• Journal of Embryo Transfer
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• v.23 no.2
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• pp.115-118
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• 2008

Glycerol is the cryoprotectant most frequently used to freeze semen in several of species. The objective of the present study was to compare the effect of three different glycerol concentrations (4, 6 or 8%, v/v) on frozen-thawed dog sperm survival rate. Ejaculates from 9 dogs collected by digital manipulation were pooled and assessed by macroscopic and microscopic criteria. Semen was divided into 3 aliquots, which were centrifuged and the sperm pellets rediluted with first Tris-glucose-citric acid extender. After 1 h cooling at $4^{\circ}C$, second extender containing 4, 6 or 8% glycerol was added, respectively. The semen was loaded into 0.25 ml straws and frozen and stored in liquid nitrogen and thawed. Sperm vigor, live:dead spermatozoa ratio using HOS test, and sperm morphology using $Spermac^{(R)}$ stain were evaluated. After thawing, there were no significant differences among groups in vigor, viability and morphology. In conclusion, the three glycerol concentrations (4, 6 or 8%) can be used successfully in cryopreservation of canine semen. Therefore the use of 4% glycerol in the extender has less toxic effect and reduces of freezing injuries.

• Journal of Embryo Transfer
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• v.28 no.1
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• pp.13-18
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• 2013

The increase in the meat quality and milk production of cows, which breed Korean Native Cow (KNC) and Holstein cow, is not improving reproductive efficiency. In this study, we examined the effect of interferon (IFN) supplementation on motility of frozen-thawed semen and pregnancy rate after artificial insemination of KNC and Holstein cow. In experiment 1, we investigated the effect of IFN-tau concentration (10,000 IU and 20,000 IU) on the percentage of total motility (TM) and progressive motility (PM) of frozen-thawed spermatozoa. In experiment 2, KNC were infused 20,000 IU IFN-tau at insemination or after insemination. In experiment 3, KNC or Holstein cow were inseminated with frozen-thawed semen and infused 20,000 IU IFN-gamma or -tau after insemination. In experiment 1, the average of TM (23.9% to 30.9%) and PM (18.5% to 21.9%) were similar between control and treatments. In experiment 2, the pregnancy rates of IFN infusing times were not different from 45.8% to 53.6%. In experiment 3, the pregnancy rates of Holstein cow infused different kinds of IFN were similar (control, IFN-gamma, IFN-tau; 42.9%, 40.5%, 48.0%). In the case of KNC, however, the pregnancy rate of control was 55.6%, which was significantly lower than that of IFN-gamma (68.9%; p<0.05). Thus, IFN is effective on the improvement of reproductive efficiency, but further study is needed.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.41-50
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• 1996

This experiment was carried out to investigate the ovarian responses of the ovulation point, ovarian weight and size, the number of ovarian follicles and collected embryos, and to study the effects of the developmental stages (oocytes, 2-4 cell. 8-16 cell and morulae), additional levels of Ficoll (0, 15, 30%) on the survival rate (FDA-test) of rat embryos frozen in vitrification solution (20% glycerol + 10% ethylene glycol + 10% sucrose). Sunanarized results was as follows; 1. The mean ovulation point per head was 7, and the weight of ovaries was 0.03g. The size of ovary was 5.9 mm(L) and 4.6 mm(W), and the number of ovarian follicles over and below 2 mm was 4.7 and 8.7, respectively. The number of the collected embryos per head was 5.5 (79%). 2. 2. The FDA score of embryos frozen in 20 G 10 E 10 S without Ficoll was 2.8 (oocyte), 2.6 (2-4 cell), 3.9 (8-16 cell) and 3.6 (morula), respectively. However, there were no significant differences among treatments. 3. The FDA score of embryos frozen in 20 G 10 E 10 S with 15% Ficoll was 3.4 (oocyte), 4.0 (2-4 cell), 4.7 (8-16 cell) and 4.8 (morulae), respectively (P>0.05). 4. The FDA score of embryos frozen in 20 G 10 E 10 S with 30 % Ficoll was 3.7 (oocyte), 3.2 (2-4 cell), 4.4 (8-16 cell) and 4.4 (morulae), respectively (P>0.05). 5. As shown in the above results, the higher survival rate was obtained in the treatment of 15% Ficoll than that of 30%. And the survival rate (FDA-test)of the oocytes and 2-4 cell stages of the rat embryos was lower than that of 8~16 cell and morulae stages. It was considered that 8-16 cell and morulae could be available for the successful freezing by vitrification of rat embryos with 15% Ficoll except for oocytes.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.9-12
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• 2011

This study was undertaken to determine whether the presence of fertility-associated antigen (FAA) in semen would influence semen characteristics and conception rate of artificial insemination in Hanwoo. The response to FAA of 36 heads of proven bull, 7 heads of young bull, and 27 heads of performance-tested bull was that one proven bull was FAA-negative and the others were FAA-positive, therefore FAA-negative bull was 1.4%. FAA-negative bull was lower in first and second semen concentrations than those of FAA-positive bull in 5,301 semen of 21 heads of proven bull, then FAA-negative bull was fewer as 11.5% in total sperm counts. The estrus of 22 heads was 70d-nonreturned in 36 cows first inseminated with frozen semen of FAA-negative bull, but that of 249 heads in 378 cows first inseminated with frozen semen of FAA-positive bull. Each conception rate was 61.1% and 65.9%, respectively. The difference of conception rates was 4.8%. These results indicate that the response of FAA to semen were influenced semen characteristics and conception rate of artificial insemination, but further investigations are needed to confirm the results.

