Programmed cell death or apoptosis is associated with changes in K+ concentration in many cell types. Recent studies have demonstrated that two-pore domain K+ (K2P) channels are involved in mouse embryonic development and apoptotic volume decrease of mammalian cells. In cerebellar granule neurons that normally undergo apoptosis during the early developmental stage, TASK-1 and TASK-3, members of K2P channels, were found to be critical for cell death. This study was performed to identify the role of K+ channels in the H2O2-induced or cryo-induced cell death of mouse and bovine embryos. Mouse and bovine two-cell stage embryos (2-cells) exposed to H2O2 for 4 h suffered from apoptosis. The 2-cells showed positive TUNEL staining. Treatment with high concentration of KCl (25mM) inhibited H2O2-induced apoptosis of 2-cells by 19%. Cryo-induced death in bovine blastocysts showed positive TUNEL staining only in the cells near the plasma membrane. Cryoprotectant supplemented with 25 mM KCl reduced apoptosis slightly compared to cryoprotectant supplemented with 5 mM KCl. However, the combination of antioxidants (β-mercaptoethanol) with 25 mM KCl significantly decreased the rate of H2O2-induced and cryo-induced apoptosis compared to treatments with only antioxidants or 25 mM KCl. These results show that blockage of K+ channel efflux for a short-time reduces H2O2- and cryo-induced apoptosis in mouse and bovine embryos. Our findings suggest that apoptosis in mouse and bovine embryos might be controlled by modulation of K+ channels which are highly expressed in a given cell type.

K+ channels are involved in the regulation of a variety of physiological functions, including proliferation, apoptosis and differentiation, in mammalian cells. Our previous study demonstrated that the blockage of K+ channels inhibits mouse early embryonic development. This study was designed to identify the effect of K+ channels during bovine embryonic development. K+ channel blockers (tetraethylammonium (TEA), BaCl2, quinine, ruthenium red and fluoxetine) were added to the culture medium during in vitro fertilization (IVF) for 6 h to first identify the short-term effect of these chemicals. Among K+ channel blockers, fluoxetine, which is used as a selective serotonin reuptake inhibitor, significantly increased the blastocyst formation rate by approximately 6% when compared to control. During the in vitro maturation (IVM) of immature oocytes and the in vitro culture (IVC) of embryos, the oocytes and embryos were exposed to fluoxetine for either a short-term (6 h) or a long-term (24 h) to compare the embryonic development in response to exposure time. The 6 h exposure to fluoxetine during IVM did not affect the blastocyst formation rate, but the rate of blastocyst formation was reduced after the 24 h exposure. On the other hand, embryonic development increased approximately 10% in both groups of embryos exposed to fluoxetine for 6 and 24 h during IVC. Taken together, fluoxetine treatment during IVF and IVC, but not IVM, enhances bovine embryonic development. These results suggest that fluoxetine-modulated signals in oocytes and embryos could be an important factor towards enhancing bovine embryonic development.

This research was investigated the relationship between the number of the transferable embryos and estrus expression rate, BCS (Body Condition Score), which affect the nutritional state of the cow, in Holstein donor cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. When BCS was 2.5, the total number of collected ova was 7.3 + 1.9, which is significantly lower (p<0.05) than the numbers 15.4 + 2.8 and 15.4 + 2.1 that were obtained when BCSs were 2.75 and 3.0, respectively. Whereas the numbers of transferable embryos were 5.2 + 1.4 when BCS was 2.5, which was smaller than the numbers 6.0 + 2.1 and 8.5 + 1.8 that were obtained when BCSs were 2.75 and 3.0, respectively; however, the differences were not significant. As for estrus induction rate, the cow groups whose BCSs were 2.75 and 3.0 showed 100.0% and 95.0%, respectively. Whereas the cow group whose BCS was 2.5 showed 57.1%, and the differences were significant (p< 0.05). As for estrous expression rate, the cow groups whose BCSs were 2.5, 2.75 and 3.0 showed 100.0%, 100.0% and 85.7%, respectively; however, the differences were not significant. According to the result of this research, it is considered that the total number of collected ova and the number of transferable embryos will be affected by the nutritional state before and after in vivo embryo production and superovulation treatment, and that although the mechanism is not clear, poor stockbreeding management and nutritional level would cause the decrease of ovum recovery rate and the number of transferable embryos in high-producing cows. On the other hand, diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.

This research was investigated the relationship, in high-producing Holstein donor cows, between the number of the transferable embryos and the blood serum concentrations of Blood Urea Nitrogen (BUN), glucose and cholesterol, which affect the nutritional state of cows. CIDRs were inserted into the vaginas of twenty two heads of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. The total numbers of ova collected from 3 experimental groups whose blood BUN concentrations were <10 mg/dl, 11~18 mg/dl and mg/dl were 8.9, 12.5 and 19.0, respectively; whereas the numbers of transferable embryos were 5.8 + 1.9, 7.9 + 2.8 and 5.2 + 1.4, respectively. When glucose concentration was <60 mg/dl, the total number of collected ova was 9.9, which was smaller than when the concentration was 60~70 mg/dl or mg/dl. When glucose concentration was 60~70 mg/dl, the number of transferable embryos was 7.1 + 2.4, which was slightly larger than the numbers 6.4 + 2.1 and 6.1 + 1.7 that were obtained when the concentrations were <60 mg/dl and mg/dl, respectively ; however, the differences were not significant (p>0.05). When cholesterol concentrations were <150 mg/dl, 150~200 mg/dl and mg/dl, the total numbers of collected ova were 11.2, 11.3 and 8.6, respectively. Whereas the numbers of transferable embryos were 7.1 + 2.1, 7.3 + 1.9 and 5.6 + 1.3, respectively ; however, the differences were again not significant (p>0.05). The result of this research showed no significant difference in ovum recovery rate and the number of transferable embryos according to major metabolite concentrations in high-producing Holstein donor cows. However, it is considered that the failure of maintaining proper nutritional status would cause the fall in in vivo embryo productivity.