본 연구는 돼지의 정자와 난소내 과립막세포에서 bisphenol S(BPS)가 생존성과 활성산소 생산에 미치는 영향을 알아보고자 연구하였다. 돼지정액은 0, 5μM BPS를 처리하여 3, 6시간동안 배양하였다. 정자의 생존성은 SYBR14/PI를 이중 염색하여 분석하였으며, 활성산소의 생산을 측정하였다. 또한, BPS(0, 5, 10, 20μM)를 과립막세포에 처리하여 24, 48, 72시간동안 처리하였다. 처리 후, 세포의 생존율과 활성산소 생산(단, 5μM BPS)을 측정하였다. 그 결과, 돼지에서 정자의 생존율은 BPS에 의해 감소하였고, 활성산소의 생산은 모든 처리시간에서 증가하였다(p<0.05). 또한 과립막세포의 생존은 BPS에 의해 억제되었고, 활성산소는 유의적으로 증가하였다(p<0.05). 이상의 결과를 토대로, BPS의 노출은 정자의 활성과 번식과 관련된 세포에 나쁜 영향을 미칠 것이다.

This study was to investigate effect of tunicamycin (TM) on sperm viability, mitochondrial activity and motility in boar semen. Collected sperm were incubated with semen extender containing 0, 1, 2, and 5 μM TM for 3, 6 and 9 h. Sperm viability was analyzed using SYBR14/PI doubling staining, and mitochondrial activity was detected using Rhodamine123/PI staining methods. Sperm viability, mitochondrial activity and motility were measured every 3 h during incubation. In results, boar sperm viability, mitochondrial activity and motility were significantly decreased in 2 and 5 μM TM groups compare to control group at all incubation time (p<0.05). In addition, mitochondrial activity and motility were significantly decreased in 1, 2, and 5 μM TM groups compare to control group at 9 h after incubation (p<0.05). These results suggest that TM can inhibit sperm viability, mitochondrial activity and motility in boar semen, and it may influence on the fertility of sperm.

Antioxidants have been added to cryoprotectant or in vitro culture medium for sperm to reduce the detrimental damage, such as reactive oxygen species. However, curcumin, an antioxidant, shows dual effect on the viability and progressive motility of bovine sperm exposed to hydrogen peroxide. Low concentration of curcumin increases sperm viability and progressive motility, whereas high concentration of curcumin reduces them. This study was performed to identify whether TREK-1 channel is related to low sperm viability and motility induced by high concentration of curcumin. Curcumin reduced TREK-1 channel activity in a dose-dependent manner. TREK-1 channel was expressed in sperm obtained from Korean native bull. Treatment with TREK-1 channel blockers, such as curcumin, fluoxetine, GdCl3, and spadin, significantly reduced sperm viability and motility (p < 0.05). However, TREK-1 channel activators showed different effect; linoleic acid showed an increase in sperm viability and motility, and wogonin did not affect them. These results show that TREK-1 channel is involved in the regulation of sperm viability and motility. In particular, high concentration of curcumin might reduce sperm viability and progressive motility of Korean native bull through blockage of TREK-1 channel.

To preserve genetic materials, cryopreservation of the semen from live animals is the main technique to establish cryo-banking system which could be used for artificial insemination and embryo transfer. However, the population of Korean black goat (KBG) becomes to dwindle in number and is now faced genetic erosion by crossbreeding with non-native breeds in small KBG farms. In this study, simple freezing method was used to preserve frozen semen from KBG using spermatozoa of cauda epididymis (CE) and electro-stimulated semen (ES). The negative effects of seminal plasma on fresh sperm was confirmed using precipitation test of Triladyl egg yolk diluent and sperm viability after thawing was compared between CE and ES spermatozoa. When seminal plasma of fresh ES semen was washed with semen washing media (SWM), the rates of live sperm shown no significant difference between CE and ES spermatozoa before freezing. However, the survival rate of frozen/thawed CE sperm was higher than ES (74.6±10.6% vs 53.8±5.2%) with significant difference (p < 0.05). The results of longevity test on frozen/thawed sperm from CE showed healthier sperm than ES. Therefore, spermatozoa from CE could be used for cryo-banking system in KBG lines. The more studies are needed to increase survival rate of ES semen.

Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with Triladyl®-egg yolk diluent and then was performed cryopreservation.There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

Cryopreservation of germ cells from genetically proven animals could be a source of restoration tools from the risk of extinction or disappearance of wanted characteristics. Using frozen semen, the genetic gains of Korean native cattle have been increased greatly for 70 years. The preservation of genetic resources as a form of frozen semen straw has limited availability due to the numbers. To circumvent this weakness of frozen semen, we tested two re-freezing methods with different initial thawing temperatures using frozen Korean proven semen and rare breed semen from albino, black and chikso breeders. It has been known that human sperm could resist to cryo-damages by repeated freeze-thaw cycles, but not for Korean proven bulls number (KPN) or for rare breeds. Total 7 frozen semem from brindled(2), black(1), Korean Albino(2) and KPN(1) bulls were used for our research. After thawing straws under 5°C/2min or 37°C/40sec with low temperature water bath and thermo jug, spermatozoa were re-diluted with triladyl diluents after first thawing and re-frozen. Sperm motilities were compared between animals and treated groups after re-thawing. Mean values of motility and viability of refrozen/thawed sperm for expansion of the number of straws were significantly higher in 5°C than in 37°C (P< < 0.05). However, the activity of viable sperm thawed at 5°C was significantly decreased after first and second thawing. It is estimated that re-freezing of frozen semen from rare Korean native cattle is possible with resistant properties of survived spermatozoa.

정자 생존성은 정자기능평가에서 중요한 부분이며 Eosin-nigrosin 염색법, Hypo osmotic swelling test법, CFDA, SYBR-14, Hoechst-33342, Calcein AM 등의 형광염색법을 통해 평가될 수 있다. 또한 Flow ctyometer를 이용, 단시간에 형광 염색된 세포를 검사하여 생 존성뿐만 아니라 정자의 여러 기능적 특성을 평가하는 기술이 최근 이용되고 있다. 이 연 구는 Flowcytometry를 사용하여 제주흑우의 동결정액에서 정자의 생존성을 평가하는 2가 지 형광염색법 간의 비교분석을 통해 정자 생존율의 차이가 있는지 알아보고자 하였다. 국립축산과학원 난지축산연구소에서 사육중인 제주흑우 2두를 인공질법을 이용하여 채 정하였으며, 정자 최종농도가 2.0×107/ml가 되도록 Triladyl-eggyolk로 희석 후 4℃에 1시 간 30분간 평형과정을 수행하였다. 그 후 스트로 정액 충전기로 정액을 충전 후 간이 액 체질소 증기 동결법으로 동결하여 LN2에 동결보존하였다. 동결보존된 정액 스트로(n=12) 를 융해하여 PBS를 이용하여 5×105/ml 농도로 희석 후 2개로 분획하였으며, 각각 Calcein Am-PI(CAM/PI)와 6-CFDA-PI(CFDA/PI)의 이중 형광염색을 실시하고 37℃, 15분간 정치 후 Flow ctyometry로 생존율을 비교 분석하였다. 분석결과 살아있는 정자 의 비율은 29.26±1.28(CAM+/PI-), 27.50±0.76(CFDA+/PI-), 약간의 생체막의 손상이 있으나 살아있는 정자의 비율은 21.67±4.92(CAM+/PI+), 23.29±2.76(CFDA+/PI+), 생체막의 손 상으로 죽은 정자의 비율은 48.44±4.18(CAM-/PI+), 48.61±2.71(CFDA-/PI+)이었으며 각 각 형광염색법간의 유의적인 차이는 없었다(SPSS v18.0 통계프로그램 사용). 본 연구결과 Flow cytometry를 사용하여 정자의 생존율을 평가할 때 Calcein AM과 6-CFDA의 차이는 존재하지 않는 점을 알 수 있었으며, 정자의 생존성 평가에 CAM/PI 방법과 CFDA/PI 방법 중 하나를 선택하여 사용 가능하다고 판단된다.

