Vitrification has been used to eliminate ice crystal formation during the cryopreservation of mammalian embryos. However, this method may introduce some problems such as loss of eggs during cryopreservation (EM grid) and damage to the zona pellucida. This study examined an alternative container (paper) for the vitrification of in vitro-produced bovine blastocysts. Bovine oocytes were aspirated from slaughterhouse ovaries and cultured in TCM-199 supplemented with 25mM NaHCO3, 10% (v:v) FBS, 0.22mM sodium pyruvate, 25mM gentamycin sulfate, 10μgmL−1 FSH (Follitropin V; Vetrepharm, Canada) and 1μgmL−1 estradiol-17β for 24h. Matured oocytes were co-cultured with sperm (1–106mL−1) treated by percoll gradient for 42–44h. Cleaved embryos were cultured in 50μL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5M ethylene glycol in CR1aa medium for 20s. The blastocyst suspensions were vitrified by one of three methods: 1) aspiration into a 0.25-mL plastic straw (10 embryos/straw), heat sealing and immediate plunging into LN2; 2) transfer of a (∼5μL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (∼5μL) drop containing 10 blastocysts onto a piece of weighing paper (5mm by 5mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5M sucrose in CR1aa at 25°C for 1min. EM grids and paper containers were warmed by transfer into 3mL of a solution of 0.5M sucrose in CR1aa medium at 25°C for 1min. Embryos were then diluted serially by transfer into 0.25 and then 0.125M sucrose solutions (1-min steps), and then rinsed and cultured in CR1aa medium supplemented with 10% FBS. After thawing, the recovery rates of embryos from EM grids, straws and paper containers were not significantly different (Table 1). Broken zonae pellucidae were observed after thawing of embryos recovered from straws and EM grids, but not from the paper container. The survival rates of blastocysts cryopreserved on EM grids and paper containers (respectively, 78.1 and 77.1%) were significantly higher (P<0.05) than that of straws (52.1%). The in vivo developmental potential of blastocysts vitrified on EM grids and paper containers was assessed by the transfer of, respectively, 102 and 3 thawed embryos into recipient cows. Pregnancy rates were, as anticipated, 28 and 67%. These results suggest that paper may be an inexpensive and useful container for the cryopreservation of mammalian embryos.
Table 1
The viability of vitrifield-thawed bovine embryos using various containers
Strategies for Highly Efficient Rabbit Sperm Cryopreservation
