought to be related to deeper epithelial folds in the preputial epithelium of mature bulls, and these folds are popularly designated as crypts. Little information has been published on the micro-architecture of the penile and preputial epithelium. Epithelial crypts are poorly defined on the penis and prepuce of the bull. In contrast, Bailli?re?s Comprehensive Veterinary Dictionary defines a crypt as a blind pit or tube-like structure on a free surface. (7) The Crypts of Liberk?hn are defined as a lumen of intestinal glands on the surface of the intestinal mucus membrane that serve to secrete or are absorptive in nature. (8) The hypotheses of this study are: 1) important differences exist between the surface architectural anatomy of the penile and preputial epithelium of bulls of different ages, 2) epithelial coverings of the penis and prepuce become thicker as bulls mature, 3) the epithelium develops an increased number of infoldings with age, and 4) the area contained within these infoldings increases with age. This study will compare the above parameters in bulls 2 years of age and bulls ? 5 years of age. 3 Chapter 2 Literature Review Penile Anatomy of the Bull The skin covers the body and protects it against injury while providing temperature regulation through heat dissipation or by surface evaporation. The skin consists of two layers, the outer epidermis and the inner dermis. The epidermis is composed of stratified squamous epithelium and the dermis is mostly connective tissue fibers. The surface of the penis and prepuce within the preputial cavity are among the very few non-haired areas of skin present on the bovine. The penis of the bull is classified as fibro-elastic and it functions as the shared outlet for the excretion of urine and the deposition of semen into the female reproductive tract during copulation. (8) The penis is cylindrical and extends forward from the ischiatic arch to the umbilical region of the abdominal wall. It is supported by the penile fascia and skin and is situated in the sheath. (9) The penis of the adult bull is almost 1 meter long with approximately a quarter of its length incorporated in a sigmoid flexure. Paired retractor penis muscles insert on the ventrum of the distal bend of the sigmoid flexure and serve to hold the penis within the prepuce during the resting state. These muscles also serve to retract the penis and prepuce into the sheath following detumescence. (10) The free portion of the non-erect penis is approximately 12cm long and lies within the caudal portion of the preputial cavity when the 4 penis is non erect. (11) Because the penis serves as the organ of copulation and ejaculation of spermatozoa, much of the penis consists of erectile tissue. The erectile tissues are composed of the corpus cavernosum penis, corpus spongiosum penis, and the glans penis. The corpus cavernosum of the penis (CCP) consists of a paired dorsal column of erectile tissue surrounded by dense connective tissue, the tunica albuginea. (8) The corpus cavernosum penis is spongy and divided into many spaces. Erection of the penis occurs with sexual stimulation which results in dilatation of the arteries that supply blood to the penis. Blood flow increases from the deep artery of the penis into the crus penis and eventually the CCP. As sexual stimulation is continued the ischiocavernosus muscles begin rhythmic contractions and occlude arterial and venous outflow of the penis creating a closed hydraulic system. Blood is then forced into the CCP from the crus penis and significantly increases the CCP pressure. Ventral to the corpus cavernosum, and partially surrounded by it, is the corpus spongiosum penis (CSP) which is a smaller column of erectile tissue that surrounds the urethra. The free portion of the penis is distal to the attachment of the prepuce and lies within the caudal portion of the preputial cavity when non-erect. The free portion is capped by a small cushion of asymmetrical, ventrally directed, slightly spiraled tissue which comprises the glans penis. (11) Sensation from the glans is necessary for intromission and ejaculation. (12) The prepuce of the bull is 35 to 40 cm long and approximately 4 cm in diameter with wide variations among breeds. (13) The prepuce receives its blood supply from branches of the internal iliac artery and is composed of an external, parietal, and visceral layer. The external portion, the sheath, is haired skin which is continuous with the abdominal skin. The external layer turns inward at the preputial opening to become the parietal prepuce. This, in turn, reflects at the fornix and continues onto the preputial attachment to the free portion of the penis as the 5 visceral prepuce. (14,15) The parietal and visceral layers of the prepuce are covered with stratified squamous epithelium, and nerve innervation is from branches of the ventral abdominal (T13, L1- 4) and pudendal (S2-4) nerve trunks. (11) As bulls age there is an increase in plasma cells in the preputial and penile epithelium that is considered to be a response from repeated infectious challenge. Bulls naturally infected with or vaccinated against antigens derived from Campylobacter fetus subsp. venerealis have an increase of specific immunoglobulins IgG 1 , IgG 2 , IgA, and IgM in the preputial and penile epithelium. These immunoglobulins are directed against antigens from C. fetus subsp. venerealis. (16) Natural infection with T. foetus also results in deposition of specific IgG 1 , IgG 2 , IgA and IgM antibodies in the preputial cavity. (17) Immunoglobulins IgG 1 , IgG 2 , IgE, and IgA directed against T. foetus are elevated in the preputial secretions post vaccination with T. foetus antigens. (18) The free portion of the penis is covered with stratified squamous epithelium which is very tightly adhered over the apex of the distal end. This epithelium transitions caudally to become more loosely attached at the junction of the free portion and prepuce allowing the epithelium to change its orientation when the penis is extended or withdrawn. (11) The most distal portion of the glans penis is encapsulated by the smooth cap-like galea glandis, while the remainder of the glans has a rough uneven epithelial surface. The epithelium of this area is characterized by a network of fine infoldings which vary from slight irregular depressions to relatively deep, closely-arranged crevices that produce a papillate appearance. (19) It has been alleged that these infoldings or crypts become deeper as a bull matures, and that this infolding of the epithelium provides a suitable protected environment