Although cryopreservation of sperm is routinely used for clinical requirement, it has some problems, such as high generation of reactive oxygen species (ROS) and cold-shock. To reduce the detrimental damage in sperm, anti-oxidants were added to cryoprotectant for sperm. Curcumin is one of anti-oxidants, which are added in cryoprotectants. However, recent studies have demonstrated that curcumin decreases sperm viability and motility. This study was performed to identify the effect of curcumin on hydrogen peroxide (H2O2)-exposed bovine sperm, which were cryopreserved-thawed. In H2O2-exposed bovine sperm, reactive oxygen species (ROS) were significantly reduced by treatment with curcumin in a dose-dependent manner (p < 0.05). Among tested concentrations of curcumin (1 to 50 μM), 30 and 50 μM curcumin showed anti-oxidant effect on H2O2-induced ROS generation. On the other hand, combination of 30 or 50 μM curcumin with anti-oxidant H2O2 increased the percentage of apoptotic sperm compared to only H2O2 treatment. Sperm viability was also decreased in the combination of 30 or 50 μM curcumin with H2O2 as judged by FDA/PI staining. H2O2–induced decrease in sperm progressive motility was recovered by treatment with 1 μM curcumin. These results show that high concentration of curcumin has anti-oxidant effect, but it has also cytotoxic effect on bovine sperm. Sperm viability and motility might be more affected by cytotoxic signals of curcumin compared to antioxidant signals.

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% eggyolk 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(H2O2) 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.

닭 정액의 동결보존기술은 유전자원 보존의 수단으로 이용될 수 있는 기술로서 고병원성 조류인플루엔자 같은 악성질병에 의한 멸실을 방지하는 매우 중요한 기술이다. 닭의 정자 는 동결보호제와 동결에 이용되는 희석액에 따른 융해 후 정자의 활성도, 수정율 및 부화율의 변이가 매우 큰 것으로 알려져 있다. 가장 널리 사용되는 동결보호제인 Glycerol은 동결 정 액 제조에 가장 많이 이용되며 융해 후 닭 정자의 생존성이 비교적 우수한 것으로 알려져 있으나, 인공수정에 직접 이용할 경우에 수정율의 저하현상이 나타난다. 본 연구에서는 동 결보호제중 하나인 N-Methylacetamide(MA)를 이용하여 한국 재래계인 오계 수탉의 정자 를 동결보존, 융해 및 인공수정을 실시하여 수정율과 부화율을 조사하였다. 복부 마사지법으 로 채취한 정액을 5℃ 저온수조에 담아 실험실로 이송하였으며 MA을 함유하지 않은 희석액 (HS-1)에 1:1의 비율로 희석하여 30분간 5℃ 온도로 유지시켰다. 2차 희석액은 14, 18 그 리고 22%의 MA가 함유된 희석제를 이용하였으며 1차 희석된 정액과 1:1 비율로 희석하여 0.5ml 스트로에 충진 하였다. 액체질소 표면 위 4 cm에 설치된 간이 지지대에 밀봉된 스트 로를 30분간 정치하여 동결하는 간이동결법을 이용하였다. 동결된 정액을 1～2주간 보관한 후, 5℃로 조절된 저온수조에서 융해하고 운동성 있는 정자와 활력이 우수한 정자의 비율 을 비교 분석하였다. 동일한 방법으로 융해한 정액으로 인공수정을 실시하고 7일 간 수정 란을 수집하여 부화기에 배양하여 7일차에 발생란을 검란하였다. 대조군에서 희석된 정액 의 경우 98.4% 수정율이 관찰되었으며 실험군에서는 7, 9 및 11% MA의 경우 각각 21.5 %, 34.7% 및 25% 수정율이 관찰되었다. 수정율에 대한 부화율은 대조군에서 88.9%를 관 찰하였고 실험군은 100%, 89.5% 및 87.5%로 관찰되었다. 이러한 결과로 유추해 볼 때, 오 계 정자의 동결보존에 유용할 수 있는 MA의 농도의 범위는 약 9% 내외로 판단되며 특히 MA는 오계 수정란의 수정율과 부화율에 미치는 영향이 낮은 것으로 사료되었다.

