• 한국가금학회지
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• 제45권4호
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• pp.237-244
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• 2018

본 연구에서는 닭 동결 정액의 융해 방법에 따라 정자 운동성의 변화도를 분석하였고, 적절한 융해방법에 대한 자료를 확보하였다. 동결정액을 융해하는 방법은 축종에 따라 서로 다른 열전달 효율을 제시하기도 하나, 닭 동결 정액에 대한 연구는 미진한 상태이다. 특히 닭 정액은 고 농도의 정액을 필요하기 때문에 이러한 요인에 대한 정자 운동성은 변이가 많은 것으로 알려져 있다. 그러므로, 닭 정액 융해에 필요한 온도는 $5^{\circ}C$임을 알 수 있었으며, 닭 농장에서 동결정액의 융해를 실시할 때, 알코올이 함유된 냉각수를 이용하게 되면 냉각수의 과냉각 생태를 방지할 수 있음을 관찰하였다. 또한, 동결정액을 이용한 인공수정을 실시할 때, 현장에서 직접 닭 정액을 융해하여 시간을 절약할 수 있으며, 인공 수정 작업시간이 30분을 넘기게 되면 정자의 운동성은 감소하여 수정율에 영향을 미치는 것으로 판단된다. 그러므로, 농가에서 동결정액을 활용할 경우, 융해에 신경을 써야 하며 정확한 방법을 적용하여야 수정란의 부화율 감소현상을 막을 수 있을 것으로 보인다. 이와 같이 본 연구에서 제시된 방법으로 동결정액을 이용할 때, 동결정액의 수정율이나 부화율의 변이를 막을 수 있고, 동결 정액을 활용한 가금종축생산 효율이 높아질 것으로 판단된다.

• 한국동물생명공학회지
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• 제34권4호
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• pp.272-279
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• 2019

The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 10⁶ cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN₂) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 10⁶ cells/mL vs. 1060.0 ± 44.3 × 10⁶ cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.

• 한국수정란이식학회지
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• 제32권1호
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• pp.1-8
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• 2017

유전자원을 동결 보존하는 것은 유전적인 개량과 우수한 종축의 재생산 및 멸종 위기종의 유전자 집단을 보존하는데 있어 중요한 방법으로 알려져 있다. 그러나 동결 유전자원의 대표적인 정액은 살아 있는 종축에서 채취하여 보존하고 있는데 동결자원은 그 수가 제한되어 있어 이용에 한계가 존재하고 있다. 이러한 단점을 극복하기 위하여 본 연구에서는 동결정액을 2가지 방법으로 융해하고 재 동결을 실시하여 그 정자의 특성을 CASA로 분석하고 생존성을 비교하였다. 일반적으로 사람의 정액은 재동결 과정을 극복하여 반복적인 동결 및 융해가 가능하다고 보고되었으나 한우 정액에 관한 재 동결 성적에 관한 연구는 자료는 찾아보기 힘들다. 본 연구에서는 칡소 2두, 흑우 1두, 백한우 2두 및 보증씨수소 1두의 동결정액을 이용하여 재 동결에 관한 연구를 실시하였으며 개체에 따라 29.7%에서 46.6%의 생존율을 얻었다. 2차 동결 방법에 있어서 생존율은 $5^{\circ}C$에서 융해하는 방법이 $37^{\circ}C$ 융해방법보다 더 높으며 변이가 낮은 것으로 관찰되었다. 그럼에도 불구하고 2가지 방법에 의한 재동결 기법은 체외 수정이나 OPU 유래 수정란 생산을 위하여 한우의 증식에 적용할 수 있을 것으로 보이며 추후 수정란 생산에 관한 연구에 도움이 될 것으로 판단된다.