for the development of a chronic carrier state of venereal diseases. (20) 6 Venereal Diseases Tritrichomonas foetus, an obligate protozoal parasite of the bovine reproductive tract, is a pyriform protozoan with a rounded anterior end and a pointed posterior end. It varies in size from 10 to 25 ?m in length and 5 to 10 ?m in width. (21) The preliminary identification of the organism is based on: 1) size and single nucleus, 2) presence of three anterior flagella and one posterior flagella, 3) a distinct undulating membrane along one side of the protozoa, and 4) characteristic motility. Ancillary testing by polymerase chain reaction is necessary for positive identification. The male is an asymptomatic carrier of T. foetus while the female suffers identifiable consequences of infection. T. foetus in bulls is found in the smegma (secretions) of the epithelial lining of the penis, prepuce, and distal urethra. (22) The organism does not invade the epithelium and fails to invoke a protective immune response by the bull. (23) T. foetus infection causes no penile or preputial lesions and does not affect libido. (22,24) Infected bulls have no observable changes in semen quality. (22) Conversely, In-vitro exposure of spermatozoa to T. foetus for one hour resulted in decreased spermatozoa motility, agglutination of sperm cells, and eventual phagocytosis of spermatozoa by the trichomonads. (25) Infection in young bulls (less than 3-4 years of age) is most often transient, with disease transmission only occurring if sexual contact with a noninfected female occurs within minutes to days following breeding of an infected female. Clearance of the organism by a young bull may be possible within 20 minutes following breeding an infected cow. (23,26) Bull to bull transmission has been theorized in a case study in which young virgin bulls were housed with infected bulls. The assumption is that the penis of uninfected bulls became infected during homosexual mounting of another animal recently mounted by an infected bull. (27) This is considered a rare 7 event, but possible if ?clean? and ?dirty? bulls are kept in close confinement. Trichomonads morphologically similar to T. foetus have been described in the feces of cattle. (28) Bulls housed together often mount and sodomize each other making fecal contamination of the preputial cavity with a non-T. foetus trichomonads possible. Differentiating non-pathogenic fecal trichomonads from T. foetus becomes an important diagnostic challenge. (29) Infection of the cow or heifer with T. foetus occurs during coitus with an infected bull. Following vaginal deposition, the organism transverses the cervix and colonizes the entire reproductive tract within 1-2 weeks. (20) Pregnancy may be established in the face of infection, as fertilization and early development of the embryo are not necessarily compromised. (30) The viability of the conceptus may be maintained until after maternal recognition of pregnancy, which occurs from days 14 to 18 after a fertile breeding, resulting in an affected cow with a prolonged interestrus interval. (31) The cause of pregnancy loss has is not been defined, but prevalent theories include: 1) overgrowth of the organism resulting in separation of the feto- maternal interface, 2) antigens from the parasite?s surface triggering a destructive immune response, and 3) a cytotoxic effect on fetal and maternal tissues. (20) Pyometra and abortion are sometimes the first signs of trichomoniasis noticed in herds, but these occur in less than 5 % of infected animals. (24) A small number of cows will abort in the second or even third trimester, and an even smaller number of cows (less than 1%) will maintain an infection through a normal gestation. (32) The few cows capable of maintaining a T. foetus infection throughout gestation represent a source of reinfection for the herd during the following breeding season. Bovine genital campylobacterosis is also a sexually transmitted disease caused by the gram negative microaerophilic rod Campylobacter fetus subsp. veneralis. Similar to Tritrichomonas foetus, this organism is an obligate parasite of the bovine reproductive tract with 8 bulls serving as asymptomatic carriers. Bulls younger than three years of age tend to be resistant to persistent infection and clear the organism, whereas mature bulls become chronically infected. It has been suggested that age related changes in the penile and preputial epithelium provide favorable habitat for C. fetus subsp. venerealis survival. (33) Clinical signs of infection with C. fetus subsp. venerealis in the cow are similar to those of trichomoniasis. Campylobacteriosis is associated with vaginitis, cervicitis, and endometritis. Infection leads to an early embryonic death as a result of the inflammatory response in the uterus and uterine tubes. (34) In closely monitored herds, an increased number of repeat breeders will be identified. The hallmark of infection is irregular and delayed return to estrus. Herd pregnancy rates are decreased, and a wide range of gestational ages may be found at the time of pregnancy examination. The asymptomatic chronic carrier state associated with either T. foetus or C. fetus subsp. venerealis infections of bulls older than 4 years of age rarely spontaneously resolves. (35) The carrier state of both organisms has been apocryphally related to the depth of the preputial and penile epithelial crypts for decades. (2,20,21) Reports of the location of T. foetus along the penis and within prepuce have varied. Hammond and Bartlett reported that trichomonads were most often found on the glans penis. (19) Rhyan et al reported trichomonads more commonly in the crypts along the midshaft of the penis. (17) Peter, in a review, states that most trichomonads are located in preputial crypts. (36) Therapy Because both of these diseases are venereally transmitted, artificial insemination of the entire herd maybe utilized to reduce or eliminate these venereal diseases from a producer?s 9 operation. Annual testing for disease and culling of positive animals, as well as using younger test negative bulls are additional tools that can be useful in reduction of disease. Vaccination against C. fetus subsp. venerealis is reported as protective and curative for the bull and cow. (33) Vaccination against T. foetus is reported to be effective in the cow (37) . In the bull, most reports state that vaccination is of limited to no value. (2,37,38) However, some reports indicate that vaccination against T. foetus may be protective in the bull. (39,40) Cobo et al noted that