• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.16 no.2
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• pp.180-184
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• 2003

The intrauterine inseminator (IUI) was developed to provide the method of depositing dog semen into the uterine body instead of the vagina. The IUI consists of a vaginal endoscope, a balloon sheath, and injection catheter. When the endoscope is inserted into the vagina and the balloon expanded with air, the cervical os becomes visible so a injection catheter can be inserted through the cervix for deposition of the frozen-thawed semen. The efficacy of the IUI device was compared to intra-vaginal artificial insemination using semen that had been collected and frozen from pooled sperm-rich fraction of ejaculates collected from two Jindo dog donors. Aliquots of semen were extended with a Tris-egg yolk diluent, centrifuged, the seminal plasma removed, the pellet resuspended with the same diluent, and cooled to $5^{\circ}C$ over a 2 h period. A Tris-egg yolk-glycerol extender was added at $5^{\circ}C$; after 1 h, semen was loaded into 0.5 ml straws, and straws were frozen in LN vapor for 5 min, and immersed in LN for storage. The final sperm concentration for freezing was approximately $100{\times}10^{6}cells/ml$. The straws were thawed at $70^{\circ}C$ for precisely 6 sec, 1.5 ml Tris-egg yolk buffer at $38^{\circ}C$ added, and the 2 ml of thawed semen was used for a single insemination using the IUI device. Each bitch was inseminated at optimal insemination point, which was estimated by vaginal epithelial cells staining and progesterone concentration analysis. Use of the IUI device resulted in 21 of 26 females giving birth to 89 pups ($4.2{\pm}1.6$ pups per litter), while intra-vaginal AI resulted in 6 of 15 females whelping a total of 17 pups ($2.8{\pm}1.2$ pups per litter). We believe the IUI device is easier to use than previously described devices used for intrauterine insemination. In our experience the expansion of the balloon has a calming effect on the bitch that aids the inseminator. These results indicate that the IUI device was able to provide high fertility with 50 million frozen sperm per insemination and two inseminations.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.153-162
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• 1992

To solve the logistical problems of the sperm penetration assay (SPA) to provide just a sufficient number of hamster ova exactly when they are needed, a new method to cryopreserve the ova has been devised (1-step dehydration and 2-step thawing). After freezing & thawing of zona-intact (ZI) and zona-free (ZF) hamster ova according to this new method, the frozen-thawed ova were compared with fresh, control ova (FO) in terms of the degree of sperm penetration in SPA using semen samples from fertile donors, subfertile, and infertile male. Each sperm sample was capacitated for 42 hours inTEST-Yolk Buffer before insemination in SPA. In fertile doner, both the penetration rate and penetration index were lower in SPA using frozen ova (ZI; 92.4%, 6.2, ZF; 63.7%, 3.9) than those of SPA using fresh ova (99.3%, 8.4). There was a significant correlation between the penetration index of SPA using FO and ZI (p<0.001), and between those of SPA using FO and ZF and ova (p<0.001). In subfertile patient, both the penetration rate and penetration index were lowered in frozen ova (ZI; 62.3%, 1.3, ZF; 21.8%, 0.4) than those of fresh ova (74.8%, 1.8). There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05 and p<0.001, respectively). In infertile patient, both the penetration rate and penetration index were ZI; 3.1%, 0.0, ZF;0.0%, 0.0, respectively. There were significant correlation between the penetration rate and penetration index in ZI ova (p<0.05).

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

• Reproductive and Developmental Biology
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• v.36 no.3
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• pp.147-154
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• 2012

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

• Clinical and Experimental Reproductive Medicine
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• v.11 no.2
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• pp.59-67
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• 1984

In vitro fertilization have been performed to know whether the frozen semen has fertilizing ability and can be used clinically. The results of cultured and developed embryos obtained are as follows: 1. The semen was frozen in three media for the good viability. The viability was more than 50% and the motility was also moderate (grade III), 2. As the 33 oocytes were collected from 45 follicles, the oocyte recovery rate was 73.3%. Among them, mature and immature ova were 5% each, and premature ova were 69.7%, When the first polar body was appeared, above ova were inseminated after adequate incubation with activated sperms. 3. The main components of three freezing medium containing egg yolk, glycerol and pyruvate respectively were the best for sperm viability, and Ham's F-10 medium was used for the fertilization and culture of eggs. 4. The results of in vitro fertilization of 33 ova, showed the second polar body developed in 12%, polyspermia in 24%, 1-cell embryo in 21% and 2-cell embryo in 9%. One mature ova developed to blastocyst via 16-cell to 32-cell embryo. The fertilization rate was 66%. 5. Above mentioned results represent that the frozen semen has fertilizing ability and can be used practically in the clinic.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.51-59
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• 2003

This study was carried out to evaluate the effect of different freezing and thawing rates on the viability, motility and acrosomal changes of frozen canine spermatozoa. The ejaculated semen was extended with Tris-egg yolk buffer containing 8% glycerol and equilibrated for 60 min after cooled to 4$^{\circ}C$ for 58 min. The straws were cryopreserved gradually by slow-cooling at different distance(6, 10 and 17 cm, respectively) from the liquid nitrogen (L$N_2$) to achieve temperature rate of 3, 8.9 and 19$^{\circ}C$ /min. Thawing of the straws was performed in a water bath fur 2 min at 37$^{\circ}C$ and 55$^{\circ}C$ , respectively. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Concentration of the ejaculated fresh semen was normal range of 3.44 $\times$ 10$^{8}$ /ml. Freezing temperature were reduced to -110, -70 and -35$^{\circ}C$, as higher distance from liquid nitrogen, 6, 10 and 17 cm, respectively. Freezing at 3$^{\circ}C$/min in distance of 17 cm from liquid nitrogen yielded better motility, viability and rate of intact acrosome than 8.9 or 19$^{\circ}C$/min and the optimal thawing was 37$^{\circ}C$ for 2 min.

