Reactive oxygen species (ROS) are the main causes of cell damage in bovine embryos in vitro. Folic acid (FA) is an antioxidant that protects cells from ROS. We studied the effect of the addition of FA to maturation and culture media on development of bovine blastocysts and their survival rate after freeze-thawing. Cell-oocyte complexes (COC) were allowed to mature in HEPES (25 mM)-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS), 0.02 AU mL−1 of FSH, and FA (0, 2.5, 25, and 50 µM) for 20 hours (20–25 COC/100-µL droplet of the medium). After 6 hours of gamete co-culture (5 × 106 sperm/mL), presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS and FA (0, 2.5, 25, and 50 μM) for 9 days (day of fertilization = Day 0). Expanded blastocysts that developed from Day 7 to 9 were frozen for further study. Each embryo was frozen in Dulbecco’s PBS (D-PBS) supplemented with 20% CS, 1.5 M ethylene glycol (EG), and 0.1 M sucrose (SUC). Embryos were equilibrated with their freezing medium for 15 min and loaded individually into a 0.25-mL straw. These straws were put into the cooling chamber of a programmable freezer precooled at −7°C. After 2 min, straws were seeded and held for 13 min at −7°C. Next, straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. Frozen embryos were thawed by allowing straws to stand in air for 7 s and warming them in a 30°C water bath for 20 s. Thawed embryos were washed twice with D-PBS supplemented with 20% fetal calf serum (FCS), which was warmed to 38°C. They were immersed into the same medium at 38°C for 10 min, and each embryo was cultured in a 20-μL droplet of TCM199 supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (TCM-199-βME) for 72 h. Embryo cleavage rate was observed at 55 h post-insemination. Blastocyst rates were analysed at 9 days post-insemination. Rates of embryos developing into reexpanded, hatching, and hatched blastocyst stages were determined after 72 h of thawing. All data were analysed by the chi-square test and Fisher’s exact test. Cleavage and blastocyst rates after insemination at 55 hours and 9 days, respectively, were not significantly different among media containing 0 μM (n = 278; 74.1% and 39.9%), 2.5 μM (n = 260; 74.2% and 45.8%), 25 μM (n = 258; 75.6% and 45.7%), and 50 μM (n = 253; 76.3% and 42.7%) FA. Survival and hatching rates of frozen and thawed expanded blastocysts after 72 h in culture were 62.5% and 56.3%, respectively, in 0 μM FA (n = 16); 85.2% and 74.1% in 2.5 μM FA (n = 27); 66.7% and 62.5% in 25 μM FA (n = 24); and 68.0% and 64.0% in 50 μM FA (n = 25). Blastocysts cultured in media containing 2.5 μM FA tended to have a higher survival rate than those cultured in media containing 0 μM FA, although this difference was not significant (P = 0.09). Inclusion of FA did not appear to influence development or post-thaw survival of bovine blastocysts produced in vitro.

Production of bovine embryos by ovum pick-up (OPU)-IVF has increased in recent years. However, numerous factors affect efficiencies of embryo production using this technology. This study was investigated the effects of different breeds on embryo production by OPU-IVF. In total, 98 OPU-IVF sessions were conducted on 41 Holstein and 27 Japanese Black cows from February 2015 to May 2017. The collected cumulus–oocyte complexes (COC) were matured for 22 h. After co-culture of COC with sperm for 6 h, the presumptive zygotes were washed and denuded by pipetting. Those zygotes were then cultured in CR1aa medium supplemented with 5% calf serum for 9 days in a micro-well culture dish. Blastocyst formation rates were analysed at 9 days post-insemination (hpi). The kinetics of embryo development was observed at 27, 31, and 55 hpi. Four factors were considered for selecting embryos to predict pregnancy competence: (1) timing of first cleavage; (2) formation of 2 blastomeres after first cleavage at 31 hpi; (3) absence of fragments after first cleavage at 31 hpi; (4) formation of 8 or more blastomeres at 55 hpi. The quality of blastocysts was compared with the proportions of embryos that were selected based on these 4 factors. Data were analysed by the Chi-squared test with Yates’ correction and Student’s t-test. Total numbers of COC from Holstein or Japanese Black cows were 1330 (n = 51) and 1543 (n = 47), respectively. However, no differences were observed in the numbers of collected and cultured COC per OPU session between Holstein (26.1 ± 20.1 and 21.7 ± 20.1) and Japanese Black (32.8 ± 24.7 and 28.7 ± 22.7) cows. The percentage of COC linked into Grade 1, 2, or 3 was 47.6, 32.1, and 10.7% for Japanese Black and 45.8, 25.6, and 12.2% for Holstein cows, respectively. The proportion of COC with expanded cumulus cells was significantly higher (P < 0.01) in Holstein (12.7%) than in Japanese Black (7.7%) cows. The number of blastocyst and transferable embryo production per OPU session in Holstein (7.5 ± 8.3 and 7.3 ± 7.7) cows was not different from that in Japanese Black (10.6 ± 9.3 and 9.8 ± 9.0) cows. Moreover, there was no difference in blastocyst formation rates between Japanese Black (36.8%) and Holstein (34.8%) cows. Percentages of embryos selected by factors 1 to 4 were 42.6, 66.8, 59.1, and 54.4% in Holstein, and 54.6, 83.2, 72.2, and 75.2% in Japanese Black cows, respectively. Proportions of blastocysts in Holstein and Japanese Black cows selected by a combination of all 4 factors were 21.6 and 36.1%, respectively. There were significant differences (P < 0.01) between the 2 breeds in terms of proportion of blastocysts selected based on individual factors as well as a combination of all 4 factors. These results suggested that the efficiency of embryo production did not differ between Japanese Black and Holstein cows. However, the quality of embryos selected by a combination of 4 factors was significantly higher in Japanese Black than in Holstein cows.

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.