• Journal of Embryo Transfer
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• v.10 no.1
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• pp.53-63
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• 1995

The effects of different protein sources (serum vs bovine serum albumin), growth factors (EGF and PDGF) and co-culture with various type of somatic cel1s (BOEC, MEF and BRL) on the in vitro development of in vitro matured / in vitro fertilized bovine oocytes were examined, and the viability of frozen/thawed embryos derived from IVM /IVF was examined. Cell numbers of blastocysts were also counted. In Experiment 1, CR$_1$aa with serum was superior to CR$_1$aa with BSA in producing morulae plus blastocysts from IVM /IVF oocytes(24.4% vs 30.4%, p>0.05). In Experiment 2, more morulae plus blastocysts(42.3%) were produced in CR$_1$aa containing long /ml EGF than in the control CR$_1$aa(33.3%). In Experiment 3, 2- to 8-cell embryos derived from IVM /IVF oocytes were randomly allotted to one of 4 culture groups : a) CR$_1$aa ; b) CR$_1$aa + ing /ml PDGF ; CR$_1$aa + Sng /ml PDGF ; CR$_1$aa + lOng /ml PDGF ; culture resulted in 21.3, 51.2, 41.4 and 45.9%(p<0.05), respectively, developing into morulae and blastocysts. In Experiment 4, 0 and Sng /ml PDGF added to CR$_1$aa coculture with BRL or BOEC yielded 47.5, 42.5, 33.8 and 41.6% morulae and blastocysts, respectively. In Experiment 5, the proportion of embryos into morulae and blastocysts was highest in CR$_1$aa with MEF coculture group(50.9%) compared to any other group(CR$_1$aa, 22.3%; CR$_1$aa+BRL, 32.9%; CR$_1$aa+BOEC, 33.8%, p>0.05). In Experiment 6, survival rate of blastocysts produced by in vitro fertilization when cryoprotectant was removed in 0.7M glycerol+0.7M sucrose and 0.7M sucrose solution for 10 min. after thawing at 2$0^{\circ}C$ (Exp. H, 58.8%) was slightly higher than when cryoprotectant was removed 10%, 6.7% and 3.3% glycerol for 10 min. after thawing at 37$^{\circ}C$ (Exp. I, 54.3%). These study indicate that growth factors and somatic cell co-culture can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number and frozen /thawed method employed this experiment was not different.

• Journal of Embryo Transfer
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• v.18 no.2
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• pp.143-149
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• 2003

This study was carried out to cryopreserve and to investigate characteristics of semen in Hanwoo. Semen was obtained from bulls selected by Daekwanryeong Branch station. Semen was collected each morning of the experiment, placed in water jacketed tubes at 37$^{\circ}C$, and trans-ported to the research laboratory within 10 minutes. Semen was extended with Egg yolk-glycerol extender to contain 50${\times}$10$^{6}$ sperm/ml. Semen was cooled over a 6h period in water jacketed tubes from about 25 to 5$^{\circ}C$, Egg yolk-glycerol extender was added in one step at 5$^{\circ}C$. Semen was aspirated into 0.5ml straws, which were sealed with powder. Egg yolk-glycerol extender, which is used in Hanwoo sperm frozen and stored, semen from 13 Hanwoo bulls collected, the postthawed percentages of motile sperm were 65.7%. In semen characteristics of Hanwoo bulls, number of bulls volume are 5.7 ml and total cell count are 975${\times}$10$^{6}$ m1 ejaculate.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.1
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• pp.36-40
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• 2007

