Viability of Porcine Embryos after Vitrification Using Water-soluble Pullulan Films

Abstract

Abstract. The efficiency of a porcine embryo vitrification method that uses water-soluble films of pullulan, a naturallyoccurring polysaccharide polymer, was compared with two other types of vitrification methods using different devices and solutions for vitrification and warming. Blastocysts collected in vivo and vitrified by the conventional straw (ST), Cryotop ® (MVC) or pullulan film vitrification (PFV) methods were stored in liquid nitrogen for a certain period of time, after which the cryoprotective agents were removed by stepwise dilution. Fresh embryos were used as controls for the non-vitrification group. The vitrified-warmed embryos were incubated in TCM199 with 0.1 mM β-mercaptoethanol and 20% fetal bovine serum for 24 h at 38.5 C in humidified air with 5% CO2 to evaluate their viability. The survival rate of embryos in the ST group (48.3%) was significantly lower than that of those in the MVC (70.7%), PFV (79.0%) and nonvitrification (94.4%) groups. The oxygen consumption rate after vitrification was significantly lower than that before vitrification in the ST group, but was not significantly different in the MVC and PFV groups. Both the oxygen consumption rates of embryos after warming and the live cell numbers in the ST group were lower than those in the MVC group, while they did not differ significantly between the PFV and MVC groups. There was a correlation between the oxygen consumption rate and the number of live cells in vitrified embryos after warming. Our results demonstrated that in vivo-derived porcine embryos could be vitrified using pullulan films. Key words: Oxygen consumption rate, Porcine embryo, Pullulan film, Vitrification (J. Reprod. Dev. 56: [279][280][281][282][283][284] 2010) reservation of porcine embryos is important for increasing the effective use of high-quality genetic resources, preventing disease transmission via animals and allowing low-cost transportation of pigs. Due to the extremely high sensitivity of porcine embryos to low temperature, it has been difficult with slow freezing methods to achieve conception rates and litter sizes equivalent to those achieved with artificial insemination [1,2]. However, it has recently become possible to produce piglets from cryopreserved embryos by using ultra-rapid vitrification methods, such as the minimum volume cooling (MVC) [3,4], open pulled straw [5], microdroplet [6] and metal mesh vitrification [7] methods.Pullulan, which is a neutral polysaccharide polymer also known as α-1,4-; α-1,6-glucan, is made from starch and consists of maltotriose units linked in an orderly manner. It has been reported that murine morulae placed on a pullulan film could be vitrified [8]. In general, stepwise dilution is required after warming of vitrified embryos, since vitrification requires a high concentration of cryoprotective agents (CPAs), which makes it difficult, due to their toxicity, to warm the embryos in a straw for direct transfer to recipients. As the pullulan film is soluble in warm water, the vitrification solution can be diluted ...

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