• 한국수정란이식학회지
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• 제26권1호
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• pp.65-70
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• 2011

희소 한우인 칡소의 정액 동결을 위해서 레시딘을 기본 희석제로 하는 AndroMed와 Tris-egg yolk extender를 사용하여 정자의 생존율과 활력 조사를 위해서 본 연구를 수행하였다. AndroMed 희석제를 사용하였을 때 생존율과 활력은 $73.4{\pm}11.2%$와 $67.9{\pm}14.6%$의 결과를 보였다. 그리고 Tris-egg yolk extender의 경우는 각각 $89.7{\pm}19.8%$와 $78.4{\pm}18.7%$ 결과를 보여 생존율에서는 Tris-egg yolk 희석제가 AndroMed 희석제를 사용하였을 때보다 유의적으로(p<0.05) 높은 결과를 보였다. 그러나 활력에서는 유의적인 차이를 보이지 않았으나 Tris-egg yolk 희석제에서 높은 경향을 보였다. 정액의 동결을 위해서 평형 시간에 따른 정자의 활력 조사를 위해서 육안적 방법과 CASA 프로그램 방법으로 조사를 실시한 결과, 처음 2시간부터 20시간까지 평형을 유도한 결과 육안적 방법보다는 CASA 프로그램 분석 방법의 결과 높은 수치를 확인할 수 있었다. AndroMed와 Tris-egg yolk extender 두 종류의 희석제로 사용된 동결 정액 사용으로 체외수정란을 생산 결과로서 분할율은 유의적인 차이를 보이지 않았으나(81.7% vs. 82.2), 배반포 발달율에서는 29.6% vs. 42.3%로서 Tris-egg yolk 희석제를 사용하였을 때 유의적으로(p<0.05) 높은 발달율을 보였다. 이러한 결과를 볼 때 희소 한우 품종인 침소 정액 동결을 위해서 Tris-egg yolk 희석제 사용이 동결된 정자의 생존율과 수정 능력 개선으로 실제적인 인공수정과 체외수정란 생산 및 가축유전자원 보존을 위해서도 효과적일 것으로 사료된다.

• 한국수정란이식학회지
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• 제18권1호
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• pp.51-59
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• 2003

본 실험은 개의 인공수정에 사용할 정자의 보존에 있어서, 개 정액 동결시 동결속도와 응해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율을 조사하였던 바 결과는 다음과 같다. 1. 본 실험에서 실험견의 사출된 평균 신선정액의 농도는 3.44$\times$$10^{8}$ /ml로 정상범위에 들었으며, 정자의 형태학적 판정에서 정상적인 정자의 농도는 평균 59.45$\pm$3.45%로 상대적인 기형율은 약 30~40% 정도 나타났으며, 이는 정상적인 상태의 개 정액이라 할 수 있다. 2 개 정액의 동결속도는 동결하는 높이가 6, 10 및 17 cm 일 때 각각 최저온도는 -11$0^{\circ}C$, 7$0^{\circ}C$, -35$^{\circ}C$로 감소하는 경향을 보였으며 이때 최저온도로 감소하는데 소요되는 시간은 각각 6분, 8분 20초 그리고 12분 50초로 결과적으로 분당 동결속도는 각각 19$^{\circ}C$/min, 8.9$^{\circ}C$/min 그리고 3$^{\circ}C$/min로 나타났다. 3. 정액을 동결속도와 융해온도에 따른 정자의 생존율, 운동성 그리고 intact acrosome의 비율은 동결속도가 3$^{\circ}C$/min일 때 가장 높았으며, 융해 온도는 37$^{\circ}C$일 때 효율성이 가장 높은 것으로 나타났다.4. 동결의 방법에 있어서는 액체질소의 표면으로부터 17 cm 높이에서 동결하는 분당 -3$^{\circ}C$의 동결속도에서 동결하여 37$^{\circ}C$에서 2분간 융해하는 방법이 가장 좋은 결과를 보였으며, 생존성과 운동성은 문제없이 사용할 수 있을 것으로 판단되나, 첨체의 intact한 비율은 저조한 결과를 나타내었다.

