This study was conducted to find out the effect that κ-Carrageenan has on the properties of dog sperm when it was added to the cryoprotectant. Extender basically was contained 1.21 g Trizma base, 0.67 g citric acid, 0.4 g glucose, 0.03 g penicillin G, 0.05 g streptomycin sulfate. Extender1 was added with 0.1%, 0.2%, 0.3%, and 0.5% carrageenan, while extender2 was supplemented with glycerol. After freezing-thawing, the motility, viability, acrosome integrity, apoptosis, and ROS (reactive oxygen specifications) of sperm were measured to analyze the effects of the supplementation of carrageenan. Total Motile (TM), Rapid Progressive Motile (RPM), Medium Progressive Motile (MPM), and Immotile were measured through the CASA system after thawing in 37 degree water. Extender with 0.2% κ-carrageenan (64.26 ± 0.49) was significantly higher than control (40.24 ± 8.27) (p < 0.05). RPMs of extender with 0.1%, 0.2% κ-carrageenan (57.64 ± 6.34, 56.47 ± 1.35) were significantly higher than the other groups (p < 0.05). Acrosome integrity was measured by dyeing to PSA-FITC with an epifluorescence microscope. Normal acrosome ratio of extender with 0.5% κ-carrageenan (61 ± 8.03) was higher than the other groups (p < 0.05). Apoptosis was measured with a FACSCalibur flow cytometer using FITC (FITC Annexin V Apoptosis Detection Kit). Treated groups of κ-carrageenan of 0.1% (0.81 ± 0.05), 0.2% (0.85 ± 0.05) were significantly higer (p < 0.05) than control. Modified SYBR/PI staining was used for determination of viability and DCF staining was used for evaluation of ROS. Viability and ROS were not significantly different from other groups. In conclusion, adding a certain concentration of carrageenan to the extender of cryopreservation, carrageenan contributes to the improvement of the sperm motility, acrosome integrity and prevention of apoptosis.

생체 유래 동해보호제인 난황 및 우유는 세균 및 바이러스에 의한 오염과 감염으로 동결융해 후 정액 성상(보존성)에 부정적 요소로 작용하여 왔다. 이에 본 연구는 렛트(rat)에 있어서 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하였다. 공시된 렛트는 18 주령 이상의 성숙된 수컷 6 마리였다. 정액채취는 렛트를 먼저 할로탄 흡입마취한 후 양측 정소의 중앙부를 1~2cm 절개하여 정소상체의 미부를 적출한 후 Androhep 희석액이 담긴 배양접시(⌀35mm)에 옮겨서 세절하여 정자를 채취하였다. 정액의 동결과정은 이장희(1993)의 방법에 준하여 실시하였다. 동결보존액으로는 기본적으로 Androhep 희석액을 사용하였으며, 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하기 위하여 2 개체의 정액을 혼합하여 난황, 두유 및 코코넛 밀크가 각각 20%씩 포함된 1 차 동결보존액으로 1 차 희석 후 정자 농도를 4x107spermatozoa/ml 로 조정하였으며 1 시간에 걸쳐 4℃까지 냉각시켰다. 냉각된 정액은 1 시간에 걸쳐 8%의 glycerol 이 포함된 2 차 동결보존액의 10%, 20%, 30%, 및 40%씩 점등하여 1:1 로 희석시켰다. 최종 희석된 정액은 별도의 glycerol 평형 시간 없이 0.5ml straw 에 충진 시켜 포장하였다. 최종 포장된 정액은 액체질소 표면 5cm 위에서 10 분간 정치시켜 예비 동결시킨 후 침지하여 동결시켰다. 동결과정 중 정자의 활력은 현미경 상의 혈구계산판을 이용하여 100 분율로 평가하였다. 2 개체로부터 채취하여 합쳐진 정액에 대해서 동결보존액의 주요 조성분인 난황, 두유 및 코코넛 밀크에 따른 동결융해 후 정자 활력은 각각 10%, 0% 및 0%로 난황이 다소 높게 나타났다. 이들 주요 조성분의 오염도 수준은 난황, 두유 및 코코넛 순으로 나타났다. 동결과정 중 활력 변화는 채취 직후 합친 상태의 활력은 평균 75% 였으며 냉각 후 2 차 희석이 완료되었을 때에는 40%, 예비동결 후에는 20% 수준, 동결 후 1 일 경과 후의 융해 후 정자 활력은 매우 저조하였다. 이상의 결과에서 동해보호제로서 식물성 동해보호제로써 두유와 코코넛 밀크의 이용 가능성이 없는 것으로 나타났다. 다만 동결 처리 과정 중 정자 활력의 급격한 손실은 정소상체 미부 정자의 미성숙 상태에 의한 내동성 부족으로 사료되었다.

