Hollow fiber vitrification provides a novel method for cryopreserving in vitro maturation/fertilization-derived porcine embryos.
In vitro matured (IVM) oocytes have been used to create genetically modified pigs for various biomedical purposes. However, porcine embryos derived from IVM oocytes are very cryosensitive. Developing improved cryopreservation methods would facilitate the production of genetically modified pigs and also accelerate the conservation of genetic resources. We recently developed a novel hollow fiber vitrification (HFV) method; the present study was initiated to determine whether this new method permits the cryopreservation of IVM oocyte-derived porcine embryos. Embryos were created from the in vitro fertilization of IVM oocytes with frozen-thawed sperm derived from a transgenic pig carrying a humanized Kusabira-Orange (huKO) gene. Morula-stage embryos were assigned to vitrification and nonvitrification groups to compare their in vitro and in vivo developmental abilities. Vitrified morulae developed to the blastocyst stage at a rate similar to that of nonvitrified embryos (66/85, 77.6% vs. 67/84, 79.8%). Eighty-eight blastocysts that developed from vitrified morulae were transferred into the uteri of three recipient gilts. All three became pregnant and produced a total of 17 piglets (19.3%). This piglet production was slightly lower, albeit not significantly, than that of the nonvitrification group (27/88, 30.7%). Approximately half of the piglets in the vitrification (10/17, 58.8%) and nonvitrification (15/27, 55.6%) groups were transgenic. There was no significant difference in the growth rates among the piglets in the two groups. These results indicate that the HFV method is an extremely effective method for preserving cryosensitive embryos such as porcine in vitro maturation/fertilization-derived morulae.
Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
Miki
M
Maehara
Hitomi
H
Matsunari
Kasumi
K
Honda
Kazuaki
K
Nakano
Yasuhiro
Y
Takeuchi
Takahiro
T
Kanai
Taisuke
T
Matsuda
Yukina
Y
Matsumura
Yui
Y
Hagiwara
Norihisa
N
Sasayama
Akio
A
Shirasu
Masashi
M
Takahashi
Masahito
M
Watanabe
Kazuhiro
K
Umeyama
Yutaka
Y
Hanazono
Hiroshi
H
Nagashima
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D001755:Blastocyst / Q000378:metabolism; D003433:Crosses, Genetic; D015925:Cryopreservation / Q000295:instrumentation / Q000662:veterinary*; D004477:Ectogenesis; D010064:Embryo Implantation; D020964:Embryo Loss / Q000517:prevention & control; D004624:Embryo Transfer / Q000009:adverse effects / Q000662:veterinary; D005260:Female; D005307:Fertilization in Vitro / Q000009:adverse effects / Q000662:veterinary*; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary*; D050498:Live Birth; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D009028:Morula / Q000166:cytology / Q000378:metabolism; D011247:Pregnancy; D011897:Random Allocation; D012662:Semen Preservation / Q000009:adverse effects; D034421:Sus scrofa / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D058989:Vitrification; D015430:Weight Gain; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein