• Asian-Australasian Journal of Animal Sciences
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• v.16 no.1
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• pp.23-28
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• 2003

The present study was undertaken to investigate the effects of PVP concentration and exposure temperature to vitrification solution on the post-thaw survival, in vitro maturation and development of immature bovine oocytes (germinal vesicle stage). The vitrification solution (VS) consisted of 40% ethylene glycol (EG)+0.5 M sucrose (S)+10% FBS. PVP was added to VS: 0%, 5% or 10%. The cumulus-oocyte complexes (COCs) were diluted in VS as one step, after 2 min the COCs were loaded in straw and vitrified by direct immersion into liquid nitrogen. For thawing, the straws were plunged into $30^{\circ}C$ water bath for 10s. After thawing, the oocytes were diluted in 0.5 M (in DPBS with 10% FBS) sucrose solution for 5 min. The survival rate (FDA-test and trypan blue) of immature bovine oocytes was measured. The survival rate was higher in 5% PVP (91.5%) than in 0% (64.2%) or in 10% PVP (79.7%). The proportion of metaphase II formation was 69.35% in control (no vitrified COCs), 9.3% in 40% EG+0.5 M S+0% PVP and 21.05% in 40% EG+0.5 M S+5% PVP (p<0.05). The effect of room temperature ($25^{\circ}C$ for 10 min) and cold temperature ($4^{\circ}C$ for 10 min) on COCs were determined in this study. After IVF, the cleavage and blastocysts rate of oocytes exposed to room temperature and cold temperature in VS+5% PVP was significantly different (2 cell: 63.20% vs 37.97%, blastocysts: 18.40% vs 2.53%). The cleavage rates of frozen-thawed oocytes were 20.53% with PVP and 22.13% without PVP (p>0.05). Two out of 151 oocytes (1.32%) developed to blastocyst stage after frozen-thawed with 5% PVP (p>0.05). Development of oocytes after frozen-thawing to the 2 cell were not significantly affected with or without PVP following IVF. However, the vitrification of immature bovine oocytes with PVP maintained the ability to develop to the blastocyst stage after IVM-IVF and IVC, while no blastocysts were obtained from oocytes vitrified without PVP. These results suggested that PVP has a protective role for vitrification of immature bovine oocytes as far as survival is concerned, however, the protection was not sufficient enough to support blastocyst formation.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.12
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• Journal of Embryo Transfer
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• v.18 no.3
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• pp.237-242
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• 2003

These studies were carried out to establish an effective in vitro embryo transfer methods by analyzing several factors. The base media were TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and CRlaa medium for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1)after IVF. Embryos were cultured in drop-culture that contained 25 embryos per 10${mu}ell$. Blastocysts cultured for 7 to 9 in vitro were transferred to recipients. The results obtained were as follows: 1. The pregnancy rate according to different region of embryo transfer were 33.8％, 48.1％, 45.0％ and 35.3％ respectively. 2. The pregnancy rate according to the parity of recipient when embryos were transferred to nulliparous (42.9％) was higher than that of 1∼3nd parlous(36.9％), however there were not show significant difference each other. 3. According to the stage of blastocyst, the pregnancy rate when middle blastocysts (MB) (45.5％) were transferred to recipients were higher than that of late blastocysts (LB) (41.0％). 4. When IVF-derived blastocysts cultured for 7 to 9 day were transferred to recipients, the pregnancy rate was higher 7 day of blastocyst than that of 8 day or 9 day of blastocyst. The results of embryo transfer according to the regions, the parity of recipient and the development stage showed that blastocyst formed for 7day transferred to nulliparou were higher pregnancy rate than others.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.111-117
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• 1996