생체 유래 동해보호제인 난황 및 우유는 세균 및 바이러스에 의한 오염과 감염으로 동결융해 후 정액 성상(보존성)에 부정적 요소로 작용하여 왔다. 이에 본 연구는 반려견에 있어서 우수한 품종의 보존과 증식을 위하여 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하였다. 공시된 반려견은 경기도 김포에 위치한 애견 번식농장에서 보유 중인 포메라니언, 시츄 및 요크셔테리어 품종 각각 2 마리씩이었다. 정액채취는 맛사지법으로 실시하였으며 채취 직후 Androhep 희석액으로 35℃ 전후의 같은 온도로 1:1 로 희석시킨 다음 서울호서전문학교 브리딩전공 실습실로 2 시간이내에 옮겨졌다. 옮겨온 직후 개체별 정액성상을 평가하였으며 동결정액 제조방법은 지달영(2004)의 방법에 준하여 실시하였다. 동결보존액으로는 기본적으로 Androhep 희석액을 사용하였으며, 동결보존액이 융해 후 정액 성상에 미치는 영향을 조사하기 위하여 품종별 2 개체의 정액을 혼합하여 난황, 우유 및 코코넛 밀크가 각각 20%씩 포함된 1 차 동결보존액으로 1 차 희석 후 정자 농도를 4x107spermatozoa/ml 로 조정하였으며 1 시간에 걸쳐 4℃까지 냉각시켰다. 냉각된 정액은 1 시간에 걸쳐 8%의 glycerol 이 포함된 2 차 동결보존액의 10%, 20%, 30%, 및 40%씩 점등하여 1:1 로 희석시켰다. 최종 희석된 정액은 별도의 glycerol 평형 시간 없이 0.5ml straw 에 충진 시켜 포장하였다. 최종 포장된 정액은 액체질소 표면 5cm 위에서 10 분간 정치시켜 예비동결시킨 후 침지하여 동결시켰다. 동결과정 중 정자의 활력은 현미경 상의 혈구계산판을 이용하여 100 분율로 평가하였다. 반려견의 품종에 따른 채취 직후의 정자 활력은 시츄, 포메라니언 및 요크셔테리어 가 각각 87.5%, 40%, 및 60%로 시추의 정자 활력이 다른 품종 보다 높았다. 동결보존액의 주요 조성분인 난황, 우유 및 코코넛 밀크에 따른 동결융해 후 정자 활력은 각각 60%, 40% 및 20%로 난황이 가장 우수하였으나. 이들 주요 조성분의 오염도 수준은 난황, 우유 및 코코넛 순으로 나타났다. 식물성 동해보호제로 코코넛은 동결융해 후 활력이 가장 낮게 나타났으나 생체 유래 물질인 난황 및 우유보다는 오염의 정도가 매우 낮게 나타났다. 이상의 결과에서 동해보호제로서 식물성 코코넛 밀크가 반려견 정액의 동결 융해 후 정자 생존성(활력)에 있어서는 생체 유래물질인 난황, 우유보다 낮았으나 오염(세균 및 바이러스 등)을 줄일 수 있는 새로운 방향을 제시한 것으로 나타났다. 코코넛 밀크를 준비하는데 불편함이 많았던 관계로 대체할 수 있는 다른 식물성 동해보호제가 탐색되어야 할 것으로 사료되었다.