Proteins and lipids not only provide a source of energy to the cell, but also play vital roles in modifying the physical properties and function of the biological membranes. In the present study, we investigated the biochemical constituents, viz. proteins and lipids, in growing oocytes of goat antral follicles during summer and winter seasons. Goat genitalia in phosphate buffered saline (pH 7.4) were brought to the laboratory within one hour of slaughter under aseptic conditions at $37^{\circ}C$. Oocytes were aspirated from normal small (<3 mm in diameter) and large (>3 mm) follicles and pooled for biochemical estimations. A significant increase in the amount of protein and lipid was observed with the growth of the oocyte. The amount of protein varied non-significantly with the season, while the amount of lipid varied significantly. The amounts of phospholipid, cholesterol, free fatty acid, and triglyceride increased with the growth of the oocyte, but no significant effect of season in these constituents was observed. Lysolecithin, sphingomyelin, and sterols were the polar lipids identified in both oocytes prepared from small follicles (small oocytes) as well as large follicles (large oocytes). In addition, the small oocytes also contained phosphatidyl serine, while large oocytes contained phosphatidyl glycerol phosphate and phosphatidyl inositol. Among non-polar lipids, triglycerides and long chain alcohols appear only in small oocytes and not in large oocytes. Monoglycerides, 1,2-diglycerides, 1,3-diglycerides and o-dialkyl glycerol ethers, fatty acids, fatty acid methyl esters, and wax esters were identified in both small and large oocytes. Information on biochemical composition of growing oocytes is relevant to oocyte and embryo competence, culture and cryopreservation.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• Clinical and Experimental Reproductive Medicine
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• v.43 no.1
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• pp.31-37
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• 2016

Objective: The aim of this study was to investigate associations between the morphology score of blastocysts and blastocoele re-expansion speed after warming with clinical outcomes, which could assist in making correct and cost-effective decisions regarding the appropriate time to vitrify blastocysts and to transfer vitrified-warmed blastocysts. Methods: A total of 327 vitrified-warmed two-blastocyst transfer cycles in women 38 years old and younger were included in this retrospective study. Results: The clinical pregnancy rate (CPR) and implantation rate (IR) of transfers of two good-morphology grade 4 blastocysts vitrified on day 5 (64.1% and 46.8%, respectively) were significantly higher than the CPR and IR associated with the transfers of two good-morphology grade 3 blastocysts vitrified on day 5 (46.7% and 32.2%, respectively). No significant differences were found in the CPR and IR among the transfers of two good-morphology grade 4 blastocysts regardless of the day of cryopreservation. Logistic regression analysis showed that blastocoele reexpansion speed after warming was associated with the CPR. Conclusion: The selection of a good-morphology grade 4 blastocyst to be vitrified could be superior to the choice of a grade 3 blastocyst. Extending the culture of grade 3 blastocysts and freezing grade 4 or higher blastocysts on day 6 could lead to a greater likelihood of pregnancy. Since re-expansion was shown to be a morphological marker of superior blastocyst viability, blastocysts that quickly re-expand after warming should be prioritized for transfer.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.80-80
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• 2003

In cryopreserved rat embryos, survival rates obtained in vitro are not always consistent with the rates obtained in vivo. To determine the optimal conditions for in vivo development to term, rat embryos at the 4-cell, 8-cell and morula stages were vitrified in EFS40 by a 1-step method and transferred into oviducts or uterine horns of recipients at various times during pseudopregnancy. Vitrified and fresh 4-cell embryos only developed after transfer into oviducts of asynchronous recipients on Day -1 to -2 of synchrony, i.e., at a point in pseudopregnancy that was 1-2 days earlier than the embryos. However, although about half the vitrified embryos transferred into oviducts on Day -1 developed to term, only a minority of embryos transferred at later times did so, whether vitrified (10-34%) or fresh (24-33%), suggesting that this may not be the most suitable stage for cryopreservation. Very few 8-cell embryos, either vitrified or fresh, developed when transferred into oviducts on Day 0 to -0.5. However, when transferred into uterine horns, high proportions of vitrified 8-cell embryos (-63%) developed to term in reasonably synchronous recipients (Day 0 to -0.5) but not in more asynchronous ones (6%; Day-1). A majority of vitrified morulae also developed to term (52-68%) in a wider range of recipients (Day 0 to -1), the greatest success occurring with recipients on Day -0.5. Similar proportions of vitrified and fresh 4-cell embryos, 8-cell embryos and morulae developed to term when there was appropriate synchronization between embryo and recipient. Thus vitrification of preimplantation stage rat embryos does not appear to impair their developmental potential in vivo.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.163-169
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• 2013

