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Hum. Reprod. Advance Access originally published online on January 12, 2009
Human Reproduction 2009 24(4):797-804; doi:10.1093/humrep/den397
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush

Saar Yavin1,2, Adaya Aroyo1,2, Zvi Roth2 and Amir Arav1,3

1 Institute of Animal Science, Volcani Centre, PO Box 6, Bet Dagan 50250, Israel 2 Department of Animal Science, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

3 Correspondence address. E-mail: arav{at}agri.huji.ac.il

BACKGROUND: Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination.

METHODS: An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN.

RESULTS: Ultrarapid cooling of murine embryos at 32 200°C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 ± 3.5%, 106/196) than cooling at 1700°C/min in 0.25 ml straws (10 ± 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains ~5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 ± 3.6%, 99/126) than cryopreservation in 100% VS (31 ± 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 ± 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4–8-cell stage formed blastocysts at rates of 68 ± 4.5 and 60 ± 3.7%, respectively, after vitrification in 87.5% VS.

CONCLUSIONS: This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Key words: blastocyst/cryopreservation/embryo/mouse/vitrification

Submitted on January 8, 2007; resubmitted on May 21, 2008; accepted on June 20, 2008.


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