• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.5
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• pp.516-523
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• 2011

To compare four populations of sea squirt Halocynthia roretzi adults, their egg quality and larval development were investigated in the hatchery. The populations were: south sea wild (SSW), south sea cultured (SSC), east sea wild (ESW) and Iwate Japan cultured (IJC). Egg quality and larval development were compared using 13 factors (fertilization rate and diameter, proximate composition, amino acid and fatty acid contents of eggs, hatching rate and various sizes of tadpole larvae) which were obtained from each population. Fertilized egg diameter, hatching rate and size of tadpole larvae were significantly different among the four populations (P<0.05). Eggs were produced with higher crude protein ($5.20{\pm}0.00%$ SSW, $4.71{\pm}0.01%$ ESW, $4.66{\pm}0.01%$ SSC and $3.96{\pm}0.01%$ IJC) and lipid ($1.22{\pm}0.01%$ ESW, $1.01{\pm}0.00%$ SSW, $0.77{\pm}0.01%$ SSC and $0.69{\pm}0.00%$ IJC,) contents from domestic wild populations than from Japanese or cultured populations. Also amino acid and fatty acid contents were different. The extent of similarity between domestic and Japanese populations (30.5% IJC:SSW, 34.3% IJC:SSC and 40.7% IJC:ESW) was relatively low but was very high between SSW and SSC (73.9%). These results may have been due to differences in the abundance of food types and environmental conditions in the four localities and consequent differences in the diets of the sea squirts.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.325-333
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• 1997

The use of hormonal stimulation in human in vitro fertilization and embryo transfer (IVF-ET) leads to increased production of embryos for ET. So to avoid high pregnancies and to allow conception in future, unstimulated cycles, cryopreservation of spare embryos is desirable. One of the improvement of cryopreservation methods is vitrification. We cryopreserved mouse day 3 embryos by vitrification using the three different vitrification solution (EFS40, VS11 and VS3a). EFS40 solution is consisted of 40% (v/v) ethylene glycol, Ficol170 30% (w/v) and 0.5M sucrose and VS11 is 6.0M ethylene glycol and 1.8M glycerol. And VS3a is 6.5M glycerol and 6% (w/v) BSA (bovine serum albumin). First we tested the toxicity of three vitrification solution by exposure to these solution during 3 min. After washing by thawing solution, the survival rates of each groups are 95.5%, 90.9% and 84.4% (EFS40, VS11 and VS3a). High percentages of them developed to expanded blastocyst and hatching embryos in culture 48hrs 94.2%, 97.7%, 100% and 97.4% (no treatment group, EFS40, VS11 and VS3a). So there is no significant differences among the each group. Second, after thawing of vitirfied embryos, the survival rates of each groups are 96.8% (slow freeze), 94.1% (EFS40), 85.5% (VS11) and 80.0% (VS3a, P vs. no freeze or EFS40 is 0.01). Vitrified embryos exhibited a high rate of development in vitro after 48hrs culture. The percentages of each group to blastocyst and hatching embryos are 88.7% (no freeze), 91.8% (slow freeze), 93.4% (EFS40), 87.7% (VS11) and 73.0% (VS3a, P vs. other group is 0.01). The results suggest that there is no significant differences in exposure of various vitrification solution and day 3 mouse embryos can be vitrified in solution EFS40 and VS11 by simple procedure.

• 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.

• Development and Reproduction
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• v.17 no.3
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• pp.221-229
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• 2013

Cathepsins are members of the multigene family of lysosomal cysteine proteinases and have regulated function in several life processes. The potential role of cathepsin F cysteine gene was expected as protease in the yolk processing mechanism during early developmental stage, but expression analysis was unknown after fertilization. The alignment analysis showed that amino acid sequence of cathepsin F from olive flounder liver expressed sequence tag (EST) homologous to cathepsin F of other known cathepsin F sequences with 87-98% identity. In this study, we examined the gene expression analysis of cathepsin F in various tissues at variety age flounder. Tissue distribution of the cathepsin F mRNA has been shown to be ubiquitous and constitutive pattern regardless of age in each group, although derived from cDNA library using liver sample. The mRNA level of cathepsin F more increased as developmental proceed during embryogenesis and early developmental stage, especially increased in the blastula, hatching stage and 3 days post hatching (dph). As a result, it may suggest that the proteolysis of yolk proteins (YPs) has been implicated as a mechanism for nutrient supply during early larval stages in olive flounder.

