• Korean Journal of Animal Reproduction
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• v.22 no.4
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• pp.375-383
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• 1998

This study was conducted to investigate the effect of $\beta$-mercaptoethanol ($\beta$-ME) and cysteamine on in vitro maturation of porcine follicular oocytes. The results obtained were summarized as follows : 1. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of $\beta$-ME for 36h, the GVBD rates were 91.6, 92.5, 91.8, 91.9 and 92.7%, respectively, and the maturation rates of the oocytes with metaphase-II were 49.6, 41.7, 32.5, 34.1 and 35.4%, respectively. Thus, lower maturation rate was shown in $\beta$-ME treated groups of 50, 100 and 200 $\mu$M as compared to control (non-treated) group (P＜0.05). After 44h of culture in the same treatments of $\beta$-ME, the GVBD rates of porcine oocytes were 91.8, 90.4, 92.5, 91.2 and 93.9%, respectively, and the maturation rates were 71.9, 58.8, 56.7, 62.2 and 56.5% respectively. All treated groups of $\beta$-ME showed lower maturation rates than the control group (P＜0.05) 2. When the immature oocytes were cultured at 0, 10, 50, 100 and 200 $\mu$M of cysteamine for 36h, the GVBD rates were 90.6, 86.3, 88.2, 87.2 and 90.0%, respectively, and the maturation rates were 53.8, 45.1, 54.4, 57.5 and 63.3% respectively. Especially, the maturation rate of 200 $\mu$M treated group was significantly higher than those of control group (P＜0.05). After 44h of culture in the same treatments of cysteamine, the GVBD rates of porcine immature oocytes were 89.5, 93.1, 85.1, 89.8 and 91.3%, respectively, and the maturation rates of the oocytes with metaphase- II were 84.2, 77.6, 66.0, 67.8 and 78.3%, respectively. Especially, the maturation rates of 50 and 100 $\mu$M treated groups were significantly lower than those of control group (P＜0.05).

• Korean Journal of Animal Reproduction
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• v.24 no.1
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• pp.97-108
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• 2000

The objectives of this study were performed to increase the efficiency of the culture conditions of embryos produced in vitro, and to assess the developmental potential after transfer of those embryos into recipients. The mean number of folliclular oocytes recovered from an ovary was 10.7. The rates of maturation and fertilization in Grade I oocytes were significantly (P＜0.05) higher than Graden and III. Developmental rate into blastocyst in the culture group of TCM-199 with BOEC were significantly higher (P＜0.05) than the groups of TCM-199 and conditioned medium (24.7% vs. 12.4% and 18.2%). The survivability of post-thawed blastocysts equilibrated for 3 min in EFS solution was significantly (P＜0.05) lower than l0 for 1 and 2 min (32.1% vs. 82.9% and 73.3%). Significantly higher (P＜0.05) survival rate in blastocysts was seen after freezing than in morulae stage embryos. Out of all 105 recipients, 49 (46.7%) were confirmed in pregnant. On pregnancy of cattle, 48 calves were born from 40 recipients. The ratio of twin and single calves was 30.5% (32/40 and 7.6% (8/40), respectively. However, the others composed of abnormal, as judging as 6 (12.2%) for abortion and 3 (6.1%) for stillbirth during the pregnant period.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.17-22
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• 1999

To investigate the maturational and development competece of porcine oocytes of different diameter groups, oocytes were obtained by aspiration from slaughterdhouse ovaries. After washing three times in NCSU23 medium, each cumulus-oocyte complex was transferred into a $8{mu}ell$ drop of the maturation medium (one oocyte per drop) under paraffin oil. The diameter without zona pellucida of oocytes was measured with micor-calibrator (Mikrometer, E. Leitz) on a screen connected to a VCR on an inverted microscope $(200\times)$. After being measured, the oocytes were divided into 6 groups according to their diameter size : <105, 105 to < 110, 110 to < 115, 115 to < 120, 120 to < 125 and > $125{\mu}{\textrm}{m}$, and in vitro maturation (IVM), fertillzation (IVF) and production (IVP) of oocytes / embryo was performed. The rates of in vitro maturation on oocytes in the greater 105 ${\mu}{\textrm}{m}$ size groups(91.8~100%) were significantly (P<0.05) higher than in the < 105 ${\mu}{\textrm}{m}$ group(66.7%). The rates of sperm penetration were significantly (P<0.05) low in < $105{\mu}{\textrm}{m}$ group (50.0%) than others groups (81.6~85.5%). But the plyspermic fertilization rate was significantly (P<0.05) higher in < $110{\mu}{\textrm}{m}$ oocytes groups than in the $110\leq{\mu}{\textrm}{m}$ size groups. The rates of cleavage and development to blastocysts rose as oocytes diameter increased, however, while oocytes over $120{\mu}{\textrm}{m}$ in diameter failed to develop to blastocysts. There results suggest that porcine oocytes have acquired full meiotic competece at a diameter of $105{\mu}{\textrm}{m}$ but not yet attended full development competence to blastocyst and that oocytes have acquired full development competence at a diameter of $110{\mu}{\textrm}{m}$.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.27-34
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• 1996

