Piglets Produced by Transfer of Vitrified Porcine Embryos after Stepwise Dilution of Cryoprotectants

Kobayashi, S.; Takei, Masami; Kano, Masatoshi; Tomita, Masakatsu; Leibo, S.P.

doi:10.1006/cryo.1997.2056

Cited by 63 publications

(22 citation statements)

References 31 publications

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“…In Method 1, conventional vitrification using a mini-straw (ST) was performed using 0.25-ml crystal straws (IMV Technologies, L'Aigle, France) as described previously [19] with some modifications. Briefly, the embryos were placed in a drop (80 μl) of equilibration solution consisting of PB1 medium with 11% (v/v) ethylene glycol (EG) and 0.5% BSA (11% EG solution) for 5 min at room temperature (20-25 C).…”

Section: Vitrification and Warming Of Embryosmentioning

confidence: 99%

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

Sakagami¹,

Yamamoto²,

Akiyama³

et al. 2010

Journal of Reproduction and Development

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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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Section: Vitrification and Warming Of Embryosmentioning

confidence: 99%

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

Sakagami¹,

Yamamoto²,

Akiyama³

et al. 2010

Journal of Reproduction and Development

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“…Previously, the birth of piglets has been reported after vitrification and warming at the unhatched blastocyst stage [1,2,3,11]. To our knowledge, there is no report of a successful transfer of vitrified/warmed morulae with intact zona pellucida.…”

Section: Introductionmentioning

confidence: 99%

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

et al. 2001

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-In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepesbuffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida. pig / embryo / morula / OPS vitrification / cryopreservationRésumé -Naissance de porcelets après transfert de morulae dans la zone pellucide intacte et vitrification par la méthode OPS. Chez la truie, 5 à 6 jours après l'insémination, il est récolté des embryons aux stades morula et blastocyste associés. Le but de ce travail est de transférer des morulae vitrifiées et d'obtenir des porcelets. Des morulae (200) sont vitrifiées par la méthode OPS après 2 étapes d'équilibration dans un mélange égal d'éthylène glycol, de DMSO et de sucrose dilués dans du TCM199 Hépès + 20 % de sérum de veau nouveau-né (TCM). Pour le réchauffement, les paillettes sont sorties de l'azote et plongées immédiatement dans du TCM + sucrose, l'élimination des cryoprotecteurs se faisant en 3 étapes. Pour évaluer la viabilité des morulae vitrifiées/réchauffées, 10 receveuses reçoivent chacune 20 morulae après transfert chirurgical. Le pourcentage de mises-bas Reprod. Nutr. Dev. 41 (2001) 267-272 267

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“…Vitrification of in vivo derived blastocytes ha s also succeeded in pigs [19]. Vitrification of IVP blastocysts has been conducted in many laboratories, but because of the low quality of IVP embryos no one has yet succeeded in producing piglets after the transfer of vitrified embryos .…”

Section: Important Technologiesmentioning

confidence: 99%

Developmental Competence of Porcine Blastocysts Produced In Vitro

Kikuchi

2004

J. Reprod. Dev.

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Abstract. The establishment of in vitro embryo production (IVP) system in pigs enables us to generate viable embryos with a quality equal to that of in vivo derived embryos. This technology contributes not only to developments in reproductive physiology and agriculture but also to biotechnologies for producing cloned or genetically modified pigs. The birth of piglets from in vitro matured and fertilized embryos at the two-to 4-cell stage was first achieved about 10 years ago, but it was only quite recently that piglets were produced after the transfer of IVP blastocysts. This improvement to the blastocyst stage of the in vitro culture system after in vitro maturation and fertilization can be expected to play a part in the development of an advanced IVP system. Here, we discuss the developmental ability of porcine embryos produced by our improved IVP system and the utilization of this technique in the new biotechnology. Key words: Pig, Embryo, In vitro production, Developmental competence, Piglet (J. Reprod. Dev. 50: [21][22][23][24][25][26][27][28] 2004) n in vitro embryo production (IVP) system includes in vitro maturation (IVM) and in vitro fertilization (IVF) of oocytes, and also in vitro culture (IVC) of the fertilized oocytes or zygotes. The establishment of a porcine IVP system enables us to generate viable embryos as well as in vivo derived embryos with less cost and in less time, and w ill contribute t o res ea rch in repr oductive physiology, agriculture, and biotechnology, including cloning and transgenesis in pigs. The in vitro developmental competence or viability of porcine IVM/IVF oocytes to the blastocyst stage was first confirmed and reported by Matiolli et al. in 1989 [1]. Further, piglets have been born from IVM/IVF embryos after IVC to the two-to 4-cell stage in 1993 [2]. Since then, some laboratories have succeeded in producing piglets from cleaved embryos at the two-to 4-cell stage after IVM/IVF and IVC for 24 to 36 h [3, 4], and from 8-cell to morula-stage embryos after IVM/IVF and IVC for 96 h [5]. However, it was only quite recently that viable piglets were generated after transfer of IVP embryos at blastocyst stage [6,7]. Here, we discuss the developmental ability of porcine embryos produced by this improved IVP system and the u t i l i z a t i o n o f t h i s t e c h n i q u e i n t e r m s o f biotechnology. Current Status of the IVM/IVF System in PigsIVM systems for porcine follicular oocytes have been improved in recent years, emphasizing the importance of the redox state and glutathione content in relation to cysteine in the maturation m e d iu m ( R ev i ew ed b y N ag ai [ 8]) an d t h e synchronization of early meiosis in oocytes [4]. Oxygen tension during IVM is also an important fac to r in cyt o plas m ic m at uri ty fo r i n v i tro development to blastocysts [7]. When cumulus-

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Piglets Produced by Transfer of Vitrified Porcine Embryos after Stepwise Dilution of Cryoprotectants

Cited by 63 publications

References 31 publications

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

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

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida

Developmental Competence of Porcine Blastocysts Produced In Vitro

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