immunoglobulins IgG 1 , IgG 2 , IgA, and IgE directed against T. foetus appeared in the preputial secretions of bulls vaccinated with T. foetus antigens. Immunoglobulins IgG 1 and IgG 2 directed against T. foetus were elevated in the serum. Vaccinated bulls demonstrated resistance to T. foetus colonization. (18) One complicating factor with bovine trichomoniasis in the United States is the lack of effective treatments with U.S. Food and Drug Administration approval. (20) Historically, the most successful treatment for bulls with trichomoniasis was systemic treatment with nitromidazole derivatives. (41) Currently, the use of nitromidazole derivatives is illegal in food-producing animals in the U.S., and no effective alternative treatments are available. The lack of effective, approved therapies for bovine trichomoniasis emphasizes the need for appropriate preventive and control measures. Prevalence Several estimates are available regarding the prevalence of trichomoniasis in different regions of North America. In 1964, Johnson reported a 7.5 % prevalence in western range bulls. (42) Later studies from Florida, Oklahoma and California found prevalence rates of 7.3, 7.8 and 4.1 %, respectively. (43-45) The Florida and Oklahoma studies sampled bulls from sale barns or abattoirs, while the California study sampled bulls from randomly selected herds. Rae et al 10 reported a 6% prevalence of T. foetus in randomly selected natural service beef herds in Florida between 1997 and 1999. (46) In a 2005-2006 study conducted by Rodning et al, the prevalence of trichomoniasis in a random subset of southern Alabama beef herds was reported to be < 1%. (47) Few or no reports exists regarding the prevalence of bovine genital campylobacterosis in the United States. Swai et al reported a 5.1% prevalence in bulls in Tanzania, and Pefanis et al reported a 28.7% in The Republic of Transkei. (48,49) Economic Impact Economic losses due to venereal disease result from increased culling, increased requirement for herd replacements, lower percentage of cows calving , and lower weaning weight secondary to late calving. (3) Fitzgerald et al in 1958 estimated an annual loss of $800 per bull infected with T. foetus in large herds. (50) In 1979, Wilson et al estimated a $2.5 million annual calf loss due to T. foetus in Oklahoma replacement heifers. (44) In 1986, Fitzgerald estimated that the total economic impact of T. foetus in the USA was $65 million annually. (4) A 1991 study by Speer et al estimated that annual losses due to T. foetus could approach $650 million in the United States. (51) A report in 2000 indicated the prevalence and economic impact of T. foetus may be greater than previously reported. (52) Chapter 3 Methods and Materials Study of Penile and Preputial Epithelium in Beef Bulls The research design (Illustration 1) for the study of penile and preputial epithelium in beef bulls focuses on sampling bulls two years of age and bulls greater than or equal to five years of age. Group 1 Biopsies from 2 yr old bulls Group 2 Biopsies from ? 5 yr old bulls Prepare and evaluate tissue biopsies via microscopic evaluation Compare penile and preputial epithelium within and between Groups 1 & 2 Illustration 1. Research Design Schematic. 11 Animals Bulls were placed into two groups of six each according to age. Group 1 (N=6) consisted of bulls two years of age (? 3 mo). Group 2 (N=6) consisted of bulls five years of age and older. Ages of both groups were determined by dentition and owner records. These age groups were selected because most bulls enter the breeding herd at 2 years of age and most are removed from the herd at 5 years of age. It is speculated that age-related penile and preputial epithelial differences will be apparent by 5 years of age. All tissue samples were collected from bulls that presented to the John Thomas Vaughn Large Animal Teaching Hospital, Auburn University College of Veterinary Medicine for routine breeding soundness exams. Written consent was obtained from cattle producers prior to tissue collection. Each bull was sampled for presence of T. foetus and C. fetus subsp. venerealis and none were found to be positive. Sampling for Tritrichomonas foetus The external preputial area was cleaned with disposable paper towels without soap or disinfectants. A new pair of exam gloves was used for each bull, and a sterile, dry, plastic infusion pipette (Equine AI pipette Butler a ) with a 12 mL syringe attached to one end was placed into the preputial fornix. The pipette tip was vigorously scraped across the penile and preputial epithelium prior to the application of negative pressure with the syringe to aspirate approximately 3 mls of the preputial smegma. The negative pressure was released before removing the pipette from the sheath to minimize unnecessary aspiration of urine or other contaminants. After removal of the pipette from the sheath, the sample was immediately placed into modified Diamond?s media (Alabama State Diagnostic Lab b ) and held at ambient 12 temperature for no more than two hours prior to the transport to the laboratory. A new sterile syringe and pipette was used for each bull. The culture media was incubated for 24 hours at 37? C before examination for trichomonads. Each sample was examined once daily for 5 days by direct light microscopy at 20x and 40x for presence of the trichomonad. Any suspect cultures were submitted for polymerase chain reaction for confirmation of T. foetus. Polymerase Chain Reaction (PCR) Verification Microscopic identification of T. foetus may be complicated by the presence of other trichomonad protozoa. (53-57) Contamination of the preputial orifice or cavity with feces likely explains the presence of these opportunistic contaminants. None of the contaminating trichomonads result in reproductive pathology in cows or bulls. (58) To avoid false-positives, all suspect samples were submitted to the Alabama State Diagnostic Laboratory for PCR verification (Appendix A). Polymerase chain reaction accurately differentiates T. foetus from non-pathogenic fecal contaminants. Sampling for Campylobacter fetus subsp. venerealis Samples for C. fetus subsp. venerealis culture were collected using sterile swabs in the preputial fornix in a similar manner to the pipette method used for T.foetus sampling. Swabs were immersed in 1 ml physiologic saline. The resulting fluid was placed on Clark?s media (Alabama State Diagnostic Lab c ) and held at 37? C for transport to the laboratory. Transported samples were inoculated onto blood agar plates and incubated under microaerophilic conditions