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Evaluation of influence of glycerol and ethylene glycol and process of freezing and thawing for complex DNA-protein of stallion spermatozoa

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Author(s):
Alessandra Cunha Brandão
Total Authors: 1
Document type: Master's Dissertation
Press: São Paulo.
Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
Defense date:
Examining board members:
José Antonio Visintin; Rubens Paes de Arruda; Luis Claudio Lopes Correia da Silva
Advisor: José Antonio Visintin
Abstract

To study the effects of freezing and thawing on DNA-protein complexes in spermatozoa, 12 ejaculate were collected from each of 6 stallions (Mangalarga Paulista breed) in each of two consecutive breeding seasons. Motility, vigor, concentration, morphology and incidence of the DNA-protein complex pathologies of spermatozoa were evaluated and compared among fresh semen, semen expose to freezing and thawing without cryoprotectants and semen exposed to the cryoprotectors glycerol and ethylene glycol left at room temperature or frozen. Incidence of the DNA-protein complex pathologies was evaluated in semen smear fixed in na ethanol-acetic-acid mixture (3:1, v/v) for 1 minute, washed in 70% ethanol for 3 minutes and air dried. To induce nuclear metachromasy, smears were treated with 4N HCL at 25ot for 20 minutes followed by rinsing in distilled water. Preparation were stained with a 0.025% toluidine blue solution in Mailvaine buffer for 15 minutes. Frequency of spermatozoa exhibiting nuclear metachromasy was determined in 1000 cells/animal by light microscopy at 1000X magnification. Motility, vigor, morphology and DNA-protein complex pathologies of spermatozoa were different for semen frozen compared to not frozen (P<0.05). For fresh semen, there were effects of seasons on the incidence of DNA-protein complex pathologies (P<0.05) For semen frozen without cryoprotectants there were significant effects seasons (P<0.05) The process of cryopreservation and thawing influences negatively the DNA-protein complexes in stallion spermatozoa. (AU)