Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush.
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 degrees C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 +/- 3.5%, 106/196) than cooling at 1700 degrees 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 approximately 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.
Yavin
Saar
S
Institute of Animal Science, Volcani Centre, PO Box 6, Bet Dagan 50250, Israel.
Aroyo
Adaya
A
Roth
Zvi
Z
Arav
Amir
A
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D002970:Cleavage Stage, Ovum / Q000166:cytology; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D003451:Cryoprotective Agents; D046149:Embryo Culture Techniques / Q000295:instrumentation / Q000379:methods*; D047108:Embryonic Development; D005260:Female; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009584:Nitrogen; D011247:Pregnancy; D012996:Solutions
D003451:Cryoprotective Agents; D012996:Solutions; D009584:Nitrogen