New technology for vitrification and field (microscope-free) warming and transfer of small ruminant embryos.
This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.
Isachenko
Vladimir
V
Servicio de Investigación Agroalimentaria, PO Box 727, Zaragoza, Spain. vovaisachenko@yahoo.com
Alabart
Jose Luis
JL
Dattena
Maria
M
Nawroth
Frank
F
Cappai
Pietro
P
Isachenko
Eugenia
E
Cocero
Maria Jesus
MJ
Olivera
Julio
J
Roche
Alberto
A
Accardo
Carla
C
Krivokharchenko
Alexander
A
Folch
Jose
J
D000818:Animals; D000831:Animals, Newborn; D001755:Blastocyst / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary*; D019855:Ethylene Glycol / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary; D005990:Glycerol / Q000494:pharmacology; D007315:Insemination, Artificial / Q000662:veterinary; D007558:Italy; D008297:Male; D011247:Pregnancy; D012756:Sheep / Q000196:embryology / Q000502:physiology*; D013030:Spain
D019855:Ethylene Glycol; D005990:Glycerol