This study was conducted to evaluate whether \"Royal jelly\" (RJ) added to Tris-buffer dilute contributed to supporting post-thaw viability and longevity of frozen canine spermatozoa. Two Japanese spitzs (2 to 4 years of age) were used as a semen donor. Semen was collected by manual masturbation and separated into 3 fractions. Only the sperm-rich fraction having sperm motility of more than 70%, containing sperm concentration of 2~410 cells/ml and having dead or abnormal spermatozoa of less than 15% was used for the experiment. Each ejaculated semen was centrifuged at 400 g for 5 min and then diluted in a Tris-buffer supplemented with 20 ml egg yolk (Ext I), 4% glycero1 and 1% Equex STM Paste (Ext II) or g1ycero1, Equex STM paste and RJ of various concentrations (Ext II-RJ). After freezing and thawing, viability of spermatozoa in Ext II -RJ containing 1% RJ immediately after thawing (67.59.6) was significantly lower than that of Ext II , Ext II -RJ containing 0.01 or 0.1% RJ (77.512.5, 78.78.2 and 80.06.3). However, Ext II-RJ containing 0.1% RJ yielded higher viability than Ext II, Ext II-RJ containing 0.01% at or 1% 1 h after thawing (69.58.1 vs. 55.012.9, 57.59.6 and 41.512.6; P<0.05). At 1 h after thawing, the viability of spermatozoa thawed in 7 (68.812.5) was significantly higher than that of spermatozoa thawed in 38 (48.816.3), although there was no difference in the viability between both groups immediately after thawing (77.59.6 and 81.38.1). Post-thaw viability and longevity of post-thaw spermatozoa in Ext II-RJ containing 0.1% RJ was higher in those in Ext II at 1 h (65.012.9 vs. 42.512.6), 2 h (52.512.6 vs. 27.517.1) and 3 h (40.014.1 vs. 20.012.1) after thawing. These results indicated that addition of 0.1% af to Tris-buffer enhanced post-thaw viability and longevity of canine spermatozoa and this additive can be used for increasing the possibility of collision between spermatozoa and ova during insemination.emination.

Artificial insemination (AI) with frozen or cooled semen is widely used in commercial fields of cattle and pig. Little is known about characteristics of canine sperm after freezing or cooling. For both practical and commercial goal, the canine semen treated with cooling and freezing should be carried out to exam the fundamentals, including sperm motility, survivability and fertilizing capacity. The aim of this study, thus, was to identify the effects of extended exposure to 4 on canine semen by motility, survivability, acrosomal changes following different duration. Fifteen ejaculates collected by digital manipulation twice per week from 3 dogs (Shih-Tzu) were divided to 16 aliquots after adding Tris-egg yolk (TE) buffer formulated by our laboratory, and cooled from 37 to 4, by ramp rate of 0.6/min. Each sample was evaluated by their motility, survivability and the acrosomal status at 0h (control), 2h, 12h and 1 d~10 d, respectively. The motility of spermatozoa was graded to 6 levels using the modified method of Seager. The survivability of sperm was assessed using an epifluorescence microscope after Fert/Light (Mole-cular Probes Inc.) staining. To estimate the proportion of the spermatozoa of intact acrosome, 200 spermatozoa were assessed in randomly selected fields, using epifluorescence microscope after FITC/PSA (Sigma) staining. At 2 h after cooling, the motility of most spermatozoa were assessed to be grade 0 and 1. At 12 h, high number of sperm were in grade 0 to 1, however, it was significantly (P<0.05) lower than that of 2 h. From 1 d to 4 d, ~50% of sperm was assessed to grade 0 to 1. On day 7, a little sperm were in grade 0 to 1. No sperm showed motility on day 10. Sperm motility was rapidly reduced by the percent of 10% of grade 0 to 1. From 2 h to 6 h, the number of live sperm was 90% and the sperm chilled for 10 days lived>50%. Acrosomal intact of spermatozoa exposed to 4 for 2 h was 51%, supposed the sperm of control was 100%. Our results suggest that 1) this is easy to transfer and preservation for short periods 2) AI can be used by semen chilled for 6-Day.

The object of this study was to find simple and effective methods for the speculation of vitality and scorsome status of bovine spermatozoa. The eosin-nigrosin staining, trypan blue staining, and naphthol yellow S-erythrosin B staining was ofter used for the speculation of vitality and/or acrosome status of bovine spermatozoa, respectively. This study has shown that the combined trypan blue-naphthol yellow S-erythrosin B staining is more accurate and effective for the examination of acrosome status and vitality of bovine spermatozoa.