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

• Korean Journal of Animal Reproduction
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• v.23 no.3
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• pp.247-256
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• 1999

The present study was performed to improve the reproductive disturbance as well as the elimination of microbiological contamination for animals bred under conventional conditions followed by in vitro fertilization and embryo transfer techniques including embryo and sperm freezing, using a mouse strain(M. m. molossinus-tt＠Kist) showing the abnormal behavior disorder derived from Korean wild mice (Mus musculus molossinus). Moreover, hematological and serum biochemical analyses were also carried out to obtain the basic data of this mouse strain The results are summarized as follows: 1. In comparison with hematological data, the numbers of RBC and platelet of this mouse strain were appeared as the higher value those that of the same aged inbred strains such as BALB/c, DBA/2, C57BL/6 and C3H /Hen. However, no differences were found in values of WBC, Hb and Ht. Moreover, total cholesterol of this strain showed a low value but triglyceride, total protein and albumin values were similar as in inbred strains. 2. The average numbers of superovulated oocytes treated with 2.5/2.5 IU and 5.0/5.0 IU of PMSG/hCG were 11.6 and 12.7, respectively. The fertilization rates of 2.5/2.5 IU PMSG /hCG treatment(87.9%) was higher than 5.0/5.0 IU treatment(52.0%) (p＜0.05) and the developmental rate of 2 cell stage embryos were 외 so appeared as higher value 99.0% and 90.6%, respectively. 3. The rates of in vitro fertilization treated with frozen sperm(24.8%) was significantly lower than of that fresh sperm(87.9%), (p＜0.05). 4. The five, six and ten heads of offspring were obtained from frozen-thawed 2 cell embryos by in vitro fertilized, 2 cell embryos from in vitro fertilized by frozen-thawed spermatozoa. and 2 cell embryos by in vitro fertilization, respectively. These offspring developed the expected disease about 2 weeks after birth, which was confirmed that the disease character of this mutant mouse strain was reliably reproduced. 5. MHV(Mouse hepatitis virus) and Staphylococcus aureus were successfully eliminated from conventional animals by in vitro fertilization-embryo transfer and the use of SPF recipient animals.

• 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.

• Journal of Embryo Transfer
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• v.21 no.2
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• pp.163-168
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• 2006

The present study was carried out to investigate in vitro development and post-thawed survivability of bovine embryos according to different ovary transport temperatures. Bovine ovaries were collected at a local slaughterhouse and were transported at 4 different temperature categories to laboratory: $7{\sim}10^{\circ}C\;(T1),\;11{\sim}17^{\circ}C\;(T2),\;18{\sim}25^{\circ}C\;(T3)$ and above $26^{\circ}C$ (control group). The cumulus-oocyte-complexes aspirated from ovaries were in vitro matured, fertilized and cultured. The rates of maturation (to metaphase II), cleavage and development to blastocysts were compared among treatment groups. Furthermore, frozen-thawed blastocysts were in vitro cultured to compare the survivability among groups. The maturation rates in the T1, T2 and T3 groups ($60.0{\sim}68.2%$) were significantly lower than that in the control group (81.8%, p<0.05). The cleavage rates in the T1 and T2 groups (52.6 and 54.5%) were significantly lower than that in the control group (83.6%, p<0.05). However, there was no difference in the development rate to blastocysts among all groups ($27.9{\sim}33.0%$, p>0.05). The survivability of frozen-thawed embryos was significantly lower in the T1 group (46.2%) than those in the T2, T3 and control groups ($68.8{\sim}7.13%$, p<0.05). In conclusion, the results suggest that ovary transport temperature at $26^{\circ}C$ may be optimal for the better in vitro development and the survival of frozen-thawed embryos produced in vitro Furthermore, exposure of ovary to temperature below $10^{\circ}C$ during transport may significantly decrease both in vitro development and survivability of frozen-thawed blastocysts.