for 5-7 days at 35-37? C. 13 Biopsy technique Each bull was appropriately restrained in a livestock chute to prevent injury to the bulls and personnel. Three separate locations were chosen for biopsy specimens: 1) the distal penis 1cm proximal to the glans, 2) 1 cm distal to the attachment of the prepuce to the free portion of the penis, and 3) the proximal prepuce 6 cm distal to the preputial orifice when the penis is extended. The penis was manually extended, held by sterile surgical gauze, and cleaned with water prior to aseptic surgical preparation. The bilateral dorsal penile nerves were anesthetized with 7 to12 mls of 2% lidocaine hydrochloride. A #22 scalpel blade was used to excise an approximately 1cm diameter sample of tissue from each target area. Each excision area was closed with #1-0 chromic gut suture material in a cruciate pattern. Anesthesia and collection of all samples were conducted by the same investigator to minimize differences of collection methods. Tissue samples were pressed onto sections of tongue depressors to reduce artifact folding during fixation (Dr. Joe Newton personal communication). Each tissue sample was immersion fixed and stored in a mixture of 4% paraformalin, 1% glutaraldehyde (Electron Microscopy Services, Hatfield, PA d ), and 150 mM phosphate buffered saline (Sigma Chemical, St Louis, MO e ) prior to processing. Light Microscopy All steps of the tissue preparation were performed under a ventilated hood in compliance with OSHA guidelines for handling potentially toxic chemicals. Tissues for light microscopy were prepared by rinsing in phosphate buffered saline (PBS) in 3 separate rinses of 20 min each to remove fixative agents. Fixative-free tissues were dehydrated with ethyl alcohol (ETOH) in graded strengths to displace water from the tissues. The following steps were performed: 1) each sample was placed in 30% ETOH and agitated at room temperature for 30 min, 2) the samples 14 were placed in a series of ascending concentrations of ETOH (50%, 70%, 85%, 100%) for 30 min each, 3) samples were washed two times in hexamethyldisilazane (HMDS, Electron Microscopy Services Hatfield, PA d ) for 30 min each to clear residual ETOH, and 4) tissue samples were placed under a fume hood overnight to allow remaining chemicals to be removed. Next, tissue samples were placed in molds for embedding in paraffin. Samples were processed in a Tissue-Tek VIP E300? (Ames Co., Inc., Elkart, IN f ) for 2-3 hrs. Each sample was cut into 7 micrometer sections using a Reichert-Jung 2040 Autocut Microtome TM, g , mounted on a glass microscope slide with resin, and allowed to dry for 20 minutes. Each section was stained with hematoxylin and eosin stain (Sigma-Chemical, St Louis, MO e ). Each biopsy sample was evaluated at 40 X magnification to describe and evaluate the morphology of the penile and preputial surface epithelium. The initial evaluation was performed at this magnification to ensure that all morphological structures could be identified. Evaluation was performed by a veterinary pathologist who was blinded as to the group and location from which the biopsy specimen was collected. Image J software (NIH.gov h ) was used by the author to determine epithelial surface area, area of infoldings, and total number of infoldings. Measurements of each parameter from each location were compared within and between groups. Scanning Electron Microscopy Tissues for scanning electron microscopy (SEM) were collected and stored in a mixture of 4% paraformalin, 1% glutaraldehyde (Electron Microscopy Service, Hatfield, PA d ), and phosphate buffered saline (Sigma Chemical, St Louis, MO e ) at 4 ? C for up to 4 weeks. The tissues were rinsed in a phosphate buffered saline (PBS) bath for 20 min with agitation. Tissues were fixed in 4% osmium tetroxide/ PBS (Electron Microscopy Services d ) for 2 hours, rinsed with PBS for 3 changes of 20 min each, and rinsed with distilled water for 20 min. 15 Tissues were dehydrated in the manner as previously described for light microscopy. All samples were stored under a vacuum to ensure complete dehydration. The samples were mounted on an aluminum specimen stud (Electron Microscopy Services, Hatfield, PA d ) and sputter coated with colloidal gold (Electron Microscopy Services, Hatfield, PA d ) prior to viewing with an EVO 50 SEM? (Zeiss XVP, USA i ). Images were captured with the EVO 50? (Zeiss XVP i ) computer and stored on removable file. Similar to light microscopy samples, surface epithelium of each sample was evaluated by a veterinary pathologist who was blinded to group and location from which biopsy specimens were collected. Differences in the surface epithelium at each anatomical location were compared within and between groups by subjective evaluation. Areas Measured The following parameters were measured for each biopsy site: 1) area of the epithelium from the basement membrane to the luminal surface, 2) area of infoldings under a tangent line drawn from the pinnacles of the surface epithelium at the edges of the infolding, and 3) total number of infoldings per linear unit of tissue. These parameters were chosen as they could be consistently measured with the Image J software. Direct measurements of epithelial thickness and depth of infolding were not repeatable. Area of Epithelium Each sample of epithelium was labeled according to the anatomical location from which it was collected. Distal refers to the penis 1 cm proximal to the galea. Middle refers to the area 1cm distal to the attachment of the prepuce to the free portion of the penis. Proximal refers to the prepuce 6 cm distal to the preputial orifice when the penis is extended. From each specimen, color images of 4080 x 3072 pixel resolution (40X magnification) were acquired with a light 16 microscope (Olympus ? BH-2 j ) and digital camera (Olympus ? DP-71 j ) with the Image J analysis program (Image J NIH.gov h ). To determine the area of the epithelium, the following steps were taken: 1) a line was drawn over the scale given in the image [analyze ? set scale to the known distance], 2) the scanned color image was converted to grayscale [image ? type ? 8-bit], 3) the automated mode was used to threshold the image [process ? binary ? make binary], 4) the edges of the epithelium were highlighted with the tracing tool, 5) the area of the epithelium was calculated [analyze ? analyze particles], and 6) minimum pa rticle size was entered as 500 pixels [toggle show outlines, check display results, and click OK]. (Illustrations 2-8) Illustration 2. Set Scale to known distance. Image at 40X magnification 17 Illustration 3. Convert to grayscale. Illustration 4. Convert to binary image. 