• Korean Journal of Animal Reproduction
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• v.25 no.3
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• pp.269-275
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• 2001

This study was carried out to investigate the general characteristics such as volume, sperm concentration, sperm motility, sperm abnormality on whole semen, RSP-S and RSP-T semen and fractional semen of small size dogs, and the effect of temperature and preservation time and cryoproservation on motility of whole and RSP-S and RSP-T semen. Multiple ejaculates were collected from small dogs by the digital manipulation of penis. 1. The volume per ejaculate semen, sperm of concentration and motility and abnormal sperm rate of 1st fractional semen were 0.65$\pm$0.09 $m\ell$, 4.52$\pm$0.35$\times$10$^{6}$ cells/$m\ell$, 15.64$\pm$3.85% and 5.50$\pm$0.62%. Also, 2nd fractional semen were 1.25$\pm$0.20$m\ell$, 3.35$\pm$0.48$\times$10$^{6}$ cells/$m\ell$, 96.25$\pm$4.65% and 4.24$\pm$0.46%. And 3rd fractional semen were 1.45$\pm$0.21$m\ell$, 3.55$\pm$0.52$\times$10$^{6}$ cells/$m\ell$, 92.82$\pm$4.24% and 4.66 $\pm$0.58%, respectively. 2. The sperm of concentration and motility and abnormal sperm rates of whole, RSP-S and RSP-T semen were 5.45$\pm$0.82$\times$10$^{6}$ cells/$m\ell$, 95.55 $\pm$4.65%, 4.58$\pm$0.45% and 4.82$\pm$0.36$\times$10$^{6}$ cells/$m\ell$, 90.10$\pm$3.42%, 6.48$\pm$0.68% and 4.55$\pm$0.45$\times$10$^{6}$ cells/$m\ell$, 93.25$\pm$3.85%, 4.82$\pm$0.58%, respectively. 3. The motility of whole, RSP-S and RSP-T semen were higher at 4$^{\circ}C$ than at 38$^{\circ}C$. When preservation temperature was at 4$^{\circ}C$, survival rates of RSP-S and RSP-T sperm were 97.54~6.25% at 1~72 hrs, 97.40~5.62% at 1~100 hrs, respectively. 4. The survival rates of slow and rapid frozen 2nd fraction, RSP-S and RSP-T semen were 67.3$\pm$4.45%, 88.8$\pm$4.46% and 46.4$\pm$3.84%, 74.4$\pm$4.20%, respectively. Survival rates was significantly higher in frozen RSP-S and RSP-T semen than that in control group(8.5$\pm$2.12%).

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.171-178
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• 2013

The purpose of this study was to evaluate the sperm viability, normal acrosome and mitochondrial activity in the frozen-thawed fowl semen by different cryoprotectants. The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at $5^{\circ}C$. After equilibration for 30 minutes, diluted chicken semen was diluted 1:1 extender containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration and was put in 0.5 mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen. Frozen semen was thawed in water bath at $5^{\circ}C$ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed with fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. In conclusion, freezing rooster semen by using 7% DMF as cryoprotectant was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotectant that has the lowest influences on sperm membranes and acrosome integrity. Therefore it could be used for freezing method of animal genetic conservation method for poultry diversity.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.3
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• pp.281-292
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• 1997

The objective of this study was to compare retrospectively the survival and pregnancy rates(PR) of cryopresered-thawed embryos obtained from intracytoplasmic sperm injection (ICSI) or conventional in vitro fertilization (IVF). Ninety-six cycles of cryopresered-thawed embryo transfer (ET) were performed in 79 patients from June, 1996 to September, 1997 and grouped as followings: 20 cycles (16 patients) inseminated by ICSI (ICSI Group) and 76 cycles (63 patients) by conventional IVF (IVF Group). Slow-freezing and rapid-thawing protocol was used with 1.5M propanediol (PROH) and 0.1M sucrose as cryoprotectant. All embryos were frozen-thawed at the two pronuclear (2 PN) stage excluding four cycles in which the early cleavage stage embryos were frozen, and allowed to cleave in vitro for one day before ET. The duration from freezing to thawing was comparable in both groups ($mean{\pm}SD$, $112.1{\pm}80.0$ vs. $124.8{\pm}140.1$ days). The age of female ($31.2{\pm}3.4$ vs. $32.6{\pm}3.3$ years) and the endometrial thickness prior to progesterone injection ($9.4{\pm}2.0$ vs. $9.3{\pm}1.8$ mm) were also comparable in both groups. There was no significant difference in the outcomes of cryopreserved-thawed ET between two groups: survival rate ($85.2{\pm}16.1%$ vs. $82.2{\pm}19.7%$), cleavage rate ($96.9{\pm}6.7%$ vs. $94.7{\pm}13.0%$), cumulative embryo score (CES, $54.5{\pm}31.1$ vs. $49.0{\pm}20.0$), preclinical loss rate (5.0% vs. 5.3%), clinical miscarriage rate (0% vs 29.4%), clinical PR per transfer (35.0% vs. 22.4%), implantation rate (9.9% vs. 5.6%), and multifetal PR (42.9% vs. 17.6%). In conclusion, human embryos resulting from ICSI can be cryopreserved-thawed and transferred successfully, and the survival rate and PR are comparable to conventional IVF.