• 한국동물생명공학회지
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• 제35권1호
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• pp.50-57
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• 2020

The objective of this research work was to know ovarian dynamics and pregnancy rate of cyclic Murrah buffalo cows with induced estrus by administration of prostaglandin F2α (PGF2α) and timed artificial insemination (TAI) with frozen thawed semen. A total of 31 female buffaloes were selected for the study. The buffalos having matured CL observed by ultrasonography were given one intra muscular injection of cloprostenol 500 ㎍ and TAI was performed using frozen thawed semen of Indian Murrah buffalo bull. Results showed that 90.32% (significantly, at p < 0.05) cows explore the sign of heat after injection of PG and 67.85% (significantly, at p < 0.05) cows were become pregnant out of 28 inseminated (TAI) cows. In the 28 inseminated (TAI) cows, average number of smaller and larger size of follicles were non-significantly (p > 0.05) higher at day 3 post PG injection, but the medium size of follicles was nonsignificantly (p > 0.05) higher at PG injection. At day 3 post PG injection the diameter of follicles was significantly (p < 0.05) higher, but the diameter of CL was significantly (p < 0.01) lower compared at PG injection. At PG injection the diameter of largest follicle was non-significantly differences (p > 0.05) in between pregnant and non-pregnant cows. But at day 3 post PG injection it was significantly (p < 0.01) higher in pregnant cows compared to non-pregnant cows. The number of small, medium, and large follicles at PG injection and at day 3 post PG injection were non-significantly (p > 0.05) difference in between pregnant and non-pregnant buffalo cows. Finally, it is concluded that the CL was effectively regresses and induced the sign of heat in buffalo cows and after AI the cows were become pregnant with significant rate. The study will help to the veterinarian and researcher to know the efficacy of PG injection and AI for reproductive efficiency in buffalo cows.

• 한국수정란이식학회지
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• 제29권1호
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• pp.13-19
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• 2014

This study was carried out to study the survival rate of thawed Hanwoo embryos frozen by the slow-rate freezing or the cryotop vitrification method. Hanwoo cumulus-oocyte complexes were recovered from ovaries at a slaughter house, matured for 20~22 hours, fertilized with Hanwoo semen for 5~6 hours, and cultured for 7~9 days in $38.5^{\circ}C$, 5% $CO_2$ incubator. For freezing, Day 7~9 blastocysts were collected. Embryos for the slow-rate freezing were equilibrated in 1.8 M ethylene glycol (EG) with Dulbecco's phosphate-buffered saline (D-PBS). Programmable cell freezer was precooled down to $-7^{\circ}C$, and the straw was seeded during 8 minutes-holding time, and was cooled to $-35^{\circ}C$ at the cooling rate of $0.3^{\circ}C/min$, and then was plunged and stored in liquid nitrogen. Embryos for the cryotop vitrification were treated in TCM199 with 0.5 M sucrose, 16% EG, 16% dimethylsulfoxide (DMSO). Embryos were then loaded individually onto cryotop and plunged directly into liquid nitrogen. The survival rates of embryos frozen by these two freezing methods were evaluated at 12 to 24h post-thawing. The survival rates of frozen/thawed Hanwoo embryos by the cryotop vitrification method ($56.86{\pm}26.53%$) were slightly higher than those by the slow-rate freezing method ($55.07{\pm}26.43%$) with no significant difference. Using the cryotop vitrification and the slow-rate freezing of Hanwoo blastocysts on Day 7 following in-vitro fertilization (IVF) treatment, the survival rates of frozen/thawed Hanwoo embryos were $72.65{\pm}18.3%$ and $79.06{\pm}17.8%$, respectively. The survival rates by the cryotop vitrification were higher than those by the slow-rate freezing on both Day 8 and 9 with significantly higher survival rate on Day 9 (p<0.05). Using the cryotop vitrification and the slow-rate freezing of Hanwoo embryos to compare between three different blastocyst stages, the survival rates of the blastocyst stage embryos were $66.22{\pm}18.8%$ and $45.76{\pm}12.8%$, respectively with higher survival rate by the vitrification method (p<0.05). And the survival rate of expanded blastocysts was higher than those of early blastocysts and blastocysts in two freezing methods with significantly higher survival rate by the slow-rate freezing method (p<0.05).

