• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.83-87
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• 2004

The objective of this study was performed to establish the in vitro culture system of porcine in vitro maturation and in vitro fertilization(IVM/IVF) embryo. These studies was to determine the effects of melatonin, nitric oxide donor(SNP), and the combination effects of SNP and melatonin in porcine IVM/IVF embryos. In routine porcine IVM/IVF procedure, oocytes were cultured for 40∼44h incubation, and the zygotes were cultured for 40∼44h in NCSU 23 medium. Then 2 to 8 cell embryos were removed cumulus cell and were allotted randomly to NCSU 23 containing different concentration of melatonin, SNP and SNP plus melatonin in 5% $O_2$, 5% $CO_2$ and 90% $N_2$ at 38.5$^{\circ}C$. Cell numbers of blastocyst were also counted using double fluorescence stain method. In NCSU 23 medium treated with melatonin 0, 1, 5 and 10 nM, the developmental rate of morula plus blastocysts were 33.3%, 39.1%, 33.3% and 27.9%, respectivly. This result show that the developmental rate of morula and blascytocys treated with 1 nM melatonin was higher than in any other groups(P<0.05). The developmental rates of morula plus blastocysts were 41.9% in 0 uM SNP, 25.6% in 50 uM and 28.4% in 100 uM, respectively. The developmental rate of morula plus blastocysts were decreased treated with SNP in NCSU 23. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM, SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), the developmental rates beyond morula stage of porcine embryos were 31.3%, 34.1%, 39.5%, 29.4% and 39.5%, respectively. The addition of SNP 50 uM plus maltonin 1 nM, developmental rates of blastocyst was higher rate than in any other groups. Cell numbers of blastocyst in NCSU 23 treated with melatonin 0, 1, 5 and 10 nM were 41.0, 42.6, 39.6 and 33.0, respectively. In combined effects of SNP plus melatonin (0, SNP 50 uM, SNP 50 uM plus melatonin 1 nM , SNP 50 uM plus melatonin 5 nM and SNP 50 uM plus melatonin 10 nM), cell numbers of developed blastocyst were 36.3, 34.6, 39.0, 39.9 and 39.0, respectively. These result show that the cell numbers of blastocyst treated with 0, 1 and 5 nM melatonin were higher than in 10 nM group(P<0.05), but cell numbers of blatocyst produced by SNP plus melatonin were not significantly difference in all experimental groups.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.5 no.1
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• pp.1-35
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• 1969

