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Effect of β-mercaptoethanol on developmental competence of fair quality bovine embryos following culture and freezing
Theriogenology
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EFFECT OF/3-MERCAPTOETHANOL ON DEVELOPMENTAL COMPETENCE OF FAIRQUALITY BOVINE EMBRYOS FOLLOWING CULTURE AND FREEZING T. Otoil, 2, N. Koyama 1, K.Yamamoto 1, N. Horikita 1, S. Tachikawa 1, and T. Suzuki 2 1Tokushima Prefectural Beef Cattle and Swine Experiment Station, Anan, Tokushima 774-0047, Japan and 2Department of Veterinary Sciences, Yamaguchi University, Yamaguchi 753-0841, Japan We investigated the effect of g-mercaptoethanol (g-ME) on the survival of fair-quality embryos frozen after in vitro culture for 24 h. Day-7 embryos at the compacted morula to early blastocyst stage, obtained by uterine flushing of supemvulated Japanese Black cows and classified as fairquality, were used in this study. The fair-quality embryos were cultured in 100 ¢tl drop (1-3 embryos/drop) of (A) TCM199 medium with 20% fetal calf serum (TCM199) alone, (B) TCM199 + g-ME (100 ¢tM), (C) Brinster's BMOC-3 medium with 20% fetal calf serum (BMOC) alone, or (D) BMOC + 13-ME at 38.5°C in 5% CO2 and air. Blastocysts that developed from fair-quality embryos following 24 h of culture in each medium were frozen with 1.8 M ethylene glycol (EG). The blastocysts were equilibrated in 1.8 MEG for 20 rain and loaded into 0.25-ml plastic straws at an embryo/straw. The straws were placed in the methanol bath of a programmable freezer at 0°C and then cooled to -6.0°C at 1 °C/rain. They were seeded at -6.0°C, cooled at a rate of 0.3 °C/min to -30°C and plunged into liquid nitrogen. The straws were thawed in air (5 sec) and then in 37°C water (10 sec). As controls, fair-quality embryos at the compacted morula to early blastocyst stage were directly frozen without in vitro culture after collection from superovulated cows, as described above. After freezing and thawing, embryos were cultured with a feeder layer of cumulus cells in the culture medium (TCM199 medium supplemented with 5% fetal calf serum and 5 ktg/ml insulin) for 72 h. The embryo survival was assessed by reexpansion of the blastocoele during a 48 h culture and the embryo development (including the hatching and hatched blastocyst) was determined during a 72 h culture. Some frozen blastocysts from fair-quality embryos following culture in TCM199 + B-ME were transferred into recipient cows by the direct transfer method without diluting the cryoprotectant. As controls, fresh fair-quality embryos without culture and fresh blastocysts from fair-quality embryos cultured in TCM199 + B-ME were transferred nonsurgically to recipient cows. As shown in Table 1, the development rates of frozen-thawed blastocysts from fair-quality embryos cultured with 13-MEwere significantly higher (P<0.05) than those without 13-ME, irrespective of the culture medium. Moreover, the development rates of frozen-thawed blastocysts from fair-quality embryos cultured with (3-ME were significantly higher (P<0.01) than those of the fair-quality embryos directly frozen without culture (non-culture). The pregnancy rates obtained with frozen cultured embryos (5/22, 22.7%) tended to be lower (P>0.05) than those of non-cultured fresh embryos (6/14, 42.9%) and cultured fresh embryos (5/10, 50%). The findings demonstrate that the inclusion of g-ME in pre-freezing culture media has a beneficial effect on the in vitro survival of frozen-thawed blastocysts, while the pregnancy rates of frozen-thawed blastocysts decreased, compared with those of fresh fair-quality embryos with or without culture. Table 1. Developmental competence of frozen-thawed blastocysts developed from fair-quality embryos following 24 h of culture in TCM199 or BMOC with 13-mercaptoethanol Culture medium No. (%) of blastocyts No. (%) of blastocysts No. (%) of blastocysts examined reexpanded hatching and hatched TCM199 20 15 (75.0) 8 (40.0)a, b TCM199 + g-ME 21 18 (85.7) a 15 (71.4) c BMOC 19 10 (52.6) b 5 (26.3) a BMOC + 13-ME 24 20 (83.3) a 16 (66.7)c, b Non-culture* 13 7 (53.8) 2 (15.4) a a,b,CValues in a column with different letters are significantly different (Chi-square, P<0.05). *'Fair-quality embryos were directly frozen without in vitro culture after collection.
