Cryopreservation of Porcine Blastocysts by Vitrification

Yoshino, Junryo; Kojima, Toshiyuki; Shimizu, Morihito; Tomizuka, Tsuneo

doi:10.1006/cryo.1993.1041

Cited by 37 publications

(8 citation statements)

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“…Different vitrification techniques have been applied to porcine embryos so far, with varying success [9][10][11][12][13][14]. The efficiency of vitrification of porcine embryos is affected greatly by the origin and the developmental stage of the embryos: the greatest numbers of piglets from the transfer of vitrified embryos have been obtained from in vivo-derived blastocysts [12,[15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Somfai

Ozawa

Noguchi

et al. 2009

Biology of Reproduction

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We report the successful cryopreservation of in vitro-produced porcine zygotes. Follicular oocytes from prepubertal gilts were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro. At 10 or 23 h after IVF, the oocytes were centrifuged to visualize pronuclei. Zygotes with two or three pronuclei were used for solid surface vitrification (SSV). Survival of vitrified-warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CPA), and untreated (control) zygotes were subjected to IVC for 6 days. Survival and developmental competence did not differ between control and CPA zygotes. The proportion of live zygotes after SSV and warming (93.4%) was similar to that in the controls (100%). Cleavage and blastocyst formation rates of SSV zygotes after vitrification (71.7% and 15.8%, respectively) were significantly lower than those of controls (86.3% and 24.5%, respectively; ANOVA P<0.05). Blastocyst cell numbers of SSV and control embryos were similar (41.2+/-3.4 and 41.6+/-3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. Storage in liquid nitrogen had no effect on the in vitro developmental competence of vitrified zygotes beyond the reduction induced by the vitrification itself. When the embryo culture medium was supplemented with 1 muM glutathione, the rate of development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of control glutathione-treated zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, vitrified zygotes were transferred to five recipients, resulting in three pregnancies and the production of a total of 17 piglets. These data demonstrate that IVM-IVF porcine zygotes can be cryopreserved at the pronuclear stage effectively without micromanipulation-derived delipation, preserving their full developmental competence to term.

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Section: Introductionmentioning

confidence: 99%

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Somfai

Ozawa

Noguchi

et al. 2009

Biology of Reproduction

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“…Vitrification has become available as an alternative method also for cryopreservation of porcine embryos (Yoshino et al, 1993). Blastocysts, expanded blastocysts, and hatched blastocysts have been shown to have high survival rates in culture following vitrification.…”

Section: Introductionmentioning

confidence: 99%

Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts

Fabian

Gjørret²,

Berthelot

et al. 2004

Molecular Reproduction Devel

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Morphological and molecular signs of injury and cell death and subsequent regeneration following vitrification of porcine blastocysts were evaluated by light (LM) and transmission electron microscopy (TEM) as well as TUNEL/propidium iodide (PI) nuclear staining followed by confocal microscopy (CSM). In vivo derived blastocysts were assigned to one of the following four groups: Controls-(1) fixed immediately after collection (C0h) and (2) after 24 hr culture in vitro (C24h) and vitrified embryos-(3) fixed immediately after vitrification and warming (V0h), and (4) after 24 hr of culture upon warming after vitrification (V24h). Observation by LM and TEM showed that the V0h embryos displayed collapse of the blastocoele cavity (BC) and cell swelling, a general distension or shrinkage of mitochondria and massive increase in the amount of vesicles, vacuoles, and secondary lysosomes (SLs). Approximately 2/3 of the V24h embryos had recovered, whereas the remaining 1/3 were degenerated. Recovered embryos displayed almost normal blastocyst morphology, except for a widening of the perivitelline space, accumulation of debris and partial distension of mitochondria, whereas degenerated embryos were disintegrated into a poorly defined mass of cells and debris including cells with abundant degeneration of mitochondria and other organelles. Both recovered and degenerated embryos displayed a persistent abundance of presence of small membrane bounded vesicles, vacuoles, and SLs. Evaluation of TUNEL/PI stained embryos showed only occasional appearance of TUNEL positive nuclei with typical apoptotic morphology in controls (C0h 0.67%, C24h 1.22%) and in the V0h embryos (0.93%). The percentage of apoptotic nuclei in embryos at V24h was significantly higher than in all other groups (2.64%). Vitrified embryos showed significantly increased appearance of DNA fragmented nuclei without typical morphological features of apoptosis (V0h 1.43%, V24h 4.30%) compared with controls (C0h 0.26%, C24h 0.45%). The observed morphological changes and increased DNA fragmentation observed immediately after vitrification and warming probably reflects a direct damaging effect of vitrification. During 24 hr of culture a portion of the embryos was able to regenerate and along with the regenerative process, apoptosis--a possible pathway for elimination of damaged cells--became evident.

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“…The results of the present study show that the postwarm survival rates of embryos vitrified in the presence of sucrose (control media) or trehalose were almost identical. Several porcine embryo vitrification studies have described the use of trehalose as a non-penetrating cryoprotectant (Lim, Quan, Lee, & Kim, 2005;Misumi et al, 2013;Uchikura et al, 2014;Yoshino, Kojima, Shimizu, & Tomizuka, 1993); however, the results presented here indicate that sucrose is equally effective.…”

Section: Discussionmentioning

confidence: 57%

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

Bartolac

Lowe

Koustas

et al. 2018

Animal Science Journal

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The aim of this study was to determine the most efficient vitrification protocol for the cryopreservation of day 7 in vitro produced (IVP) porcine blastocysts. The post-warm survival rate of blastocysts vitrified in control (17% dimethyl sulfoxide + 17% ethylene glycol [EG] + 0.4 mol/L sucrose) and commercial media did not differ, nor did the post-warm survival rate of blastocysts vitrified in medium containing 1,2-propandiol in place of EG. However, vitrifying embryos in EG alone decreased the cryosurvival rate (55.6% and 33.6%, respectively, p < .05). Furthermore, the post-warm survival rates of blastocysts vitrified with either trehalose or sucrose as the non-penetrating cryoprotectant did not differ. There was also no significant difference in post-warm survival of blastocysts vitrified in control (38°C) media and room temperature (22°C) media with extended equilibration times, although when blastocysts were vitrified using control media at room temperature, the post-warm survival rate increased (56.8%, 57.3%, 72.5%, respectively, p < .05). The findings show that most cryoprotectant combinations examined proved equally effective at supporting the post-warm survival of IVP porcine blastocysts. The improved post-warm survival rate of blastocysts vitrified using media held at room temperature suggests that the cryoprotectant toxicity exerted in 22°C media was reduced.

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Cryopreservation of Porcine Blastocysts by Vitrification

Cited by 37 publications

References 0 publications

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Live Piglets Derived from In Vitro-Produced Zygotes Vitrified at the Pronuclear Stage1

Ultrastructure and cell death of in vivo derived and vitrified porcine blastocysts

Effect of different penetrating and non‐penetrating cryoprotectants and media temperature on the cryosurvival of vitrified in vitro produced porcine blastocysts

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