• 대한수의학회지
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• 제38권4호
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• pp.882-897
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• 1998

This experiment was conducted to predict the effects of motional characteristics on the fertility of Korean native cattle(KNC) by using CASA technology and in vitro fertilization system. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system(SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber(Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The oocytes collection, in vitro maturation, IVF, in vitro culture and determination of the cleavage rate were performed by the technique, as described by Hwang et al (1997). Statistical analysis was done by linear regression with use of the Sigma plot program on a IBM personal computer. The cleavage rate in vitro fertilized oocyte was significantly correlated(P<0.05) with MOT, VCL, VSL, VAP, ALH, BCF and MAD, but not CON, LIN, STR, WOB, DNM, DNC and HYP in regressional analysis. The results show that some kinematic characteristics of frozen-thawed semen by CASA can be predict the fertility in in vitro model system.

• Reproductive and Developmental Biology
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• 제31권3호
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• pp.139-143
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• 2007

본 연구는 체세포를 이용하여 생산된 복제 한우 수소의 번식능력을 검토하기 위해 실시하였다. 복제 한우 수소(C-38 및 C-39) 또는 일반 한우 종모우로부터 정액을 채취하여 정자의 수 및 동결 전 후의 생존성 등을 살펴보았으며, 정자의 운동성 등은 computer assisted sperm analysis(CASA)를 이용하여 측정하였다. 또한, 이들의 수정 능력을 확인하기 위하여 체외수정과 인공수정을 각각 실시하였다. 정액 성상에서는 복제 수소들과 일반 종모우 간에 정액의 양, 정자의 농도 및 동결융해 후의 생존성 등에서 차이가 나타나지 않았다. CASA를 이용한 분석에서 운동성, 곡선 운동 속도(VCL), 직선운동 속도(VSL) 및 평균 진행 속도(VAP) 등은 복제 수소의 정액이 일반 종모우의 정액에 비하여 유의적으로 높았다 (p<0.05).체외수정에 따른 수정란의 분화율 및 배반포로의 발달율은 복제 수소와 일반종모우 간에 차이가 나타나지 않았다. 복제소 정액(C-38)을 이용하여 인공수정을 한 5두의 체세포 복제 대리모에서 암 수 각각 한 두씩의 건강한 복제 후대 송아지 2두를 생산하였다. 이상의 결과를 종합하여 보면, 실험에 공시된 복제 수소 개체간의 차이가 나타나기는 하였지만, 복제 수소는 정액 성상과 정자의 운동성 등에서 일반 종모우와 차이가 없었으며, 또한 인공수정을 통해 송아지를 생산함으로써 정상적인 번식능력이 있음을 확인하였다.

• Animal Bioscience
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• 제37권7호
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• pp.1177-1184
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• 2024

Objective: Semen cryopreservation is an effective method of preserving genetic material, particularly in native chicken breeds facing a substantial decline. In this study, we evaluated the quality of frozen/thawed rooster semen treated with different concentrations of oral administrations of black ginger (Kaempferia parviflora: KP) extract and determined its fertility. Methods: Thirty-two Thai native roosters (Pradu Hang Dum, 42 weeks old) were used in this study. The treatments were classified into four groups according to the concentration of KP extract administered to the roosters: 0, 100, 150, and 200 mg/kg body weight. The quality of fresh semen was analyzed before cryopreservation. Post-thaw sperm quality and fertility potential were determined. Also, lipid peroxidation was determined. Results: The results showed that sperm concentration and movement increased in roosters treated with 200 mg/kg of KP extract (p<0.05). The malondialdehyde (MDA) in the roosters receiving 200 mg/kg KP extract was lower than that in the other but had an insignificant difference within the KP treatment groups (p>0.05). The highest MDA levels were observed in the control group (p<0.05). The percentage of motile sperm (total motility and progressive motility) after semen thawing was higher in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). MDA levels decreased significantly in roosters that received 150 and 200 mg/kg KP extract than in those that received 100 mg/kg KP extract and the control (p<0.05). Fertility and hatchability were greater in the KP150 and KP200 groups than in the KP100 and control groups (p<0.05). Conclusion: The optimal amount of KP extract influencing initial sperm quality was determined to be 200 mg/kg. However, 150 mg/kg was the optimal low dosage of KP extract administration that maintained sperm quality and fertility following semen cryopreservation.

