Part Ⅰ. Effect of OPU (Ovum Pick-Up) Duration on the Rate of Collected Ova and In Vitro Produced Blastocyst Formation This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of 3...
Part Ⅰ. Effect of OPU (Ovum Pick-Up) Duration on the Rate of Collected Ova and In Vitro Produced Blastocyst Formation This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of 3~4 days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH for another 3~4 days. The mean numbers of retrieved oocytes were remained constantly up to 3 months (6.0 ± 0.5, 6.2 ± 0.7, 5.2 ± 0.6), but significantly decreased at over 4 to 6 months (3.7 ± 0.5, 2.8 ± 0.4, 1.2 ± 0.2) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months (2.2 ± 0.3, 2.5 ± 0.3 and 2.3 ± 0.4) were significantly higher than those in 4 to 6 months (0.9 ± 0.2, 0.8 ± 0.2 and 0.3 ± 0.2, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos. Part Ⅱ. Effect of Early Pregnant Heifer as Donor on the Ovum Pick-Up Derived Oocyte Aspiration and Embryo Production This study was carried out to evaluate the effect of early pregnant cow as donor for Ovum Pick-Up (OPU) derived oocyte aspiration and embryo production in Holstein heifers. Four non-pregnant and 2 pregnant Holstein heifers were used as donor and then carried out total 17 OPU sessions for 10 weeks (2 times per week). Recovered cumulus-oocyte-complexes (COCs) were classified into 4 grades by oocyte cytoplasm and cumulus cells and matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol in 5% CO2 and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3 days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes in the early stage pregnant and non-pregnant heifers were 13.0 ± 4.3 and 10.6 ± 3.9, 5.4 ± 3.4 and 7.7 ± 3.6 per session, respectively. Rate of collected oocyte from aspirated follicles were 59.2% and 50.5%, respectively. The average number of good quality oocytes (Grade I and II) in the early stage pregnant and non-pregnant heifers was 3.7 ± 2.7 and 4.9 ± 2.6 (Mean ± SD). Cleavage and blastocyst developmental rates in Grade I and II were 22.2% and 25.5%, and then 1.7 ± 0.9 and 1.4 ± 1.1 blastocyst per session, respectively. In conclusion, OPU technology can be used in early stage pregnant and non-pregnant heifers without any problem and so applied OPU derived embryo production to maximize the ability of genetically valuable females. Part Ⅲ. Comparison of Korea Native Hanwoo with Holstein of OPU derived Embryo The objective of this study was to evaluate the comparison of production efficiency of oocytes and OPU (ovum pick-up) derived embryos of Hanwoo with Holstein. The OPU session of each species (6 cows) was carried out from the Hanwoo (106 sessions) and Holstein (114 sessions) at intervals of 3~4 days (2 times per week) for 3 months. Cumulus-oocyte-complexes (COCs) retrieved were classified into 4 grades by the status of oocyte cytoplasm and cumulus cells. The COCs collected were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol-17β in 5% CO2 and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes were not significantly different between Hanwoo and Holstein species (10.4 ± 0.42 vs. 11.4 ± 0.41 and 7.5 ± 0.38 vs. 6.1 ± 0.37 per session). But the collection rate of oocytes from aspirated follicles were significantly higher in Hanwoo (72.8%) than that in Holstein (53.6%) (p < 0.05). Furthermore, the average number of good quality oocytes (Grade Ⅰ and Ⅱ) was 5.9 ± 0.28 and 4.1 ± 0.27 (Mean ± SE), and the cleavage rate and the development rate to blastocysts was significantly (p < 0.05) higher in Hanwoo (40.0%) than Holstein (21.6%). The OPU derived embryos of Honwoo were transferred 83 times into 52 recipients and then 42 calves were produced from 44 pregnancy recipients. In conclusion, the efficiency of OPU derived embryo was significantly different between Hanwoo and Holstein species. In vitro culture system for OPU derived embryo production should be optimized for industrialization and the improvement of livestock. Part Ⅳ. Effect of Recipient Breeds on Gestation Length and Birth Weight of Offspring Derived from OPU Korea Native Hanwoo Embryos This study evaluated the effect of recipient`s breed on the gestation length and birth weight of Hanwoo calves produced by Hanwoo ovum pick up (OPU) derived embryos. Embryos of OPU derived Hanwoo cows were transferred into the Hanwoo and Holstein recipients at 6~7 days of estrus cycle. Gestation length was expressed in days from the day of embryo transfer to the birth day of offspring, and birth weight of offspring was recorded within 24 h of birth. Breed of recipient cows (Hanwoo and Holstein) has no effect on overall gestation length (280.9 ± 6.2 vs. 284.4 ± 9.8 days) recipient cows. Birth weight of Hanwoo calves did not differ when Hanwoo recipients (26.5 ± 1.3 vs. 23.3 ± 1.2 kg) were used for embryo transfer. However, male calves were heavier (p<0.05) at birth than that of female calves when embryos were transferred into the Holstein cow (33.5 ± 4.9 vs. 27.8 ± 4.9 kg). In conclusion, the present results demonstrated that breed of recipient cows have no gestation length and birth weight of OPU derived Hanwoo calves. However, gestation length and birth weight of male calves were higher in both Hanwoo and Holstein recipient cows.
