Freezing of boar semen: study of penetrating and nonpenetrating cryoprotectants, freezing methods and freezability molecular marker

Apesar do s??men su??no congelado estar dispon??vel comercialmente deste 1975, o seu uso tem ocorrido somente em ocasi??es espec??ficas, pois, em rela????o ao uso de s??men refrigerado, requer duas a tr??s vezes maior n??mero de espermatoz??ides por dose, o processamento do s??men ?? trabalhoso, o tamanho da leitegada ?? diminu??do em um a tr??s leit??es por parto e a taxa de pari????o ?? menor. Dessa forma, a grande maioria das insemina????es artificiais nessa esp??cie utiliza s??men dilu??do e acondicionado no estado l??quido entre 15 e 18 ??C. Os objetivos desta tese foram: avaliar diferentes diluidores de resfriamento, metodologias de congelamento, temperaturas de centrifuga????o, uso de crioprotetores intracelulares a base de amidas e extracelulares baseados em lipoprote??na de baixa densidade, al??m do estudo de fatores prot??icos presentes no plasma seminal nos resultados de avalia????es in vitro e in vivo de s??men su??no congelado-descongelado. Foram executados cinco experimentos com avalia????es da qualidade do s??men congeladodescongelado, quatro experimentos com avalia????es in vitro (motilidade e integridade de membrana) e um experimento com fertiliza????o in vivo. O tratamento utilizado como controle foi o congelamento com glicerol na concentra????o de 3%. A melhor curva de resfriamento ap??s a coleta at?? 15 ??C, foi atingida com o tempo de 120 min. Foi identificado um fator de 26 kDa no plasma seminal, cuja aus??ncia proporcionou melhores resultados de integridade de membrana plasm??tica ap??s o descongelamento. O uso do crioprotetor intracelular dimetilacetamida na concentra????o de 5%, proporcionou nas avalia????es in vitro, resultados superiores ao tratamento controle (glicerol 3%), n??o diferindo na avalia????o in vivo. O presente estudo demonstrou a rela????o de componentes do plasma seminal com a qualidade esperm??tica, al??m de apresentar um novo protocolo de congelamentodescongelamento de s??men su??no baseado no uso de dimetilacetamida 5%.Although boar semen is available in the frozen format since 1975, its use has been restricted to very specific situations for many reasons: it requires spermatozoa concentration per dose twice to three times higher than that for cooled semen; semen processing is labor intensive; and farrowing rate and litter size are reduced. Thus, most of the artificial inseminations in swine are performed at the day of semen collection or at most at the following day, using liquid semen conditioned between 15 and 18 ??C. The aims of this thesis were: to evaluate distinct extenders for cooling semen, methods for freezing, centrifugation temperatures, the use of amides as penetrating cryoprotectants and of low density lipoprotein as nonpenetrating cryoprotectants, and the presence of specific protein factors in seminal plasma in the results that could be associated with boar semen freezability. Five experiments have been executed: four with evaluations in vitro (motility and cell membrane integrity); and one with fertilization in vivo. The control treatment was the freezing with glycerol at 3% concentration. The semen processing method that provide more efficient results consisted of semen cooling during 120 min up to a centrifugation temperature of 15 ??C. We also identified a 26 kDa factor in the seminal plasma that is associated with maintenance of the integrity of the sperm cell membrane post-thawing. Additionally, parameters of semen quality in vitro with the use of dimetilacetamide at 5% were better than those observed with the most used penetrating cryoprotectant (glycerol 3%) as control, although no difference between those cryoprotectants was observed in vivo. This study demonstrated the association of components in the seminal plasma with the sperm quality, and presenting an alternative protocol of semen freezing-thawed boar semen based in the use of dimetilacetamide at 5%