본 연구는 말 냉장정액의 정자 성상 향상을 위한 기초 연구로 국립축산과학원 난지축산연구소에서 한라마(더러브렛×제주마) 수말 정액의 보존시간대별 정자 성상을 분석하기 실시하였다. 본 연구의 공시마는 국립축산과학원 난지축산연구소에서 사육・보유하고 있는 한라마(더러브렛×제주마) 씨수마 2 두를 공시하였으며, 씨수마의 정액채취는 채취 시마다 같은 시간인 오전 9 시에서 10 시 사이에 실시하였다. 정액채취는 독성이 제거된 50ml 플라스틱 일회용 용기를 인공질(INRA model, IMV, French)에 장착하여 사용하였으며, 자외선 차단용 커버로 용기 주변을 보호하였다. 인공질은 45∼50℃의 온수를 이용하여 인공질 내부 온도가 38℃가 되도록 유지하였으며, 삽입을 원활하게 하기 위해 젤을 도포하였다. 정액은 채취 직후 10 분 이내 실험실로 운반하여 겔(gel) 제거 후 부피, 농도, 운동성 등을 조사하였다. 정장 물질 제거를 위하여 INRA96 희석제로 1:1 희석 후 50ml 튜브에 40ml 씩 분주한 후 400×g 에서 10 분 원심분리하여 seminal plasma 및 부유액을 제거하고 하층의 펠렛(정자괴)을 회수하였다(Cochran 등, 1984). 회수한 정자 펠렛은 최종 농도를 50×106/ml 로 희석하여 24h, 48, 72, 96 동안 혐기상태로 4℃냉장고에 보관하여 각각의 정자성상을 분석하였다. 말 정액은 INRA 96 희석제와 희석제에 Egg Yolk 3%가 처리 후 시간대별 정자성상을 분석하였다. 2 차 희석 후 INRA 96 과 Egg Yolk 3% 처리구에서 총 운동성은 각각 89.18%, 91.81%를 보였으며, 96 시간이 경과한 후에 정자 총 운동성은 INRA 96 이 80.71%, Egg Yolk 3% 처리구는 88.54%로 유의적인 차이를 보이지는 않았다. 본 연구의 기초 데이터를 바탕으로 정장제거 원심분리 조건, 정장제거 필터링 조건 등 정자 생존율 향상 연구 등 냉장정액의 시간대별 정자 성상분석을 통한 장기보존 방법 등의 연구가 수행되어야 할 것으로 생각된다.

This study was conducted to determine the effect of pentoxifylline levels on sperm motility, survival rate, sperm membrane integrity of frozen semen and fresh-extended equine semen in Jeju cross-bred horses. As a result of sperm characteristic comparison depending on pentoxifylline levles at 30 minutes post-thaw, the progressive motilities were 53.25±2.87 (4mM pentoxifylline) and 50.28±2.14 (8mM pentoxifylline) and significantly higher compared to the control group(40.09±5.15) and other treatment group (16mM pentoxifylline, 41.27± 2.82). The progressive fast motility were 22.44±1.62 (4mM pentoxifylline,) and 22.74±3.07 (8mM pentoxifylline) and significantly higher compared to the control group (13.47±1.48) and other treatment group (16mM pentoxifylline, 14.66±3.68) (p<0.05). As a result of sperm characteristic comparison depending on pentoxifylline levles at 30 minutes post-thaw were 68.96±1.64 (4mM pentoxifylline) and 67.90±6.72 (8mM pentoxifylline) and significantly higher compared to the control group (53.48±4.84) and other treatment group (16mM pentoxifylline, 58.14±2.65) (p<0.05). In conclusion, these results suggest that treatment groups with 4mM and 8mM pentoxifylline were higher compare to equine seperm mobility and the control group and treatment groups with more than 16mM pentoxifylline has a negative effect on sperm characteristics. After thawing, the total motility in post-thawed equine sperm has increased by 10 percent for 1 hour. these results suggest that pentoxifylline contributes to the improvement of the equine sperm motility and characteristics in post-thawed semen.

