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.

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.

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