These experiments were conducted to examine the effects of theophylline, pentoxifylline and heparin on frozen-thawed Hanwoo sperm for enhancing motility and viability of sperm. Frozen-thawed semen collected from one bull was treated in TALP(tyrode-albuminlactate-pyruvate) containing varous concentrations of theophylline and pentoxifylline. After incubated at 5% CO2 in air atmosphere for 6 hours, the motility of sperm after the treatments was characterized by CASA(computer aided semen analysis) system. When monitored notility(MOT) and curvilinear velocity(VCL), theophylline and pentoxifylline exerted their optimal action at the concentration of 30 mM and 3 mM, respectively. No difference of sperm motility was observed when the sperm was treated with both substances compared with a single treatment of each substance. Comparison was then made for evaluating the effect of theophylline and / or pentoxiophylline on the motility and viability of significant treatment effects of each substance, high MOT and VCL values were detected in sperm treated with theophylline. In the case of sperm viability examined by an eosin-nigrosin staining, however, a significant decrease was found after the combined treatment of theophylline+pentoxyphilline than after the treatment with heparin alone or no treatment(P<0.05). In conclusion, theophylline, pentoxiphylline or heparin can be used for enhancing the motional characteristics and viability of frozen thawed Hanwoo semen. Considering characteristics of these substances, theophyline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

This study was conducted to investigate the effects of vitrification solution and developmental stage on the survival rate of vitrified-thawed human blastocyst embryos. Human blastocyst embryos were cryopreserved by vitrification using EFS and GE solution, and their survival rates were examined after thawing and further culture. EFS solution was consisted of 40% ethylene glycol, 18% Ficoll 70 and 0.3M sucrose. GE solution was consisted of 25% glycerol and 25% ethylene glycol. Embryos were exposed to EFS and GE solution by 2 steps and 3 steps, respectively, and plunged into liquid nitrogen after loading into 0.25ml plastic straws. Blastocysts were classified into 4 groups in accordance with their developmental stage: into 1) EEB, 2) MEB and 3) EdB, of blastocysts developed on day 5, and 4) 6d-Bla(the blastocysts which formed on day 6). The blastocysts at each stage were vitrified by GE solution and cryopreserved in LN2. After thawing them, we examined their survival rates, respectively. The resulted of this study were as follows: 1. The survival rate of blastocysts vitrified by GE solution was 64.4%, significantly higher than that (5.7%) vitrified by EFS solution (P<0.001). 2. When the blastocysts were vitrified by GE solution according to each developmental stage, the survival rates of EEB, MEB, EdB and 6d-Bla were 65.9%, 65.9%, 73.2% and 58.1%, respectively. In conclusion, the cryopreservation of human blastocysts by vitrification is likely to have a marked advantage in terms of cost, work and time as compared to the conventional slow freezing in IVF-ET programs.

1997년 11월부터 1998년 6월까지 총 4회에 걸쳐 군산인근 해역을 대상으로 Vibrio 속의 분포와 특성을 조사하였고, 분리동정된 Vibrio 속 3종(V. anguillarum, V. vulnificus, V, metschnikovii)을 대상으로 해수에서 수온 변화에 따른 생존을 관찰하였다. 조사 기간 중 총 해양 종속영양 세균의 분포는 평판도말법으로는 1.2±0.6×103~2.0±1.5×104

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 for one min. (p<0.01) than those thawed at 2 or 37 for one min. The sperm viability was not significant at the diluent temparature of 2or 37 after thawing: but the sperm viability was higher in thawing diluent at 2 than in that at 37. 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 for one min. and then diluted in mBTS medium at 2or 37. 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.

본 연구는 서로 다른 수명 패턴을 갖고 있는 누에성충 중 두가지 타입을 이용하여 수행되었다. 장명품종(성충 수명이 15일 이상, LLS), 단명 품종(성충 수명이 5일 미만, SLS). 누에성충의 노화 생리를 밝히기 위해 장단명 암수에 있어서의 지방체 미세구조를 비교해 보았다. 단명품종에 있어서는, 암컷에는 조면소포체 및 글리코겐 과립이 세포질내에서 다량 확인된 반면, 수컷에는 활면소포체만이 세포질내에서 발견되었다. 또한 단명품종에 있어서는, 성충 3일째 이후 미토콘드리아의 용적이 비대해지는 경향을 보였으며, 많은 지방구 퇴화가 관찰되었다. 단명품종에 반해 장명품종에서는, 성충 5일째의 암컷은 비교적 정상적인 미토콘드리아와 핵막이 관찰되었다. 성충 15일째에 이르러서야 대부분의 세포막이 사라졌고 미토콘드리아가 비정상적으로 비대해졌다. 장명품종 수컷(성충 10일째)의 세포질내에서 다량의 지방과립이 관찰되었으며, 이 개체는 성충 15일째에 세포내용물이 모두 고갈되어 사망하였다. 따라서, 금후 수명에 관한 조직 연구를 위해서는 조직의 변화상을 관찰하기 용이한 단명품종이 적절할 것으로 사료된다.

본 연구는 돼지 정액의 동결과정동안 flow cytometric 분석에 의한 정액내 생존정자의 비율을 조사하여 주관적으로 평가되는 활력 및 정상첨체율(normal apical ridge ; NAR)과 비교하여 정자의 손상과 생존성에 대한 적절한 평가법을 찾기 위하여 실시하였다. 동결과정 중 정액채취, 냉각, 예비동결 및 동결융해 후에 flow cytometric 분석에 의한 정자 생존율은 각각 93.03.6, 85.13.9, 28.96.8 및 26.15

본 연구는 소 체외성숙, 체외수정 유래 배반포의 초자화 등결 시 동해방지제의 평형방법과 응 해 후 동해방지제의 희석방법이 수정란의 생존성에 미치는 영향을 검토하고자 실시하였다. 초자화 동결액은 20% FBS(Gibco)가 첨가된 D-PBS에 20% glycerol, 20% ethylene glycol, 3/8 M sucrose, 3/8 M deftrose 가 함유된 GESD를 사용하였다. 동결 전 평형방법은 3단계 (El), 2단계 (E2), 1단계(E