This experiment was designed to evaluate the effect of transforming growth factor-$\beta$ (TGF-$\beta$) and insulin-like growth factor-I (IGF-I) in bovine oocyte maturation in the presence or absence of serum on subsequent fertilization and embryo development. In addition, various concent rations of these growth factors were evaluated for the ability to promote development of eight-cell stage embryos to the blastocyst stage. Cumulus-oocyte complexes were recovered from 2 to 6 mm follicles obtained from slaughterhouse ovaries and cultured at 38.5$^{\circ}C$ for 24 hours in TCM-199 (HEPES Modification) with or with out 20 % fetal bovine serum (FBS) to which the following growth factors were added TGF$\beta$ IGF-l or TGF $\beta$ + IGF-I, all at 10 ng/ml each. The matured oocytes were fertilized in IVF-TL medium with frozen-thawed semen at a concentration of 1 ${\times}$ 10$^6$ cells/ml of fertilization medium following Percoll separation. After 24 hours of sperm-egg incubation, the embryos were transferred to CZB medium without glucose for 48 hours and then cultured in TCM-199 with 20% fetal bovine serum (FBS) for 96 hours. The addition of growth factors to IVM medium in the presence of serum had no effect on cleavage and subsequent embryo devlopment to blastocyst. In the absence of serum, TGF- improved cleavage and development to blastocyst compared to control's(p<0.05) and no synergistic effeet of IGF-I + TGF-$\beta$ was observed. In the second experiment, eight-cell embryos obtained by in vitro maturation (IVM) in TCM-199 + 20% FBS without growth facrors and in vitro fertil-ization (IVF) were cultured in the in vitro cuiture (IVC) medium supplemented with 5, 10 ng/ml TGF-$\beta$ or 5, 10, 50, 100 ng/ml IGF-I. Cleavage rate and development to the blastocyst stage was observed during seven days of incubation. The supplementation of 10 ng/ml TGF-$\beta$ to lVC medium for eight-cell embryos improved development to blastocyst (p<0.05) compared to control. In conclusion, these data indicate that the supplementation of growth factors to IVM medium in the presence of serum does not influence cleavage and subsequent embryo development. However, significantly more oocytes matured in serum-free TCM-199 and eight-cell embryos cultured in lVC medium developed to blastocyst with supplementation of 10ng/ml TGF-$\beta$.

• Korean Journal of Animal Reproduction
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• v.26 no.3
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• pp.215-221
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• 2002

Antioxidants(N-acetyl-1-cysteine, ebselen and glutathione) and growth factors(EGF, PDGF) were studied as a mean of increasing the development of porcine embryos produced by in vitro maturation (IVM) and in vitro fertilization(IVF). Porcine embryos developed to the 2~8 cell stage after IVF were cultured fer 6 to 7 days at 38.5$^{\circ}C$ in NCSU 23 medium containing antioxidants plus growth factors. Cell numbers of blastocysts were counted by fluorescence staining method. The developmental rate beyond morula stages in NCSU 23 containing NAC(1nm) or NAC(1nm) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 28.1, 32.3 and 35.3%, respectively. NAC plus PDGF group was slightly higher than control group(P>0.05). The developmental capacity in NCSU 23 containing ebselen(10 $\mu$M) or ebselen(10 $\mu$M) plus EGF(100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 17.8, 36.9 and 40.3%, respectively Ebselen plus growth factor groups were significantly higher than control group(P<0.05). The developmental capacity in NCSU 23 containing glutathione(100 $\mu$M) or glutathione(100 $\mu$M) plus EGF (100ng/$m\ell$) or PDGF(5ng/$m\ell$) were 24.1, 30.5 and 27.7%, respectively. There were not difference in all experimental groups(P>0.05). In all experimental groups, there was no significantly differences on the cell number of blastocysts, but ebselen plus growth factor groups were significantly higher than control group. These studies indicate that antioxidants plus growth factors can increase the proportion of embryos that developed beyond morulae stage.

