Comparison of canine sperm quality under different temperature storage

• Autor(a) principal: Borges, Paulo Alexandre Paulos
• Data de Publicação: 2014
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10348/2871
• topic: Sémen; Análise do sémen; Marcadores moleculares; Criopreservação

Successful gamete cryopreservation is essential to preserve the genetic pool of several species. The canine semen is known to possess a certain resilience to chilling procedures, which has a limited interest for long term gamete preservation; however, when frozen, canine sperm suffers an important decrease in quality. This feature determined the development of different extenders aiming the protection of the cell and to increase the success of canine sperm freezing/thawed procedures. In parallel, development of new techniques allowing a more objective evaluation of putative sperm fertility were also developed. However, those methods do not explain why some stud males are “good freezers” while other are “bad” freezers, when both were selected at start as adequate for freezing. Further, no clear definition exists on the cut-off values for specific seminal parameters to distinguish between a fertile and infertile stud, although the analysis of semen is regularly performed to assess the male fertility. In this study we plan to evaluate cold treatment associated changes on sperm cells by using two different approaches. For that, semen was collected from 8 dogs frequently used as semen donors. Freshly ejaculated samples were assess (Control group) before its use for chilling (Treatment A) or freezing (Treatment B), using routine procedures. For treatment A, three different times were considered: at 1.5h, at 4h and at 24h of chilling (respectively times 1, 2 and 3). Sperm characteristics were assessed by the convention methods, CASA and hypoosmotic test (HOST) for motility and movement parameters, velocity fractions, cell morphology and membrane integrity. Additionally, a molecular approach was tried by the study of heat sock protein 70 (HSP70) immunoexpression in the spermatozoa. HSP70 has protective effects over cells under conditions of thermal or proteotoxic stress. In the study presented here, the more conservative tests confirmed that chilling is not an aggressive procedure for dog semen in comparison to freezing, provided that the samples have a minimum acceptable quality at start. In frozen/thawed samples it was found a decrease in sperm motility and an increase in the percentage of static cells, along with a loss of the sperm membrane integrity. In parallel, a decrease in HSP intensity of immunostaining and a dislocation of the immunoreaction towards the flagellum were observed in the frozen samples (treatment B), while for treatment A no significant changes were found in the pattern of HSP immunoexpression. The use of molecular marker may reprosent an increase value in the study of the mechanisms underlaying the cenine sperm sensitivity to cryopreservation.