• 한국동물생명공학회지
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• 제37권2호
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• pp.113-120
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• 2022

Sperm cryopreservation is a fundamental process for the long-term conservation of livestock genetic resources. Yet, the packaging method has been shown, among other factors, to affect the frozen-thawed (FT) sperm quality. This study aimed to develop a new mini-straw for sperm cryopreservation. In addition, the kinematic patterns, viability, acrosome integrity, and mitochondrial membrane potential (MMP) of boar spermatozoa frozen in the developed 0.25 mL straw, 0.25 mL (minitube, Germany), or 0.5 mL (IMV technologies, France) straws were assessed. Post-thaw kinematic parameters were not different (experiment 1: total motility (33.89%, 32.42%), progressive motility (19.13%, 19.09%), curvilinear velocity (42.32, 42.86), and average path velocity (33.40, 33.62) for minitube and the developed straws, respectively. Further, the viability (38.56%, 34.03%), acrosome integrity (53.38%, 48.88%), MMP (42.32%, 36.71%) of spermatozoa frozen using both straw were not differ statistically (p > 0.05). In experiment two, the quality parameters for semen frozen in the developed straw were compared with the 0.5 mL IMV straw. The total motility (41.26%, 39.1%), progressive motility (24.62%, 23.25%), curvilinear velocity (46.44, 48.25), and average path velocity (37.98, 39.12), respectively, for IMV and the developed straw, did not differ statistically. Additionally, there was no significant difference in the viability (39.60%, 33.17%), acrosome integrity (46.23%, 43.23%), and MMP (39.66, 32.51) for IMV and the developed straw, respectively. These results validate the safety and efficiency of the developed straw and highlight its great potential for clinical application. Moreover, both 0.25 mL and 0.5 mL straws fit the present protocol for cryopreservation of boar spermatozoa.

• 한국수정란이식학회지
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• 제24권3호
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• pp.199-205
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• 2009

This study was carried out to investigate the effective genetic resources preservation system using the frozen boar semen. The porcine oocytes were matured for 44 hours in NCSU-23 medium with or without 10% Porcine Follicle Fluid (PFF), 0.5 ${\mu}g/ml$ porcine FSH, 0.5 ${\mu}g/ml$ equine LH, 1.0 ${\mu}g/ml$ 17 $\beta$-estradiol ($E_2$) and 10 ng/ml Epidermal Growth Factor (EGF) under mineral oil at $38.5^{\circ}C$ in humidified atmosphere of 5% $CO_2$ in air. After 44 h of culture, the oocytes were inseminated with frozen-thawed semen and fresh semen prepared with mTBM medium for 6 h. Later, set of 50 presumptive zygotes were transferred into 4-well dish (500 ${\mu}l$) of IVC medium. for embryos freezing, slow-freezing and vitrification methods were used as a cryopreservation. Differences among treatments were analyzed using General Linear Model Procedure by SAS Package (version 6.12) differences were considered significant when p<0.05. Following IVF and IVC, the rates of cleavage and blastocysts formation were significantly higher (p<0.05) in hormone supplemented group than that of hormone-free group (25.7 vs, 12.1). The development rates to cleavage and blastocysts were significantly higher in PZM-5 group than NCSU-23 group (60.3%, 46.6% vs 27.4%, 11.1%). Further improvement was achieved when PZM-5 was supplemented with FBS. Cleavage rates was significantly higher in fresh semen source group than frozen semen (66.7% vs 43.7%). However in blastocysts rates was similar two groups. Post-thaw survival rates of embryos were 1.2% and 2.2% in slow-frezing and vitrification groups, respectively. The results of our study suggest that it is still possible to improve the culture conditions and boar semen cryopreservation for enhance reproductive technology and animal genetic resources conservation.