• Korean Journal of Animal Reproduction
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• v.23 no.4
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• pp.281-291
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• 1999

The objective of this study was to optimize the freezing/thawing method of in vitro produced Hanwoo blastocysts. Day 7 blastocysts after IVF were vitrified using EFS40 (40% ethylene glycol, 18% ficoll, 0.3 M sucrose and 10% FBS added m-DPBS) as a freezing solution and electron microscope (EM) grid (V-G) or straw (V-S) as an embryo container. In both method, freezing/thawing were treated by 2-step, treatment time was required in V-G method and V-S method, for 2 min / 3 min and 3.5 min / 10 min, respectively. Embryo survival was assessed as re-expanded and hatched rates at 24 h and 48 h after warming, respectively. The results obtained in these experiments were summarized as follows: when the effect of exposure in vitrification solution and chilling injury from freezing procedure on in vitro produced expanded blastocysts were examined, at 24 h after warming, embryo survival in exposure group (100.0%) was not different compared to that in control group (100.0%), although those results were significantly different with two vitrified groups (V-G: 87.8, V-S: 77.8%) (P＜0.001). However, at 48 h after warming, hatched rates of V-G group (67.8%) were significantly higher than those of V-S group (53.3%) (P＜0.05). In addition, this hatched rate in V-G group was not different with that in exposure group (73.3%). When the effects of embryo developmental stage (early, expanded and early hatching blastocysts) and embryo container (EM grid and straw) to the in vitro survival of vitrified-warmed day 7 Hanwoo blastocysts were simultaneously examined, fast developed embryos were indicated the better resistance to freezing than delayed developed one, irrespective of embryo containers (early; 57.1 ＆ 24.4%, expanded; 84.7 ＆ 60.6%, early hatching; 91.7 ＆ 80.0%) (P＜0.001). Especially, in expanded and early hatching blastocysts, embryo survival of V-G group (67.8, 95.0%) was significantly higher than those of V-S group (53.0, 65.0%) at 48 h post warming, respectively (P＜0.05, P＜0.001). Therefore, this study indicates that Hanwoo blastocysts can be cryopreserved more simple, efficient and successful by vitrification method using EM grid.

• 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.

• Journal of Embryo Transfer
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• v.17 no.3
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• pp.211-218
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• 2002

These studies were carried out to investigate the effects of the addition of amino acids and FBS as source of exogenous nitrogen fixation added to medium on in vitro production of blastocyst derived from bovine follicular oocytes. The base medium was TCM-199 solution for in vitro maturation(IVM) of bovine follicular oocytes and Fer-TALP solution for in vitro fertilization(IVF) and YS solution for in vitro culture(IVC). IVC used the fertilized oocytes of 24-hr culture (day 1) after IVF. Rmbryos were cultured in drop-culture that contained 25 embryos per 10 ${mu}ell$. The results obtained are as follows: 1 The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with NEAA derived from MEM alone were higher than those of YS solution without NEAA. 2. The developmental rates of fertilized oocytes to blastocyst that developed from YS solution with EAA derived RPMI 1640 alone were significantly higher than those of YS solution without EAA (p<0.05). 3. When added to EAA on day 5 after NEAA supplementation on day 1, the developmental rates of hatched blastocyst and blastocyst to hatched blastocyst were improved. 4. When removed to EAA on day 3, day 4 and day 5 from medium after NEAA and EAA supplementation on day 1. the developmental rates of blastocyst to hatched blastocyst were reduced. 5. When added to FBS as source of exogenous nitrogen fixation, the developmental rates of blastocyst and hatched blastocyst that developed from the later culture higher(day 5) than those of the early culture.

