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The effect of Ca++ levels in the freezing media on in vitro survival of bovine embryos
THERIOGENOLOGY
THE EFFECT OF CA++ LEVELS IN THE FREEZING MEDIA ON -IN VITRO SURVIVAL OF BOVINE EMBRYOS
K. R. Bondioli and P. C. Me&es Granada Genetics, Inc., Marquez, Texas
77865
A total of 86 embryos was collected from 13 Holstein donors as Embryos Were 7-day embryos (day zero equals behavioral estrus). collected in Dulbecco's phosphate-buffered saline (PBS) supplemented with 2% calf serum (CS) and held from time of collection until freezing in PBS enriched with 1 g/l glucose, 36 mg/l sodium pyruvate and 10% CS. All transferable embryos, graded morphologically as excellent to poor, from each donor animal were randomly assigned to Embryos were transferred into Hank's one of three treatments. balanced salts solution containing 0, .30, or .90 mM CaC12 and .47 bovine serum albumin (BSA). Following a 10 min equilibration time embryos were transferred to Hank's balanced salts solution containing After 40 min 0, .30, or .90 mM CaC12, .4$ BSA and 10% glycerol. equilibration with the glycerol solutions embryos were loaded into .25 ml straws. Straws were placed directly into an alcohol bath at -60~ and seeded after l-2 min. After seeding, straws were cooled from -6oc to -28OC at .3OC/min, cooled from -28OC to -35OC at .lOC/min and plunged into liquid nitrogen. Embryos were thawed by plunging straws into a 30°C water bath. Cryoprotectant was removed In the first step embryos were by a 3-step sucrose method. transferred into a solution consisting of a I:1 ratio of a 10% glycerol solution and an isosmotic sucrose solution both of which After 5-7 min, embryos were were prepared in PBS + .4% BSA. transferred to a solution consisting of a I:1 ratio of the isosmotic sucrose solution and PBS + -4% BSA. After 5-7 min, embryos were transferred to PBS + ,496 BSA. All embryos were cultured in Hams FIO + 10% CS at 37OC in a humidified atmosphere of 95% air and 5% C02. Blastocyst expansion and hatching were used as criteria for development. Data were analyzed by chi square procedures. Of 29 embryos frozen in media with no CaC12 5 (17%) developed. When the freezing media contained .30 mM CaC12 7 of 29 (24%) developed and 8 of 28 (29%) developed after being frozen in media While chi square analysis showed no containing .go mM Cac12. significant differences, there was a consistent trend towards reduced in vitro development when the CaC12 level of the freezing media was -reduced. This observation warrants further investigation considering the propensity for some media commonly used for freezing to form a calcium phosphate precipitate when frozen.
JANUARY
1985 VOL. 23 NO. 1
181
THE EFFECT OF CA++ LEVELS IN THE FREEZING MEDIA ON -IN VITRO SURVIVAL OF BOVINE EMBRYOS
K. R. Bondioli and P. C. Me&es Granada Genetics, Inc., Marquez, Texas
77865
A total of 86 embryos was collected from 13 Holstein donors as Embryos Were 7-day embryos (day zero equals behavioral estrus). collected in Dulbecco's phosphate-buffered saline (PBS) supplemented with 2% calf serum (CS) and held from time of collection until freezing in PBS enriched with 1 g/l glucose, 36 mg/l sodium pyruvate and 10% CS. All transferable embryos, graded morphologically as excellent to poor, from each donor animal were randomly assigned to Embryos were transferred into Hank's one of three treatments. balanced salts solution containing 0, .30, or .90 mM CaC12 and .47 bovine serum albumin (BSA). Following a 10 min equilibration time embryos were transferred to Hank's balanced salts solution containing After 40 min 0, .30, or .90 mM CaC12, .4$ BSA and 10% glycerol. equilibration with the glycerol solutions embryos were loaded into .25 ml straws. Straws were placed directly into an alcohol bath at -60~ and seeded after l-2 min. After seeding, straws were cooled from -6oc to -28OC at .3OC/min, cooled from -28OC to -35OC at .lOC/min and plunged into liquid nitrogen. Embryos were thawed by plunging straws into a 30°C water bath. Cryoprotectant was removed In the first step embryos were by a 3-step sucrose method. transferred into a solution consisting of a I:1 ratio of a 10% glycerol solution and an isosmotic sucrose solution both of which After 5-7 min, embryos were were prepared in PBS + .4% BSA. transferred to a solution consisting of a I:1 ratio of the isosmotic sucrose solution and PBS + -4% BSA. After 5-7 min, embryos were transferred to PBS + ,496 BSA. All embryos were cultured in Hams FIO + 10% CS at 37OC in a humidified atmosphere of 95% air and 5% C02. Blastocyst expansion and hatching were used as criteria for development. Data were analyzed by chi square procedures. Of 29 embryos frozen in media with no CaC12 5 (17%) developed. When the freezing media contained .30 mM CaC12 7 of 29 (24%) developed and 8 of 28 (29%) developed after being frozen in media While chi square analysis showed no containing .go mM Cac12. significant differences, there was a consistent trend towards reduced in vitro development when the CaC12 level of the freezing media was -reduced. This observation warrants further investigation considering the propensity for some media commonly used for freezing to form a calcium phosphate precipitate when frozen.
JANUARY
1985 VOL. 23 NO. 1
181