Bulk vitrification of in vitro produced bovine zygotes without reducing developmental competence to the blastocyst stage. 2022

Tamás Somfai, and Kazuko Ogata, and Kumiko Takeda, and Yuji Hirao
Division of Dairy Cattle Feeding and Breeding Research, Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, 305-0901, Japan. Electronic address: somfai@affrc.go.jp.

Cryopreservation of mammalian zygotes can be advantageous since it enables their flexile use in time and space for alternative purposes such as genome editing. Here we report a simple, quick and inexpensive vitrification protocol for in vitro produced bovine zygotes which enables their bulk preservation. Slaughterhouse-derived oocytes were subjected to in vitro maturation and fertilization (IVF). Ten h after IVF, cumulus-enclosed zygotes were equilibrated in 2% (v/v) ethylene glycol + 2% (v/v) propylene glycol for 13-15 min then vitrified in groups of 52-100 in 2 μL microdrops of 17.5% (v/v) ethylene glycol + 17.5% (v/v) propylene glycol supplemented with 0.3 M sucrose and 50 mg/mL polyvinylpyrrolidone. The presence of cumulus cells is important for the success of the process. Therefore, we applied a modified IVF protocol using a short (30 min) co-incubation interval which allowed zygote culture with attached cumulus cells until vitrification and even reduced polyspermy rates without affecting the total fertilization rate. Vitrified zygotes were similar to their non-vitrified counterparts in terms of survival, post-warming development to the blastocyst stage and blastocyst quality measured by cell numbers and cryo-survival. In conclusion, our vitrification protocol integrated with the modified IVF system enabled the quick cryopreservation of bovine zygotes in large groups without reducing their developmental competence to the blastocyst stage.

UI MeSH Term Description Entries
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D009865 Oocytes Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM). Ovocytes,Oocyte,Ovocyte
D001755 Blastocyst A post-MORULA preimplantation mammalian embryo that develops from a 32-cell stage into a fluid-filled hollow ball of over a hundred cells. A blastocyst has two distinctive tissues. The outer layer of trophoblasts gives rise to extra-embryonic tissues. The inner cell mass gives rise to the embryonic disc and eventual embryo proper. Embryo, Preimplantation,Blastocysts,Embryos, Preimplantation,Preimplantation Embryo,Preimplantation Embryos
D002417 Cattle Domesticated bovine animals of the genus Bos, usually kept on a farm or ranch and used for the production of meat or dairy products or for heavy labor. Beef Cow,Bos grunniens,Bos indicus,Bos indicus Cattle,Bos taurus,Cow,Cow, Domestic,Dairy Cow,Holstein Cow,Indicine Cattle,Taurine Cattle,Taurus Cattle,Yak,Zebu,Beef Cows,Bos indicus Cattles,Cattle, Bos indicus,Cattle, Indicine,Cattle, Taurine,Cattle, Taurus,Cattles, Bos indicus,Cattles, Indicine,Cattles, Taurine,Cattles, Taurus,Cow, Beef,Cow, Dairy,Cow, Holstein,Cows,Dairy Cows,Domestic Cow,Domestic Cows,Indicine Cattles,Taurine Cattles,Taurus Cattles,Yaks,Zebus
D003451 Cryoprotective Agents Substances that provide protection against the harmful effects of freezing temperatures. Cryoprotective Agent,Cryoprotective Effect,Cryoprotective Effects,Agent, Cryoprotective,Agents, Cryoprotective,Effect, Cryoprotective,Effects, Cryoprotective
D005307 Fertilization in Vitro An assisted reproductive technique that includes the direct handling and manipulation of oocytes and sperm to achieve fertilization in vitro. Test-Tube Fertilization,Fertilizations in Vitro,In Vitro Fertilization,Test-Tube Babies,Babies, Test-Tube,Baby, Test-Tube,Fertilization, Test-Tube,Fertilizations, Test-Tube,In Vitro Fertilizations,Test Tube Babies,Test Tube Fertilization,Test-Tube Baby,Test-Tube Fertilizations
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D015053 Zygote The fertilized OVUM resulting from the fusion of a male and a female gamete. Fertilized Ovum,Ovum, Fertilized,Fertilized Egg,Egg, Fertilized,Eggs, Fertilized,Fertilized Eggs,Zygotes
D015925 Cryopreservation Preservation of cells, tissues, organs, or embryos by freezing. In histological preparations, cryopreservation or cryofixation is used to maintain the existing form, structure, and chemical composition of all the constituent elements of the specimens. Cryofixation,Cryonic Suspension,Cryonic Suspensions,Suspension, Cryonic
D058989 Vitrification The transformation of a liquid to a glassy solid i.e., without the formation of crystals during the cooling process. Glass Transition,Glass-Liquid Transition,Liquid-Glass Transition,Glass Liquid Transition,Liquid Glass Transition,Transition, Glass,Transition, Glass-Liquid,Transition, Liquid-Glass

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