The objective of this study was to investigate the efficiency of nicotinic acid during in vitro fertilization (IVF) in frozen-thawed bull sperm . The ejaculated semen was diluted with Triladyl containing 20% egg-yolk and cryopreserved in liquid notrigen. The frozen sperm was thawed for 45 seconds in the 38℃ water bath. Sperm was diluted with IVF medium (Bovine-Oviduct medium; BO) containing 0, 15, 30 and 60 mM nicotinic acid (NA), which were incubated at 39℃, 5% CO2 for 0, 0.5, 1, 2 and 4h. The characteristics of frozen-thawed sperm were estimated with SYBR14/PI double staining for viability, FITC-PNA/PI for outer acrosomal membrane damage and Rhodamine123/PI for mitochondrial integrity using flow cytometry. And the sperm ability was analysed by Coomassie brilliant blue (CBB) staining for acrosome reaction state and Rose bengal staining for abnormality. Acrosome reaction and abnormality were analyzed using a microscope. In results, sperm viability was significantly higer in 30 mM group than 0 and 15 mM groups at 1 and 2 h (p<0.05). Outer acrosomal membrane damage was significantly lower in 30 mM group than 0 and 15 mM groups at 1, 2 and 4 h (p<0.05). And mitochondrial integrity was significantly higher in 30 mM group than 0 and 15 mM groups at 2 and 4 h (p<0.05). Also, acrosome reaction was significantly lower in 30 mM than 0 and 15 mM groups at 1 and 2 h (p<0.05) and abnormality was lower NA groups than 0 group at 1 h (p<0.05). In couclusion, we suggest that using the thawing medium containing NA for sperm dilution can be benefical for IVF in bulls

This study was designed to determine whether low-density lipoporoteins (LDL) extracted from egg yolk in extender improve the function of Korean Jeju Black Bull semen. The semen was cryopreserved with 5% ethylene glycol (EG) or 7% glycerol (G) extenders containing 10% egg yolk (EY), 4% LDL and 5% EY or 8% LDL. Frozen-thawed sperm were evaluated sperm motility, viability, membrane integrity and acrosome integrity. Post-thawed sperm motility has been significantly higher (p<0.05) in 4% LDL + 5% EY (; EG and ; 7% G) than 8% LDL (; EG and ;G). Treatment of 4% LDL + 5% EY-EG () has been significantly improved sperm viability compared to other treatments except 10% EY - EG. Moreover, in membrane integrity, swollen sperm ratio has been only significantly increased (p<0.05) in 4% LDL + 5% EY - EG () among all treatments. In assess to detect acrosome integrity, especially, AR pattern ratio has been significantly decreased (p<0.05) in 4% LDL + 5% EY - EG among all treatments. In sperm viability as time passes, between 4% LDL + 5% EY and 10% EY, there was no significant difference, but 8% LDL was significantly decreased sperm viability in EG (1 and 2 hrs) and G (30 min, 1, 2, 5 and 12 hrs) extender. However, there were no significant differences among all treatments except 8% LDL-G in sperm membrane integrity. 8% LDL-G has been significantly decreased swollen sperm ratio at 5 hrs after thawed. It is concluded from these results that 4% LDL + 5% EY to the freezing extender showed more positive effect on the frozen-thawed spermatozoa in Korean Jeju Black bull.

This study was designed to determine whether low-density lipoproteins (LDL) from egg yolk and taurine, hypotaurine and trehalose as antioxidant in extender improve the freezability and fertility of Korean Jeju Black Bull semen. The semen was cryopreserved with tris egg yolk extenders containing 7% glycerol and treated 4% LDL, 20 mM taurine, hypotaurine and trehalose. Frozen-thawed sperm were evaluated motility, viability, membrane, and acrosome integrity and sperm penetration ability. The results were compared to semen cryopreserved in tris egg yolk extender only as control. Frozen-thawed semen evaluation cleary indicated that the addition of LDL and LDL-antioxidants (taurine, hypotaurine and trehalose) combination were significantly improved (p<0.05) the viability (%; with staining test using eosin-Y) compared to control spermatozoa. Also, in membrane integrity (%; with supravital hypo-osmotic swelling test), not only LDL-antioxiants combination but also LDL were significantly increased (p<0.05) the swelled sperm using HOST compared to control. Sperm acrosome integrity state was classified by CTC (chlortetracycline) staining test. F pattern was significantly increased in LDL-antioxidant combination than control (p<0.05) and B pattern was not significantly differences among all treatments and control. However, AR pattern was significantly decreased in LDL-antioxidants combination than control (p<0.05). Pronucleus formation and sperm penetration index (SFI) were significantly increased in LDL and LDL-antioxidants combination than control (p<0.05). Especially, LDL-taurine significantly improved pronucleus fomation and SFI than LDL (p<0.05). It was concluded that LDL and LDL-antioxidants in extender improved the freezability and fertility of Korean Jeju Black bull spermatozoa.

