Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide

Ishimori, H.; Saeki, Kazuhiro; Inai, Maya; Nagao, Yoshikazu; Itasaka, J.; Miki, Yoshinobu; Seike, Noboru; Kainuma, H.

doi:10.1016/0093-691x(93)90279-e

Cited by 62 publications

(27 citation statements)

References 19 publications

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“…Next they were expelled into 10 µl of vitrification medium for 0.5 min (20% EG, 20% DMSO, 0.6 M sucrose) and loaded into a 0.25-ml plastic straw (IMV, L'Aigle, France) that was preloaded with 10 µl of vitrification medium and 200 µl of 1 M sucrose with an air partition separating the two liquid portions [12]. The straw was immediately heat-sealed and placed on a floating board in liquid nitrogen (LN) vapor for 2 min before plunged and stored in LN for several days.…”

Section: Vitrificationmentioning

confidence: 99%

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Improved Survival of Vitrified In Vivo-Derived Porcine Embryos

Ushijima

Yoshioka

Esaki

et al. 2004

Journal of Reproduction and Development

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Abstract. An efficient cryopreservation protocol for porcine morulae was investigated with three types of vitrification having different cooling rates (Exp. 1). Survival of embryos vitrified after removal of cytoplasmic lipid droplets was also examined by means of the minimum volume cooling (MVC) method (Exp. 2). In Exp. 1, the morula stage embryos were vitrified with a 0.25 ml plastic straw (ST-method), gel loading tip (GLT-method) and the MVC-method, respectively, and stored in liquid nitrogen after which they were warmed in sucrose solutions with cryoprotectants being subsequently removed in a stepwise manner. In Exp. 2, morulae were centrifuged with 7.5 µg/ml cytocharasin B at 12000 × g for 20 min to polarize the cytoplasmic lipid droplets that were then removed from the embryos by micromanipulation (delipation). Both those delipated at the morula stage and the intact embryos at the morula to blastocyst stages were vitrified by the MVC-method. In vitro survival of the vitrified embryos was assessed in both experiments by culturing in NCSU-23 + 10% FCS for 48 h. In vitro developments of vitrified embryos after warming to blastocysts were 20% (6/30) for the ST-method, 39% (18/46) for the GLT-method, and 60% (26/43) for the MVC-method. Embryo survival was further improved by vitrification after delipation (95%, 35/37) compared to intact vitrified morulae (24/42, 57%, P<0.001) and blastocysts (23/31, 74%, P<0.05). Moreover, the number of cells in blastocysts (92 ± 25) derived from the delipated-vitrified morulae was comparable to those derived from intact control non-vitrified embryos (103 ± 31). Our results demonstrate that vitrified porcine morulae have the highest survival when using the MVC-method in conjunction with delipation.

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Section: Vitrificationmentioning

confidence: 99%

“…Three vitrification methods, i.e., straw (ST) [12], gel loading tip (GLT) [13], and minimum volume cooling (MVC) methods [14], were compared in Exp. 1 ( Fig.…”

Section: Vitrificationmentioning

confidence: 99%

Improved Survival of Vitrified In Vivo-Derived Porcine Embryos

Ushijima

Yoshioka

Esaki

et al. 2004

Journal of Reproduction and Development

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“…Day 6.5-8 embryos were biopsied and then vitrified either by a GL-Tip method [27] or by a VSED-straw method [46] with minor modification. Pregnancy rate f o l l o w i n g t r a n s f e r o f s e x -i d e n t i f i e d a n d cryopreserved embryos in GL-Tip vitrification method (46.9%, 61/130) was higher than that in modified VSED-straw method (37.2%, 119/320), and was similar to that of fresh embryos (51.4%, 73/142).…”