• Reproductive and Developmental Biology
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• 제37권3호
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• pp.97-102
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• 2013

The objective of this study was to develop of semen transport system for cryopreservation and fertility in bull sperm. The ejaculated semen were diluted with Triladyl containing 20% egg-yolk for transportation. Diluted semen was transported by three methods that there were wrapping tissue (Tissue), sinking under $30^{\circ}C$ water (Water) and sinking between warm water and air (Air) methods. Semen was transported within 2 hours in $0.3^{\circ}C$. For this study, the freezing of diluted semen were added with Triladyl containing 20% egg-yolk. And frozen-thawed sperm were estimated with SYBR14/PI double stain for viability, FITC-PNA/PI double stain for acrosome reaction analysis and Rhodamine123 double stain for mitochondrial intact assessment. In results, live sperm (SYBR+/PI-) in Air treatment group ($43.3{\pm}4.7%$) was significantly (p<0.05) higher than other treatment groups (Tissue: $16.3{\pm}2.7%$ and Water: $27.5{\pm}3.1%$), dying sperm (SYBR+/PI+) in Air treatment group ($55.6{\pm}4.7%$) was significantly lower than other treatment groups (Tissue: $77.6{\pm}3.2%$ and Water: $67.6{\pm}3.3%$) (p<0.05). Acrosome reaction in Air treatment group ($0.2{\pm}0.1%$) within live sperm (PI negative region) was significantly (p<0.05) lower than other treatment groups (Tissue: $0.7{\pm}0.2%$ and Water: $0.5{\pm}0.1%$), the acrosome reaction in Air treatment group ($28.6{\pm}2.8%$) within all sperm also was significantly lower than other treatment groups (Tissue: $44.2{\pm}1.8%$ and Water: $36.2{\pm}2.0%$) (p<0.05). And mitochondrial intact in Air treatment group within live ($97.1{\pm}0.4%$) and all ($61.9{\pm}3.3%$) sperm were significantly higher than other treatment groups (Tissue: $85.2{\pm}3.3%$, Water: $87.8{\pm}2.9%$ within live sperm and Tissue: $49.28{\pm}3.7%$, Water: $42.0{\pm}3.1%$ within all sperm) (p<0.05). Therefore, we suggest that transportation by sinking method between warm water and air was beneficial to improvement of fertility in frozen-thawed in bull semen.

• 한국가축번식학회지
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• 제12권2호
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• pp.112-119
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• 1988

These studies were conducted to investigate the effects of culture temperature and time on the in vitro maturation and semen type and media on the in vitro fertilization of bovine follicular oocytes, and to asses in vitro fertilization rate of oocytes cultured by extraffollicular method following fertilization in vitro, or transfer into the pseudopregnant rabbit oviduct or uterus. The bovine oocytes recovered from follicles were cultrued for 18 hrs or 72hrs at 38$^{\circ}C$ with 5% CO2 in moist air. Flesh-diluted(2 folds) and frozen-thawed semen in 0.5ml straw from a fertile bull were used. In order to obtain capacitation of spermatozoa were treated with bovine follicular fluids(BFF) and Inophore A(IA). The results obtained were summarized as follows: 1. The oocytes were classified as "A, B, C, D and Degenerative" depending morphological integrity and those were 62.0%, 12.0%, 17.2%, 5.9% and 3.0% of the total oocytes harvested, respectively. 2. The oocytes matured to metaphase II were significantly increased between 24-48hrs of incubation and at 37-39$^{\circ}C$ with 5% CO2 in moist air. 3. The in vitro fertilization rate following transferred into rabbit oviduct or uterus with bull semen and in vitro matured oocytes were higher ligation than non-ligation of oviduct or uterus. 4. The in vitro fertilization rate of oocytes matured in vitro were higher neat than frozen semen and treatment of IA than BFF on the capacitation of spermatozoa. 5. The effects of semen types and media on in vitro fertilization of oocytes matured in vitro were higher fertilization rate of neat than friozen semen, and media was not significant.