The objective of this study was to examine the cell number of Total, ICM and TE cells of bovine blastocysts according to development progression cultured in CR1 medium, which was reported as successfully supporting medium for preimplantaion bovine embryo development to the blastocyst stage, by differential labelling of the nuclei with immunosurgery and polynucleot-ide-specific fluorochromes. Blastocysts were obtained at day 8 after in vitro fertilization and classified to early, middle, expanded stage according to the developmental morphology; blastocoel expansion and zona thickness. Also, bias tocysts in the same category were divided into two parts to check the Total cell number by using bisbenzimide only and ICM, TE and Total cell number by using immunosurgery and two polynucleotide-specific fluorochromes. 1) The development rate of blastocysts at day 8 after in vitro fertilization was 29.3% and classified bIas tocysts to early, middle, expanded and hatching stage were 8.7, 9.9, 7.6 and 3.1%, respectively. 2) The numbers of total blastomere using bisbenzimide in the classified blastocysts to early, middie and expanded were 46.9${\pm}$8.6, 66.2${\pm}$12.5 and 122.8 ${\pm}$ 14.4, respectively. This indicated that CR1 is a appropriate culture medium for bovine embryo development. 3) The count of ICM and TE cell number by using differential labelling with immunosurgery and polynucleotide-specific fluorochromes in the classified blastocysts to early, middle and expanded; ICM cell numbers of were 12.8${\pm}$5.9, 26.3${\pm}$8.4 and 35.5${\pm}$15.0, respectively and TE cell numbers were 30.5${\pm}$5.0, 4 41.3${\pm}$8.2 and 81.1${\pm}$13.4, respectively. These results presented that the increase of ICM and TE cell numbers averaged two and three doublings between early and expanded blastocyst stage and also total cell number counted from ICM nuclei and TE nuclei by using differential label-ling showed the increase pattern with development advance level and the results were similar to total cell number obtained from bisbenzimide treatment only. Therefore, the differential labelling of ICM and TE nuclei in situ is a very useful technique to evaluate embryo qualities and can be used as an indicator on study of preim-plantation embryo development.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.313-321
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• 1998

This study was conducted to investigate the effects of equilibration and dilution methods on the survival rate of vitrified IVM-IVF bovine blastocysts. Vitrification solution was composed with 20% glycerol, 20% ethylene glycol, 3/8 M sucrose and 3/8 M dextrose in D-PBS supplemented with 20% FBS (GESD). Embryos were equilibrated in 1 of 3 methods: 3-step (El), 2-step (E2), or 1-step (E3), and after loading into 0.25-ml straws, were plunged into liquid nitrogen. After warming in water bath at 2$0^{\circ}C$, cryoprotectants were diluted in 1 of 3 methods: 1) D1(VS+1/2 M sucrose, 1/2 M sucrose and l/4 M sucrose), 2) D2 (1/2 M sucrose and 1/4 M sucrose), or 3) D3(1/2 M sucrose only). All procedures except warming were conducted at room temperature. Survival and hatching rates of blastocysts and expanded blastocysts following equilibration methods were 50 and 83.6%, and 27.8 and 67.3%, respectively in El, which were significantly higher (P〈0.01) than those of E2 (16.7 and 23.2%, and 7.4 and 12.5%, respectively) and 23 (0 and 3.7%, and 0 and 0%, respectively). Survival and hatching rates of expanded blastocysts were significantly (P〈0.01) higher than those of blastocysts in El. Survival rates of blastocysts and expanded blastocysts following dilution methods were 52% and 80.6% in D2, which were significantly higher (P〈0.05) than those of D1 (29.6 and 48.3%) and D3 (47.2 and 63.8%). Hatching rates of blastocysts were similar in D1, D2 and D3, however in expanded blastocysts, that of D2(61.3%) was significantly higher (P〈0.01) than that of D1(34.5%). Survival rates of expanded blastocysts in D1 and D2, and hatching rates in D2 and D3 were significantly higher(P〈0.01) than those of blastocysts. These results indicate that the viability of vitrified blastocysts was improved by the several steps of equilibration, and by 2-steps dilution after warming, independently of their stage of development. The results also indicated that the expanded blastocysts are more profitable to vitrification than blastocysts.

• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.231-238
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• 1999

The objective of this study was to examine the effect of developmental stage and embryo age of in vitro produced bovine blastocysts after vitrification and thawing. In vitro cultured day 8 blastocysts after IVF were equilibrated 20% ethylene glycol (EG) for 3 min. and were vitrified using EFS40, which is consisted of 40% EG, 18% ficoll, 0.3M sucrose and 10% FBS added in mDPBS for 30 sec. before being plunged into $LN_2$. Also, survival in vitro was assessed by re-expansion and hatching or hatched at 24 hand 48 h postwarming, respectively. The results obtained in these experiments were summarized as follows; 1) When the embryos were cultured for 8 day after IVF, 41.0% of the cleaved embryos developed to the blastocysts (early; 7.6%, expanded; 22.9%, hatching; 4.6% and hatched; 5.9%). 2) When the embryos were exposed or vitrified to the freezing solution, the re-expansion of vitrified embryos (73.3%) was significantly lower than that of control and exposed embryos (100, 97.0%) (p<0.05). But the formation rate of hatching or hatched blastocysts of vitrified embryos (66.7, 46.7%) at 48h after thawing was similar to that of exposed embryos (66.7, 39.4%) but not control (100, 100%) (p<0.01). However, in the total cell numbers of those developed hatched blastocysts, there were not significantly different among the treatment groups. 3) When the embryo survival rates by different developmental stage were examined, the re-expansion was not different among the groups $(64.5{\sim}75.6%)$. After warming 48 h, the hatching and hatched formation of early blastocysts (25.8, 9.7%) was significantly lower than those of expanded (69.7, 39.4%) and hatching blastocysts (53.3, 43.3%) (p<0.05). 4) In addition, when the expanded blastocysts at day 7, 8 and 9 were vitrified, the re-expansion of day 8 and 9 embryos was significantly lower than that of day 7 (day 7; 93.9%, day 8; 75.8% and day 9; 87.5%) (p<0.05). However, the rates of development to hatched blastocysts were no difference among the groups (day 7; 36.4%, day 8; 36.4% and day 9; 31.3%). These results suggested that in vitro produced expanded or hatching blastocysts can be efficiently cryopreserved by the two-step vitrification method using EFS40.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.141-148
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• 1999

This study was to test whether the viability of bovine hatched blastocysts (HBs) can be maintained after vitrification and thawing. The HBs were produced in vitro at Day 9 and Day 10 after IVF, and they were classified to small (S-HBs; ø$\leq$300 ${\mu}{\textrm}{m}$) and large(L-HBs; ø＞300 ${\mu}{\textrm}{m}$) on the basis of embryo diameter using eyepiece micrometer. As freezing solution, we used EFS35 which consisted of 35% ethylene glycol (EG), 18% ficoll, 0.3 M sucrose and 10% FBS added in mDPBS. Vitrification was taken by two-step method, the HBs were equilibrated in 10% EG for 5 minutes and then shortly exposed in EFS35 and plunged into L$N_2$for 30~45 sec. After thawing, the survival rates were assessed by the re-expansion of the blastocoel during 2 h and 16 h of culture. The results obtained in these experiments were summarized as follows; 1) When the blastocysts(40.8%) recovered at Day 8 after IVF were further cultured for 24 h(Day 9 after IVF) and 48 h(Day 10 after IVF), the rates of HBs were 20.5% and 6.7%, respectively. Also, the total cell number of HBs on Day 9 was significantly higher than that of HBs on Day 10 (p＜0.01). 2) When the effects of freezing solution to the survival of Day 9 L-HBs were examined, the rate of vitrified group (75.7%) was significantly lower than 100% of control and exposed group(p＜0.05). 3) When the survival rates of vitrified HBs according to size and developmental age were examined, the data of L-HBs (75.5%) and S-HBs(63.6%) on Day 9 were slightly higher than those of L-HBs(64.3%) and S-HBs(60.7%) on Day 10. 4) Also, when the in vitro survival of Day 9 HBs was evaluated under different culture condition after thawing, the result in culture medium only (79.3%) was significantly higher than 43.2% in co-culture group (p＜0.05). These results demonstrated that bovine HBs can be successfully cryopreserved by two-step vitrification method using EFS35.