• 한국수정란이식학회지
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• 제30권1호
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• pp.7-15
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• 2015

The objective of this study was to investigate the efficiency of nicotinic acid on sperm cryosurvival and fertilization ability in frozen-thawed boar semen. Boar semen was collected by glove-hand method and was frozen using freezing solution treated to 0, 5, 10 and 20 mM of nicotinic acid. The frozen sperm for sperm characteristic analysis was thawed such as viability, acrosome reaction, and mitochondrial integrity. The frozen-thawed sperm was estimated by SYBR14/PI double staining for viability, FITC-PNA/PI double staining for acrosome reaction and Rhodamine123/PI double staining for mitochondrial integrity using a flow cytometry. The embryo was estimated in vitro development and DCFDA staining for reactive oxygen species assessment. As results, frozen-thawed sperm viability was significantly higher in 5 and 10 mM ($61.1{\pm}1.5%$,$64.7{\pm}2.0%$) of nicotinic acid than other groups (0 mM, $52.1{\pm}2.3%$; 20 mM, $47.8{\pm}5.1%$, P<0.05). The live sperm with acrosome reaction was significantly higher in 5 and 10 mM of nicotinic acid ($26.1{\pm}1.8%$, $24.9{\pm}1.5%$) than other groups (0 mM, $35.3{\pm}0.8%$; 20 mM, $36.5{\pm}1.9%$, P<0.05). The live sperm with mitochondrial integrity was significantly higher in 5 and 10 mM ($84.2{\pm}3.6%$, $88.4{\pm}2.3%$) of nicotinic acid than other groups (0 mM, $77.3{\pm}4.4%$; 20 mM, $73.3{\pm}3.6%$, P<0.05). Blastocyst rate of in vitro development was significantly higher in 10 mM ($17.0{\pm}1.3%$) of nicotinic acid than other groups (0 mM, $9.4{\pm}0.5%$; 5mM, $12.6{\pm}0.8%$; 20 mM, $5.0{\pm}1.0%$, P<0.05). Moreover, total cell number was higher in 5 and 10 mM ($53.6{\pm}2.9%$, $57.9{\pm}2.8%$) of nicotinic acid than other groups (0 mM, $41.0{\pm}1.4%$; 20 mM, $23.2{\pm}2.8%$, P<0.05). Hydrogen peroxide in embryos was lower in 5 mM nicotinic acid ($0.7{\pm}0.1%$) than other groups (0 mM, $1.0{\pm}0.1%$; 10mM, $0.9{\pm}0.0%$; 20 mM, $1.4{\pm}1.0%$, P<0.05). In conclusion, nicotinic acid-treated semen improves cryosurvival and quality of spermatozoa. Also, the fertilized oocytes with nicotinic acid improve quality of embryo and blastocyst formation.

• 한국가금학회지
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• 제41권1호
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• pp.21-27
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• 2014

본 연구는 닭 정액의 동결 보존을 위하여 비 글리세롤성 동결 보호제 중 MA 농도가 정자의 생존율과 융해된 동결 정자를 인공수정을 실시하여 생산된 수정란의 수정율, 부화율을 조사하고자 실시하였다. 동결 보조제로써 MA의 효율성은 7%, 9% 및 11% 범위에서 동결을 실시하였을 때, 정자의 생존율은 $35.16{\pm}6.12%$, $67.83{\pm}15.3%$ 및 $66.2{\pm}16.3%$로 관찰되었으며, 융해된 정자를 인공수정을 실시하여 생산된 수정란의 수정율은 21.5%, 34.7% 및 25%로 관찰되었으며, 수정된 수정란의 부화율은 100%, 89.5% 및 87.5%로 관찰되었다. 대조군으로써 신선 정액은 수정율이 96.0%로 관찰되었고, 부화율은 92.2%로 관찰되었다. 9% MA를 이용한 간이 동결법으로 생산된 동결 정자를 이용하여 3주간 수정란을 검사하였을 때, 수정율은 비록 35.3%로 관찰되었으나, 부화율은 90.3%로 관찰되었다. 이러한 결과에 따르면, 9% 농도로 MA 동결 보호제를 이용할 경우, 동결 및 융해된 정자를 이용하여 생산된 수정란에서 수정율을 감소시킬 수 있음을 보여주고 있으며, 부화율에는 영향을 주지 않음을 추정할 수 있다. 그러므로 수정율과 부화율에 나쁜 영향을 미치지 않고 가금 유전자원의 보존에 중요한 요인이 될 수 있는 적절한 농도의 MA 동결 보호제 범위는 7~9% 농도로 추정된다.

