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Ultrarapid freezing and transfer of mouse morulae
THERIOGENOLOGY
ULTRARAPJD FREEZING AND TRANSFER OF MOUSE MORULAE A. Mezzalira and M.I.B. Rubin Deparmmento de Clfnica de Grandes Animais Universidade Federal de Santa Maria 97119 - Santa Maria, RS - Brazil Mouse morulae (n = 684) were frozen in glycerol 3.0, 4.0 and 5.OM plus 0.25 or OSM lactose, both with and without pre-treatment in a factorial 3x2x2 design. The embryos were exposed for 5 min to O.lml glycerol pre-treatment in the same concentration used for the freezing procedure. When pre-treatment was not used the embryos were directly exposed to the freezing solution and placed in 0.25ml straws in groups of 6-13. The straws were maintained in liquid N2 vapour for 1 min and then plunged into liquid N2 After thawing in a water bath at 37°C for 20 seconds, glycerol was removed by exposure for 5 min to O.lml lactose at the same concentration used in the freezing solution. The embryos were cultured for 48h in modified. Whitten’s medium (Thadani, 1982, J.Exp.Zool. 219: 277- 283) + 20% FCS at 37°C with 5% Ct&. The ‘in vitro’ development was not altered by pre-treatment when only this factor was assessed (R>O.O5). Increasing concentrations of glycerol (3.0, 4.0 and 5.OM) decreased embryo survival (93.1, 81.1 and 69.5 96, respectively), which was more significant with lactose at 0.25M than at 0.5M. A signitkant interaction was observed among glycerol concentration, pre-treatment, and time of assessment. Just after thawing, no difference in embryo development was observed among the three glycerol concentrations. After 24 and 48h of culture, survival rates decreased linearly with glycerol concentrations of 3.OM (93.6 and 88.8%) and 4.OM (76.2 and 73.5%). In 5.OM glycerol, survival rates decreased also with time, but with a quadractic relationship, being respectively 54.2 and 61.4% at 24 and 48h. Variance analysis was performed by split-split plot design. Embryos frozen in 3.OM glycerol + 0.5M lactose, with and without pretreatment, were transferred to one uterine horn, and fresh embryos to the contralateral horn. Results on day 10-14 of gestation are shown below. The data demonstrate that 3.OM glycerol + lactose 0.25M or 0.5M are useful for ultrarapid freezing of mouse embryos.
Treatments
Embryos transferred Implantation sites n n (%)
Normal fetuses n (%)
Frozen without RT*
90
41 (45)
Frozen with PT*
147
84 (57)
27 (30)a 48 (33)a
Control (fresh)
185
107 (58)
94 (51)b
RT* pretreatment; a,b different superscripts denote significant difference by Chisquare test (P
JANUARY
1992 VOL. 37 NO. 1
257
ULTRARAPJD FREEZING AND TRANSFER OF MOUSE MORULAE A. Mezzalira and M.I.B. Rubin Deparmmento de Clfnica de Grandes Animais Universidade Federal de Santa Maria 97119 - Santa Maria, RS - Brazil Mouse morulae (n = 684) were frozen in glycerol 3.0, 4.0 and 5.OM plus 0.25 or OSM lactose, both with and without pre-treatment in a factorial 3x2x2 design. The embryos were exposed for 5 min to O.lml glycerol pre-treatment in the same concentration used for the freezing procedure. When pre-treatment was not used the embryos were directly exposed to the freezing solution and placed in 0.25ml straws in groups of 6-13. The straws were maintained in liquid N2 vapour for 1 min and then plunged into liquid N2 After thawing in a water bath at 37°C for 20 seconds, glycerol was removed by exposure for 5 min to O.lml lactose at the same concentration used in the freezing solution. The embryos were cultured for 48h in modified. Whitten’s medium (Thadani, 1982, J.Exp.Zool. 219: 277- 283) + 20% FCS at 37°C with 5% Ct&. The ‘in vitro’ development was not altered by pre-treatment when only this factor was assessed (R>O.O5). Increasing concentrations of glycerol (3.0, 4.0 and 5.OM) decreased embryo survival (93.1, 81.1 and 69.5 96, respectively), which was more significant with lactose at 0.25M than at 0.5M. A signitkant interaction was observed among glycerol concentration, pre-treatment, and time of assessment. Just after thawing, no difference in embryo development was observed among the three glycerol concentrations. After 24 and 48h of culture, survival rates decreased linearly with glycerol concentrations of 3.OM (93.6 and 88.8%) and 4.OM (76.2 and 73.5%). In 5.OM glycerol, survival rates decreased also with time, but with a quadractic relationship, being respectively 54.2 and 61.4% at 24 and 48h. Variance analysis was performed by split-split plot design. Embryos frozen in 3.OM glycerol + 0.5M lactose, with and without pretreatment, were transferred to one uterine horn, and fresh embryos to the contralateral horn. Results on day 10-14 of gestation are shown below. The data demonstrate that 3.OM glycerol + lactose 0.25M or 0.5M are useful for ultrarapid freezing of mouse embryos.
Treatments
Embryos transferred Implantation sites n n (%)
Normal fetuses n (%)
Frozen without RT*
90
41 (45)
Frozen with PT*
147
84 (57)
27 (30)a 48 (33)a
Control (fresh)
185
107 (58)
94 (51)b
RT* pretreatment; a,b different superscripts denote significant difference by Chisquare test (P
JANUARY
1992 VOL. 37 NO. 1
257