• Reproductive and Developmental Biology
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• 제35권3호
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• pp.377-383
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• 2011

본 연구에서는 제주흑우의 유전자원 보존과 증식을 위한 안정적인 동결정액 제조법을 수립하기 위하여 제주흑우의 정액 동결 제조에 있어 동결 융해 후 ethylene glycol이 정자의 운동성, 생존율 그리고 첨체에 미치는 영향에 대하여 알아보고자 수행하였다. 제주흑우의 동결정액제조시 5%의 ethylene glycol의 첨가가 $72.5{\pm}5.00%$의 활력과 $54.88{\pm}0.66%$의 생존율 그리고 $46.00{\pm}2.40%$의 정자막 온전성을 나타내 3%와 5%의 ethylene glycol을 첨가한 실험구보다 유의적으로 높은 결과를 나타냈다(p<0.05). 또한, 7% glycerol을 사용한 실험구와 비교하였을 때 5% ethylene glycol을 첨가하였을 때 유의적으로 높은 생존율과 정자막 온전성을 나타냈다(p<0.05). 또한, 예비 동결 조건이 정자의 성상에 미치는 영향에 있어서는 3 cm, 5분 예비동결 실험구에서 생존율과 정자막 온전성이 유의적으로 감소하였다(p<0.05). Ethylene glycol이 정자의 첨체 양상에 미치는 영향에 있어서는 5%와 7%의 ethylene glycol을 사용하였을 때 효과적으로 첨체가 보호되어 F pattern의 비율이 유의적으로 높게 나타났으며(p<0.05), 조기 첨체 반응도 유의적으로 낮은 수준을 보였다(p<0.05). 그러나 7%의 glycerol과 비교하였을 때는 유의적인 차이를 나타내지 않았으며, 5%와 7% ethylene glycol 사이의 유의적 차이도 나타나지 않았다. 예비 동결 조건이 정자의 첨체 양상에 미치는 영향에 있어서는 역시 정자의 생존율과 정자막 온전성과 같이 3 cm, 5분 예비동결 시 유의적인 F pattern의 감소와 조기 첨체 반응에 의한 AR paattern의 증가를 볼 수 있었다(p<0.05). 이와 같은 결과는 본 연구에서 사용된 ethylene glycol과 예비동결 조건의 조합으로 나타난 결과를 활용하여, 희소가축의 생식세포 보존 및 유전자원 확립을 위한 중요한 자료가 될 것이며, 생존율과 운동성 증대를 위한 다양한 희석제를 활용한 연구가 필요할 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제19권2호
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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.

• 한국수정란이식학회지
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• 제28권3호
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• pp.193-198
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• 2013

This study was designed to evaluate the effect of L-cysteine on sperm characteristics and oocyte cleavage in vitro in Korean native cattle. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk and/or 0, 5, 10 and 20 mM L-cysteine before cryopreservation. The viability in frozen-thawed sperm were estimated by SYBR14/PI double stain, acrosome damage with FITC-PNA, mitochondria intact with Rhodamin123 and hydrogen peroxide($H_2O_2$) level with carboxy-DCFDA by flow-cytometry. The developmental capacity was also assessed with cleavage rates in oocytes fertilized in vitro by frozen-thawed sperm. In results, the sperm viability was significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). In addition, acrosome damage was significantly decreased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). The mitochondria intact was also significantly increased in 10 mM and 20 mM concentrations of L-cysteine than other groups (p<0.05). On the other hand, the cleavage rates were significantly increased in 0 mM, 5 mM and 10 mM groups than 20 mM concentration of L-cysteine (p<0.05). The oocyte degeneration of oocytes were significantly decreased in 0 mM, 5 mM and 10 mM groups than in 20 mM L-cysteine group (P<0.05). However, there are no significantly differences among the L-cysteine treatment groups. We suggest that concentration of 10 mM L-cysteine have beneficial impact for sperm cryopreserved in Korean native cattle. This result also could be recommended for artificial insemination program if supported by an improvement in the fertility results and required further study.

• 한국수정란이식학회지
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• 제30권3호
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• pp.121-128
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• 2015

The objective of this study was to evaluate the efficiency of sperm cryosurvival in boar sperm separated by Percoll containing antioxidant enzymes. The boar semen was collected into a pre-warmed ($37^{\circ}C$) thermos bottle by gloved-hand method and was separated by 65% Percoll with superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) before freezing. The frozen sperm was thawed at $38.5^{\circ}C$ for 45 sec in water-bath for sperm characteristic analysis. The sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction, Rhodamine123/PI double staining for mitochondrial integrity and were analyzed using flow cytometry. In results, sperm viability, acrosome reaction and mitochondrial integrity were improved in separated sperm groups compared with unseparated sperm by Percoll (UP) group. Especially, viability was significantly higher in sperm separated by Percoll containing 400 IU CAT group compared with other groups (P<0.05). And acrosome reaction was decreased in sperm separated by Percoll with 300 IU SOD, 400 IU CAT and 0.5 mM GSH groups compared with other groups, however, there were no significantly difference mitochondrial integrity among sperm separated by Percoll with antioxidant enzymes. In conclusion, we suggest that use of Percoll containing antioxidant enzymes for sperm separation will be beneficial for sperm cryopreservation in pigs.