This study was carried out to investigate artificial insemination (AI) failure status and frozen semen characteristics in Korean proven bulls‘ number (KPN) semen used for AI of Hanwoo cows in Gangwon East region (Gangneung, Donghae, Taebaek, Samcheok, Sokcho, Yangyang, Goseong). Among semen used for AI, AI failure rate showed lowest at KPN506 (27.6%), whereas highest at KPN593 (77.2%). Correlations of AI failure in between Korean proven bulls semen and cows was 0.2941, which means that AI failure rate of Korean proven bulls semen may have respectable effect on reproduction of Hanwoo cow. In addition, present study was conducted to investigate spermatozoal viability rate, ruptured acrosome rate and active mitochondria in frozen Korean proven bulls semen with flow cytometry. The semen of KPN593 showed significantly (p<0.05) higher viability rate in KPN593 (30.49%) than that in KPN637 (37.34%). Furthermore, percentage of ruptured acrosome was lower in KPN637 as 21.37% than in KPN637 (21.37%), but it was not statistically significant. In conclusion, these results indicate that choice of Korean proven bulls semen may correlate positively with conception rate in Hanwoo cow. Therefore, KPN with high AI failure rate might be avoid to increase conception rate and characteristics of frozen semen might be evaluated before its use for AI.

한우(韓牛, Korean Cattle, Bos taurus coreanae)는 모색에 따라 황소, 칡소, 흑소로 나 누어지고 있다. 칡소는 현재 1,700여두가 전국에 사육되고 있는 것으로 추정하고 있다. 그러나 종모우로 활용 가능한 수소의 숫자가 매우 적어 근친의 위험도가 높다. 본 연구 에서는 우량 칡소를 선발하기 위하여 성장단계별 체형을 측정하고, 12개월 및 30개월 령 에 신선정액과 동결융해 정액의 활력을 비교하였다. 칡소의 발육단계별 체형은 체고, 흉위, 십자부고, 체장, 흉심, 흉폭, 고장, 요각폭, 곤폭, 좌골폭을 각각 6개월, 12개월, 18개월, 24개월 및 36개월 이상으로 구분하여 측정하였다. 정액 채취는 인공질을 이용하여 채취하였고, 정액동결용 완충액은 Triladyl를 이용하였다. 칡소 수소의 성장단계별 체형을 조사한 결과, 12개월령 흉위, 체고, 십자부고, 체장, 흉 심, 흉폭, 고장, 요각폭, 곤폭 및 좌골폭이 각각 평균 156.0 cm, 113.2 cm, 118.6 cm, 122.6 cm, 59.5 cm, 31.7 cm, 43.0 cm, 34.7 cm, 36.6 cm 및 19.8 cm였고, 30개월령에 는 각각 평균 214.8 cm, 140.5 cm, 140.5 cm, 179.3 cm, 83.2 cm, 48.6 cm, 62.3 cm, 53.9 cm, 51.0 cm 및 34.7 cm로 조사되었다. 칡소 육성우의 모색을 조사한 결과, 전체 호반 무늬를 가진 개체는 9.6%, 부분 호반 무늬는 59.0%, 호반 무늬 없이 갈색인 개체는 20.5% 그리고 흑색인 개체는 10.8%였다. 15개월령 칡소와 30개월령 칡소의 신선 및 동 결융해 정액의 활력을 조사한 결과, 15개월령 수소의 정액량이 평균 2.3ml로서, 30개월 수소의 정액량의 평균 5.0ml로서 유의차가 인정되었다(p<0.05). 채정 직후의 신선정자의 생존율은 15개월 및 30개월 수소가 각각 평균 93.7% 및 88.3%, 운동성은 각각 97.0% 및 88.3%로서 운동성은 15개월령이 유의하게 높았다(p<0.05). 한편 동결융해 정자의 생 존율은 각각 평균 56.0% 및 58.0%였고, 운동성은 각각 평균 64.0% 및 70.7%로서 차이 가 없었다. 본 연구를 통하여 칡소의 동결정액 생산을 위한 체형이 우수한 개체의 선발이 가능하 였으나, 대량 증식을 위한 추가적인 연구가 필요할 것으로 생각된다.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