Attempts were made to obtain the fundamental knowledge on the quantitative constitution status of leaves and stem of elongating node-part, and the relationships between these morphological characteristics along with the nitrogen contents of leaves and grain yield were examined varing application amounts of nitrogen in rice plant. I. The agronomic characteristics of leaves and nodes of elongation node-part (4-node parts from the top of stem) were observed at heading stage with 20 leading rice varieties of Kang Won district. The results are summarized as follows: 1. Leaf area magnitude of the flag and the fourth leaf was smaller than that of the second and the third with the average value of flag leaf 18.61 $cm^2$, the second leaf 21.84 $cm^2$, the third 21.52 $cm^2$ and the fourth 18.56 $cm^2$. The weight of leaf blade showed an isotonic tendency with the magnitude of leaf area with the value of the flag leaf 97.0 mg, the second leaf 117.1 mg, the third 115.4 mg, and the fourth 95.3 mg. The weight of each leaf sheath was remarkably larger at the higher node-part than at the lower node-part of the stem with the value of flag leaf sheath 176.3 mg, the second 163.7 mg, the third 163.4 mg and the fourth 123.9 mg. Accordingly, the total leaf weight of each part was larger at the second and the third leaf than at the first and the fourth. Total plant weight of each part (weight of leaf blade, leaf sheath, and culm) also was larger at the middle node-part. 2. Coefficients of variation for the varietal differences of the morphological characteristics of elongating node-part were 12.75% for the leaf area, 15.29% for the weight of leaf blade, 15.90%, for the weight of leaf sheath, 11.42% for the weight of internode, 15.45% for the leaf weight (leaf blade ＆ leaf sheath) and 13.24% for the straw weight. And these coefficient values of the most characteristics were, on the whole, smaller at the second and the third node-part than at the first and the fourth node-part, but the coefficient value of the internode weight was rather small at the third and fourth node-part. 3. Constitutional ratio of each plant organ to the total plant weight in term of dry matter weight (excluding head and root wight) was 39.2% for the leaf sheath, 34.2% for the culm, 26.6% for the leaf blade. And ocnstitutional ratio of leaf sheath in term of dry matter weight was larger at the higher position in contrast with that of culm. 4. Average weight ration of leaf blade to culm, leaf sheath to culm, leaf blades to sheath and the leaf blades to culm plus leaf sheath were 77.7 %, 114.5%, 67.9% and 36.2%, respectively. With regard to the position of the plant organ, the weight ratio of leaf blade to culm and that of leaf sheath to culm were larger at higher part in contrast with that of leaf blade to leaf sheath. 5. Generally, there founded deep relationships between grain yield and each morphological characteristics of plant organ of elongating node-part as follows; Correlation coefficient between total area of 4 leaves (from flag to the fourth leaf) and grain yield was ${\gamma}$=0.666$^{**}$ In regard to the position of leaves, correlation coefficient values of flag, the second, the third and the fourth leaf were ${\gamma}$=0.659$^{**}$, ${\gamma}$=0.609$^{**}$, ${\gamma}$=0.464$^{*}$ and ${\gamma}$=0.523$^{*}$, respectively. Correlation coefficient between total weight of leaf blades and the grain yield was ${\gamma}$=0.678$^{**}$. In regard to the position of leaves, that of flag leaf was ${\gamma}$=0.691$^{**}$, and ${\gamma}$=0.654$^{**}$ for the second leaf, ${\gamma}$=0.570$^{**}$ for the third, and ${\gamma}$=0.544$^{**}$ for the fourth. Correlation between the weight of leaves (blade weight plus sheath weight) and the grain yield showed similar values. In the relationship between plant weight and grain yield there also was significant correlation, but with highly significant value only for the first node-part. There appeared correlation between total weight of leaf sheath and grain yield with the value of ${\gamma}$=0.572$^{**}$ and in regard to the position of each leaf sheath the values were ${\gamma}$=0.623$^{**}$ for the flag leaf, ${\gamma}$=0.486$^{**}$ for the second leaf, ${\gamma}$=0.513$^{**}$ for the third, ${\gamma}$=0.450$^{**}$ for the fourth. However, there was no significant correlation between culm weight and grain yield. 6. With respect to in gain yield, varietal differences in magnitude of leaf area, weight of leaf blade, leaf weight per unit area, weight of leaf sheath, culm weight, total leaf and stem weight were larger in the case of high yielding varieties and decreased in accordance with decreasing yield. And this tendency also was shown in the varietal differences of magnitude of each part. Variation in magnitude of each part for the leaf area, weight of leaf blade, culm weight was significantly small in high yielding varieties compared to low yielding varieties. 7. Plant constitutional ratio of each organ of the elongating node-part in term of weight magnitnde varied to som extent according to varieties indicating leaf blade 27.6%, leaf sheath 39.5%, culm 32.9% in the case of high yielding varieties, leaf blade 25.5%, leaf sheath 38.1%, culm 36.4% in the case of low yielding varieties, and medium yielding varieties showed intermadiate values. 8. Far higher values of the weight ration of leaf blade to culm and leaf sheath to culm were given to the high yielding varieties compared to low yielding varieties. And medium yielding varieties showed intermadiate values. II. Effects of application rate of nitrogen on the morphological characteristics of the elongating node-part, nitrogen content of leaf blade, and their relation with the grain yield of the rice were observed with 3 rice varieties; Shin No.2, Shirogane, and Jinheung varying application amounts of nitrogen as 8kg, 12kg and 16kg per 10 are. 1. As for the variation of morphological magnitude s affected by the amounts of nitrogen application, total leaf area (4 leaves from the flag leaf) increased to 16.5% at 12kg N plot, and about 30% at 16kg N polt compared to 8kg N plot and total weight of leaf blade also increased to similar extent, respectively, in contrast with weight of leaf sheath increasing 4.9% and 7.8%, respectively. However, the weight of culm decreased to 1.5% and 11.2%at the 12kg N plot and 16kg N plot, respectively, and these decreasing rate was noted at the nodes of lower part. 2. As for the verietal differences in variation of morphological magnitude as affected by the amount of nitrogen fertilization, leaf area coefficient value of variation of the total leaf area was 15.40% for Shin No. 2, 12.87% for Shirogane, and 10.99% for Jinheung. With respect to the position of nodes, the largest variation of leaf blade magnitude was observed at the fourth for Shin No. 2, the second for Shirogan, and flag leaf for Jinheung. And there also was an isotonic varietal difference in the weight of leaf blade. Variation in total culm weight showed varietal differences with the coefficient value of 7.72% for Shin No.2, 12.11% for Shirogane, and 0.94% for Jinheung. There also was varietal differences in the variation according to the position of nodes. 3. Variation of each elongating node-part related to the fertilization amount decreased with the increase of fertilization amount in the items of leaf area, weight of leaf sheath, culm weight, but weight of leaf sheath varied more at heavier fertilization than at others. 4. Constitutional ratio of each organ excluding head also varied with fertilization amount; constitutional ratio of leaf blade increased much with the increasing amount of fertilization in contrast with the response of culm eight. However, constitutional ration of the weight of leaf sheath was not much affected. 5. Lower value of the ration of leaf blade to culm was given to the 8kg N per 10 are plot, and the ratio of leaf blade to leaf sheath decreased with the increasing amount of fertilization in contrast with the increase in the ratio of leaf sheath to culm. however, the ration of leaf blade to culm plus leaf sheath decreased. 6. With the increase of nitrogen fertilization, leaf area, weight of leaf blade and leaf sheath increased. Accordingly, grin yield also increased to some extent. It was noted that culm weight was changed inversely to the changes in grain yield, but the degree of this variation varied with varietal characteristics. 7. Nitrogen content of leaves at heading and fruiting stage varied with the fertilization amount, and average nitrogen content of leaves of the varieties used 2.19%, 2.49% and 2.74% at the plot of 8kg N, and 12kg N and 16kg N per 10 are, respectively, at heading time, and 0.80%, 0.92% and 1.03% at each plot at fruiting stage. Thus, nitrogen content of leaves increased much with the increasing amount of fertilization, and higher value was given to the leaves on the higher position of elongating node-part. 8. There also was variation of nitrogen content of leaves in accordance with the varieties. However higher grain yield was obtained from the plants retaining higher nitrogen content in leaves at heading or fruiting stage.