• 한국임상수의학회지
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• 제17권2호
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• 한국수정란이식학회지
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• 제30권3호
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• pp.137-142
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• 2015

Cryopreservation of boar semen is continually researched in reproductive technologies and genetic resource banking in breed conservation. For evaluating the boar semen quality, sperm motility (MOT) is an important parameter because the movement of spermatozoa indicates active metabolism, membrane integrity and fertilizing capacity. Various researches have been trying to improve the quality of semen post-thawed in boar. Recently, polymorphism (g.158T>C) of phospholipase C zeta (PLCz) gene reported to be significant association with MOT. This study was conducted to evaluate the PLCz gene as a positional controlling for motility and kinematic characteristics of post-thawed boar semen. To results, The g.158 T>C SNP of PLCz was significantly associated with frozen semen motility and kinematic characteristics. g.158 T>C SNP was high significantly associated with MOT, VCL, VSL and VAP (p<0.0001, p=0.0002, p<0.0001 and p<0.0001, respectively). Therefore, we suggest that the intron region of the porcine PLCz, may be used as a molecular marker for Duroc boar post-thawed semen quality, although its functional effect was not defined yet. Whether the association is due to the candidate gene or not require further verification. Thus, it will be of interest to continue association studies in the regions surrounding those genes.

• 한국가축번식학회지
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• 제23권2호
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• pp.165-174
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• 1999

본 연구는 동결융해 후 정자의 생존성에 영향을 미치는 요인을 찾기 위하여 수행하였다. 동결융해 후 생존성에 대한 요인으로써 동결보존액, 동해보호제, 예비동결법 및 동결소요시간을 비교하였다. 동결과정중 정액의 질을 평가하기 위하여 활력, NAR 및 생존율을 조사한 결과는 다음과 같다. 돼지정액을 BF5, LYE, Soejima 및 modified Soejima 보존액으로 동결하였을 때 동결융해 후 정자활력은 M-Soejima 보존액이 44.5$\pm$6.4%로 다소 높았다. M-Soejima 보존액의 2차 회석액에 caffeine(2mM), heparin(l00 ${\mu}\ell$/$m\ell$) 및 caffeine＋heparin 를 첨가하였을 때 동결융해 후 활력은 caffeine 첨가구가 61.7% 로 가장 높았으며, 단독 혹은 혼합첨가가 첨가하지 않은 대조구보다 유의적으로 높은 활력을 나타내었다 (p＜0.05). M-Soejima 보존액에 동해보호제로서 glycerol(Gly), ethylene glycol(EG), propylene glycol(GP), Gly＋EG 및 Gly＋PG을 첨가하였을 때 동결융해 후 활력 및 NAR 율은 Gly＋PG의 혼합첨가시 (31.3%/39.5%) 가 다른 첨가구보다 다소 높았으며 생존율은 Gly＋EG 첨가구가 21.2% 로 다른 첨가구보다 다소 높았다. BE5와 M-Soejima 보존액으로 straw 및 pellet 동결법으로 동결하는 동안 dry ice-pellet, dry ice-straw 및 L$N_2$vapor-straw 법으로 예비동결하였을 때 각각 22.8, 47.5, 52.5% 및 42.5, 47.5, 57.5% 의 활력을 나타내었다. 또한 M-Soejima 보존액의 straw 법으로 l차 희석부터 동결완료까지 소요되는 시간을 2, 5 및 7시간으로 하였을 때 동결융해 후 활력 및 생존율은 처리간에 큰 차이가 없었으나, NAR 율은 처리시간이 길어질수록 다소 높은 경향을 나타내었다. 이러한 결과로 미루어 볼 때 동결융해 후 활력, NAR 및 생존율을 높이기 위해서는 caffeine 이 첨가된 M-Soejima 보존액에 동해보호제로 glycerol과 propylene glycol 또는 ehtylene glycol 을 사용하여 2시간 동안 빠르게 동결처리된 정액이 다소 유리할 것으로 사료되었다.

• 한국임상수의학회지
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• 제19권1호
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• pp.73-79
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• 2002

To investigate the usability of frozen canine embryos for embryo transfer in the dog, 19 donors, 3 recipients, and 6 male dogs were used for the experiment. Natural mating or artificial insemination was performed for breeding the bitches in natural estrus. Vaginal smear test along with progesterone titre test were performed to detect the appropriate mating time and the bitches were bred twice during 3-6days following LH surge. Embryo collection was done on 8, 9-11, 12-13 days after the second mating to collect morula and blastocyst. Embryos were frozen using a programmable freezer and preseued in LNE tank. Embryos were thawed in 37$^{\circ}C$ water for 15 seconds and transferred into each uterine horn within 30 minutes. Embryos were collected from 13 bitches of 19 donors(68.4%) and the collected embryos were from between 9 and 13 days after 2nd mating. Embryos were produced both by natural mating(60.0%, 9115) and AI with frozen semen(100.0%, 4/4). Embryos were collected from the donors weighed between 2.5 and 30 kg and their age was from 1.5 to 3 years. 52 embryos were collected from 13 donors and the mean number of embryos was four. The stage of embryos was from 2-cell to gastrula and morulae were colledted mostly from 10 to 11 days after 2nd mating. Embryos were collected evenly from each uterine horn and the rate of embryo collection for the number of corpus luteum was 83.9%. Embryos were transferred to 3 recipients(morula 8, blastocyst 1, gastrula 8), however, no offspring was produced.