• 한국수정란이식학회지
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• 제28권3호
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• pp.297-302
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• 2013

Cryopreservation of epididymal spermatozoa offers a potential tool for rescuing genetic material from males of genetically elite populations. Castration, catastrophic injury, sudden death or any other event that makes semen collection or mating impossible may prematurely terminate a stallion reproduction. Stallion epididymal spermatozoa vary widely in the loss of progressive motility, acrosomal integrity, and viability during freezing and thawing. The objective of this work was to investigate the effect of (1) freezing package types on cryopreservation efficiency, (2) thawing temperatures (37, 56 or $70^{\circ}C$) on Computer Assisted Sperm Analysis (CASA) parameters and (3) post-thawing incubation time (0, 1, 2 or 4h) on castrated stallion epididymis. Post-thawed sperm motility ranged between 59.69% and 64.28% ($56^{\circ}C$ and $37^{\circ}C$) in various thawing temperatures. When stallion epididymis sperm was frozen, straw was better than freezing tube on VCL (Velocity of Curvilinear Line) and VAP (Velocity of Average Path) parameter. Higher percentage of motility was observed at $37^{\circ}C$ thawing temperature even though no significant difference was observed among various temperatures. The motility, VCL, ALH (Amplitude of Lateral Head displacement), VAP, BCF (Beat-Cross Frequency) and STR (Straightness index) parameter of post-thawed sperm were significantly decreased by increasing the incubation time for all thawing temperatures. The present study showed that type of freezing package (Straw vs. Freezing tube) was not significantly different on cryopreservation efficiency. Furthermore, stallion epididymal spermatozoa frozen-thawed at $37^{\circ}C$ for 1 min resulted the highest proportion of motility and velocity movement. In addition, motility and viability of frozen-thawed stallion epididymal spermatozoa were also decreased by incubation.

• Clinical and Experimental Reproductive Medicine
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• 제46권4호
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• pp.147-151
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• 2019

Objective: The aim of this study was to investigate DNA fragmentation status in human spermatozoa according to specific tail swelling patterns determined via hypo-osmotic swelling test (HOST). Methods: Frozen semen samples from 21 healthy donors were thawed and prepared by the swim-up technique for use in intracytoplasmic sperm injection. The semen samples were treated for 5 minutes as part of the HOST procedure and then underwent the sperm chromatin dispersion test using a Halosperm kit. DNA fragmentation status (large halo, medium halo, small halo, no halo, or degraded) and the specific tail swelling pattern ("a"-"g") were assessed at the level of a single spermatozoon. A total of 42,000 spermatozoa were analyzed, and the percentage of spermatozoa without DNA fragmentation (as evidenced by a large or medium halo) was assessed according to the specific tail swelling patterns observed. Results: The HOST examinations showed that > 93% of spermatozoa across all types displayed no DNA fragmentation. The percentage of spermatozoa without DNA fragmentation was 100% in type "d", 98.67% in type "g", and 98.17% in type "f" spermatozoa. Conclusion: We found that the type "d" spermatozoa displayed no DNA fragmentation, but the other types of spermatozoa also displayed very low rates of DNA fragmentation. This result may be associated with the processing of the spermatozoa by density gradient centrifugation and the swim-up technique.

• 한국수정란이식학회지
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• 제16권2호
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• pp.133-138
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• 2001

개의 인공수정에 사용할 정자의 보존방법을 확립하기 위하여, 동결속도와 응해 온도를 설정하여 적절한 동결방법을 정립하고자 본 실험을 실시하여 다음과 같은 결과를 얻었다. 동결의 방법에 있어서는 액체질소의 표면으로부터 17 cm 높이에서 동결하는 -3$^{\circ}C$/min의 동결속도로 실시하여 37$^{\circ}C$에서 2분간 응해하는 방법이 가장 좋은 결과를 보였다. 생존성과 운동성에 있어서의 차이는 없지만 첨체의 intact한 비율은 약간 낮은 결과를 보였으며, 이의 보완을 위해, 액체질소 표면으로부터 10cm와 17cm의 높이를 세분화하여 동결속도를 설정한 후보다 나은 정액동결방범을 찾는다면 동결정액을 이용한 인공수정의 수태율은 더욱 향상될 수 있을 것으로 사료된다.