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.173-183
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• 2004

This study was carried out to examine the effects of epidermal growth factor (EGF) and the number of cumulus-oocyte complexes (COCs) on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). COCs were collected from antral follicles of porcine ovaries collected from abattoir, and were maturated in modified NCSU-23 (mNCSU-23) with 10% pFF, 0.6 mM cysteine, 50 ${\mu}mM{\beta}-mercaptoethanol$, 1 mM dbcAMP, 10 IU/mL PMSG and 10 IU/mL hCG, which was supplemented with or without 10 ng/mL EGF and into which 50 or 15 COCs per droplet was put. Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium (mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU- 23. The results are as follows. 1.In the result of IVM, 10 ng/mL EGF supplement duplicated the percentage of C4 group of COCs(41% vs 81%). But the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, and there was not a significant interaction between the two factors, either. 2. In the result of IVF, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet, or was not a significant interaction between the two factors, in the rate of sperm penetration, in the percentage of oocytes with male pronucleus (MPN), and in the rate of polyspermy. 3. In the result of IVD, there was not significantly different between control and EGF supplemented, or between the number of COCs per culture droplet in the percentage of cleaved oocytes. There was not significantly different between the number of COCs per culture droplet, but between control and EGF supplemented (p<0.01) in the percentage of blastocysts, the number of inner cell mass (ICM), trophectoderm (TC) and total cells. There was no significant interaction between the two factors anywhere. These results suggested that 10 ng/mL EGF supplement into mNCSU-23 for IVM was effective in the production of more as well as better blastocysts during IVD through increasing the number of cells in those.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.4
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• pp.285-291
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• 2008

