- Email: [email protected]
Nonsurgical transfer and the survival of frozen-thawed bovine embryos supplemented with raffinose
THERIOGENOLOGY
NONSURGICAL TRANSFER AND THE SURVIVAL OF FROZEN-THAWED BOVINE EMBRYOS SUPPLEMENTED WITH RAFFINOSE D.W. Richards, J.D. Sikes and C.N. Murphy Department of Dairy Science 162 Animal Science Research Center University of Missouri Columbia, MO 65211 One hundred and sixty-seven bovine embryos were frozen in two types of media. Media in Treatment A contained modified phosphate buffered saline (PBS) with 20% raffinose (20 gm/lOO), 20% newborn heat-treated calf serum, 1.0 mM Hepes buffer and 1.4 M glycerol. Media in Treatment B was similar but without raffinose. Embryos were prepared for freezing by the addition of glycerol in three steps; .46 M, .93 M and 1.4 M at 10 min. intervals, for both treatments. Embryos were loaded in .25 ml plastic straws and were frozen in a wide-mouth liquid nitrogen tank using a thermos containing ethyl alcohol and a source of bubbling nitrogen gas. The cooling rate is controlled by the amount of ethyl alcohol, liquid nitrogen and the opening of the thermos. The straws were submerged in an ethyl alcohol bath (310 ml) with a portion of the column (1 cm) being exposed to the nitrogen vapor, which causes spontaneous crystallization and automatic seeding of the rest of the column containing the embryo upon sufficient cooling. Straws were allowed to equilibriate at seeding temperature for 5 to 10 min. at -6'C. Embryos were cooled at a rate of 0.3'Cfmin. to -35"C, followed by rapid cooling from 35'C to -8O"C, then plunged into liquid nitrogen (-196°C). The straws were thawed in 35-37°C water. Concentration of glycerol was decreased by pipetting the embryos into .93 M glycerol + .3 M sucrose-enriched PBS for 5 min., .46 M glycerol + .3 M sucroseenriched PBS for 5 min. and finally into enriched PBS solution. Survival of bovine embryos following freezing and thawing: In Vitro -Developed B;;;;;c,st I;;)
Treatment A (raffinose + glycerol) B (glycerol) TOTALS:
22140
(55)
50184
(60)
In Vivo -Pregnancies/ Transfers (X) 18140 (45) 14143 (33) -32183
(39)
Embryos at the morula stage did not tolerate freezing and thawing as well as those at the blastocyst stage, 49% vs. 77% (P ~.05), respectively, as evident by hatching. The survival of bovine embryos was increased in both -In Vitro and -In Vivo in treatments containing raffinose, but was not significantly different from controls.
JANUARY
1988 VOL.
29 NO.
1
295
NONSURGICAL TRANSFER AND THE SURVIVAL OF FROZEN-THAWED BOVINE EMBRYOS SUPPLEMENTED WITH RAFFINOSE D.W. Richards, J.D. Sikes and C.N. Murphy Department of Dairy Science 162 Animal Science Research Center University of Missouri Columbia, MO 65211 One hundred and sixty-seven bovine embryos were frozen in two types of media. Media in Treatment A contained modified phosphate buffered saline (PBS) with 20% raffinose (20 gm/lOO), 20% newborn heat-treated calf serum, 1.0 mM Hepes buffer and 1.4 M glycerol. Media in Treatment B was similar but without raffinose. Embryos were prepared for freezing by the addition of glycerol in three steps; .46 M, .93 M and 1.4 M at 10 min. intervals, for both treatments. Embryos were loaded in .25 ml plastic straws and were frozen in a wide-mouth liquid nitrogen tank using a thermos containing ethyl alcohol and a source of bubbling nitrogen gas. The cooling rate is controlled by the amount of ethyl alcohol, liquid nitrogen and the opening of the thermos. The straws were submerged in an ethyl alcohol bath (310 ml) with a portion of the column (1 cm) being exposed to the nitrogen vapor, which causes spontaneous crystallization and automatic seeding of the rest of the column containing the embryo upon sufficient cooling. Straws were allowed to equilibriate at seeding temperature for 5 to 10 min. at -6'C. Embryos were cooled at a rate of 0.3'Cfmin. to -35"C, followed by rapid cooling from 35'C to -8O"C, then plunged into liquid nitrogen (-196°C). The straws were thawed in 35-37°C water. Concentration of glycerol was decreased by pipetting the embryos into .93 M glycerol + .3 M sucrose-enriched PBS for 5 min., .46 M glycerol + .3 M sucroseenriched PBS for 5 min. and finally into enriched PBS solution. Survival of bovine embryos following freezing and thawing: In Vitro -Developed B;;;;;c,st I;;)
Treatment A (raffinose + glycerol) B (glycerol) TOTALS:
22140
(55)
50184
(60)
In Vivo -Pregnancies/ Transfers (X) 18140 (45) 14143 (33) -32183
(39)
Embryos at the morula stage did not tolerate freezing and thawing as well as those at the blastocyst stage, 49% vs. 77% (P ~.05), respectively, as evident by hatching. The survival of bovine embryos was increased in both -In Vitro and -In Vivo in treatments containing raffinose, but was not significantly different from controls.
JANUARY
1988 VOL.
29 NO.
1
295