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Survival of mouse morulae vitrified in media containing antifreeze protein type 1
Theriogenology
349
SURVIVAL OF MOUSE MORULAE VITRIFIED lN MEDIA CONTAINING ANTIFREEZE PROTEIN TYPE 1 B.Leboeuf’, W.M.C.Maxwell* and G.Evans* ’ INRA-SEIA 86480 Rouille, France. * Department of Animal Science, University of Sydney, NSW 2006, Australia. Antifreeze proteins (AFP) help prevent Antarctic fishes from freezing at low temperature. Vitrification is a rapid method for low temperature preservation of embryos. The aim of this study was to evaluate the effect of AFP type I on survival and in vitro development of preimplantation mouse embryos vitrified in a solution of ethylene glycol and glycerol. Morulae were obtained from superovulated inbred Quackenbush Swiss mice by flushing the reproductive tracts with M2 medium (Quinn P. et al., 1982, J.Repro.Fert., 66, 161-168). The embryos were transferred to Kasai culture medium (Kasai M. et al., 1978, Jap. J. Anim. Repro., 24, 19-24), at 37°C under 5% CO*, then randomly allocated to treatments. The basic vitrification solution was VSll: 3.5M ethylene glycol+3.5M glycerol (Ali J. and Shelton J.N., 1993, J. Reprod. Fert., 98, 459-465), + AFP type 1 (A/P Proteins Pty Ltd). Toxicity of the AFP was first tested by exposure of embryos to M2 medium containing 0 (control), 0. I, 1.O, or 10 mg/ml of AFP for 30 min. at room temperature, after which embryos were washed twice and cultured in Kasai medium for 48h (37°C 5% COz). The experiment comprised a control and 3 vitrification treatments: VS 11, VS 11+AFP (10 mg AFP/ml), VS 11+AFP+EXP (10 mg AFP/ml after embryo exposure to 1 mg AFP/ml M2 medium at room temperature for 10 min). VSl 1 solution preparation, vitrification and thawing procedures were performed according to the method of Ali & Shelton. After warming, embryos were transferred to Kasai medium and cultured for 48h (37°C 5% CO*). No toxic effect of AFP type 1 was observed; morulae were not affected during in vitro culture by exposure to the different doses of AFP at room temperature. Compared with the control group, vitrification reduced embryo survival rate and number of embryos developing to hatched blastocysts after vitrification and re-warming (Table 1). Prolonged exposure to VSl1 supplemented with AFP type I before vitrification did not improve the post-warming in vitro development rate. These data suggest that AFP type 1 has no protective effect on mouse morulae during vitrification, although previous results from Rubinsky et al.(l992, Cryobiology, 29, 69-79) and Shaw J. (1994, Cryobiology, 31, 570) indicate that factors such as type of AFP, purity, concentration and exposure time of AFP could affect the efficacy of AFPs during vitrification. Table 1. In vitro survival of preimplantation morulae stage embryos after vitrification in medium VS 11 suplemented with AFP type I (4 replicates). Treatment Control VSll VSll + AFP VSll +AFP+EXP
n embryos 65 51 49 48
Survival rate (%) after vitrification : Hatched Blast0 48 h survival 93.8 a 72.5 b 61.2b 70.8 b
“b Values in the same column with different superscripts differ (PcO.01)
78.5 52.9 40.8 43.8
a b b b
349
SURVIVAL OF MOUSE MORULAE VITRIFIED lN MEDIA CONTAINING ANTIFREEZE PROTEIN TYPE 1 B.Leboeuf’, W.M.C.Maxwell* and G.Evans* ’ INRA-SEIA 86480 Rouille, France. * Department of Animal Science, University of Sydney, NSW 2006, Australia. Antifreeze proteins (AFP) help prevent Antarctic fishes from freezing at low temperature. Vitrification is a rapid method for low temperature preservation of embryos. The aim of this study was to evaluate the effect of AFP type I on survival and in vitro development of preimplantation mouse embryos vitrified in a solution of ethylene glycol and glycerol. Morulae were obtained from superovulated inbred Quackenbush Swiss mice by flushing the reproductive tracts with M2 medium (Quinn P. et al., 1982, J.Repro.Fert., 66, 161-168). The embryos were transferred to Kasai culture medium (Kasai M. et al., 1978, Jap. J. Anim. Repro., 24, 19-24), at 37°C under 5% CO*, then randomly allocated to treatments. The basic vitrification solution was VSll: 3.5M ethylene glycol+3.5M glycerol (Ali J. and Shelton J.N., 1993, J. Reprod. Fert., 98, 459-465), + AFP type 1 (A/P Proteins Pty Ltd). Toxicity of the AFP was first tested by exposure of embryos to M2 medium containing 0 (control), 0. I, 1.O, or 10 mg/ml of AFP for 30 min. at room temperature, after which embryos were washed twice and cultured in Kasai medium for 48h (37°C 5% COz). The experiment comprised a control and 3 vitrification treatments: VS 11, VS 11+AFP (10 mg AFP/ml), VS 11+AFP+EXP (10 mg AFP/ml after embryo exposure to 1 mg AFP/ml M2 medium at room temperature for 10 min). VSl 1 solution preparation, vitrification and thawing procedures were performed according to the method of Ali & Shelton. After warming, embryos were transferred to Kasai medium and cultured for 48h (37°C 5% CO*). No toxic effect of AFP type 1 was observed; morulae were not affected during in vitro culture by exposure to the different doses of AFP at room temperature. Compared with the control group, vitrification reduced embryo survival rate and number of embryos developing to hatched blastocysts after vitrification and re-warming (Table 1). Prolonged exposure to VSl1 supplemented with AFP type I before vitrification did not improve the post-warming in vitro development rate. These data suggest that AFP type 1 has no protective effect on mouse morulae during vitrification, although previous results from Rubinsky et al.(l992, Cryobiology, 29, 69-79) and Shaw J. (1994, Cryobiology, 31, 570) indicate that factors such as type of AFP, purity, concentration and exposure time of AFP could affect the efficacy of AFPs during vitrification. Table 1. In vitro survival of preimplantation morulae stage embryos after vitrification in medium VS 11 suplemented with AFP type I (4 replicates). Treatment Control VSll VSll + AFP VSll +AFP+EXP
n embryos 65 51 49 48
Survival rate (%) after vitrification : Hatched Blast0 48 h survival 93.8 a 72.5 b 61.2b 70.8 b
“b Values in the same column with different superscripts differ (PcO.01)
78.5 52.9 40.8 43.8
a b b b