18 Illustration 5. Tracing tool to locate edges of the epithelium. Illustration 6. Analyze particles 19 Illustration 7. Set particle size, display outlines and results. Illustration 8. Outline of area and results of area calculated. 20 Area of infoldings Each of the three samples (Proximal, Middle, Distal) were evaluated for area of infoldings to determine differences within and between groups. To determine the area of the infoldings, the following steps were taken: 1) a line was drawn over the scale given in the image [analyze ? set scale to the known distance], 2) the a rrow tool was selected, and a tangent line was drawn that touches the tissue at exactly two points determined by visual observation to be the pinnacles of the tissue on either side of the fold. The tangent line touched the pinnacles but did not intersect the tissue, 3) the polygon tool was selected and used to trace the borders of the fold, 4) the threshold area was outlined [image ? adjust ? threshold], 5) the color image was converted to a binary image [process ? binary ? make binary], 6) the outlined area was calculated [analyze ? analyze particles], and 7) minimum particle size was entered as 500 pixels [toggle show outlines, check display results, and click OK]. (Illustrations 9- 14) Illustration 9. Set scale to known distance 21 Illustration 10. Using arrow tool to draw tangent line. Illustration 11. Tracing the outline of the fold 22 Illustration 12. Adjusting image to threshold. Illustration 13. Converting to binary image 23 Illustration 14. Analyzing particles Total Number of Infoldings The total number of infoldings per linear unit was recorded by Image J software (NIH.gov h ). Results were compared within and between groups. Statistical Analysis All measurements of light microscopy slides were collected with Image J software (NIH.gov h ). Results of the area of epithelium, area of folds, and total number of infoldings per linear unit were entered into a Microsoft Excel? k spread sheet and analyzed with the SAS analytical system (SAS software, SAS Institute l ) using an ANOVA procedure. The ANOVA procedure compared results within and between age groups. A value of p ? 0.05 indicated a significant difference. 24 Examination for Crypts Samples from each biopsy site were examined with electron microscopy for presence of structures that met the criteria for crypts as previously described. (7) (Illustration 18) Examinations were performed with a scanning electron microscope at 31-170X magnification level. The surface epithelium of all three segments from each bull was evaluated. Because software for measurement was not available, electron microscopy samples were subjectively evaluated (Illustrations 15-17). Illustration 15. Scanning electron image proximal sample 2yr old (31X magnification). Infoldings are indicated by arrows. 25 Illustration 16. Scanning electron image middle sample 6yr old (31X magnification). Infoldings are indicated by arrows. 26 Illustration 17. Scanning electron image distal sample 5yr old (90 X magnification). Infoldings are indicated by arrows. 27 Illustration 18. Scanning electron image of intestinal crypts (Skrzypek et al 59 ). Crypts are indicated by arrows. (170 X magnification) 28 Chapter 4 Results This study was conducted from December 2008 to March 2009. Two age groups were compared (Table 1), and a total of 36 samples were collected from 12 bulls in each age group. Samples were evaluated using Image J software (NIH.gov h ). These results were analyzed by ANOVA. Group # 1 Group # 2 Angus 25 mo Angus 26 mo Angus 25 mo Brahman 24 mo Angus 27 mo Table 1. Age and breed of bulls Angus 26 mo Angus 6 yrs Angus 5 yrs Angus 12 yrs Angus 5 yrs Angus 7 yrs Angus 6 yrs 29 Three parameters were evaluated for each sample. The area of the epithelium was measured and compared within and between groups. No significant difference in related epithelial area was detected between groups (p = 2.96, Illustration 19). The ranges of the epithelial area in ?M 2 for group 1 are: distal 0.4 x 10 6 ? 1.3 x 10 6 , middle 0.3 x 10 6 ? 1.3 x 10 6 , and proximal 0.2 x 10 6 - 1.8 x 10 6 . The ranges of the epithelial area in ?M 2 for group 2 are: distal 0.3 x 10 6 - 1.9 x 10 6 , middle 0.3 x 10 6 ? 1.6 x 10 6 , and proximal 0.4 x 10 6 ? 1.7 x 10 6 (Table 2). The mean (means ? standard deviation) epithelial areas in ?M 2 for group 1 are: distal 0.8 x 10 6 ? 0.3 x 10 6 , middle 0.7 x 10 6 ? 0.2 x 10 6 , and distal 1.0 x 10 6 ? 0.3 x 10 6 . The mean (means ? standard deviation) epithelial areas in ?M 2 for group 2 are: distal 0.8 x 10 6 ? 0.3 x 10 6 , middle 0.7 x 10 6 ? 0.3 x 10 6 , and proximal 0.8 x10 6 ? 0.3 x 10 6 (Illustration 19). Group Range (?M 2 ) 1. 2yr old (? 3 mo) distal 0.4 x 10 6 ? 1.3 x 10 6 middle 0.3 x 10 6 ? 1.3 x 10 6 proximal 0.2 x 10 6 ? 1.8 x 10 6 2. ? 5yr old distal 0.3 x 10 6 ? 1.9 x 10 6 middle 0.3 x 10 6 ? 1.6 x 10 6 proximal 0.4 x 10 6 ? 1.7 x 10 6 Table 2. Range of epithelial area. 30 Mean Epithelial Area 0 0.2 0.4 0.6 0.8 1 1.2 Distal Middle Proximal Anatomical Location Ar e a ( 1 0 6 ? M 2 ) 2 yr old ? 5 yr old a b c d e f Illustration 19. Mean epithelial area and standard deviation (a-f) per site. Area between groups is not different (p= 2.96) a) sd ? 0.3 x 10 6 b) sd ? 0.3 x 10 6 , c) sd ? 0.2 x 10 6 , d) sd ? 0.3 x 10 6 , e) sd ? 0.3 x 10 6 , f) sd ? 0.3 x 10 6 The area encompassed by the infoldings was measured and compared within and between groups. No significant difference was detected between groups (p = 2.67, Illustration 20). The ranges of the area encompassed by the infoldings in ?M 2 for group 1 are: distal 0.009 x 10 6 - 0.7 x 10 6 , middle 0.002 x 10 6 - 0.3 x 10 6 , and proximal 0.006 x 10 6 - 0.8 x 10 6 . The ranges of the area encompassed by the infoldings in ?M 2 for group 2 are: distal 0.006 x 10 6 - 0.8 x 10 6 , middle 0.003 x 10 6 - 0.3 x 10 6 , and proximal 0.004 x 10 6 ? 1.0 x 10 6 (Table 3). The mean (means ? standard deviation) of the areas encompassed by the infoldings in ?M 2 of group 1 are: distal 0.1 x 10 6 ? 0.1 x 10 6 , middle 0.1 x 10 6 ? 0.03 x 10 6 , and proximal 0.2 x 10 6 ? 0.1 x 10 6 . The mean (means ? standard deviation) of the areas encompassed by the infoldings in ?M 2 of group 2 are: distal 0.1 x 10 6 ? 0.09 x 10 6 , middle 0.07 x 10 6 ? 0.05 x 10 6 , and proximal 0.1 x 10 6 ? 0.1 x 10 6 (Illustration 20). 31 Group Range ?M 2 1. 2 yr old (? 3 mo) distal 0.9 x 10 4 ? 0.8 x 10 6 middle 0.003 x 10 6 ? 0.3 x 10 6 proximal 0.007 x 10 6 ? 0.9 x 10 6 2. ? 5 yr old distal 0.007 x 10 6 ? 