This study was undertaken to find out the effect of cholesterol and serum albumin on sperm ability and lipid peroxidation levels period to the liquid storage of miniature pig sperm. Ejaculated semen from miniature pigs was collected by gloved-hand method into a pre-warmed () thermos bottle, and extended with Modena solution {with and without BSA, methyl-beta-cyclodextrin (-cholesterol) and cholesterol loaded cyclodextrin (+cholesterol)}. Each semen was assessed for viability (SYBR-14/PI staining) and acrosome intactness, intensity and capacitation status by chlorotetracycline (CTC) staining at 1, 3, 5, 7 and 10 days of storage. At for the effects of cholesterol and serum albumin on lipid peroxidation, semen were incubated with (), and lipid peroxidation level were measured by flow cytometry using the lipid peroxidation reporter probe . The result, lipid peroxidation level in sperm added with cholesterol were lower in compared to the added sperm with serum albumin. Also, added cholesterol to sperm had significant (p<0.05) higher viability when storage for 7 and 10 days and lower when 10 days of storage percentage of acrosome-reacted sperm (AR pattern) in acrosome state as say result compared to other treated groups. In conclusion, role of cholesterol during lipid storage in miniature pig spermatozoa was protected boar spermatozoa from lipid peroxidation prior to lipid storage. Addition serum albumin during lipid storage in sperm may be induce sperm membrane damage by lipid peroxidation. Therefore, addition of cholesterol to miniature pig sperm will be lead to extension of liquid storage periods.

This study was undertaken to determine whether the presence of fertility-associated antigen (FAA) in semen would influence semen characteristics and conception rate of artificial insemination in Hanwoo. The response to FAA of 36 heads of proven bull, 7 heads of young bull, and 27 heads of performance-tested bull was that one proven bull was FAA-negative and the others were FAA-positive, therefore FAA-negative bull was 1.4%. FAA-negative bull was lower in first and second semen concentrations than those of FAA-positive bull in 5,301 semen of 21 heads of proven bull, then FAA-negative bull was fewer as 11.5% in total sperm counts. The estrus of 22 heads was 70d-nonreturned in 36 cows first inseminated with frozen semen of FAA-negative bull, but that of 249 heads in 378 cows first inseminated with frozen semen of FAA-positive bull. Each conception rate was 61.1% and 65.9%, respectively. The difference of conception rates was 4.8%. These results indicate that the response of FAA to semen were influenced semen characteristics and conception rate of artificial insemination, but further investigations are needed to confirm the results.

This study was performed to investigate the characteristics within ages and freezing tolerance of spermatozoa in Jindo Dog. Experimental animals were selected 12 herds within 1～8 year’s old and collected semen for 2 times in a week. Collected semen was evaluated whole volume and sperm number with CASA system (SIAS, Medical Supply, Korea). Then seminal plasma were separated and diluted with modified Tris-egg yolk extender and added 4, 6 and 8% glycerol for 4 times to final concentration and equilibrated for 1.5 hrs. Before and after freezing, equilibrated semen were evaluated the survival rates. Total volume of sperm at 1～2 year old group is as 5.2×108 cells/ ml largest and there were no significance among groups. The motility of 1～2 year old group is highest as 90.9% and there were significance among groups. Abnormal sperm showed similar among groups. The survival rate in terms of pre-freezing and post-freezing were decreased all levels of glycerol and reveled 87.0% to 64.5% in 4%, 87.5% to 51.9% in 6% and 73.4% to 29.7% in 8%, there were significant difference among the groups (p<0.05). These results suggest that the optimal sperm-freezing methods in Jindo Dog are utilized with modified Tris egg-yolk extender with 4% glycerol and were improve the reproductive activity by these methods.

