101 Effect of Nitrocooler Negative Pressure and Recovery Interval on Cryotolerance of Bovine in Vitro-Produced Embryos

Abstract

Exposing bovine embryos and porcine oocytes to hydrostatic pressure has been shown to increase cryosurvival, possibly by a resulting expression of stress tolerance proteins. This study aimed to evaluate the effect of the negative pressure stress condition (a 5-min-long embryo exposure to a negative pressure) and the interval between vacuum exposure and vitrification (40 min or 2 h) on survival of bovine in vitro-produced (IVP) embryos. The negative pressure was achieved with the same apparatus used previously for the cryopreservation of embryos (Nitrocooler; Mezzalira et al. 2009 Reprod. Fert. Dev. (1) 134), in which a negative pressure (vacuum) is applied to liquid nitrogen to increase the cooling rate through the slush phenomenon, except that in this study, the vacuum was applied to the chamber without liquid nitrogen, at room temperature. Grades 1 and 2 bovine IVP expanded blastocysts were allocated to 1 of 5 experimental groups: embryos in vitro-cultured as fresh (control) or after vitrification (Vitri); and embryos subjected to the negative pressure for 5 min and then in vitro-cultured as fresh (NP-fresh) or after vitrification performed 40 min (NP-Vitri-40 min) or 2 h (NP-Vitri-2h) following the vacuum exposure. Embryos were vitrified in pulled glass micropipettes in a solution with 20% ethylene glycol + 20% dimethylsulfoxide + 20% fetal bovine serum and rewarmed in decreasing sucrose concentrations (Mezzalira et al. 1999 Acta Scientiae Veterinariae 27 262-262). In vitro culture was carried out in all treatments for 72 h for the assessment of re-expansion and hatching rates (Table 1), which were analyzed by the chi-square test, for P < 0.05. No differences in re-expansion rates were observed between groups. However, the vitrification of embryos after 2 h of exposure to a 5-min-long negative pressure (NP-Vitri-2h) improved embryo survival expressed by a higher hatching rate than for embryos vitrified without vacuum exposure (Vitri) or after 40 min following the 5-min-long exposure to vacuum. In addition, hatching rates in group NP-Vitri-2h were similar to those for fresh embryos (control and NP-fresh). Our results indicated that a short exposure of embryos to a negative pressure can improve cryotolerance following vitrification, which is dependent on the time interval between NP exposure and cryopreservation. Bovine IVP embryos should be allowed to recover for at least 2 h after NP exposure before the increase in cryotolerance is achieved. Table 1.Effect of negative pressure on re-expansion and hatching rates of fresh and vitrified

Células Fetais Bovinas De Cultivo Primário Submetidas a Diferentes Pressões Negativas Antes Do Congelamento Em Palhetas

“…Já está bem documentado o efeito positivo da aplicação de pressão hidrostática em diferentes estruturas, como sêmen de suínos (10) , sêmen de bovinos (11) , embriões de camundongo (12) e embriões ovinos (13) , bem como do efeito positivo da pressão negativa em embriões bovinos criopreservados (8) . Todavia, ao contrário do esperado, neste estudo não foi observado aumento da criotolerância das LIPOSKI, D.M.…”

“…A submissão das células fetais à pressão negativa três horas antes do congelamento reduziu a sobrevivência das células no pós-descongelamento. Em contraste com esses dados, a submissão à pressão negativa de embriões bovinos duas horas antes da vitrificação aumentou a taxa de eclosão após o reaquecimento (8) . Já, quando a submissão foi realizada 40 minutos antes da vitrificação, não foi verificado qualquer efeito sobre a eclosão.…”

“…dependente do tempo entre a aplicação do estresse e a criopreservação (8) . Entretanto, não existem estudos investigando o efeito gerado pela pressão negativa no desempenho pós-descongelamento de células somáticas, especialmente em palhetas.…”