• 한국수정란이식학회지
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• 제14권2호
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• pp.131-137
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• 1999

This experiment was carried out to investigate the effects of osmolarity of thawing diluents, seminal plasma added in thawing diluents on the sperm viability and the effects of thawing temperature, the temparature of the thawing diluents on the sperm viability and acrosomal morphology of boar spermatozoa by the straw method. The result obtained were summarized as follows: 1. The sperm viablilty after thawing of the frozen semen was shown greater in the high osmolarity(392~492mOsm) than low osmolarity(300mOsm) in thawing diluent. The added levels of seminal plasma in thawing diluent did not affect the viability of frozen-thawed boar semen. 2. In terms of thawing temperature, the sperm viability was shown higher in the frozen semen thawed at 5$0^{\circ}C$ for one min. (p<0.01) than those thawed at 2$0^{\circ}C$ or 37$^{\circ}C$ for one min. The sperm viability was not significant at the diluent temparature of 2$0^{\circ}C$or 37$^{\circ}C$ after thawing: but the sperm viability was higher in thawing diluent at 2$0^{\circ}C$ than in that at 37$^{\circ}C$. However, the effects of thawing temperature and diluent solution on normal acrosomal rate were not significant. 3. Cleavage rates of oocytes fertilized with frozen semen were 46.4% and 43.3%, respectively, which were thawed at 5$0^{\circ}C$ for one min. and then diluted in mBTS medium at 2$0^{\circ}C$or 37$^{\circ}C$. To sum up, the sperm viability was shown greater at the high of thawing diluents of frozen boar semen. In terms of thawing conditions, the sperm viability was shown greater, when semen was thawed at a high temperature for a short time and then diluted at the same temperature as that in the straw.

• 한국발생생물학회지:발생과생식
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• 제1권1호
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• pp.29-36
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• 1997

Experiments were performed to study the effects of diluents, cryoprotectant, equilibration time, thawing temperature and addition of BSA and egg yolk. Among the various diluents, Alsever's solution was the best for sperm cryopreservation. A combination of Alsever's solution and 15% ethylene glycol showed the better results than others did. Sperm activity indection and survival rate gradually decreased with the equilibration time. The appropriate thawing temperature was 30 ${\pm}1^{\circ}$C. These results indicate that sperm cryopreservation methods can be developed in tiger puffer.

• 한국발생생물학회지:발생과생식
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• 제21권1호
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• pp.79-91
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• 2017

The aim of this study was to compare the efficacy of cryopreservation methods for ex situ conservation of spermatozoa from far eastern catfish, Silurus asotus. The spermatozoa activity index (SAI) and hatching rates were higher in spermatozoa stored in Alserver's solution than those of spermatozoa stored in glucose solution. The SAI and hatching rates in all experimental groups gradually decreased with increasing duration of storage. Additionally, the SAI and hatching rates gradually decreased with increasing thawing temperatures at all storage durations (P<0.05). Based on the SAI and hatching rates, our results suggest that the optimal cryopreservation conditions of catfish spermatozoa involve storage in Alserver's solution with 15% ethylene glycol, and thawing at $25^{\circ}C$. Cryopreservation of spermatozoa is a useful and reliable technique for conserving gene resources and for artificial propagation of far eastern catfish.

• Fisheries and Aquatic Sciences
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• 제24권2호
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• pp.63-77
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• 2021

The aim of this research was to investigate different factors, including cryoprotective agents (CPAs), diluents, dilution ratios, equilibrium times, freezing rates, and thawing methods to optimize cryopreservation protocols for large yellow croaker (Larimichthys crocea). The parameters evaluated were sperm motility, sperm activity index (SAI), survival rate, and DNA damage. Different types of CPAs, such as dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), methanol, and glycerol, were tested for sperm preservation. The highest motility, SAI, and survival rate were observed when EG was used. Different diluents such as Stein's solution, Hank's balanced salt solution, marine fish Ringer's solution, artificial seminal plasma (ASP) of small yellow croaker, and Cortland solution were investigated. The highest post-thaw motility was observed upon using ASP as the diluent. Different concentrations of EG were then mixed with ASP to identify the optimal EG concentration. Experimental results showed that the motility (70.33 ± 1.20%), SAI (5), and survival rate (78.30 ± 0.42%) of post-thaw sperm were optimum when 10% EG and ASP were used as the CPA and diluent of cryopreservation, respectively. Post-thaw sperm motility was high at equilibration times below 150 s and at an optimum dilution ratio of 1:1 (sperm: CPA + diluent) and was not significantly different compared with fresh sperm motility. The freezing rate was found to be slow below -10℃/min. The thawing temperature of 45℃ was identified as ideal. The percentage of tail DNA in post-thaw sperm at 10% EG and ASP was also investigated and was found to have more significant DNA damage than that in fresh sperm but significantly lower damage than that in post-thaw sperm at EG concentrations of 5%, 15%, and 20% (p < 0.05). The cryopreservation protocols obtained in this study will be useful in large yellow croaker hatcheries.

