103 A NEW PAPER CONTAINER FOR THE VITRIFICATION OF BOVINE EMBRYOS

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 25 mM NaHCO3, 10% (v:v) FBS, 0.22 mM sodium pyruvate, 25 mM gentamycin sulfate, 10 µg mL-1 FSH (Follitropin V; Vetrepharm, Canada) and 1 µg mL-1 estradiol-17ß for 24 h. Matured oocytes were co-cultured with sperm (1–106 mL-1) treated by percoll gradient for 42–44 h. Cleaved embryos were cultured in 50 µL CR1aa medium containing 0.4% BSA for 5 days. Blastocysts were exposed to 5.5 M ethylene glycol in CR1aa medium for 20 s. 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 (~5 µL) drop containing 10 blastocysts onto a EM grid and immediate plunging into LN2; or 3) transfer of a (~5 µL) drop containing 10 blastocysts onto a piece of weighing paper (5 mm by 5 mm; VWR, West Chester, PA, USA) and immediate plunging into LN2. Straws were thawed by holding in air for 10 s and then transfer into 37°C water. The embryos were recovered from the straw and transferred into a solution of 0.5 M sucrose in CR1aa at 25°C for 1 min. EM grids and paper containers were warmed by transfer into 3 mL of a solution of 0.5 M sucrose in CR1aa medium at 25°C for 1 min. Embryos were then diluted serially by transfer into 0.25 and then 0.125 M 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

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