• Journal of Aquaculture
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• v.9 no.2
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• pp.133-140
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• 1996

Hybird and allotriploid between female rainbow trout (Oncorhynchus mykiss) and male coho salmon (O. kisutch) were produced by artificial fertilization and heat shocks. Hatching and survival rate of allotriploid at 2 month after hatching was $77.6\%$ and $54.5\%$ respectively, and these rates clearly exceeded those of their hybrid. Cell and nuclear sizes of the erythrocyte of hybrid were intermidiate of their parents and those of allotriploid were larger than thier hybird. The somatic chromosome number of viable hybrid was 2n = 60 and that of allotriploid was $90\~93$ with chromosomal polymorphism. Allotriploid karyotpe was constituted by two sets of rainbow trout chromosome and one set of coho salmon chromosome.

• International Journal of Industrial Entomology and Biomaterials
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• v.26 no.1
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• pp.61-66
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• 2013

Baculum elongatum has several peculiarities such as parthenogenesis, unique external features, changing body color, and dropping oviposition, which makes it a potential economically useful insect. Oviposition was performed by females without fertilization by the sperm of males. The oviposition period was $42.2{\pm}22.7$ days and the number of eggs per female was $109.5{\pm}70.5$ eggs. The hatch rate was 73.3% at $25^{\circ}C$ and 66.7% after low temperature treatment ($8^{\circ}C$ for 60 days). In nature, B. elongatum overwinters as an egg, but it can be assumed from the results that cold temperatures were not required for hatching. The hatch rate was 98.2% in the treatment using floral foam, fermented sawdust, and leaves. The developmental period was $100.9{\pm}4.2$ days for eggs, $55.3{\pm}4.6$ days for nymphs, and $49.7{\pm}16.0$ days for adults. The length of the eggs was $0.33{\pm}0.0$ cm and the lengths of the nymphs were clearly distinguishable according to the instar stage. Clover(Trifolium repens) was an excellent diet as it was similar to the host plant and could be used as an alternative diet. The rate of reaching adulthood for the insects was 66.7% on an artificial diet containing 25% acacia leaves. For sustainable mass rearing of nymphs or adults of B. elongatum, a natural diet could be used such as acacia (Robinia pseudoacacia), white oak (Quercus aliena), chestnut (Castanea crenata var. dulcis), and bush clover (Lespedeza bicolor), or an alternative diet such as clover or artificial diet.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.2
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• pp.87-92
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• 2011

Objective: This study aimed to determine the safety and clinical effect of artificial shrinkage (AS) in terms of assisted hatching of fresh blastocysts. Also, we evaluated the correlation between patient age and the effect of AS on clinical outcome. Methods: Two AS methods, using a 29-gauge needle and laser pulse, were compared. Seventy-three blastocysts were shrunk using a 29-gauge needle and the same number of other blastocysts were shrunk by a laser pulse. We evaluated the shrunken blastocysts hourly and considered them viable if they re-expanded >70%. Blastocyst transfer cycles (n=134) were divided into two groups: a control group consisted of the cycles whose intact embryos were transferred (n=100), while the AS group consisted of the cycles whose embryos were replaced following AS (n=34). The implantation and pregnancy rates of the control group and AS group were compared ($p$ <0.05). Results: The re-expansion rates of the 29-gauge needle and laser pulse AS groups were similar (56 [76.7%] vs. 62 [84.9%], respectively). All of the remaining shrunken blastocysts were re-expanded within 2 hours. There was no degeneration of shrunken blastocysts. The total and clinical pregnancy rate of the AS group (23 [67.6%]; 20 [58.8%], respectively) was significantly higher than that of the control group (47 [47.0%]; 39 [39.0%], respectively). In the older patient group, there was no difference in the clinical outcomes between the AS and control groups. Conclusion: These results suggest that AS of blastocoele cavity, followed by the transfer, would be a useful approach to improve the clinical outcome in cycles in which fresh blastocyst stage embryos are transferred.

• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.329-338
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• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.8
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• pp.1117-1123
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• 2009

This study investigated the effects of IGF-I and EGF on the development of blastocysts or hatched blastocysts during the in vitro culture of embryos from immature porcine oocytes. After the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs) and their culture in vitro in PZM3 medium, we examined the embryo development rate for 168 h. When different concentrations of IGF-I (0, 1, 10, 20 ng/ml) were supplemented to fertilized porcine embryos in vitro, there were no significant differences in cleavage rate, blastocyst development rate or blastocyst hatching rate among the treated groups. On the other hand, when different concentrations of EGF (0, 1, 10, 20 ng/ml) were supplemented to the in vitro culture medium, blastocyst development rate was highest in the group in which EGF was not supplemented and, specifically, it was higher than in the 20 ng/ml treatment group (p<0.05). When 10 ng/ml IGF-I and 1 ng/ml EGF were supplemented separately or simultaneously, there were no significant differences among the treated groups in blastocyst hatching rate and the number of cells in each condition. This study demonstrated that the addition of IGF-I and EGF into PZM3 medium did not enhance development of the blastocyst stage and total cell number in blastocysts.