• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.143-152
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• 1992

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.171-177
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• 1998

The combination of testicular sperm extraction (TESE) with ICSI can achieve normal fertilization and pregnancy rate and is effective method in obstructive and non-obstructive azoospermic patients. But, when pregnancy was not occurred, repeated testicular biopsies are not evitable. Therefore, in this study, we observed the survival rate of testicular spemratozoa and spermatozoa extracted from the seminiferous tubules after cryopreserved-thawed used for next IVF cycle with ICSI. In a total of 23 cases, obstructive azoospermia was 17 cases and non-obstructive azoospermia was 6 cases. In obstructive azoospermia, after thawing, motile spermatozua was observed in 13 cases (76.5%). The fertilization rate with 2PN was 67.6% and 5 pregnancies (29.4%) were achieved. In non-obstructive azoospermia, motile spermatozoa was observed in 2 case (33.3%) after thawing. The fertilization rates with 2PN was 53.7% and 3 pregnancies (50.0%) were achieved. A comparison of the results of motile spermatozoa after thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 3 cases (60.0%) and 12 cases (66.6%), respectively. The fertilization and pregnancy rates of thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section were 69.4% and 20.0%, 62.5% and 38.8%, respectively. Conclusively, thawed testicular spermatozoa and spermatozoa extracted from the thawed seminiferous tubule section can achieve normal fertilization and pregnancy and cryopreservation of testicular spermatozoa and seminiferous tubule may avoid repetition of testicular biopsies in azoospermic patients in whom the only source of spermatozoa is the testis.

• Clinical and Experimental Reproductive Medicine
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• v.30 no.2
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• pp.185-188
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• 2003

Objective: To report tow cases of successful pregnancies following long term cryopreserved spermatozoa prior to bone marrow transplantation (BMT) for chronic myelogenous leukemia (CML) and severe aplastic anemia (SAA). Materials and Methods: Case report. Results: With the first case, after cryopreservation of semen from 25 year-old man with CML prior to BMT, his wife is being pregnant by intracytoplasmic sperm injection (ICSI) using thawed spermatozoa. With the second case, 28 year-old man with SAA became father by ICSI using banked spermatozoa before BMT. Conclusion: These cases support that men with malignancy have the chance of fathering their own genetic children. It is important therefore, to increase the awareness of clinicians especially oncologists and patients themselves to the new developments in preserving fertility for cancer patients.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.19-24
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• 2008

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.329-334
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• 2011

This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.11
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• pp.1527-1533
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• 2001

Garole is a prolific, rare, less known and small size Indian sheep breed found in low and humid Sunderban region of West Bengal. Although information on stored Garole ram liquid semen upto 24 h is available, but there is a need to further investigate the short-term and long-term preservability of Garole ram semen for extensive utilization of this valuable germplasm by artificial insemination. The aim of the present study was to apply computer-assisted sperm analysis technique for assessing the motion characteristics of Garole ram semen stored (i) in liquid state at refrigeration temperature for short-term preservation upto 48 h and (ii) in frozen state at $-196^{\circ}C$ for long-term preservation after packaging in mini straws. Short-term preservation had a significant effect on motility (p<0.01) as the motility progressively decreased from 90.1% at 0 h to 85.5% and 73.2% after 24 and 48 h of storage, respectively. Although the decline in rapid moving sperms was also significant (p<0.01) on storage but the decrease was more pronounced at 48 h as compared to 24 h of storage period. Storage of chilled semen had also a significant effect on % linearity (p<0.05), % straightness (p<0.01), sperm velocities (p<0.01), amplitude of lateral head displacement (p<0.01) and beat frequency (pO.Ol) of spermatozoa. The replication had a significant effect for all the variables except average path and straight line velocity. However, the interactions of short-term storage and replication were non-significant for most of the variables except % of medium moving sperms, sperm velocities and beat frequency. On long-term preservation of Garole ram spermatozoa under controlled conditions the mean post-thaw recovery of 70.4 and 71.4% motile spermatozoa was achieved having 48.8 and 48.9% of rapidly motile spermatozoa, respectively in both the replicates. The effect of replication on cryopreservation was significant (p<0.05) on amplitude of lateral head displacement and beat frequency, but there was no significant effect on motility, rapidly motile spermatozoa, linearity, straightness and sperm velocities of frozen-thawed spermatozoa. It can be concluded from these results that an average 70% motility can be achieved on storage of Garole ram semen in chilled liquid state upto 48 h or in liquid nitrogen after freezing under controlled conditions in straws. However, further studies are required to evaluate the fertility of short-term and long-term preserved Garole ram semen for extensive use of this prolific sheep breed.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.21 no.9
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• pp.57-63
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• 2020

To develop simple acrosome staining of horse spermatozoa, this study tested the binding properties of Coomassie brilliant blue G or R on the sperm smears after 3.7% paraformaldehyde (PF) or 35% methanol (MT) fixation. After being fixed with PF and stained with 0.05, 0.1, or 0.2 % of CBB G or R for 2 min, horse spermatozoa were examined for their intact acrosome status. The intact acrosome of fresh horse spermatozoa were 62.6% and 61.5% with 0.05% of the G and R CBB solution, but 80.2 and 79.7% with G type and 78.1 and 76.0% with R type. On the other hand, when MT was used for fixation, the acrosome reacting sperm ratio was 3.5%, but was 9.0% in the case of PF. These results show that the intact acrosome of horse sperm could be judged using a 0.1~0.2% CBB G or R staining technique. PF would be an essential fixative for examining acrosome reacting horse spermatozoa. This method could be used to identify sperm with a damaged acrosome during low-temperature storage or cryopreservation for artificial insemination of horses.

• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1160-1168
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• 2018

Objective: This study was conducted to compare morphological defects, viability, motility (MOT), fertility (F), and hatchability (H) in four Korean native chicken breeds (KNCBs), and to evaluate whether defective segments of spermatozoa are associated with MOT, F, and H. Methods: Four KNCBs, including Korean Ogye (KO), Hwangbong (HB), Hyunin Black (HH), and Hoengseong Yakdak (HY) were used. White Leghorn (WL) was used as a control. Nine cocks from each breed were randomly assigned into three groups. Semen was collected by abdominal massage method. Eosin-nigrosin staining method was used to identify live-dead spermatozoa. Different segments and specific morphological defects of spermatozoa were identified using 4', 6-diamidino-2-phenylidole and MitoTracker Red CMXRos. F and H rates were evaluated following artificial insemination (AI). Results: KO had the highest MOT rate compared to HY. Viable normal sperm rates of KO and HH were high and comparable with WL. HY spermatozoa had the highest viable abnormal sperm (VAS) or morphological defect rate followed by HB. Likewise, HB spermatozoa had the highest dead sperm (dead) rate compared to KO, HY, and WL. Bent, coiled, detached, broken, and knotted were common identified specific morphological defects for all breeds. Most morphological defects were at the head and tail in all breeds. VAS showed strong negative correlation with MOT (r = -0.697) and F (r = -0.609). Similarly, defective tail was negatively correlated with MOT (r = -0.587), F (r = -0.797), and H (r = -0.448). The F and H rates of KO and WL were comparable. Conclusion: These data indicate that most identified specific morphological defects are at the head and tail. VAS and defective tail were associated with poor motility, F, and H. KNCBs showed more morphological defects than WL. Finally, these results will facilitate successful AI and semen cryopreservation.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.1
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• pp.59-65
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• 2000

Objective: The purpose of this study was to determine the important factors affecting survival and pregnancy rate in frozen-thawed embryo transfer cycles. Methods: we performed retrospective analysis in 738 cycles of frozen-thawed embryo transfers, in relation to the insemination methods, the freezing stage of embryo, patient's age, infertility factors and the origin of injected sperm in ICSI cycles. After conventional IVF or ICSI, the supernumerary PN stage zygotes or multicellular embryos were cryopreserved by slow freezing protocol with 1,2-propanediol (PROH) as a cryoprotectant. Results: The survival rates of thawed embryos were 69.3% (1585/2287) in conventional IVF group and 71.7% (1645/2295) in ICSI group. After frozen-thawed embryo transfers, 27.0% (92/341) and 32.0% (109/341) of pregnancy rates were achieved in conventional IVF and ICSI group, respectively. There were no significant difference in the survival and pregnancy rates according to the insemination methods, the freezing stage and patient's age. However, the pregnancy rate (36.2%) of male factor infertility was significantly higher than the tubal (27.2%) and other female factor infertility (22.9%). In ICSI group, the origin of injected sperm did not affect the outcome of frozen-thawed embryo transfer cycles. Conclusion: The present study demonstrates that acceptable clinical outcomes can be achieved after the transfer of frozen-thawed embryos regardless of the stage of embryos for freezing, the patient's age and the origin of injected sperm.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.1
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• pp.25-31
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• 2001

연구목적: $Isolate^{(R)}$ gradient와 swim-up 방법이 정자의 형상 및 정밀정자형태 (strict morphology)에 미치는 영창을 비교분석하고, 이러한 정자처리방법이 정자의 동결-융해과정에 미치는 영향을 비교하고자 하였다. 연구재료 및 방법: 20명의 정상 정자를 대상으로 하였으며 각각의 정자는 두 가지 정자처리방법으로 나누어 정자의 형상과 정밀정자형태를 컴퓨터를 이용한 정자자동분석기를 통하여 측정하였고, 동결보호제의는 TYB 용액을 사용하였으며, 동결 및 융해는 cryo Magic사의 기계를 사용하였다. 통계는 SPSS PC+(version7.0)를 이용하였으며 통계학적인 유의성은 p<0.05로 하였다. 결과: 정자의 농도는 $Isolate^{(R)}$ gradient 처리군이 swim-up 처리군보다 유의성 있게 높았으나 ($51.2{\pm}40.1,\;156.6{\pm}64.3$), 운동성 VCL, VSL, VAP, Linearity, 및 ALH는 swim-up 처리군에서 유의성 있게 높았다. 정밀 정자형태는 swim-up 처리군과 $Isolate^{(R)}$ gradient 처리군에서 차이가 없었다 ($53.7{\pm}6.8$ vs $50.3{\pm}9.1%$). 동결-융해과정 중 두 가지 정자 처리군에서 정자의 형상들은 swim-up 처리군에서 전반적으로 높은 양상을 보였으나, 정밀정자형태는 $Isolate^{(R)}$ gradient 처리군이 swim-up 처리군 보다 감소율이 컸지만 두 군간에 유의한 차이는 없었다 ($12.8{\pm}8.5$ vs $8.6{\pm}6.6$). 결론: 정상 정자에서 swim-up 방법이 $Isolate^{(R)}$ gradient 방법보다 정자 회수율은 우수하였으나, 동결-융해과정 중 정밀정자형태에는 차이가 없어 두 방법을 상호보완적으로 사용할 수 있을 것으로 사료된다.