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EFFECT OF/3-MERCAPTOETHANOL ON DEVELOPMENTAL COMPETENCE OF FAIRQUALITY BOVINE EMBRYOS FOLLOWING CULTURE AND FREEZING T. Otoil, 2, N. Koyama 1, K.Yamamoto 1, N. Horikita 1, S. Tachikawa 1, and T. Suzuki 2 1Tokushima Prefectural Beef Cattle and Swine Experiment Station, Anan, Tokushima 774-0047, Japan and 2Department of Veterinary Sciences, Yamaguchi University, Yamaguchi 753-0841, Japan We investigated the effect of g-mercaptoethanol (g-ME) on the survival of fair-quality embryos frozen after in vitro culture for 24 h. Day-7 embryos at the compacted morula to early blastocyst stage, obtained by uterine flushing of supemvulated Japanese Black cows and classified as fairquality, were used in this study. The fair-quality embryos were cultured in 100 ¢tl drop (1-3 embryos/drop) of (A) TCM199 medium with 20% fetal calf serum (TCM199) alone, (B) TCM199 + g-ME (100 ¢tM), (C) Brinster's BMOC-3 medium with 20% fetal calf serum (BMOC) alone, or (D) BMOC + 13-ME at 38.5°C in 5% CO2 and air. Blastocysts that developed from fair-quality embryos following 24 h of culture in each medium were frozen with 1.8 M ethylene glycol (EG). The blastocysts were equilibrated in 1.8 MEG for 20 rain and loaded into 0.25-ml plastic straws at an embryo/straw. The straws were placed in the methanol bath of a programmable freezer at 0°C and then cooled to -6.0°C at 1 °C/rain. They were seeded at -6.0°C, cooled at a rate of 0.3 °C/min to -30°C and plunged into liquid nitrogen. The straws were thawed in air (5 sec) and then in 37°C water (10 sec). As controls, fair-quality embryos at the compacted morula to early blastocyst stage were directly frozen without in vitro culture after collection from superovulated cows, as described above. After freezing and thawing, embryos were cultured with a feeder layer of cumulus cells in the culture medium (TCM199 medium supplemented with 5% fetal calf serum and 5 ktg/ml insulin) for 72 h. The embryo survival was assessed by reexpansion of the blastocoele during a 48 h culture and the embryo development (including the hatching and hatched blastocyst) was determined during a 72 h culture. Some frozen blastocysts from fair-quality embryos following culture in TCM199 + B-ME were transferred into recipient cows by the direct transfer method without diluting the cryoprotectant. As controls, fresh fair-quality embryos without culture and fresh blastocysts from fair-quality embryos cultured in TCM199 + B-ME were transferred nonsurgically to recipient cows. As shown in Table 1, the development rates of frozen-thawed blastocysts from fair-quality embryos cultured with 13-MEwere significantly higher (P<0.05) than those without 13-ME, irrespective of the culture medium. Moreover, the development rates of frozen-thawed blastocysts from fair-quality embryos cultured with (3-ME were significantly higher (P<0.01) than those of the fair-quality embryos directly frozen without culture (non-culture). The pregnancy rates obtained with frozen cultured embryos (5/22, 22.7%) tended to be lower (P>0.05) than those of non-cultured fresh embryos (6/14, 42.9%) and cultured fresh embryos (5/10, 50%). The findings demonstrate that the inclusion of g-ME in pre-freezing culture media has a beneficial effect on the in vitro survival of frozen-thawed blastocysts, while the pregnancy rates of frozen-thawed blastocysts decreased, compared with those of fresh fair-quality embryos with or without culture. Table 1. Developmental competence of frozen-thawed blastocysts developed from fair-quality embryos following 24 h of culture in TCM199 or BMOC with 13-mercaptoethanol Culture medium No. (%) of blastocyts No. (%) of blastocysts No. (%) of blastocysts examined reexpanded hatching and hatched TCM199 20 15 (75.0) 8 (40.0)a, b TCM199 + g-ME 21 18 (85.7) a 15 (71.4) c BMOC 19 10 (52.6) b 5 (26.3) a BMOC + 13-ME 24 20 (83.3) a 16 (66.7)c, b Non-culture* 13 7 (53.8) 2 (15.4) a a,b,CValues in a column with different letters are significantly different (Chi-square, P<0.05). *'Fair-quality embryos were directly frozen without in vitro culture after collection.