0.8 x 10 6 middle 0.003 x 10 6 ? 0.3 x 10 6 proximal 0.005 x 10 6 ? 1.0 x 10 6 Table 3. Range of epithelial fold area. Mean Area of Infoldings 0 0.05 0.1 0.15 0.2 0.25 Distal Middle Proximal Anatomical Locations Area (10 6 ?M 2 ) 2 yr old ? 5 yr old A B B A A,B A,B a b c d e f Illustration 20. Mean area of infoldings and standard deviations (a-f). Area of infoldings between age groups are not different (p = 2.67) a) sd ? 0.1 x 10 6 b) sd ? 0.09 x 10 6 , c) sd ? 0.03 x 10 6 , d) sd ? 0.05 x 10 6 , e) sd ? 0.1 x 10 6 , f) sd ? 0.1 x 10 6 A) Mean area of infoldings within each group was greater in proximal sites than in middle sites (p = 0.025) B) Mean area of infoldings within each group was greater in distal sites than in middle sites (p = 0.028) 32 The number of infoldings per 3256 ?M of epithelium was compared between age groups. These numbers were compared to related portions. No significant difference in total fold numbers was detected between age groups (p = 0.69). The number of infoldings per 3256 ?M for each site within group 1 is: distal 74, middle 55, and proximal 76. The number of infoldings per 3256 ?M site within group 2 is: distal 69, middle 64, and proximal 85. The mean number (mean ? standard deviation) of infoldings per site for group 1 is: distal 12.3 ? 3.8, middle 9.2 ? 3.4, and proximal 12.7 ? 5.8. The mean number (mean ? standard deviation) of infoldings per site for group 2 is: distal 12.3 ? 6.7, middle 10.6 ? 5.3, and proximal 14.1 ? 11.4 (Illustration 21). Total # of Infoldings 0 10 20 30 40 50 60 70 80 90 Distal Middle Proximal Anatomical Location # F o l d s / 32 56 m i cr o n s 2 yr old ? 5 yr old p = 0.69 Illustration 21. Total number of folds / 3256?M and standard deviations (a-f). c a b d e f a) Mean 12.3 sd ? 3.8, b) Mean 12.3 sd ? 6.7, c) Mean 9.2 sd ? 3.4, d) Mean 10.6 sd ? 5.3 e) Mean 12.6 sd ? 5.8 f) Mean 14.2 sd ? 11.4 33 Electron microscopic evaluation Tissue samples were examined by electron microscopy for presence of epithelial crypts at 31 ? 170X magnification. Several images of intestinal crypts have been published. (59) (Illustration 18) Infoldings of epithelium were present on all sections that were viewed, but no structures comparable to intestinal crypts were located in any sections examined. 34 Chapter 5 Discussion Shortly after the initial description of Tritrichomonas foetus, a report in 1941 by Abelein noted that there were numerous infoldings present on the surface of the penis. (60) The author suggested that Tritrichomonas foetus established long term infection in these crevices. (19) It has been stated that presence of deeper crypts in older bulls allows the development of chronic infection with C. fetus subsp. venerealis. (2) For decades it has been assumed that older bulls have deeper crypts than younger bulls, but little documentation exists to support this conclusion. Chronic infections can be established in older bulls. (35) Christensen et al, indicated that the herd prevalence of trichomoniasis could be reduced by exclusive use of younger bulls, suggesting that chronic trichomoniasis is primarily a problem of older bulls. (61) Other studies have shown that young bulls (< 2yrs) may be susceptible to chronic infections with T. foetus. (41) This study was undertaken to evaluate the differences in the penile and preputial epithelium between different aged bulls at three different anatomical locations. The study was not intended to prove or disprove that folds of the epithelium exist. An early hypothesis of this study was that the epithelial layer of the penis and prepuce becomes thicker as the bull matures, which results in deeper infoldings. The area of the surface of the epithelium was not different between groups (p = 2.96, Illustration 19). The second objective of this study was to determine the area encompassed by the epithelial folds and to compare differences within and between groups. There was no statistical difference in area contained within the infoldings between groups. This was an unexpected 35 finding contrary to the widely held belief that older bulls would likely have increased areas of infolding. (p= 2.67, Illustration 20). In both groups the area contained within the infoldings was greater at the proximal and distal sites as compared to the middle. (p = 0.02) The final assumption tested was that the total number of infoldings was different between age groups. The results of this study do not support this assumption. The total number of folds did not differ between the two groups (p=0.69, Illustration 21). Previous studies compared breed prevalence of trichomoniasis and campylobacterosis. (48,49,62) The bulls in the current study were predominantly Angus, consistent with the population of beef bulls currently used in Alabama. A recent retrospective review of breeding soundness evaluations revealed that 72% of the 1,076 bulls presented to the Auburn University Food Animal Theriogenology service for breeding soundness evaluation from 2006 to 2008 were Angus. (63) Based upon visual examination of sections prepared for electron microscopy, there is an absence of structures that can be classified as crypts. Due to the absence of the crypt or crypt like structures, the author proposes that the term epithelial folds or infoldings be used as the preferred descriptive term. Collectively, these findings indicate that chronic venereal infection of the bull is not likely due to the presence of increased epithelial area, increased area within epithelial folds, or increased density of epithelial folds of the prepuce and free portion of the penis. Other differences in the penile and preputial environment must exist to allow establishment of persistent infection in mature bulls. A review by Felleisen suggests that carbohydrate receptors and lectin molecules play a role in the development of venereal infection. (65) It is possible that the penile and preputial epithelium of older bulls have a higher concentration of these receptors, 36 creating a favorable environment for chronic infection. Early studies by Hammond et al noted that distribution of T. foetus in the preputial cavity was correlated with the distribution of smegma. (19) The quantity and consistency of smegma may favor the development of venereal disease in older bulls. Architectural characterization and electron microscopic evaluation of penile and preputial epithelium may enhance future studies of the pathophysiology and immunology of venereal infections in bulls. 37 References 1. Hafez ESE. Anatomy of Male Reproduction. In: Reproduction in Farm Animals. 