Improved Survival of Vitrified In Vivo-Derived Porcine Embryos

Self Cite

Abstract. The aim of the present study was to clarify the overall efficiency of porcine somatic cell nuclear transfer (SCNT) by incorporating cryopreservation of the cloned embryos before transfer. The SCNT embryos reconstructed with preadipocytes and in vitro-matured (IVM) oocytes were cultured to harvest morula stage embryos; they were then subjected to delipation (removal of cytoplasmic lipid droplets) and vitrification. After warming and culture, the embryos developing to blastocysts were transferred to recipients to obtain cloned piglets. From 372 reconstructed embryos, 188 (50.5%) reached the morula stage and 117 (31.5%) developed to blastocysts after vitrification. Transfer of 98 (26.3%) morphologically normal blastocysts gave rise to 6 (1.6%) piglets, including 1 stillborn. The efficiency of the cloned piglet production was comparable with that obtained using SCNT embryos without cryopreservation (2.7%, 17/ 635). Here, we demonstrate that porcine somatic cell cloning can be performed without a significant reduction in efficiency even when the SCNT embryos are cryopreserved before transfer. Key words: Pig cloning, Somatic cell nuclear transfer, Vitrification (J. Reprod. Dev. 57: [312][313][314][315][316] 2011) n recent years, pigs have often been used as large laboratory animals in biomedical research [1][2][3][4][5][6][7], and genetically engineered pigs are highly desired in rapidly advancing research areas, such as regenerative medicine, organ transplantation, analysis of intractable genetic disorders and stem cell therapy [for review, see 7]. For the production of genetically engineered pigs, including gene knockout pigs, somatic cell cloning is a key technology [8][9][10][11][12][13][14][15]. Therefore, if a reliable cryopreservation technique for cloned embryos can be established, the production and application of various specially designed pigs will advance further.We conducted the present study to elucidate the conditions that could achieve a high survival rate of cloned porcine embryos after cryopreservation in a stable manner. We previously showed that, for the cryopreservation of porcine IVM-derived embryos, it is effective to combine vitrification and delipation, a process for reducing the amount of lipid droplets in the embryonic cytoplasm [16,17]. We have also confirmed in our previous studies that delipation of porcine in vitro-produced embryos at the morula stage is less injurious than when done at the early cleavage stages or the blastocyst stage [16][17][18][19]. In addition, it was shown that vitrification of delipated porcine embryos at the morula stage results in a high postwarming survival rate [16,18,19]. In conjunction with these studies, we carried out a study of the vitrification of porcine cloned embryos at the morula stage. The overall efficiency of cloned pig production by SCNT with cryopreservation (i.e., how many cloned piglets could be produced from all of the SCNT embryos prepared via cryopreservation) was clarified.In the cryopreservation experiment, the SCNT embryos ...

supporting

confidence: 80%

Cloned Porcine Embryos can Maintain Developmental Ability after Cryopreservation at the Morula Stage

Nakano

Matsunari

Nakayama

et al. 2011

Self Cite

“…The Cryotop method requires a maximum microdroplet volume of 0.1 µl of vitrification solution [13]. The Cryotop method is very effective for cryopreserving embryos and has been used in various mammalian species, including humans [19,32], cattle [28], mice [33], pigs [34,35] and rabbits [36]. Thus, it is appropriate to consider the 0.1 µl maximum volume as standard for MVC methods.…”

Section: Discussionmentioning

confidence: 99%

Hollow Fiber Vitrification: A Novel Method for Vitrifying Multiple Embryos in a Single Device

Matsunari

Maehara

Nakano

et al. 2012

Self Cite

Abstract. Current embryo vitrification methods with proven efficacy are based on the minimum volume cooling (MVC) concept by which embryos are vitrified and rewarmed ultrarapidly in a very small amount of cryopreserving solution to ensure the high viability of the embryos. However, these methods are not suitable for simultaneously vitrifying a large number of embryos. Here, we describe a novel vitrification method based on use of a hollow fiber device, which can easily hold as many as 40 mouse or 20 porcine embryos in less than 0.1 µl of solution. Survival rates of up to 100% were obtained for mouse embryos vitrified in the presence of 15% DMSO, 15% ethylene glycol and 0.5 M sucrose using the hollow fiber vitrification (HFV) method, regardless of the developmental stage of the embryos (1-cell, 2-cell, morula or blastocyst; n = 50/ group). The HFV method was also proven to be effective for vitrifying porcine in vitro-and in vivo-derived embryos that are known to be highly cryosensitive. For porcine embryos, the blastocyst formation rate of in vitro maturation (IVM)-derived parthenogenetic morulae after vitrification (48/65, 73.8%) did not decrease significantly compared with non-vitrified embryos (59/65, 90.8%). Transfer of 72 in vivo-derived embryos vitrified at the morula/early blastocyst stages to 3 recipients gave rise to 29 (40.3%) piglets. These data demonstrate that the HFV method enables simultaneous vitrification of multiple embryos while still adhering to the MVC concept, and this new method is very effective for cryopreserving embryos of mice and pigs. Key words: Cryopreservation, Hollow fiber, Mouse embryos, Pig embryos, Vitrification (J. Reprod. Dev. 58: [599][600][601][602][603][604][605][606][607][608] 2012) E mbryo cryopreservation is widely and routinely used both for research in reproductive biology and for the development of practical applications in animal industries and human reproductive medicine. The cryopreservation of oocytes and embryos is essential for the long-term preservation of valuable genetic resources in experimental and livestock animals [1][2][3][4][5], and the cryopreservation of embryos improves pregnancy rates in assisted reproductive technology applications [6][7][8]. Various protocols have been developed for the cryopreservation of animal embryos, and many are used in research and clinical applications [1,5,9].In recent years, embryo vitrification has been applied to an increasing number of animal species, and high post-cryopreservation embryo viability has been achieved [10,11]. The basic concept of vitrification is as follows: a solution containing a high concentration of a cryoprotective agent (CPA) is rapidly cooled, causing it to transform from the liquid phase to the solid phase without forming ice crystals. As a result, cells suspended in the solution are preserved in an ultra-low temperature glassy (amorphous) material [10,12]. To create this glassy state, a solution containing a high concentration of CPA (normally 4-6 M) needs to be cooled ultrarapidly. Howeve...

“…Therefore, the number of embryos that can be vitrified in one operation has been limited. Ushijima et al [4] reported that the MVC method allows simultaneous vitrification of 8 to 12 embryos. In pigs, it is desirable to cryopreserve larger numbers of embryos together because 15 to 20 embryos should be transferred into a recipient at one time.…”

Section: Discussionmentioning

confidence: 99%

“…In Method 2, the MVC method was conducted using a Cryotop ® and commercial vitrification and warming solutions (VT101 and VT102, Kitazato BioPharma, Shizuoka, Japan) by the method previously reported [3,4]. The embryos were contracted by dehydration in an equilibration solution (ES of VT101, Kitazato BioPharma) and kept at room temperature (25 C) for 5 to 10 min until they regained their volume to some extent.…”

Section: Vitrification and Warming Of Embryosmentioning

confidence: 99%

“…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].…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Sakagami¹,

Yamamoto²,

Akiyama³

et al. 2010

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 ...