Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production.
We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42 °C during warming prevented temperature drops in a medium below 34.0 °C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38 °C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38 °C and 42 °C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.
Animal Breeding and Reproduction Research Division, NARO Institute of Livestock and Grassland Science, Tsukuba, Ibaraki, Japan.
Tamás
T
Somfai
Pathology and Pathophysiology Research Division, National Institute of Animal Health, Tsukuba, Ibaraki, Japan.
Koji
K
Yoshioka
Animal Development and Differentiation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Yamaguchi, Japan.
Fuminori
F
Tanihara
Animal Development and Differentiation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Hiroyuki
H
Kaneko
Animal Development and Differentiation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan.
Junko
J
Noguchi
Graduate School of Veterinary Sciences, Azabu University, Sagamihara, Kanagawa, Japan.
Naomi
N
Kashiwazaki
Food and Fertilizer Technology Center, Taipei, Taiwan.
Takashi
T
Nagai
Animal Development and Differentiation Research Unit, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan; The United Graduate School of Veterinary Science, Yamaguchi University, Yamaguchi, Yamaguchi, Japan.
Kazuhiro
K
Kikuchi
D000818:Animals; D001755:Blastocyst / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods; D004624:Embryo Transfer; D047108:Embryonic Development; D005306:Fertilization; D005307:Fertilization in Vitro / Q000379:methods; D066298:In Vitro Techniques; D009865:Oocytes / Q000166:cytology; D013552:Swine; D058989:Vitrification