A protocol used for splitting mouse embryos into two halves.

8-16 cell embryos and early blastocysts were obtained from the oviducts and anterior portion of uterine horns of albino mice at 70 and 90 hr after LH injection respectively. Splitting of embryos was done by using two microtools attached to a micromanipulator unit (Research Instruments Ltd, UK). After bisection, each pair of the half embryos is transferred to a dish containing 2 ml of T-6 medium and cultured in CO2 incubator (at 39 degrees C, 95% RH and 5% CO2 in air mixture). Splitting of blastocysts as compared to 8-16 cell embryos was found difficult (35.48% vs 52.44%, respectively). 38.88% of bisected 8-16 cell embryos and 11.36% of bisected blastocysts developed on 48 hr culture. Information on splitting mouse embryos and their subsequent development in culture are significant in view of using the technique for commercial application and for research in developmental biology of animal embryos.

Survival of mouse embryos after being frozen in glycerol-sucrose mixture.

Random bred female albino mice (6-8 weeks old) were used as a source of embryos. 8- to 16 cell embryos were dehydrated in glycerol-sucrose mixture in 0.25 ml straws at room temperature. Straws were cooled at the rate of 5 degrees C/min to -7 degrees C. Seeding was induced by touching the out side of the straw at -7 degrees C. Straws were further cooled at 0.5 degree C/min down to -35 degrees C and then plunged into liquid N2. Thawing of straws was done by direct transfer into water at 35 degrees C. Frozen-thawed embryos were cultured in a CO2 incubator maintained at 39 degrees C. Out 190 embryos (8-16 cell) initially frozen, 169 (88.94%) were recovered on thawing. 158 (93.5%) out of 169 were apparently normal and used for culture. 75 (47.46%) developed to morulae/early blastocysts and 72 (45.56%) to expanded blastocysts on 24 and 48 hr culture respectively. In conclusion, the incorporation of sucrose in the freezing medium at a concentration of 0.25 M has led us to propose a freezing, thawing and transfer method without dilution of glycerol. The technique being quite simple is worth trying in farm animals where importance of this technique in non-surgical transfer of frozen-thawed embryos will be a boon.

Cryopreservation of mouse embryos at -196 degrees C by vitrification.

Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U. PMSG and 5 I.U. hCG given 48 hr apart. Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS). Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2. After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish. After 3 washings in culture medium the embryos were kept in CO2 incubator for further development. In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture. Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).