Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts.
BACKGROUND
Vitrification using the cryoloop procedure was evaluated for preservation of non-human primate blastocysts by comparing survival results from two different cryoprotectant mixtures with prior results from controlled rate cooling.


METHODS
Rhesus monkey blastocysts were produced by intracytoplasmic sperm injection of mature oocytes from cycling females stimulated with recombinant human hormones. Morphologically well-formed blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and 3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 micromol/l Ficoll in TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid nitrogen, the removal of cryoprotectants was accomplished in the presence of a 3-step series of decreasing sucrose concentrations in TH20. Surviving embryos were co-cultured on buffalo rat liver cells.


RESULTS
Of 16 blastocysts vitrified via Procedure A, 38% survived with minimal lysis and only one hatched in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85% survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow cooling, 36% survived and 6% hatched. Transfer into three recipients, each with two embryos vitrified with Procedure B, resulted in a successful twin-term pregnancy.


CONCLUSIONS
Modified cryoloop vitrification with a final solution of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.
Yeoman
R R
RR
Andrology/Embryology Laboratory, Department of Obstetrics/Gynecology, Oregon Health Sciences University, Portland, Oregon 97201, USA. yeomanr@ohsu.edu
Gerami-Naini
B
B
Mitalipov
S
S
Nusser
K D
KD
Widmann-Browning
A A
AA
Wolf
D P
DP
D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004338:Drug Combinations; D004624:Embryo Transfer; D019855:Ethylene Glycol; D005260:Female; D005990:Glycerol; D008253:Macaca mulatta; D011247:Pregnancy; D018873:Pregnancy Rate; D051381:Rats; D011915:Rats, Inbred BUF; D012996:Solutions
D003451:Cryoprotective Agents; D004338:Drug Combinations; D012996:Solutions; D019855:Ethylene Glycol; D005990:Glycerol