The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition ( for 20 sec, 45 sec and for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE (Viability : , NAI : ), TLE (, ) extender significantly(p<0.05) increased than that in LEY (, ) extender thawed at for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE (, ) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE () while that in LEY () is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted (30×106 spermatozoa/ml) in different extenders. Semen was stored at 17℃ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection. The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significantly different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were and respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at was significantly lower than that at 0 hr (<), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

본 실험은 돼지정액의 동결보존을 위한 희석액과 냉각속도 및 동해방지제의 적정농도를 결정하기 위해 실시하였으며, 얻어진 결과는 다음과 같다. 1. 에서 까지의 냉각속도에서는 LEY 희석액에서 분당 로 냉각하는 것이 생존율과 정상 첨체율에서 가장 높은 결과를 얻었다. 2. LEY 희석액이 BF5와 M-Soejima 희석액보다 정자를 동해로부터 보호하는 능력이 우수하였다. 3. LEY 희석액에 첨가하는 glycerol의 농도는 3 또는 가 의 glycerol

본 연구는 종모돈의 정액성상 동결-융해 후 정자의 생존성 그리고 혈청 중 FSH, LH, estradiol-17β 및 testosterone 농도에 미치는 품종과 계절의 영향을 조사하여 우수한 종모돈의 선발을 위한 기초자료를 얻고자 실시하였다. 요크셔종이 듀록종보다 봄, 여름, 가을, 겨울에서 정액량이 많았으며, 정액농도에서는 차이가 없었다. 계절별 정액량은 듀록 및 요크셔종에서 봄철이 여름, 가을 및 겨울철에 비하여 많았고, 정자농도는 차이가 없었다. 듀록종과 요크셔종에서 각각 봄철에 생산한 정자가 여름, 가을 및 겨울철에 생산한 정자보다 동결-융해 후 정자운동성 및 정상첨체 비율이 높았다. 한편 듀록종과 요크셔종에서 동결-융해 후 정자운동성은 모든 계절에서 요크셔종이 높게 나타났으나, 정상첨체에서는 차이가 없었다. 혈청 중 FSH의 농도를 비교한 결과 요크셔종이 듀록종보다 모든 계절에서 낮은 농도를 나타내었다. 그러나 두 품종 모두에서 각각 계절 간에 차이가 없었다. 혈청 중 LH와 estradiol-17β의 농도를 비교한 결과 요크셔종과 듀록종 간에 차이가 없었다. 또한 두 품종 모두에서 계절 간에 차이가 없었다. 종모돈의 품종별, 계절별 혈청 중 testosterone의 농도를 비교한 결과 요크셔종이 듀록종보다 모든 계절에서 높게 나타났다. 또한 두품종 모두에서 각각 봄철이 여름, 가을 및 겨울철에 비하여 혈청 중 testosterone의 농도가 높은 것으로 나타났다. 이상의 결과를 종합하여 보면, FSH의 농도가 낮을수록 정액생산량이 높은 것으로 나타났으며, 혈청 중 testosterone의 농도가 높을수록 동결-융해 정자의 운동성 및 정상첨체의 비율이 높은 것으로 나타났다.

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 /ml로 정상범위에 들었으며, 정자의 형태학적 판정에서 정상적인 정자의 농도는 평균 59.453.45%로 상대적인 기형율은 약 30~40% 정도 나타났으며, 이는 정상적인 상태의 개 정액

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/min의 동결속도로 실시하여 37에서 2분간 응해하는 방법이 가장 좋은 결과를 보였다. 생존성과 운동성에 있어서의 차이는 없지만 첨체의 intact한 비율은 약간 낮은 결과를 보였으며, 이의 보완을 위해, 액