• 대한수의학회지
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• 제42권1호
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• pp.35-43
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• 2002

Cryopreservation is commonly used for an efficient utilization of semen, oocytes and embryos but has disadvantage in the survival, development of the post-thawed eggs. The high risk in the survival, development of eggs after thawing is thought to be caused by inappropriate internal regulation of $Ca^{2+}$ and/or formation of intracellular ice crystals. In this experiment, we tested whether the $Ca^{2+}$ current (iCa), a decisive factor to $Ca^{2+}$ entry, was altered in post-thawed oocytes by using whole cell voltage clamp technique. The quality and survival rates of the oocytes derived from both fresh and frozen groups were examined by morphology and FDA-test. Vitrified oocytes (VOs) were incubated for 4 hr after thawing and then donated to this experiment. Ethyleneglycol-ficoll-galactose (EFG) was used as a cryoprotectant for vitrification. The membrane potential was held at -80 mV and step depolarizations of 250 ms were applied from -50 mV to 50 mV in 10 mV increments. The survival rates showed a higher in VOs vitrified with EFG containing $Ca^{2+}$ than in VOs vitrified with EFG under the $Ca^{2+}$-free condition (82.0% vs 14%). In group with/without $Ca^{2+}$, the survival rates were significantly (P<0.01) difference. In the fresh metaphase II oocytes (FOs), current-voltage (I-V) relationship showed that iCa began to activate at -40 mV and reached its maximum at -10 mV. With same voltage pulses, inward currents were elicited in VOs. I-V relationships observed in VOs were similar to those in FOs. Time constants of activation and inactivation of the inward current shown in VOs were not different to those in FOs. This accordance in I-V relations and time constants in FOs with those in VOs indicates that the inward currents in FOs are unaltered by vitrification and thawing. Therefore, vitrification with EFG does not play as a factor to deteriorate $Ca^{2+}$ entry across the membrane of the oocytes.

• 한국수정란이식학회지
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• 제26권3호
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• pp.181-186
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• 2011

Spermatozoa sorted by flow cytometry have been successfully used to produce offspring in domestic animals and are commercially available for cattle. Also sheath fluid is the important environment for viability of sex-sorted sperm in flow cytometry. The aim of this study was to investigate whether or not HEPES (N-2-hydroxyethylpiperazine-N'-2-Ethanesulfonic acid) has any effect on the viability in sex-sorted Hanwoo (Korean native cattle) sperm. In this study, the semen was collected from Hanwoo of Hoengseong Livestock Cooperation by artificial vagina method then pooled and subjected to cryopreservation in straws. Sperm were cultured for 0, 30, 60 and 120 min with 0, 2.5, 5, 7.5 and 10 mM of HEPES added to the sheath fluid and incubated at 4, 20 and 38$^{\circ}C$, respectively. For the cytometric analysis the frozen-thawed semen was extended with 5 mM HEPES extender to final concentration ($2{\times}10^7$ spermatozoa) at 4, 20 and 37$^{\circ}C$. Sperm viability was assessed with SYBR-14 and propidium iodide (PI) staining. This study shows that the viability of sperm was decreased with prolongation of incubation time in all of test. But the viability of sperm which were treated with 38$^{\circ}C$ was gently decreased than that of treated with other temperature. The viability of the control was sharply decreased (p<0.05) than all of the HEPES treatment group at 60 to 120 min in 38$^{\circ}C$. X-sexed sperm was more sensitive than Y-sexed sperm to temperature during f10w cytometry (p<0.05). In conclusion, the results of this study suggest that the sheath fluid with 5 mM HEPES has effect on maintenance of viability after sperm sexing at 37$^{\circ}C$ in Hanwoo.

• Asian-Australasian Journal of Animal Sciences
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• 제31권8호
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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.