• Journal of Embryo Transfer
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• v.26 no.3
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• pp.219-222
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• 2011

Eight female Himalayan tahrs (Hemitragus jemlahicus) were estrus-synchronized, and transcervically inseminated with frozen-thawed semen in September, 2009, about 2 to 3 months earlier than their natural breeding season. Intravaginal progesterone-releasing devices were inserted into vaginas of six Himalayan tahrs on September 7, and the other two on September 8 to suppress luteal function of ovaries. The devices had been placed deep inside the vagina prior to withdrawal on September 23. A day before CIDR removal, a combination of PMSG 400 IU and hCG 200 IU was intramuscularly injected. Forty hours later, frozen-thawed semen was transcervically inseminated. Pregnancy diagnosis was performed 39 days later by analyzing progesterone level of serum. Every treatment was done under anesthesia inducted by xylazine injection. In conclusion, vaginal discharge of cervical mucus, hormonal changes induced by implant-typed or muscularly injectable hormones and widening of cervix enough to insert an insemination gun into uterine body were achieved in non-breeding season. Moreover, the first inseminated Himalayan tahr, 36 hours after CIDR removal was assumed to be pregnant but the fetus may have been lost due to the use of anesthetic drug.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.269-277
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• 1994

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes These experiments were conducted to examine the effect of sperm factor on the IVF and IVD, and the effect of coculture with somatic cells on the IVD of embryos. Although the concentration of epididymal sperm for IVF did not affect on cleavage rate, but 5 x 105 sperm/mi showed the highest cleavage rate(48.7%) and the developmental potential of IVF oocytes from this concentration was also greatly higher (P$^{\circ}C$-stored sperm for l2hrs and the cleavage rate from fresh sperm was significantly higher (P<0.05) than that from frozen sperm, but the developmental potential after IVF was slightly high from the frozen sperm. The cleavage rate of IVF oocytes cocultured with oviductal epithelial cells and cumulus cells was 76.3% and 72.9%, respectively. There was no difference between two coculture systems but this rate was significantly higher(P<0.05) than that of medium alone(42.0%).

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.103-103
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• 2002

This study was conducted to investigate cryopreservation of pearl oyster, Pinctada fucata martensii larvae. Four cooling rates (-0.25, -0.5, -0.75 and -1.0$^{\circ}C$/min.) were used to examine a proper cooling rate during cryopreservation of trochophores before seeding temperature (-12$^{\circ}C$). Seven developmental stages (early and late trochophores, early and late D-shaped larvae and early, middle and late umbo stage larvae) and different sugars (fructose, glucose and sucrose) were used to investigate optimal larval stage and effective sugar in cryopreservation of larvae. The survival rates of frozen-thawed trochophores increased at cooling rate of -1.0$^{\circ}C$/min. As larval developing, survival rate of frozen-thawed larvae increased, except umbo stage larvae, and especially late D-shaped larvae highly survived as 91%. Addition of sugar revealed positive effect on cryopreservation in this experiment and 0.2 M glucose and sucrose mixed with 2.0 M dimethyl sulfoxide significantly enhanced survival rate of larvae (P<0.05). The results of our study indicate that desirable cooling rate, developmental stages of larvae and effective sugar far cryopreservation of pearl oyster, P. fucata martensii larvae are -1$^{\circ}C$/min, late D-shaped larvae and 0.2 M glucose and sucrose, respectively.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.104-104
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• 2002

This study was performed to find out the optimal larval stage among trochophore, D-shaped and umbo stage larvae and the desirable protective additive such as fructose, glucose, sucrose and trehalose with cryoprotectant for cryopreservation of surf clam, Spisula sachalinensis larvae. Dimethyl sulfoxide and ethylene glycol were used as cryoprotectant and each cryoprotectant was made to 2.0 M with previous protective additives. The larvae were immersed in the preparations waited for 15 minutes to reach equilibration, and then frozen in a program freezer (-35$^{\circ}C$) and liquid nitrogen (-196$^{\circ}C$). The freezing rate of 1.0$^{\circ}C$ /min. was used for cryopreservation of trochophores before seeding temperature (-12$^{\circ}C$). The survival rate of frozen-thawed larvae increased as larval developing and that of umbo stage larvae was the highest as 96.1 ${\pm}$ 1.0%. The presence of lower concentration of disaccharides as sucrose or trehalose significantly enhanced survival rate when mixed with cryoprotectants (P<0.05). The results of our study indicate that desirable developmental stages of larvae and protective additive for cryopreservation are the umbo stage larvae and 0.2 M sucrose, respectively.

• Journal of Embryo Transfer
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• v.7 no.1
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• pp.13-19
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• 1992

This study were carried out to investigate the effective concentration of cryoprotective agents and sucrose by one-step straw method, and to determine the optimum thawing temperature and equilibration time of frozen porcine embryos. The porcine embryos foflowing dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined by FDA test. The results are sunnnarized as follows : 1. The survival rates of porcine embryos after ultrarapid frozen4hawing in the freezing medium with a various concentration of glycerol, DMSO and propanediol added 0.25M sucrose were higher survival rate than those of sucrose concentration of 0.50M. 2. The survival rates of porcine embryos after ultrarapid ftozen4hawing in the freezing medium added 0.25M and 0.SOM sucrose were higher survival rate than those of sucrose concentration of 0.75M and 1.00M. 3. The temperature thawed at 2$0^{\circ}C$ and 3$0^{\circ}C$ resulted in a significantly higher embryos survival rate after 72 hrs in culture than did at 35$^{\circ}C$. 4. The equilibration time on the survival rate of porcine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time(10~20 min.).

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.193-202
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• 1995

In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.