• Asian-Australasian Journal of Animal Sciences
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• 제15권11호
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• pp.1553-1558
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• 2002

This study was carried out to obtain informations regarding the effect of N-acetyl-D-glucosamine in the LEY (lactoseegg yolk) diluent according to incubation time in 5 ml maxi-straw and the effects of freezing rate, thawing temperature and thawing time in the LEN (lactose-egg yolk and N-acetyl-D-glucosamine) diluent on acrosome morphology and motility of frozen-thawed boar sperm. The study showed that the LEN diluent was higher post-thaw NAR (normal apical ridge) acrosome than the LEY diluent for 0.5 h incubation at 37$^{\circ}C$. However, there were no differences between the LEN and LEY diluents on post-thaw sperm motility according to incubation time. The straws frozen from 5.0 cm (20$^{\circ}C$/min) to 17.0 cm (1$^{\circ}C$/min) above the liquid nitrogen surface did not show any significant differences on post-thaw sperm motility. However, the straws frozen above 5.0 cm from the liquid nitrogen surface were higher NAR acrosome than those frozen above 17.0 cm. The post-thaw percentages of motile sperm and NAR acrosome were significantly higher (p<0.05) for the maxi-straws submerged for 40 or 45 sec in a 52$^{\circ}C$ water bath than for 30, 35, 50 or 55 sec. The mean sample temperatures of maxi-straws after 40 or 45 sec submersion were 20.7 or 26.4$^{\circ}C$. In conclusion, the sample temperature of the thawed semen was very important for post-thaw sperm survival in the LEN diluent of 5 ml maxi-straw. When the temperature of the thawed semen was 20.7$^{\circ}C$, the percentages of motile sperm and NAR acrosome were highest.

• Asian-Australasian Journal of Animal Sciences
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• 제22권12호
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• pp.1614-1619
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• 2009

The present study was conducted to observe the effect of increased osmolality of basic tris extender supplemented with trehalose and sucrose on post-thawing quality (motility, progressive motility, viability, the rate of acrosome abnormality, total abnormality and membrane integrity) of Markhoz goat spermatozoa. Fresh semen samples were evaluated for motility and sperm concentration. Only semen samples with motility more than 70% and sperm concentration higher than $3{\times}10^{9}$ sperm/ml were used for cryopreservation. In Exp. 1, trehalose (50, 75 or 100 mM) and sucrose (40, 60 or 80 mM) were added to a basic tris diluent. Based on the results of experiment 1, the goal of Exp. 2 was to investigate the combinational effects of the highest and lowest concentrations ($T_{100}+S_{80}$ or $T_{50}+S_{40}$) of trehalose and sucrose. As the control, semen was diluted and frozen in the tris diluent without trehalose or sucrose. The results in Exp. 1 showed that all evaluated spermatozoa characteristics improved significantly after freezing and thawing (p<0.05) and at the same time the increase of trehalose and sucrose concentrations in basic extenders was seen, with the best results obtained for extenders containing 70 and 100 mM trehalose and 80 mM sucrose. Comparing these results with those of control diluents, the effects of supplementation were significantly (p<0.05) better. In Exp. 2, the results showed no significant differences (p>0.05) between $T_{100}+S_{80}$ and $T_{50}+S_{40}$ extenders, but the results of $T_{50}+S_{40}$were slightly better than obtained with $T_{100}+S_{80}$ diluents. Furthermore, the results of this experiment indicated that the sperm characteristics in the isotonic control extender were significantly (p<0.05) lower than examined extenders. In conclusion, the results of this study indicated that goat sperm can tolerate hypertonic trehalose and sucrose solutions better than isotonic control diluents in the freezing period. In particular, these positive effects have been shown for acrosome integrity, which is very important for the fertilization capacity of sperm. The data indicated that addition of trehalose plus sucrose to the freezing extender can be recommended for cryopreservation of goat spermatozoa, but more data is needed on pregnancy rate, acrosome reaction and IVF to ascertain the real effect.