Objective: This study was performed to investigate whether cumulus morphology and oocyte diameter influence on in vitro maturation (IVM) of human germinal vesicle (GV) stage oocytes obtained from stimulated in vitro fertilization (IVF) cycles. Methods: Forty-one GV stage oocytes were obtained from 21 patients who received ovarian hyperstimulation and IVF. According to cumulus morphology before denudation, GV oocytes were classified into oocytes with dispersed cumulus cells (CCs) or compacted CCs. The diameters of denuded oocytes, both including and excluding the zona pellucida, were measured. All oocytes were cultured in commercial IVM medium. Maturation was defined as extrusion of the first polar body and the matured oocytes were inseminated by ICSI method. Results: Overall maturation and fertilization rate were 56.1% and 73.9%. Matured oocytes had significantly higher proportion of oocytes with dispersed CCs compared to oocytes failed to mature (91.3% vs. 55.6%, p=0.023). There were no significant differences of oocytes outer ($155.7{\mu}m$ vs. $152.4{\mu}m$, NS), inner ($114.3{\mu}m$ vs. $113.4{\mu}m$, NS) diameters and zona thicknesses ($41.3{\mu}m$ vs. $39.1{\mu}m$, NS) between matured and not-matured oocytes. In-vitro maturation rate of oocytes with dispersed CCs was significantly higher than which of oocytes with compacted CCs (67.7% vs. 20.0%, p=0.044). Oocyte diameters (outer and inner) and thicknesses were not related with maturational competence. Conclusion: Our results suggest that in vitro maturational competence of GV stage oocytes obtained from stimulated IVF cycles is closely associated with the cumulus morphology but not oocyte diameter.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.87-92
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• 1998

This study was carried out to investigate in vitro/in vivo development of vitrified mouse oocytes. Mouse oocytes were vitrified using EFS30, 35 and 40 (30, 35 and 40% ethylene glycol, 18% ficoll and 0.5 M sucrose in M2 medium). After being exposed or vitrified-thawed, oocytes of normal morphology were inseminated in vitro by $1-2\times10^6/ml$ of epididymal sperm. The rates of fertilization, in vitro/in vivo development and cell number (inner cell mass and tropectoderm cell) of blastocysts in each treatment group were examined. The results obtained in these experiments were summarized as follows: The cleavage rates were obtained in EFS35 containing 35% ethylene glycol higher than in EFS30 and EFS40. The development rate of vitrified-thawed oocytes to two-cell stage after in vitro fertilization (51.1%) was significantly different compared to that of exposed to vitrification solution without cooling (60.0%) and control (68.2%) (p<0.05). However, there were no differences in the blastocyst formation from the cleaved embryos among groups (75.0, 73.3 and 80.0%). Also, the mean number of cells per blastocysts of vitrified group $(92.5{\pm}2.9)$ was similar to that of the exposed $(98.5{\pm}5.3)$ and control $(100.9{\pm}4.8)$. In vivo development of the blastocysts derived from vitrified-thawed oocytes resulted in fetal development (50.7%) and implantation rates (80.0%) which are very similar to those of control (58.2, 78.2%). These results suggest that mouse oocytes could be cryopreserved using vitrification solution (EFS35) based on ethylene glycol.

• Asian-Australasian Journal of Animal Sciences
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• v.30 no.9
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• pp.1245-1252
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• 2017

Objective: Phellodendron amurense (P. amurense) and Humulus japonicus (H. japonicus) are closely involved in anti-oxidative response and increasing antioxidant enzymes activities. However, the effects of their extracts on development of preimplantation bovine embryos have not been investigated. Therefore, we investigated the effects of P. amurense and H. japonicus extracts on developmental competence and quality of preimplantation bovine embryos. Methods: After in vitro fertilization, bovine embryos were cultured for 7 days in Charles Rosenkrans amino acid medium supplemented with P. amurense ($0.01{\mu}g/mL$) and H. japonicus ($0.01{\mu}g/mL$). The effect of this supplementation during in vitro culture on development competence and antioxidant was investigated. Results: We observed that the blastocysts rate was significantly increased (p<0.05) in P. amurense ($28.9%{\pm}2.9%$), H. japonicus ($30.9%{\pm}1.5%$), and a mixture of P. amurense and H. japonicus ($34.8%{\pm}2.1%$) treated groups compared with the control group ($25.4%{\pm}1.6%$). We next confirmed that the intracellular levels of reactive oxygen species (ROS) were significantly decreased (p<0.01) in P. amurense and/or H. japonicus extract treated groups when compared with the control group. Our results also showed that expression of cleaved caspase-3 and apoptotic cells of blastocysts were significantly decreased (p<0.05) in bovine blastocysts derived from both P. amurense and H. japonicus extract treated embryos. Conclusion: These results suggest that proper treatment with P. amurense and H. japonicus extracts in the development of preimplantation bovine embryos improves the quality of blastocysts, which may be related to the reduction of ROS level and apoptosis.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.191-197
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• 2016

