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Effect of β-mercaptoethanol on development of bovine blastocysts
Theriogenology
204
EFFECT OF B-MERCAPTOETHANOL ON DEVELOPMENT OF BOVINE BLASTOCYSTS J. R. Giles Department of Animal Science Cornell University, Ithaca, New York 14853-4801 USA The in vitro process of blastocyst “hatching” or escape from the zona pellucida is often considered a measure of embryo quality. The proportion of bovine embryos, derived from in vitro matured and fertilized oocytes, that undergo this process often is small. This low proportion of hatched blastocysts may be because bovine embryos are often cultured from the one-cell to the blastocyst stage in the same medium. Since the embryo requirements may be different at various stages of development, we examined effects of media and their components on hatching. In these studies, bovine embryos, derived from in vitro matured and fertilized oocytes, were cultured in KSOM containing 0.1% bovine serum albumin (KSOM/BSA) under a humidified atmosphere of 5% COZ: 5% 0,: 90% NZ at 39” C for the first 10 days of development (Insemination on day 0) and were then allocated to treatment media and cultured for an additional 3 days under a humidified atmosphere of 5% CO2 in air at 39” C. All media contained 100 units/ml of penicillin and 100 pg/ml streptomycin. In study 1, blastocysts (n=45 total) were allocated on day 10 to KSOM/BSA, KSOM + 10% fetal bovine serum (KSOM/FBS), medium 199 + 10% FBS (199/FBS), Menezo’s & + 10% FBS (B2/FBS) or embryonic stem cell (ESC) medium containing Dulbecco’s modified Eagle’s medium (DMEM; no pyruvate, high glucose) + 10 % FBS + 100 PM Bmercaptoethanol (&ME) + 2 mM glutamine + 2 mM MEM non-essential amino acids (NEAA). The percentages of cultured blastocysts that hatched by day 13 for the five media were 11, 0, 56, 44 and 67%, respectively, and were greater (P sO.05) for blastocysts cultured in 199/FBS, BZ/FBS and ESC compared to KSOM/BSA or KSOM/FBS. In study 2, bovine blastocysts (n=81 total) were allocated on day 10 to ESC, DMEM + 0.1% BSA or DMEM + 10% FBS. The percentages of cultured blastocysts that hatched were 68,21 and 12%, respectively, for the three media and were greater (P 10.05) for ESC medium, indicating that one or more components added to DMEM was beneficial for blastocyst hatching. In study 3, variouscomponents normally added to DMEM to prepare ESC medium were selectively eliminated and day 10 blastocysts (n=62 total) were cultured in these modified media. The five media were: ESC, ESC without R-ME, ESC without NEAA, ESC without glutamine or ESC without B-ME, NEAA and glutamine. The percentages of hatched blastocysts by day 13 were 62, 38, 50, 71 and 40%, respectively, and were not different among media. Comparing media with or without D-ME revealed that a greater (P10.05) percentage of blastocysts hatched following culture in the presence of R-ME (59%) than in the absence of DME (36%). These data indicate that embryonic stem cell medium may be useful for the culture of bovine embryos and that B-mercaptoethanol may be an important component of this medium. These studies confirm the work of Takahashi and et al. (1993; Biol. Reprod. 49:228-232) who observed that the inclusion of low-molecular-weight thiol compounds, including B-ME, in culture medium is beneficial for the in vitro development of bovine embryos. In addition, the present studies extend these observations to ESC medium which is simpler than modified TCM 199.
204
EFFECT OF B-MERCAPTOETHANOL ON DEVELOPMENT OF BOVINE BLASTOCYSTS J. R. Giles Department of Animal Science Cornell University, Ithaca, New York 14853-4801 USA The in vitro process of blastocyst “hatching” or escape from the zona pellucida is often considered a measure of embryo quality. The proportion of bovine embryos, derived from in vitro matured and fertilized oocytes, that undergo this process often is small. This low proportion of hatched blastocysts may be because bovine embryos are often cultured from the one-cell to the blastocyst stage in the same medium. Since the embryo requirements may be different at various stages of development, we examined effects of media and their components on hatching. In these studies, bovine embryos, derived from in vitro matured and fertilized oocytes, were cultured in KSOM containing 0.1% bovine serum albumin (KSOM/BSA) under a humidified atmosphere of 5% COZ: 5% 0,: 90% NZ at 39” C for the first 10 days of development (Insemination on day 0) and were then allocated to treatment media and cultured for an additional 3 days under a humidified atmosphere of 5% CO2 in air at 39” C. All media contained 100 units/ml of penicillin and 100 pg/ml streptomycin. In study 1, blastocysts (n=45 total) were allocated on day 10 to KSOM/BSA, KSOM + 10% fetal bovine serum (KSOM/FBS), medium 199 + 10% FBS (199/FBS), Menezo’s & + 10% FBS (B2/FBS) or embryonic stem cell (ESC) medium containing Dulbecco’s modified Eagle’s medium (DMEM; no pyruvate, high glucose) + 10 % FBS + 100 PM Bmercaptoethanol (&ME) + 2 mM glutamine + 2 mM MEM non-essential amino acids (NEAA). The percentages of cultured blastocysts that hatched by day 13 for the five media were 11, 0, 56, 44 and 67%, respectively, and were greater (P sO.05) for blastocysts cultured in 199/FBS, BZ/FBS and ESC compared to KSOM/BSA or KSOM/FBS. In study 2, bovine blastocysts (n=81 total) were allocated on day 10 to ESC, DMEM + 0.1% BSA or DMEM + 10% FBS. The percentages of cultured blastocysts that hatched were 68,21 and 12%, respectively, for the three media and were greater (P 10.05) for ESC medium, indicating that one or more components added to DMEM was beneficial for blastocyst hatching. In study 3, variouscomponents normally added to DMEM to prepare ESC medium were selectively eliminated and day 10 blastocysts (n=62 total) were cultured in these modified media. The five media were: ESC, ESC without R-ME, ESC without NEAA, ESC without glutamine or ESC without B-ME, NEAA and glutamine. The percentages of hatched blastocysts by day 13 were 62, 38, 50, 71 and 40%, respectively, and were not different among media. Comparing media with or without D-ME revealed that a greater (P10.05) percentage of blastocysts hatched following culture in the presence of R-ME (59%) than in the absence of DME (36%). These data indicate that embryonic stem cell medium may be useful for the culture of bovine embryos and that B-mercaptoethanol may be an important component of this medium. These studies confirm the work of Takahashi and et al. (1993; Biol. Reprod. 49:228-232) who observed that the inclusion of low-molecular-weight thiol compounds, including B-ME, in culture medium is beneficial for the in vitro development of bovine embryos. In addition, the present studies extend these observations to ESC medium which is simpler than modified TCM 199.