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution Ⅰ: 11% Lactose or Triladyl + egg yolk; solution Ⅱ: solutionⅠ + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CTC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p< 0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

The relationships of scrotal circumference (SC) to semen characteristics and the conception rate (70 days-nonreturn rate) of artificial insemination in farm were studied with 137 heads of bull in Hanwoo. The average and range of SC were cm and 26.0~52.5 cm, respectively. Hanwoo bulls were classified with SC, divided into 34 cm below group, 34~39 cm group, and 39 cm over group. The 5,487 semen records of 43 heads of bull from July. 1. 2007 to June. 30. 2008. were used to determine the relationships between SC and semen characteristics. The semen concentration and total sperm number of each group were 11.18, 16.68, and , and 69.83, 101.64 and /ejaculate. The bulls with 34 cm or more SC were higher than the bulls with 34 cm below in semen concentration and total sperm number (p<0.01). But between SC and semen volume have no significant relationship (p>0.05). The 9,862 mating records of 44 farm with 137 heads of bull were used to determine the relationships between SC and conception rate. The conception rate of 1st artificial insemination were 73.31, 74.16, and 77.33 % in each group. Also SC was positively correlated with pregnancy rate (r=0.12, p=0.17). These results indicate that SC correlates positively with semen characteristics, and maybe with pregnancy rate in Hanwoo.

The objectives of this study was to evaluate the efficiency of the bacteria eliminated sperm by percoll gradient method on sperm quality and embryo cleavage in vitro in pig. The semen of miniature pig collected by gloved-hand method pre-warmed (37℃) in thermos bottle, and separated by 65% percoll. Analysis of sperm ability was estimated by examining viability, capacitation and acrosome reaction using chlortetracycline (CTC) and the abnormality. Also, fertility of sperm was monitored with cleavage rate of embryo after IVF using separated and un-separated sperm by percoll. The result, viability of separated sperm was significantly(p<0.05) higher(83.6±2.0 vs 59.0±4.4%) than un- separated sperm. The results of CTC analysis showed the percentage of F- and B-patterned separated sperm was higher in separated that than un-separated sperm. On the contrary, the percentage of AR-patterned form un-separaed sperm was significantly(p<0.05) higher(13.6±0.8 vs 8.1±0.6%) than separated sperm. Also, abnormality of un-separated sperm was significantly(p<0.05) higher(20.2±0.4 vs 16.8±2.8%) than separated sperm. However, the cleavage rates of embryo using separated sperm by percoll and un-separated sperm had not significantly difference on 2 cell stage(9.25 vs 11.88%), 4 cell stage(26.76 vs 24.51%) and >4 cell stage(63.99 vs 63.61%) at 48h of IVF. Therefore, the sperm separated by percoll method showed improvement in sperm quality than un-separated sperm in miniature pig.

This study was carried out to investigate the general characteristics, such as volume, pH, sperm motility and sperm concentration of the semen collected from Beagle dogs (age 24～48 months, weight 10～15 kg) by using the method of digital manipulation of the penis, and the effect of preservation temperature and time on motility of fresh semen. Multiple ejaculates were collected from 4 male Beagles. The average volume, pH, motility and sperm concentration of the second fraction (contained with small volume of the third fraction) per ejaculation were 2.94±0.24(SD) ml, 6.43±0.42(SD), 97.04±3.50(SD)% and 1.67±0.23(SD)×108 cells/ml, respectively. Average semen volume per ejaculate, semen pH, sperm motility and sperm concentration of the first fraction from the ejaculate were 1.24±0.20(SD) ml, 6.03±0.26(SD), 11.30±4.02(SD)% and 7.25±1.02(SD)×105 cells/ml. Those of second fraction were 2.52± 0.32(SD) ml, 6.32±0.31(SD), 96.25±3.52(SD)% and 2.35±0.35(SD)×108 cells/ml. Those of third fraction were 2.71±0.27 (SD) ml, 6.52±0.20(SD), 95.65±2.78(SD)% and 5.72±0.29(SD)×107 cells/ml. Motility of semen was higher at 17℃ preservation temperature than 5℃ or 36℃ during preservation period. When preservation temperature was 17℃, motility was 96.54±2.05(SD)% at 1 h, 90.20±3.90(SD)% at 6 h, 89.05±2.01(SD)% at 12 h, 78.21±3.50(SD)% at 18 h, 45.24±6.25 (SD)% at 24 h and 30.75±17.24(SD)% at 30 h, respectively.