• Asian-Australasian Journal of Animal Sciences
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• v.15 no.4
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• pp.485-493
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• 2002

Different factors may affect the sensitivity of porcine oocytes during cryopreservation. The effect of two methods (cooling and vitrification), four cryoprotectants [glycerol (GLY), 1, 2-propanediol (PROH), dimethyl sulfoxide (DMSO) or ethylene glycol (EG)] and two vitrification media (1 M sucrose (SUC)+8 M EG; 8 M EG) on the developmental capacity of porcine oocytes at the germinal vesicle (GV) stage or after IVM at the metaphase II (M II) stage were examined. Survival was assessed by FDA staining, maturation and cleavage following IVF and IVC. A toxicity test for different cryoprotectants (GLY, PROH, DMSO, EG) was conducted at room temperature before cooling. GV and M II-oocytes were equilibrated stepwise in 1.5 M cryoprotectant and diluted out in sucrose. The survival rate of GV-oocytes in the GLY group was significantly lower (82%, p<0.01) than that of the other group (92 to 95%). The EG group achieved a significantly higher maturation rate (84%, p<0.05) but a lower cleavage rate (34%, p<0.01) than the DMSO group and the controls. For M II-oocytes, the survival rates for all groups were 95 to 99% and the cleavage rate of the GLY group was lower than the PROH-group (21 vs 43%, p<0.01). After cooling to $10^{\circ}C$, the survival rates of GV-oocytes in the cryoprotectant groups were 34 to 51%, however, the maturation rates of these oocytes were low (1%) and none developed after IVF. For M II-oocytes, the EG group showed a significantly higher survival rate than those of the other cryoprotectant groups (40% vs 23-26%, p<0.05) and the cleavage rates of PROH, DMSO and EG group reached only 1 to 2%. For a toxicity test of different vitrification media, GV and M II-oocytes were equilibrated stepwise in 100% 8 M EG (group 1) and 1 M SUC + 8 M EG (group 2) or equilibrated in sucrose and then in 8 M EG (SUC+8 M EG, group 3). For GV-oocytes, the survival, maturation and cleavage rates of Group 1 were significantly lower than those in group 2, 3 and control group (p<0.05). For M II-oocytes, there were no differences in survival, maturation and cleavage rates between groups. After vitrification, the survival rates of GV and M II-oocytes in group 2 and 3 were similarly low (4-9%) and none of them matured nor cleaved after in vitro maturation, fertilization and culture. In conclusion, porcine GV and M II-oocytes do not seem to be damaged by a variety of cryoprotectants tested, but will succumb to a temperature decrease to $10^{\circ}C$ or to the process of vitrification, regardless of the cryoprotectant used.

• Asian-Australasian Journal of Animal Sciences
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• v.11 no.4
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• pp.398-403
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• 1998

The objective of the present study was to examine the developmental rates, and the stage of development in relation to time since fertilization, of in vitro produced buffalo embryos. Buffalo cumulus-oocyte complexes obtained from slaughterhouse ovaries were matured and fertilized in vitro. The fertilized oocytes (n = 248) were then co-cultured with buffalo oviductal epithelial cells and evaluated for the developmental stages on Days 2, 4, 6, 7, 8, 9 and 10 post-insemination. The peak of 4-cell stage embryos was observed on Day 2 (63.7 %), whereas Day 4 was marked by peaks of 6-8-cell stage embryos (20.9%) and 16-cell stage embryos to early morulae (50%). On Days 6, 7, 8, 9, and 10 post-insemination, 49.5, 48.3, 38.3, 33.8 and 33.4% embryos were found to be at morula/compact morula stages, 8.8, 12.5, 25.4, 6.0 and 1.2% at early blastocyst/blastocyst stages, 0, 6.8, 7.2, 15.3 and 2.0% at expanded blastocyst stage and 0, 1.6, 4.8, 19.3 and 38.5% hatching/hatched blastocyst stages, respectively. The peaks of early blastocyst/blastocyst, expanded blastocyst and hatching/hatched blastocyst stages were observed on Days 8, 9 and 10, respectively. The percentages of oocytes which initially became arrested and subsequently degenerated were 3.6, 4.8, 10.4, 14.5, 21.3 and 24.5% on Days 4, 6, 7, 8, 9 and 10 post-insemination, respectively.