• 한국양식학회지
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• 제20권3호
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• pp.173-177
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• 2007

희석액과 동해방지제로 각각 ASP와 DMSO를 사용하여 1년 동안 냉동보존한 후 해동한 강도다리(Platichthys stellatus) 정자의 운동속도는 10% 농도에서 가장 빨랐다. Methanol의 경우 $10{\sim}20%$ 범위에서 유의한 차이를 보이지 않았지만 15%에서 가장 높은 값을 보였다. Glycerol을 동해방지제로 사용하였을 때는 첨가농도가 높아질수록 정자의 운동성과 운동속도가 낮아졌다. 희석액으로 SS를 사용한 경우도 ASP와 비슷한 결과를 보였다. 투과형전자현미경으로 관찰한 냉동하지 않은 신선한 강도다리 정자는 머리, 중편부 및 꼬리로 구성되어 있으며, 치밀한 핵질로 충만한 구형의 머리에는 첨체구조가 관찰되지 않았다. 동해방지제 없이 냉동보존한 경우, 대부분의 정자들이 머리가 찌그러지거나 부분적으로 수축되는 경우가 많았으며 머리의 원형질막이 이탈되고 염색질이 과립상으로 변하거나 균질화되지 않았다. 반면에 냉동보존을 위해 동해방지제를 사용한 경우 부분적으로 정자의 구조적 손상이 관찰되었으나 대부분의 정자는 냉동과 해동과정에서 손상을 입지 않았다.

• 한국임상수의학회지
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• 제11권2호
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• pp.545-552
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• 1994

This experiment was carried out to investigate the renditions to maintain good post-thaw motility and viability of canine spermatozoa when the semen was frozen using methanol. The semen from two male dogs which had been proven to be fertile in the previous one year was treated with different compositions of semen diluent and was frozen at different freezing temperatures, When canine semen was frozen at-2$0^{\circ}C$, -6$0^{\circ}C$, or -8$0^{\circ}C$, the spermatozoa frozen and stored at -2$0^{\circ}C$ showed very low post-thaw motility and viability from day 2 to 7 and showed no viability since day 15 after freezing. The spermatozoa frozen and stored at -6$0^{\circ}C$ or -8$0^{\circ}C$ showed higher post-thaw motility and viability on day 2, 1, 15 and 30 after freezing than that frozen and stored at-2$0^{\circ}C$(p<0.01), with no difference between two groups. Among different composition groups of the semen diluents of control(tris + egg yolk + glycerol), egg yolk-free, 히ycerol-free, and tris-free, Prior to freezing, the egg yolk-free diluent showed significantly love. motility and viability than the other diluents(p<0.05). On each thawing day (from day 2 to 15 after freezing), control group showed considerably higher motility and viability than the other groups(p<0.01). The canine spermatozoa frozen and stored at -6$0^{\circ}C$ and -8$0^{\circ}C$ showed gradual decrease of motility from day 2 to 30 after freezing and the spermatozoa of these two groups thawed on day 30 showed considerably love. motility than those thawed on day 2 after freezing, respectively(p<0.01). These results indicate that the freezing temperature of either -6$0^{\circ}C$ or -8$0^{\circ}C$ can be applicable to the freezing method using methanol and also all of the components of the semen diluent including cryoprotectant, buffer and cold-shock buffer are very important to maintain motility and viability of canine spermatozoa in the freezing and thawing procedure.

• 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.

• 한국양식학회지
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• 제11권1호
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• pp.67-75
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• 1998

감성돔 (Acanthopagrus schlegeli) 정자의 냉동 보존시 정자의 생리활성과 보존효과를 비교하기 위하여, 순환여과 사육시스템에서 사육한 전장 $25.9{\pm}1.7$ cm, 체중$292.8{\pm}53.7$ g의 성어로부터 얻은 정액을 재료로 하여 희석액별, 동해방지제별 해동후 정자의 활성, 생존율 및 알에 대한 수정능력을 평가하였다. 희석액별 냉동보존에서 해동정자의 생존율은 3% sodium citrate에서 $80{\pm}1.4$%로 가장 높았으나, 수정률은 5.4% glucose에서 $63.4{\pm}4.4$%로 가장 높았다. 동해방지제별 냉동보존에서 해동정자의 수정률은 5~15% glycerol을 동해방지제로 사용하였을 때, 50.1~69.4%로 DMSO 보다 나은 효과를 보였다. 감성돔 정자의 냉동보존을 위한 적정 희석액과 동해방지제는 5% glucose와 5% glycerol이었다. 냉동하지 않은 감성돔 정자의 머리는 구형으로 직경 $1.5{\pm}0.1$ ${\mu}$m, 길이 $1.3{\pm}0.1$${\mu}$m였으며, 치밀한 핵질로 채워져 있었다. 편모는 전형적인 9+2 구조를 나타냈다. 그러나 냉동후 해동시킨 정자중에서는 염색질의 과립화, 세포막의 이탈에 의한 그 용적이 커지는 구조변화를 보였다.