title: Efficient cryopreservation of mouse embryos by modified droplet vitrification (MDV).
abstract: The aim of this study was to assess modified droplet vitrification (MDV) for the cryopreservation of early developmental mouse embryos. Mouse embryos were equilibrated in holding solution for 3 min followed by immersion in vitrification solution for 30-45 s, and then three embryos per 3-μL vitrification droplet were directly dropped into liquid nitrogen. Vitrified embryos were warmed to examine their developmental potential both in vitro and in vivo. The results demonstrated that MDV vitrified and warmed embryos had a survival rate of 98.1-99.6% (P>0.05); however, blastocyst development post warming and culture in vitro demonstrated that vitrified 4-celled, 8-celled, 16-celled, morulae, and blastocyst embryos had significant higher developmental potentials (94.7-99.5%) than those from zygotes (9.2%) and 2-celled embryos (85.7%) (P<0.05). Compared to CryoLoop and CryoTech vitrification, MDV showed similar results with regards to rates of survival, blastocyst development, but with the higher hatching rate (76.1% vs. 64.0-67.3%) (P<0.05). Cryopreservation by MDV resulted in a similar blastocyst developmental potential in 4-celled and 16 celled embryos from ICR (94.7-99.5%), C57BL/6J (94.7-96.4%), and their crossbred F1 strain (97.9-98.9%) (P>0.05). After embryo transfer of vitrified ICR embryos from 4-celled, 16-celled, morulae and blastocyst stage, 40.7-43.7% of the embryos developed into live offspring (P>0.05), but MDV vitrification resulted in the highest birth rate (43.8%) compared to CryoLoop (38.3%) and CryoTech (35.4%) (P<0.05), when 4-celled mouse embryos were used for vitrification. Our study clearly demonstrated that MDV is the most efficient vitrification to cryopreserve embryos at least 4-celled and advanced stages, which can be used to preserve important mouse genomes from different strains and different developmental stages.
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
authors.forename: Liyou
authors.initials: L
authors.lastname: An
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
authors.forename: Shiwei
authors.initials: S
authors.lastname: Chang
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
authors.forename: Yeshu
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Li
authors.affiliation: Lannuo Biotechnologies Wuxi Inc., Wuxi 214000, PR China.
authors.forename: Bowen
authors.initials: B
authors.lastname: Xu
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China.
authors.forename: Fenli
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Lannuo Biotechnologies Wuxi Inc., Wuxi 214000, PR China.
authors.forename: Lan
authors.initials: L
authors.lastname: Yang
authors.affiliation: ARSIAL, Rome 00162, Italy.
authors.forename: Giorgio Antonio
authors.initials: GA
authors.lastname: Presicce
authors.affiliation: Jiangsu Key Laboratory for Molecular and Medical Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing 210046, PR China; Renova Life Inc., College Park, MD 20742, USA. Electronic address: fuliangd@njnu.edu.cn.
authors.forename: Fuliang
authors.initials: F
authors.lastname: Du
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D015925:Cryopreservation / Q000379:methods*; D046149:Embryo Culture Techniques; D004624:Embryo Transfer; D047108:Embryonic Development; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D009028:Morula / Q000166:cytology; D058989:Vitrification; D015053:Zygote / Q000166:cytology
keywords: Birth rate; CryoLoop; CryoTech; Cryopreservation; Developmental potential; Embryo transfer; Embryos; Modified droplet vitrification; Mouse