• Journal of Animal Science and Technology
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• 제54권2호
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• pp.95-101
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• 2012

본 연구는 희소 한우인 제주흑우의 정액 동결을 위하여 동결보호제로서 glycerol과 amide 계열의 동결보호제인 5%의 DMA, DMF 그리고 7%의 MF를 이용하여 동결 융해 후 정자의 운동성, 생존율, 정자막 온전성 및 정자의 첨체 양상의 변화 조사를 위하여 수행하였다. 제주흑우의 정액을 동결 한 결과 glycerol과 DMF를 사용하였을 때 운동성은 $64.00{\pm}9.62$와 $59.00{\pm}5.48$로 DMA와 MF에 비해 유의적으로 높았으며(p<0.05), 생존율 역시 glycero과 DMF 처리구에서 $58.25{\pm}6.63$과 $53.05{\pm}3.77$로 유의적으로 높게 나타났다(p<0.05). 그러나 glycerol과 DMF 처리구 간의 유의적 차이는 나타나지 않았다. 정자막 온전성 검사(HOST)에 있어서도 역시 glycerol과 DMF 처리에서 정자 미부의 팽창 비율 역시 $45.12{\pm}25.08$과 $44.95{\pm}8.58$로 유의적으로 높았다(p<0.05). 정자의 첨체막 양상 변화에 있어 F pattern의 비율이 MF 처리시 다른 처리구에 비하여 유의적으로 낮았으며(p<0.05), B pattern의 비율은 DMA, DMF 및 MF의 사용시 glycerol에 비하여 유의적으로 증가하였다(p<0.05). AR pattern의 비율에 있어서는 DMF를 제외한 amide의 사용은 F pattern의 감소와 조기 첨체반응에 의한 유의적인 AR pattern의 증가를 볼 수 있었다(p<0.0.5). 그러나 DMF의 사용은 glycerol 보다 AR pattern의 비율이 유의적으로 낮았다(p<0.05). 동결 융해 후 시간에 따른 정자의 생존율과 정자미부 팽창 비율에 있어서 융해 초기에는 glycerol에서 DMF 보다 높은 생존율과 정자 미부 팽창율을 나타냈으나, 1시간 이후부터는 DMF에서 glycerol 보다 높은 생존율과 정자 미부 팽창율을 나타내었다. 이러한 결과는 희소 가축의 생식세포 보존 및 유전자원 확립을 위한 중요한 자료가 될 것이며, 제주흑우 및 희소 가축의 안정적이고 효율적인 동결 정액 제조 연구에 있어 중요한 정보를 제공할 것으로 평가된다.

• 한국가축번식학회지
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• 제25권4호
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• pp.409-419
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• 2001

본 실험은 PF과 FPP가 소와 사람 동결 정자에 첨가되었을 때 응해 후 정자의 체외 생존성, 운동성 그리고 intact acrosome 향상에 기여할 수 있는지의 여부를 조사하고자 실시하였다. 사람의 정액은 TYB 동결 배양액을 사용하여 초 급속 동결하였다 PF과 FPP 첨가 효과는 각각의 시약이 동결-응해된 소나 사람 정자의 현미경적 조사에 의한 운동성에 미치는 영향과 Coomassie brilliant blue 염색방법에 의한 intact acrosome의 비율에 미치는 영향으로 조사하였다. Bovine 동결 응해 정자에 PF을 첨가하여 운동성을 조사하였던 바, 5 mM 처리군 (50.0%)이 대조군 (34.0%) 보다 유의하게 높은 운동성을 보여주었다 (F＜0.05). 동결 응해된 소 정자에 FPP를 농도에 따라 처리하여 intact acrosome을 조사하였던 결과, 50 nM 치리춘 (49%)이 대조군과 25 nH 처리군 (30.0, 38.0%) 보다 유의하게 많은 intact한 acrosome을 유지하였다 (P＜0.01). 사람 정자에서 동결에 앞서 PF을 농도에 따라 첨가하여 동결 응해 후 운동성을 조사한 결과, 5.0 mM 처리군 (51.0%)이 대조군과 2.5 mM (39.0, 40.0%) 처리군의 운동성보다 높았다 (P＜0.01). 사람 정액의 모든 동결 처리과정 (동결전, 동결, 응해후)에서 50 nM (75.5%) FPP 첨가가 intact acrosome percentage 유지하는데 유의한 효과가 있었다 (대조군: 45.0; 25 nM: 53.0; 100 nM: 68.0%) (P＜0.01). PF와 FPP 첨가하여 사람 정자의 동결융해 후 운동성과 intact acrosome에 미치는 영향을 동시에 비교해본 결과, 운동성에서는 PF 처리군이 약간 높지만 intact acrosome rate는 FPP 처리군의 결과 (65.0%)가 PF 처리군 (43.0%)보다 유의하게 높았다 (P＜0.05). 따라서 본 실험은 동결-융해된 소 정자에 PF이나 FPP 첨가는 정자의 운동성이나 intact acrosome 비율을 좀더 개선시킬 수 있고, 특히 사람 정자는 동결 전 과정에 FPP를 첨가하는 것이 정자의 체외 생존성, 운동성 그리고 intact acrosome을 유의하게 향상시킬 수 있다는 것을 시사한다.

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.