• 한국수정란이식학회지
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• 제21권1호
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• pp.77-83
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• 2006

본 연구는 개 동결 정액 융해 시 straw 크기 및 융해 속도가 융해 정자의 질(quality)에 미치는 영향을 조사하고 최적의 융해 조건을 조사하는데 그 목적이 있다. 정상적인 번식능을 가진 비글 수컷 5마리에서 정액을 채취하여 원심 분리하여 정장을 버리고 남은 정자에 동결보호제인 glycerol이 첨가된 tris-glucose-egg yolk extender를 첨가하여 동결하고 액체질소에 보관한 후 융해하였다. 동결 융해 조건에 따른 효과를 알아보기 위해 straw는 0.25 ml과 0.5 ml크기를 사용하였고 융해 조건은 $75^{\circ}C$에 10초, $55^{\circ}C$에 12초 및 $37^{\circ}C$에서 120초로 하여 융해 후 정자의 활력도(vigor), 운동성(motility), Hypo-osmotic test(HOS test)를 이용한 생존성(viability) 및 $SperMac^{\circledR}$ 염색을 하여 정자의 membrane integrity를 비교 조사하였다. 조사 결과 0.5 ml 크기의 straw를 사용한 경우 $37^{\circ}C$ 융해가 $55^{\circ}C,\;75^{\circ}C$ 융해보다, 0.25 ml 크기의 straw를 사용한 경우에는 $37^{\circ}C,\;55^{\circ}C$ 융해가 $75^{\circ}C$ 융해보다 유의적으로 높은 활력 지수 및 생존성을 보였다(P<0.05). Straw크기에 따라 비교하였을 경우 0.5 ml 군에서 유의적으로 높은 활력도, 생존성 및 membrane integrity를 보였다(P<0.05). 결론적으로 개 정액이 동결 및 융해 시 0.5ml straw를 이용하여 동결한 후 $37^{\circ}C$에서 120초 동안 융해하는 것이 최적의 조건임이 사료된다.

• Reproductive and Developmental Biology
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• 제29권4호
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• pp.235-239
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• 2005

This study was conducted to test whether the transgenic cattle pass the transgene to their progeny through germ cells, and whether the transgene is expressed in the mammary gland of ransgenic cows. Two male ransgenic calves were born from IVF-derived embryos injected with bovine $\beta-casein/human$ lactoferrin fusion gene and then grew up to be reproducible. Semen was collected from a transgenic bull after 18 mon of age and then frozen. Bovine oocytes matured in vitro were fertilized with spermatozoa of the transgenic bull and cultured in $50\;{\mu}L$ drops of CRlaa medium supplemented with 3 mg/mL BSA. After 48 h of culture, cleaved embryos were determined for the presence of transgenes by DNA polymerase chain reaction (PCR). Proportion of transgene positives among bovine embryos fertilized with sperm of the transgenic bull was $20.9\%$ (28/134). One of transgenic bulls did not produce transgenic sperm. Out of 34 calves produced from recipient heifers inseminated with semen of the other bull, 3 $(8.8\%)$ were transgenic animals (2 females and 1 male). Thus, one transgenic bull showed a low transmission frequency below Mendelian levels in both the IVF-derived embryos and his progeny. It was demonstrated by Southern blot that copy numbers of the transgene in the transgenic progeny enhanced about 1.8 times as compared to those of the founder bull The results demonstrate that the transgenic bull carrying human lactoferrin gene could pass his transgene to the progeny through germ cells, although he is a germ-line mosaic.

• Reproductive and Developmental Biology
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• 제36권1호
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• pp.1-6
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• 2012

This study evaluated a sexed sperm ability to produce embryos by flow cytometer. Hanwoo bulls sperm were separated to X and Y sperm via Hoechst 33342 stained with near UV laser or performed the pre-sorted without near UV laser beam in flow cytometry. Pre-sorted sperm had significantly higher viability ($84{\pm}1.15%$, $p$<0.05) compared to other sorted groups in frozen-thawed semen. For fresh semen, pre-sorted sperm had the higher viability ($79{\pm}3%$, $p$<0.05) than those of the X and Y sperm ($44.7{\pm}1.67$ and $41.7{\pm}1.2%$) separated by differences of DNA content. On the other hand, pre-sorted and X sperm sorted according to differences in DNA content had significantly higher viabilities ($24.3{\pm}1.2$ and $25.7{\pm}0.9%$, $p$<0.05) compared to that of the sorted Y sperm ($13.7{\pm}1.2%$) in the hypoosmotic swelling test. The proportion acrosome reaction in the sorted X sperm was higher ($55.0{\pm}1.7$ and $45.0{\pm}1.5%$) than those of the sorted Y-sperm ($32.3{\pm}0.9%$, $p$<0.05). However, the sperm morphologies of the sorted groups were not significantly differences. In conclusion, the sex-sorting procedure by flow cytometry affected some characteristics of Hanwoo sperm. Further study is needed to determine the optimal procedures to enhance male and female embryos and sorting accuracy.