Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods

Abstract

Caprine preantral follicles within ovarian fragments were exposed to or vitrified in the presence of sucrose, dimethyl sulfoxide (DMSO), ethylene glycol (EG), or various combinations thereof. The fragments were cryopreserved by using either a conventional (CV) or a solid-surface vitrification (SSV) protocol, and the cryoprotectants were removed by equilibrating vitrified ovarian fragments in "warming solution" consisting of minimum essential medium and heat-inactivated fetal calf serum (MEM(+)) followed by washes in MEM(+) with or without sucrose. Histological analysis of follicle integrity showed that the percentages of normal follicles in ovarian fragments vitrified in sucrose mixed with EG and/or DMSO (CV method) or mixed with EG or DMSO (SSV method) followed by washes in MEM(+) plus sucrose were similar to those of controls (ovarian fragments fixed without previous vitrification). Unlike for MEM(+) (supplemented or unsupplemented by sucrose) and DMSO followed by washes in the absence of sucrose, the percentages of normal follicles found after exposure to cryoprotectant did not significantly differ from that found after vitrification, indicating that follicular degeneration was attributable to a toxic effect of cryoprotectants and not to the vitrification procedure. The viability of preantral follicles after the CV and SSV procedures was investigated by using calcein-AM and the ethidium-homodimer as "live" and "dead" markers, respectively. In both tested vitrification procedures, the highest percentages of viable follicles were observed when a mixture of sucrose and EG (70.3% for CV and 72.4% for SSV) was used. Preantral follicles were also vitrified (either by CV or SSV) in sucrose and EG and then cultured for 24 h, after which their viability was compared with that of cultured fresh and uncultured vitrified follicles. The viability of these follicles was maintained after SSV, but not after CV. Thus, the viability of caprine preantral follicles can be best preserved after SSV in a mixture of sucrose and EG, followed by washes in medium containing sucrose.

Short-term preservation of canine preantral follicles: Effects of temperature, medium and time

“…Notwithstanding previous studies on preservation of preantral follicles provided important knowledge on this issue, approaches were limited to analysis of morphology, which is not always correlated with the viability of follicles (Santos et al, 2007). Therefore, in the present work, viability assessment using the trypan blue dye exclusion test was employed.…”

Successful in vitro culture of pre-antral follicles derived from vitrified murine ovarian tissue: oocyte maturation, fertilization, and live births

“…Lower concentrations of cryoprotectant are required in freezing media for slow cooling, which reduces the risk of toxic and osmotic damage to cells, but this is insufficient to prevent ice crystal formation jeopardizing cell survival during the freezing procedures (Vajta 2006). Vitrification, though still regarded as a relatively novel freezing method, has recently been reported as an effective alternative method for the cryopreservation of ovarian tissue in various species, including mouse (Wang et al 2009), rat (Deng et al 2009), pig (Gandolfi et al 2006), goat (Santos et al 2007), sheep (Al-aghbari & Menino 2002, Courbiere et al 2006, monkey (Yeoman et al 2005), and human (Keros et al 2009, Zhou et al 2010. It combines a high cooling rate with a high concentration of cryoprotectant in the vitrification media, which rapidly dehydrates the cells and prevents water from precipitating as ice (Vajta 2006).…”

Live offspring from vitrified blastocysts derived from fresh and cryopreserved ovarian tissue grafts of adult mice

“…Using this method, tissue samples are normally placed in a very small volume of vitrification solution and cooled on a sterile metal surface to maximize the cooling rate, and minimize the potential for contamination of samples by LN 2 . Measures of survival of vitrified ovarian tissue after cryopreservation have included morphological examination (histology), viability assessment by viability staining or development in vitro or in vivo culture (Yeoman et al 2005, Santos et al 2007, Aerts et al 2008. There are, however, limited data on the effects on the function of ovarian tissue vitrified using solid surface vitrification systems.…”