Historically, Korea old cattle had been consisted with various lines of coat color brindle, black and white-brown breeds or more. The two rare lines of black and white coat color are maintained for animal resources and preserved critically. The present study was carried out to evaluate potential usage of cysteamine supplementation during in vitro matration (IVM) and in vitro culture/production of embryo (IVP) by transvaginal ultrasound-guided follicle aspiration (Ovum Pick-Up: OPU) for the establishment of cryo-banking system. Immature slaughterhouse-derived cumulus-oocyte complexes (SL-COCs) were matured in IVM medium supplemented with 0, 0.1, 0.3 or 0.9 mM cysteamine, and then cultured in mSOF-BAS for 8 days after in vitro fertilization. The treatment of 0.1 mM cysteamine on SL-COCs showed higher rate of blastocyst, so OPU-derived COCs from rare breeds were matured in TCM media supplemented with or without 0.1 mM cysteamine, FSH and 5% FBS. The embryos were evaluated their developmental stages on day 8. During IVM, cysteamine treatment significantly increased the embryo production rate of slaughterhouse-derived COCs (19.6% vs. 30.5%). The presence of cysteamine during IVM of OPU-derived COCs from rare Korean cattle breeds (albino white and black line) also increased embryo production rates than those from SL-COCs (27.4% vs. 41.9% and 36.4%). With these results, cysteamine treatment during IVM is one of key factors IVP of blastocysts to establish banking system of endangered rare Koarean cattle with OPU derived transferable blastocysts.

• Korean Journal of Animal Reproduction
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• v.11 no.1
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• pp.73-84
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• 1987

This experiment was carried out to investigate the effects of hormone addition(FSH, HCG, estrogen and progesterone) and composition (BSA and FCS) of mKRB on the in vitro maturation and fertilizability of follicular oocytes of the Korean native cattle. The ovaries were removed at a slaughterhouse, returned to laboratory in a thermostat (30-35$^{\circ}C$) within 4 hr, and collected by aspirating normal follicles which had diameters of 1 to 6 mm. The oocytes with cumulus cells were cultured for 8, 16, 24 and 30 hr in a modified KRB solution containing BSA or FCS and hormones. The in vitro matured oocytes in mKRB containing FCS, FSH and steroids were transferred in the rabbit uterus for examination of their in vivo fertilizability with bovine sperm preincubated 4 to 6 hr in the rabbit uterus. 1. The mean number of oocytes collected per cattle was 6.5 from 1-3mm follicles, 1.3 from 4-6mm follicles, and total was 7.7. 2. The meiotic division at 16hr-cuture in the oocytes from 1-3mm follicles was slightly stimulated by the addition of FSH in mKRB + BSA solution compared with the control. At 30hr-culture, their maturation rates(%Met II) were also increased by FSH of 1 $\mu\textrm{g}$/ml(38.4%) and 5$\mu\textrm{g}$/ml(35.7%) as compared with the control (21.4%). The maturation rate at 30hr-culture in the oocytes from 4-6mm follicles was 53.8% and 57.1% by the FSH addition of 1$\mu\textrm{g}$/ml and 5$\mu\textrm{g}$/ml, respectively. These rates were similar with the control(57.1%), but higher than those of oocytes from 1-3mm follicles. 3. The meiotic division at 16hr-culture in the oocytes from 1-3mm follicles was stimulated by the HCG addition of 1IU/ml and 5IU/ml. However, the maturation rate at 30hr-culture was greatly decreased by the HCG addtion (26.6% and 13.3%) compared with the control(53.3%) and these rates (30.8%) in the oocytes from 4-6mm follicles were also lower than that fo the control(58.3%). 4. Low maturation rate (37.5%) of the oocytes cultured in mKRB containing BSA and 5IU/ml HCG was increased (55.0%) when 15% FCS with HCG was added to mKRB instead of BSA. 5. When 16hr-cultured oocytes in mKRB containing BSA and gonadotropins (5$\mu\textrm{g}$/ml FSH and 5IU/ml HCG) were transferred in the medium without gonadotropins and recultured for 16hr, the maturation rate of HCG-treated oocytes was greatly improved. 6. The maturation rates of oocytes were greatly affected by steroids. The combined addition of FCS+FSH+estrogen or +progesterone to mKRB increased the maturation rate compared with the combination of BSA+FSH or FCS+FSH in mKRB. 7. The fertilization rate, presence of pronuclei, was increased by the combination of FCS+FSH+p in mKRB as compared with that (5.6%) of BSA+FSH and the rates of FCS+FSH+steroids ranged from 12.5 to 17.6%.