6th Edition Philadelphia: Lea & Febiger; 1993.p 3-19. 2. BonDurant RH. Venereal Diseaes of Cattle: Natural History, Diagnosis, and the Role of Vaccines in their Control. Vet Clin North Am Food Anim Pract 2005;21:(2)383-408. 3. Clark BL, Dufty JH, Parsonson IM. The effect of Tritrichomonas foetus on calving rates in beef cattle. Aust Vet J 1983;60:71-4. 4. Fitzgerald PR. Bovine trichomoniasis. Vet Clin North Am Food Anim Pract 1986;277:(2)82. 5. Mickelsen WD, Paisley LG, Anderson PB. Survey of the prevalence and types of infertility in beef cows and heifers. J Am Vet Med Assoc 1986;189:51-4. 6. Parsonson IM, Clark BL, Dufty JH. Early pathogenesis and pathology of Tritrichomonas foetus infection in virgin heifers. J Comp Pathol 1976;86:59-66. 7. Blood DC, Studdert VP. Bailli?re?s Comprehensive Veterinary Medical Dictionary. Tindall Company; 1995.p 240. 8. Samuelson DA. Textbook of Veterinary Histology. Saunders Company; 2007. p339. 9. Roberts SJ, Veterinary Obstetrics and Genital Diseases. Ann Arbor: Edwards Brothers 1971. p609. 10. Beckett SD, Wolfe DF, Anatomy of the Penis, Prepuce, and Sheath In: Large Animal Urogenital Surgery. Williams and Wilkins; 1998.p201. 11. Dyce KM, Sack WO, Wensing CJG. Textbook of Veterinary Anatomy. 2 nd ed. W.B. Saunders Company; 1987.p278. 12. Izumi T. Neuro-Anatomical Studies on the Mechanism of Ejaculation Reflexes In the Bull. Special Bulletin of Ishikawa Prefecture of Agriculture. 1980;9:45-85. 13. Bellenger CR. A Comparsion of Certain Parameters of the Penis and Prepuce in Various Breeds of Beef Cattle. Res.Vet.Sci. 1971;12,299-304. 14. Bacha WJ, Bacha LM. Color Atlas of Veterinary Histology. 2 nd ed. Philadelphia: Lippencott Williams and Wilikins,2000.p205. 38 15. Bone JF. The Vascular System In: Animal Anatomy and Physiology. 3 rd Ed. Prentice Hall; Englewood Cliff, New Jersey;1988:p 233-288. 16. Flowers PJ, Ladds PW, Thomas AD, et al. An immunologic study of the bovine prepuce. Vet Pathol 1982;20:189-202. 17. Rhyan JC, Wilson KL, Wagner B, et al. Demonstration of Tritrichomonas foetus in the external genitalia and of specific antibodies in preputial secretions of naturally infected bulls. Vet Pathol 1999;36:406-411 18. Cobo ER, Corbeil LB, Gershwin LJ, et al. Preputial cellular and antibody responses of bulls vaccinated and/or challenged with Tritrichomonas foetus. Vaccine 2010:28;361-370 19. Hammond DM, Bartlett DE. The distribution of Trichomonas foetus in the preputial cavity of infected bulls. Am J Vet Res 1943;4:143-149. 20. BonDurant RH. Pathogenesis, diagnosis and management of trichomoniasis in cattle. Vet Clin North Am Food Anim Pract 1997;13:345-61. 21. BonDurant RH, Honigberg BN. Trichomonads of veterinary importance. In: Kreier JP, editor. Parasitic Protozoa. 2 ed. San Diego: Academic Press;1994.p111-88. 22. Parsonson IM, Clark BL. Dufty J.H. The pathogenesis of Tritrichomonas foetus infection in the bull. Aust Vet J 1974;50:421-3. 23. Morgan BB. Studies on the trichomonad carrier-cow problem. J Anim Sci 1944;3:437. 24. Anderson ML. Barr BC. Conrad PA. Protozoal causes of reproductive failure in domestic ruminants. Vet Clin North Am Food Anim Pract 1994;10:439-61. 25. Benchimol M, Rosa IA. Fontes RS, Dias AJB. Trichomonas adhere and phagocytose Sperm cells: adhesion seems to be a prominent stage during interaction. Parasitol Res 2008;102:597-604. 26. Clark BL, Dufty JH, Parsonson RD, Parsonson IM. Studies on the transmission of Tritrichomonas foetus. Aust Vet J 1977;53:170-2. 27. Parker S, Campbell J, McIntosh K, Gajadhar A. Diagnosis of trichomoniasis in ?virgin? bulls by culture and polymerase chain reaction. Can Vet J 2003;44:732-734. 28. Castella J, Munoz E, Ferrer D, Gutierrez JF. Isolation of the trichomonad Tetratrichomonas buttreyi in bovine diarrhoeic faeces. (Hibler et al, 1960; Honigberg, 1963) 1997;70, 41-45. 29. Campero CM, Rodriguez Dubra C, Bolondi A, et al. Two step (culture and PCR) diagnostic approach for differentiation of non- T. foetus trichomonads from genitalia of virgin beef bulls in Argentina. Veterinary Parasitology 2003;112:167-175. 39 30. Abbitt B: Trichomoniasis in Cattle. In: Morrow DA. (ed): Current Therapy in Theriogenology, ed 1. Philadelphia, WB Saunders,1980:482-488. 31. Bazer FW, Thatcher WW, Hansen PJ. et al: Physiological mechanisms of pregnancy recognition in ruminants. J Reprod Fertil 43: (suppl)39-47,1991. 32. Skirrow S. Identification of trichomonad-carrier cows. J Am Vet Med Assoc 1987;191:553-4. 33. Yaeger MJ, Holler LD. Bacterial causes of bovine infertility and abortion. In: Younquist R.S., editor. Current Therapy in Large Animal Theriogenology. 2 nd ed. Philadelphia; W.B. Saunders Company; 2007.p391. 34. Hoffer MA, Bovine Campylobacterosis: A Review. Can Vet J 1981;22:327-330. 35. Clark BL, Parsonson IM, Dufty JH, Experimental infection of bulls with Tritrichomonas foetus Aust Vet J 1974;50(5):189-191. 36. Peter D. Bovine venereal diseases. In: Youngquist R.S., editor. Current therapy in large animal theriogenology. 1 ed. Philadelphia: W.B. Saunders Company; 1997. p.355-63. 37. Herr S, Ribeiro LM,Claassen E, et al. A reduction in the duration of infection with Trichomonas foetus following vaccination in heifers and the failure to demonstrate a curative effect in infected bulls. Onderstepoort J Vet Res 1991;58(1):41-5. 38. Soto P, Parma AE. The immune response in cattle infected with Tritrichomonas foetus. Vet Parasitol 1989;33(3-4):343-348. 39. Clark BL, Dufty JH, Parsonson IM. Immunisation of bulls against trichomoniasis. Aust Vet J 1983;60:178-179. 40. Clark BL, Emery DL, Dufty JH. Therapeutic immunization of bulls with the membrane and glycoproteins of Tritrichomonas foetus. Aust Vet J 1984;61:65-66. 41. Skirrow S, BonDurant RH,Farley J, et al. Efficacy of ipronidazole against trichomoniasis in beef bulls. J Am Vet Med Assoc 1985;187(4):405-7. 42. Johnson AE. Incidence and diagnosis of trichomoniasis in western beef bulls. J Am Vet Med Assoc 1964;145:1007-1010. 43. Abbitt B, Meyerholz GW. Trichomonas foetus infection of range bulls in South Florida. Vet Med Sm Anim Clin 1979;74:1339-1342. 44. Wilson SK, Kocan AA, Gaudy ET, et al. The prevalence of trichomoniasis in Oklahoma beef bulls. Bov Pract 1979;14:109-110. 45. BonDurant RH, Anderson ML, Blanchard P, et al. Prevalence of trichomoniasis among California beef herds. J Am Vet Med Assoc 1990;196:1590-1593. 40 46. Rae DO, Crews JE, Greiner EC, et al. Epidemiology of Tritrichomonas foetus in beef bull populations in Florida. Theriogenology 2004;61:605-618. 47. Rodning SP, Wolfe DF, Carson RL.et al. Prevalence of Tritrichomonas foetus in a small subpopulation of Alabama beef bulls. Theriogenology 2008;69:212-217. 