• 한국가축번식학회지
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• 제13권1호
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• pp.18-25
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• 1989

This study was carried out to investigate the general semen characteristics of the Korean native goat and the effect of temperature, incubation time, dilution rate, freezing rate and glycerol concentration on motility and NAR (normal apical ridge) acrosome of fresh and frozen Korean native goat spermatozoa. 1. Average semen volume per ejaculate, motility, concentration and pH of fresh Korean native goat spermatozoa were 0.19${\pm}$0.09 ml, 94.5${\pm}$0.47%, 26.17${\times}$108${\pm}$1.68/ml and 6.63${\pm}$0.18, respectively. 2. Motility and NAR acrosome of fresh spermatozoa during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 3. Motility and NAR acrosome of spermatozoa diluted 1:4 during incubation were higher at 22$^{\circ}C$ than at 5$^{\circ}C$ or 37$^{\circ}C$(P<.01). 4. Motility and NAR acrosome of spermatozoa during incubation were higher for samples diluted 1:1, 1:2, or 1:4 than for samples diluted 1:6(P<.01). 5. Motility and NAR acrosome of post-thaw spermatozoa were higher at freezing rate of 12$^{\circ}C$/min than at freezing rate of 1$^{\circ}C$/min or 24$^{\circ}C$/min when glycerol concentration was 9%(P<.01).

• 한국가축번식학회지
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• 제12권1호
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• pp.15-19
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• 1988

This study was carried out to investigate the fertility and farrowing date in post-weaning sows using PG600 and inseminated with frozen semen. A total of 48 sows of Landrace, Large White and Duroc after 7-week lactation were used at the Chungnam Provincial Animal Breeding Station. The results obtained were summarized as follows: 1. Motility had no significant differences between the breeds, but NAR acrosome was highest in Landrace, followed by Duroc and Large White(p<.01). 2. Interval from weaning to estrus and length of estrus were, respectively, 3.7 days and 52.6hours of sows treated with PG600, and 6.5 days and 53.8 hours for control sows. The average interval from weaning to onset of estrus was significantly(p<.01) shorter by 2.8 days in PG600 treated sows compared to control sows. 3. In Landrace, Duroc and Large White, farrowing rate and number of pigs born alive per litter were 55.0%, 10.0; 43.8%, 8.1; and 16.7%, 3.5, respectively. Average pig weight at birth and survival rate at 56 days had no significant differences between the breeds. 4. Farrowing rate, number of pigs born alive per litter, average pig weight at birth and survival rate at 56 days were, respectivey, 45.8%, 101, 1.56kg and 94.5% for sows treated with PG600, and 37.5%, 7.0, 1.66kg and 93.8% for control sows. Sows treated with PG 600 had an average of 3.1 more pigs at farrowing compared to control sows.

• 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.

• Reproductive and Developmental Biology
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• 제35권1호
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• pp.61-65
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• 2011

The objective of this study was to examine the effect of various discontinuous Percoll washing conditions on motile sperm recovery rate and motion kinematics. Frozen semen samples from 3 bulls (0.5 ml plastic straws, 6% glycerol in egg yolk-Tris-glycerol extender) were thawed in $37^{\circ}C$ water bath for 1 min. After thawing, the mixed semen samples were randomly allocated to 12 treatment groups. Briefly, the spermatozoa were centrifuged for three different time lengths (10, 20, and 30 min) at two gravities ($300{\times}g$ and $700{\times}g$) through two concentrations of discontinuous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll and 2 ml 90%: 2 ml 45% Percoll to remove extender, debris, and dead spermatozoa. Motile sperm recovery rate and motion kinematics were evaluated by computer assisted sperm analyzer using Makler counting chamber. Sperm motility (%) and motile sperm recovery rate showed similar pattern in all treatment groups. However, sperm motility (%) and motile sperm recovery rate were highest at $700{\times}g$ for 30 min through a discontionous Percoll density gradient of 1 ml 90%: 1 ml 45% Percoll. There were no significant differences in motion kinematics after various Percoll washings. These results suggest that force of centrifugation, centrifugation time, and Percoll volume significantly affect motile sperm recovery rate.