Effects of Pentoxifylline on sperm quality and uterine hemodinamics in equine

Author(s)

Roberta Harue Tsunoda

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2013-08-08

Examining board members

Eneiva Carla Carvalho Celeghini; Rubens Paes de Arruda; Luciano Andrade Silva

• Advisor: Eneiva Carla Carvalho Celeghini

Abstract

The post breeding uterine inflammatory response occurs due to remove spermatozoa and contaminants; however, delayed uterine clearance has been identified, in equines, as the underlying cause of persistent post breeding endometritis. Pentoxifylline acts as a phosphodiesterase inhibitor and increases sperm motion characteristics, and it has been reported to participate as a rheologic agent, improving blood flow to compromised tissues, and as an immunomodulatory agent. The purpose of this study was to examine the effects of pentoxifylline added to the seminal extender after thawing, on sperm motion, plasmatic, acrosomal and mitochondrial membranes and chromatin integrities, as well as the uterine inflammatory response on mares after artificial insemination. Ten batches of a unique stallion were cryopreserved (100x106 spermatozoa/0.5 mL straws), in an automated system (TK 3000) and storaged in cryogenic cylinder for posterior use for laboratory analysis and artificial insemination. For laboratory analysis two semen straws of each batch were thawed (37°C/30 seconds) and diluted in skim milk based extender (control) or diluted in skim milk extender containing pentoxifylline (7.18 mM). Aliquots of each treatment were maintained in dry water bath (37°C) and analyzed at four moments: 5 (T0), 30 (T30), 60 (T60) and 120 minutes (T120) post dilution and incubation. Samples were analyzed for sperm motion characteristics, through Computer-Assisted Semen Analysis (CASA), plasmatic, acrosomal and mitochondrial membranes through fluorescent probes association (PI, H342, FITC-PSA and JC-1), spermatic morphology through differential interference contrast and chromatin integrity through Toluidine blue. To examine the effect of extender containing or not pentoxifylline on uterine inflammatory response 15 mares were divided into 5 groups: Group Ccontrol (control): no uterine deposition of semen, or extender, just mimicking the AI procedure; Group SM Extender: deposition of skim milk based extender (Botusemen®, Botupharma); Group SM Extender + PTX: deposition of skim milk based extender containing Pentoxifylline; Group Semen: deposition of semen diluted with extender only; Group Semen + PTX: deposition of semen diluted with extender containing Pentoxifylline (7,18 mM). Uterine hemodynamics were examined through color and spectral mode ultrasonography on moments: immediately before ovulation induction (approximately 30 hours before AI, T-30), immediately before artificial insemination (TAI) and 2 (T2), 6 (T6), 12 (T12), 24 (T24) and 48 (T48) hours after artificial insemination. Samples for endometrial cytology were collected, through cytobrush at T6 moment. Statistical analysis was performed using the analysis of variance (ANOVA), and the range was compared by LSD test, SAS 9.3 version (2010). Pentoxifylline addition did not exert effect on progressive motility, however, pentoxifylline increased path velocity, progressive velocity and curvilinear velocity when compared to controls. There were no significant differences on the percentage of cells presenting intact plasma membrane, intact acrosome and high mitochondrial membrane potential (IPIAH). No statistical difference was found on uterine blood flow of mares after treatments. However, endometrial cytology showed a major inflammatory response on groups containing Pentoxifylline when compared to controls. In conclusion, pentoxifylline exerted a positive effect on velocity parameters, but do not influence cellular membranes and chromatin. Furthermore, when mares were inseminated with semen containing pentoxifylline, an intense inflammatory response was observed. (AU)

• FAPESP's process: 12/14927-6 - Pregnancy rate, inflamatory response and uterine blood flow affected by the effect of Pentoxifylline and sêmen quality assessed by fluorecent probes
• Grantee: Roberta Harue Tsunoda
• Support Opportunities: Scholarships in Brazil - Master