• Journal of Korean Society of Forest Science
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• v.77 no.4
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• pp.361-370
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• 1988

This study was carried out for observation of growth, mycorrhizal formation and nutrient absorption of Pinus thunbergii seedlings treated with two soil mixtures and various nitrogen levels after inoculation with mycorrhizal fungus, Pisolithus tinctorius. 1. Seedlings grown on vermiculite applied with $50-150{\mu}g/ml$ nitrogen levels were well developed with pinnate type and cluster-like mycorrhizae. But seedlings on sandy loam had monopodial type in addition to the above-mentioned two types. 2. Optimum fertilization level for mycorrhizal formation is 50 or $150{\mu}g/ml$ N that showed best mycorrhizal formation of $86.4({\pm}3.14)%$ or $73.0({\pm}7.21)%$, respectively, but increased nitrogen levels decreased formation of mycorrhizal short roots. Seedlings applied with $450{\mu}g/ml$ nitrogen level decreased in net assimilation rate (NAR) and crop growth rate(CGR) during early growth of the seedlings, and they were increased since Aug, when nutrient application was stopped. 9. Inorganic nutrient absorption was increased more in seedlings grown on vermiculite and inoculated growth medium than those grown on sandy loam and noninoculated one, and it was gradually increased with increasing nitrogen increasing nitrogen level until $350{\mu}g/ml$. But $450{\mu}g/ml$ nitro gen level rather reduced absorption of nutrient.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.131-137
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• 1997

This study was carried out to investigate the in vivo development rates of vitrified-thawed mouse expanding, hatching and hatched blastoc ysts(BL). In vitro fertilization produced blastocysts were vitrified in EFS40(40% ethylene glycol, 30% Ficoll and 0.3 M sucrose in phosphate buffer saline containing 10% FBS). Expanding a and hatching blastocysts were equilibrated in 20% ethylene glyco](EG) for 5 min. before exposure to EFS40 at 25°C for 1 min., they were then vitrified in liquid nitrogen. Hatched blastocysts which cultured in m-CR1 medium supple mented 0.4% bovine serum albumin on day 5. were equilibrated in 10% EG for 5 min. and then vitrified in EFS40 for 30 sec. After thawing, re-expanding blastocysts were transferred to recipients(3 day of pseudopregnant) on one or both uterine horns(6-8 embryos per a horn). Preg¬n nancy rates of recipients and implantation were a assccessed by autopsy on 15 gestation. The res¬u ults obtained in these experiments were summar¬1 ized as follows; 1) The pregnancy and live fetus rates, for vitrified expanding BL(77.8 and 25.0%) and hatching BL(77.8 and 26.4%)n vitro were not significantly difference in those of control BL (66.7 and 42.9%: 83.3 and 40.4%), respectively, 2) in vitro development of vitrified hatched BL was 34.0%. and 3) in vivo developmental rate of vitrified hatched BL was only 33.3%. These results suggested that proposed rapid vitrification p procedures used EFS40 cryoprotectant can be effectively performed in mouse expanding Ihatching blastocysts and that mouse blastocysts a after being hatched from zona pellucida can be successfully cryopreserved.