Swine seminal plasma for ram sperm cryopreservation

O interesse pelo uso da insemina????o artificial (IA) em ovinos vem crescendo, em fun????o do avan??o no melhoramento gen??tico. Entretanto, a criopreserva????o causa danos aos espermatoz??ides, diminuindo seu potencial fertilizante, quando a IA ?? feita com s??men congelado, pela via cervical. Uma alternativa para proteger ou recuperar a c??lula dos danos da criopreserva????o ?? a adi????o de plasma seminal (PS), que cont??m v??rios fatores produzidos pelos test??culos, epid??dimos e gl??ndulas acess??rias do macho, com potencial de prevenir a capacita????o prematura e danos gerados pelo congelamento. A adi????o de PS antes do congelamento seria ben??fica para os processos de sele????o e congelamento. A suplementa????o do diluente com PS ovino e su??no foi associada com aumento na motilidade esperm??tica, ap??s a segunda hora de incuba????o sob condi????es in vitro, assim como quando usado na refrigera????o, congelamento e descongelamento. Este estudo teve como objetivo testar a adi????o de PS su??no (20%) ao diluente para congelamento do s??men ovino e avaliar os par??metros seminais in vitro p??s-descongelamento. Ejaculados de quatro machos ovinos e tr??s machos su??nos foram coletados para forma????o de amostras combinando PS de v??rios machos (pools). Uma fra????o das amostras de PS foi destinada a quantifica????o de prote??nas. Seis coletas dos quatro machos ovinos foram colhidas e dilu??das em Tris-gema de ovo-glicerol, para congelamento, compondo tr??s tratamentos: controle (sem PS); inclus??o de 20% de PS ovino; e inclus??o de 20% PS su??no. Ap??s o descongelamento, as amostras foram submetidas a um teste de termo resist??ncia durante cinco horas. Avalia????es de qualidade esperm??tica foram realizadas a cada duas horas. Tamb??m foram realizadas an??lises por citometria de fluxo para as avalia????es de integridade de acrossoma e integridade de membrana. Para os tratamentos controle, PS ovino e PS su??no, n??o foram observadas diferen??as (P > 0.05) quanto a motilidade (30,0 ?? 2,0; 30,4 ?? 2,0 e 24,6 ?? 2,0 respectivamente), integridade de membrana (37,5 ?? 2,6; 40,9 ?? 2,6 e 31,4 ?? 2,6, respectivamente), fun????o mitocondrial (70,0 ?? 1,7; 61,8 ?? 1,7 e 63,6 ?? 1,7, respectivamente) e integridade de DNA (91,2 ?? 3,1; 96,5 ?? 3,1 e 93,6 ?? 3,1, respectivamente). A integridade do acrossoma foi maior (P < 0.05) com inclus??o de PS su??no (59,3 ?? 3,5) em compara????o com o controle (46,7 ?? 3,5), mas ambas as m??dias foram similares (P > 0.05) ?? observada para o PS ovino (56,7 ?? 3,5). Conclui-se que apesar do beneficio do PS su??no a 20% para a integridade de acrossoma, n??o se obteve resultados positivos nas demais avalia????es de qualidade seminal.There is a growing interest in using artificial insemination (AI) in sheep due to the potential for genetic improvement. However, cryopreservation damages spermatozoa, decreasing their fertilizing potential when frozen semen is deposited in the cervix. Sperm damages attributed to cryopreservation may be minimized by the addition of seminal plasma (SP), which contains several factors produced by the testis, epididhymis and accessory glands with potential to prevent premature capacitation. Supplementation of SP prior to freezing would be beneficial for various processes of periods of the selection and freezing process, reported before freezing would be beneficial for the processes of selection and freezing. The supplementation of extenders with SP from ram and boars is associated with increased sperm motility after incubation in vitro, as well as when used for cooling, freezing and thawing. The objectives of this study were to test the addition of 20% boar SP to the extender to freeze ram sperm and to evaluate parameters of sperm quality after thawing. Ejaculates from four rams and three boars were collected to form pooled SP samples. A fraction of each pooled sample was used for protein quantification. Six samples from four rams were collected and diluted in Tris-egg yolk - glycerol for freezing, forming three treatments: control (no SP); inclusion of 20% ram SP; and inclusion of 20% boar SP. After thawing, the samples were subjected to a thermal stress test for five hours. Sperm quality was assessed every two hours. Analyses by flow cytometry were done to evaluate the integrity of acrosome and membrane integrity. For the control, ram SP and boar SP treatments, the evaluated parameters of sperm quality were: motility (30.4 ?? 2.0, 24.6 ?? 2.0 and 30.0 ?? 2.0, respectively); membrane integrity (37.5 ?? 2.6; 40.9 ?? 2.6 and 31.4 ?? 2.6 respectively); mitochondrial functionality (70.0 ?? 1.7; 61.8 ?? 1.7 and 63.6 ?? 1.7); and DNA integrity (91.2 ?? 3.1; 96 5 ?? 3.1 and 93.6 ?? 3.1 respectively). For those parameters, no significant differences were observed across treataments (P > 0.05). However, addition of boar SP to the extenders was related to greater acrosome integrity (59, 3 ?? 3.5) than that of the control (46.7 ?? 3.5) (P < 0.05), although both means were similar (P > 0.05) to that observed for the treatment with ram SP (56.7 ?? 3.5). Despite of the benefit on acrosome integrity related to addition of boar SP, no other positive effects were observed for post-thawing ram sperm viability