Section: Cryopreservation Of Sex-identified Embryosmentioning

confidence: 99%

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

Tominaga

2004

Journal of Reproduction and Development

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Abstract. My research awarded includes contributions to cryopreservation and sexing of bovine embryos produced in vitro and in vivo, as follows; (1) In vivo-derived morulae and blastocysts were cryopreserved in the presence of 10% glycerol, and the embryos were transferred into recipients after two-step dilution of glycerol in straw, with a practically acceptable pregnancy rate. (2) The survival rate of 16-cell stage embryos frozen in the medium with ethylene glycol was higher than that with DMSO or 1,2-propanediol. Addition of linoleic acid-albumin to culture medium enhanced the survival rate of post-thaw bovine 16-cell stage in vitro-produced (IVP) embryos. (3) Polarization of cytoplasmic lipid droplets by centrifugation of 2-cell stage embryos was found effective to increase freezing tolerance in 16-cell stage embryos developed from the centrifuged embryos, because blastomeres of 16-cell stage embryos were mostly lipid-free. (4) The usefulness of gel-loading tip (GLTip) as a container for ultra-rapid vitrification was demonstrated in IVP embryos from 2-cell to blastocyst stages, with a higher in vitro survival than the conventional two-step freezing. (5) PCR analysis for sexing of in vivo-derived Day-7 embryos indicated that male embryos developed faster and graded higher than female embryos. But such correlation between genetic sex and embryonic development was not found in IVP embryos obtained from individual cows. (6) Addition of 0.1-1.0% deproteinized hemodialysate product from calf blood to culture medium increased the producing efficiency of demi-embryos with good quality. Female embryos rather than male embryos required a longer time to repair after bisection. (7) In vivo-derived bovine embryos after biopsy for sexing by PCR analysis and subsequent vitrification using GL-Tips are available to practical use in the field. (8) Introduction of primer extension preamplification-PCR and purification of DNA product before standard sexing PCR of biopsy samples from Day 3-4 in vitro-derived embryos allowed accurate sex determination, and Day-7 blastocysts developed from Day 3-4 embryos were cryopreserved by GLTip vitrification without a loss of their viability. Thus the field application of bovine embryo transfer is in part supported by improvements of technologies in embryo cryopreservation and sex predetermination.

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“…However, vitrification has not been widely adopted by ET practitioners for commercial use in cattle [7,8]. In general, vitrification requires gradual cryoprotectant dilution in a laboratory setting [9,10] because it uses a high concentration of cryoprotectants. Therefore, it is difficult to perform vitrification in the field.…”

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confidence: 99%

In-straw Cryoprotectant Dilution for Bovine Embryos Vitrified Using Cryotop

et al. 2011

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Abstract. The aim of this study was to develop an in-straw dilution method suitable for 1-step bovine embryo transfer of vitrified embryos using the Cryotop vitrification-straw dilution (CVSD) method. The development of embryos vitrified using the CVSD method was compared with those of embryos cryopreserved using in-straw vitrificationdilution (ISVD) and conventional slow freezing, outside dilution of straw (SFODS) methods. In Experiment 1, in vitroproduced (IVP) embryos cryopreserved using the CVSD method were diluted, warmed and exposed to the dilution solution at various times. When vitrified IVP embryos were exposed to the dilution solution for 30 min after warming, the rates of embryos developing to the hatched blastocyst stage after 72 h of culture (62.0-72.5%) were significantly lower (P<0.05) than those of embryos exposed to the solution for 5 and 10 min (82.4-94.3%), irrespective of supplementation with 0.3 M sucrose in the dilution solution. In Experiment 2, the rate of embryos developing to the hatching blastocyst stage after 48 h of culture in IVP embryos cryopreserved using the SFODS method (75.0%) was significantly (P<0.05) lower than those of embryos cryopreserved using the CVSD and ISVD methods (93.2 and 97.3%, respectively). In Experiment 3, when in vivo-produced embryos that had been cryopreserved using the CVSD, ISVD and SFODS methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception and delivery rates among groups. In Experiment 4, when IVP embryos derived from oocytes collected by ovum pick-up that had been cryopreserved using the CVSD and ISVD methods and fresh embryos were transferred to recipient animals, no significant differences were observed in the conception rates among groups. Our results indicate that this simplified regimen of warming and diluting Cryotop-vitrified embryos may enable 1-step bovine embryo transfer without the requirement of a microscope or other laboratory equipment. Key words: Bovine, Conception rate, Cryotop, Embryo transfer, Vitrification (J. Reprod. Dev. 57: [437][438][439][440][441][442][443] 2011) ince the first successful cryopreservation of bovine embryos [1], cryopreservation of bovine embryos has been widely used commercially. A recent worldwide inventory revealed that more than 250,000 bovine in vivo-produced embryos have been used for embryo transfer (ET) after freezing and thawing [2]. However, the pregnancy rate of frozen-thawed embryos is slightly lower than that of fresh embryos [3]. And the pregnancy rate of frozen-thawed in vitro-produced (IVP) embryos is also significantly lower than that of fresh IVP embryos. Therefore, it is necessary to develop an embryo cryopreservation method to obtain higher conception rates. Recent reports have confirmed that vitrification of embryos, especially IVP embryos, is at least as efficient as conventional slow freezing [4][5][6][7]. Vitrification reduces the time commitment and equipment expense associated with cryopreservation compared with con...

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Vitrification of bovine embryos in a mixture of ethylene glycol and dimethyl sulfoxide

Cited by 62 publications

References 19 publications

Improved Survival of Vitrified In Vivo-Derived Porcine Embryos

Improved Survival of Vitrified In Vivo-Derived Porcine Embryos

Cryopreservation and Sexing of <i>In Vivo</i>- and <i>In Vitro</i>-Produced Bovine Embryos for Their Practical Use

In-straw Cryoprotectant Dilution for Bovine Embryos Vitrified Using Cryotop

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