48. Swai ES, Hulsebosch J, Van der Heijden W. Prevalence of genital campylobacterosis and trichomoniasis in crossbred breeding bulls kept on zero-grazed smallholder dairy farms in the Tanga region of Tanzania. J S Afr Vet Assoc 2005 Dec;76(4):224-7. 49. Pefanis SM, Herr S, Venter CG, et al. Trichomoniasis and campylobacterosis in bulls in the Republic of Transkei. J S Afr Vet Assoc 1988 Sep;59(3):139-40. 50. Fitzgerald PR, Johnson AE, Thorne JL, et al. Trichomoniasis in range cattle. Vet Med 1958;53:249-52. 51. Speer CA, White MW. Better diagnostics and control could save beef industry $650 million annually. Large Animal Vet 1991;46:p18. 52. Crews, JE, Rae, DO, Donovan, GA, et al. Prevalence of and factors associated with Tritrichomonas foetus in bull populations in the state of Florida (USA). In: Proceedings of theWorld Buiatrics Conference, Punta del Este, Uruguay, 2000 December 4?8, pp.781? 794. 53. Taylor MA, Marshall RN, Stack M. Morphological differentiation of Tritrichomonas foetus from other protozoa of the bovine reproductive tract. Brit Vet J 1994;150:73-80. 54. BonDurant RH, Gajadhar A, Campero CM, et al. Preliminary characterization of a Tritrichomonas foetus-like protozoan isolated from preputial smegma of virgin bulls. Bov Pract 1999;33:124-7. 55. Cobo ER, Campero CM, Mariante RM, et al. Ultrastructural study of a tetratrichomonad species isolated from preputial smegma of virgin bulls. Vet Parasitol 2003;117:195-211. 56. Felleisen RSJ, Lambelet N, Bachmann P. et al. Detection of Tritrichomonas foetus by PCR and DNA enzyme immunoassay based on rRNA gene unit sequences. J Clin Microbiol 1998;36:513-519. 57. Hayes DC, Anderson RR, Walker RL. Identification of trichomonadid protozoa from the bovine preputial cavity by polymerase chain reaction and restriction fragment length polymorphism typing. J Vet Diagn Invest 2003;15:390-4. 58. Cobo ER, Canton G, Morrell E, et al. Failure to establish infection with Tetratrichomonas sp. in the reproductive tracts of heifers and bulls. Vet Parasitol 2004;120:145-50. 59. Skrzypek T. Valverde Piedra J.L., Skrzypek H., et al. Intestinal villi structure during the development of pig and wild boar crossbreed neonates. Livestock Science 2007;109;(1-3) :pp 38-41. 41 60. Abelein R, Trichomonadenseuche beim Bullen und ihre Behandlung. Berl. Munich. Tier Wchnschr., July 25, 1941:357-362 61. Christensen HR, Clark BL, Parsonson IM, Incidence of Tritrichomonas foetus in young replacement bulls following introduction into an infected herd. Aust Vet J 1977;53:132-134. 62. Rae DO, Chenoweth PJ, Genho PC. Prevalence of Tritrichomonas foetus in a bull population and effect on production in a large cow-calf enterprise. J Am Vet Med Assoc 1999;214:1051-1055. 63. Carson RL. Breeding soundness exam of the bull. Short Course Society For Theriogenologists meeting, St. Louis Mo. 2008 64. Rae DO, Crews JE. Tritrichomonas foetus. Vet Clin North Am Food Anim Pract 2006;22(3):595-608. 65. Felleisen SJ. Host parasite interaction in bovine infection with T.foetus. Microbes and Infection. 1999;1:807-816. 42 Appendix A Reprinted with permission Dr. K. Farmer Thompson Bishop Sparks Diagnostic Lab Alabama Department of Agriculture and Industries 43 44 45 46 47 48 49 50 51 Appendix B Individual Bull Epithelial Area Range and Mean Distal Middle Proximal Bull # & Age Range ?M 2 Mean ?M 2 Range ?M 2 Mean ?M 2 Range ?M 2 Mean ?M 2 47 / 6yr old 882584 - 1936671 1340585 395088 - 833352 607728 634064 - 1133975 849104 48 / 5yr old 426944 - 1378165 734631 544824 - 974387 790637 650157 - 1799887 1104763 49 / 12yr old 384084 - 996036 747372 398172 - 847979 618233 470269 - 1150715 679985 50 / 5yr old 423180 - 1478482 1004658 752971 - 1634229 1132880 562485 - 1083634 821854 51 / 2yr old 572725 - 1042728 753338 389473 - 1046252 594487 251116 - 1832740 907015 52 / 2yr old 461594 - 936462 711866 502833 - 929470 718505 1058627 - 1522066 1190887 53 / 2yr old 545074 - 1291795 858087 708662 - 1145499 832032 629531 - 1100649 933549 54 / 7yr old 522978 - 915460 659214 708662 - 114549 832032 506954 - 686292 572387 55 / 6yr old 605503 - 1131074 872921 364399 - 796347 630623 605494 965749 101 / 2yr old 540479 - 1373499 943946 540338 - 968423 810046 1059679 - 1508778 1305229 102 / 2yr old 776480 - 1301775 1132321 358493 - 810975 574748 563783 - 1579383 1124585 103 / 2yr old 595344 - 982672 735344 598221 - 1354708 949096 401061 - 883053 677620 52 Appendix C Individual Bull Infolding Area Range and Mean Distal Middle Proximal Bull # & Age Range ?M 2 Mean ?M 2 Range ?M 2 Mean ?M 2 Range ?M 2 Mean ?M 2 47 / 6yr old 6544 - 810495 164436 3464 -52306 31563 4680 -730462 234342 48 / 5yr old 17360 - 402147 155535 28238 - 208921 854887 10004 -61802 27452 49 / 12yr old 87481 - 745203 52738 8903 -39163 25614 7142 -771173 214601 50 / 5yr old 13954 - 99886 52738 3359 - 298434 135512 7107 -59028 27711 51 / 2yr old 12629 - 181163 93192 42826 - 192462 141280 6509 -806099 157558 52 / 2yr old 9495 - 240145 118589 29767 - 235954 118404 111913 - 759245 366862 53 / 2yr old 38745 - 328114 157448 25708 - 219693 97294 34399 - 117680 76039 54 / 7yr old 32721 - 234905 113752 18420 - 53914 34145 73901 - 244595 159248 55 / 6yr old 73873 - 392790 241356 87302 - 307513 146425 24241 - 247292 79049 101 / 2yr old 69842 - 731661 315065 98350 - 349685 165393 86871 - 488983 205962 102 / 2yr old 125988 - 783692 398433 37791 - 126860 84069 21220 - 891456 494694 103 / 2yr old 9857 - 275985 116472 34567 - 209739 113652 15719 - 210566 122926 53 Appendix D Endnotes a. Butler Schein Animal Health, Dublin, OH b. Diamond?s Media Reagents 1. Bacto Agar 1gram 2. Bacto Peptone (trypticase peptone) 4.0 grams 3. Bovine serum 20 ml 4. Distilled or deionized water 18ml 5. L-ascorbic acid .04grams 6. L-cysteine hydrochloride .2grams 7. Maltose 1.0 grams 8. Penicillin G solution (100,000 units per milliliter) 2ml 9. Potassium phosphate dibasic K 2 HPO 4 .16 grams 10. Potassium phosphate monobasic KH 2 PO 4 .16 grams 11. Yeast extract 2.0 grams 12. Streptomycin sulfate solution (0.2 gram per milliliter) 1ml c. Clark?s Media Reagents. 1. 5-Fluorouracil ( 300?g/ml) 3.0ml 2. Brilliant Green (50?g/ml) .1ml 3. Calf serum 100ml 4. Cycloheximide (100?g/ml) 1.0ml 5. Polymixin B sulphate (100 IU/ml ).1ml d. Electron Microscopy Services, Hatfield, PA k. Microsoft Excel? e. Sigma Chemical, St Louis, MO l. Statistical Analysis System, Cary, NC f. Tissue-Tek VIP E300? Ames Co, Elkart, IN g. Reichert-Jung 2040 Autocut microtome? Depew, NY h. Image J software NIH.gov i. EVO 50 SEM? Zeiss XVP, USA j. Olympus BH-2, DP-71 ? Olympus America Inc 54