Part Ⅰ. Effect of OPU (Ovum Pick-Up) Duration on the Rate of Collected Ova and In Vitro Produced Blastocyst Formation This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of 3~4 days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH for another 3~4 days. The mean numbers of retrieved oocytes were remained constantly up to 3 months (6.0 ± 0.5, 6.2 ± 0.7, 5.2 ± 0.6), but significantly decreased at over 4 to 6 months (3.7 ± 0.5, 2.8 ± 0.4, 1.2 ± 0.2) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months (2.2 ± 0.3, 2.5 ± 0.3 and 2.3 ± 0.4) were significantly higher than those in 4 to 6 months (0.9 ± 0.2, 0.8 ± 0.2 and 0.3 ± 0.2, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos. Part Ⅱ. Effect of Early Pregnant Heifer as Donor on the Ovum Pick-Up Derived Oocyte Aspiration and Embryo Production This study was carried out to evaluate the effect of early pregnant cow as donor for Ovum Pick-Up (OPU) derived oocyte aspiration and embryo production in Holstein heifers. Four non-pregnant and 2 pregnant Holstein heifers were used as donor and then carried out total 17 OPU sessions for 10 weeks (2 times per week). Recovered cumulus-oocyte-complexes (COCs) were classified into 4 grades by oocyte cytoplasm and cumulus cells and matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol in 5% CO2 and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3 days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes in the early stage pregnant and non-pregnant heifers were 13.0 ± 4.3 and 10.6 ± 3.9, 5.4 ± 3.4 and 7.7 ± 3.6 per session, respectively. Rate of collected oocyte from aspirated follicles were 59.2% and 50.5%, respectively. The average number of good quality oocytes (Grade I and II) in the early stage pregnant and non-pregnant heifers was 3.7 ± 2.7 and 4.9 ± 2.6 (Mean ± SD). Cleavage and blastocyst developmental rates in Grade I and II were 22.2% and 25.5%, and then 1.7 ± 0.9 and 1.4 ± 1.1 blastocyst per session, respectively. In conclusion, OPU technology can be used in early stage pregnant and non-pregnant heifers without any problem and so applied OPU derived embryo production to maximize the ability of genetically valuable females. Part Ⅲ. Comparison of Korea Native Hanwoo with Holstein of OPU derived Embryo The objective of this study was to evaluate the comparison of production efficiency of oocytes and OPU (ovum pick-up) derived embryos of Hanwoo with Holstein. The OPU session of each species (6 cows) was carried out from the Hanwoo (106 sessions) and Holstein (114 sessions) at intervals of 3~4 days (2 times per week) for 3 months. Cumulus-oocyte-complexes (COCs) retrieved were classified into 4 grades by the status of oocyte cytoplasm and cumulus cells. The COCs collected were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml FSH and 1 mg/ml estradiol-17β in 5% CO2 and over 99% humidity for 24 h. After 24 h co-incubation with post-thaw sperm, the presumed zygotes were cultured in CR1aa medium with 4 mg/ml BSA for 3days and then changed CR1aa medium with 10% of FBS for another 3~4 days. The Mean number of aspirated follicles and collected oocytes were not significantly different between Hanwoo and Holstein species (10.4 ± 0.42 vs. 11.4 ± 0.41 and 7.5 ± 0.38 vs. 6.1 ± 0.37 per session). But the collection rate of oocytes from aspirated follicles were significantly higher in Hanwoo (72.8%) than that in Holstein (53.6%) (p < 0.05). Furthermore, the average number of good quality oocytes (Grade Ⅰ and Ⅱ) was 5.9 ± 0.28 and 4.1 ± 0.27 (Mean ± SE), and the cleavage rate and the development rate to blastocysts was significantly (p < 0.05) higher in Hanwoo (40.0%) than Holstein (21.6%). The OPU derived embryos of Honwoo were transferred 83 times into 52 recipients and then 42 calves were produced from 44 pregnancy recipients. In conclusion, the efficiency of OPU derived embryo was significantly different between Hanwoo and Holstein species. In vitro culture system for OPU derived embryo production should be optimized for industrialization and the improvement of livestock. Part Ⅳ. Effect of Recipient Breeds on Gestation Length and Birth Weight of Offspring Derived from OPU Korea Native Hanwoo Embryos This study evaluated the effect of recipient`s breed on the gestation length and birth weight of Hanwoo calves produced by Hanwoo ovum pick up (OPU) derived embryos. Embryos of OPU derived Hanwoo cows were transferred into the Hanwoo and Holstein recipients at 6~7 days of estrus cycle. Gestation length was expressed in days from the day of embryo transfer to the birth day of offspring, and birth weight of offspring was recorded within 24 h of birth. Breed of recipient cows (Hanwoo and Holstein) has no effect on overall gestation length (280.9 ± 6.2 vs. 284.4 ± 9.8 days) recipient cows. Birth weight of Hanwoo calves did not differ when Hanwoo recipients (26.5 ± 1.3 vs. 23.3 ± 1.2 kg) were used for embryo transfer. However, male calves were heavier (p<0.05) at birth than that of female calves when embryos were transferred into the Holstein cow (33.5 ± 4.9 vs. 27.8 ± 4.9 kg). In conclusion, the present results demonstrated that breed of recipient cows have no gestation length and birth weight of OPU derived Hanwoo calves. However, gestation length and birth weight of male calves were higher in both Hanwoo and Holstein recipient cows.
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#Ovum Pick-Up 체외수정 공란우 체외성숙 Hanwoo Holstein
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