• 한국수정란이식학회지
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• 제14권2호
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• pp.131-137
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• 1999

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$0^{\circ}C$ for one min. (p<0.01) than those thawed at 2$0^{\circ}C$ or 37$^{\circ}C$ for one min. The sperm viability was not significant at the diluent temparature of 2$0^{\circ}C$or 37$^{\circ}C$ after thawing: but the sperm viability was higher in thawing diluent at 2$0^{\circ}C$ than in that at 37$^{\circ}C$. 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$0^{\circ}C$ for one min. and then diluted in mBTS medium at 2$0^{\circ}C$or 37$^{\circ}C$. 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.

• 한국수정란이식학회지
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• 제16권1호
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• pp.53-60
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• 2001

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 2~4$\times$10$^{8}$ cells/ml and having dead or abnormal spermatozoa of less than 15% was used for the experiment. Each ejaculated semen was centrifuged at 400 $\times$ 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 (67.5$\pm$9.6) was significantly lower than that of Ext II , Ext II -RJ containing 0.01 or 0.1% RJ (77.5$\pm$12.5, 78.7$\pm$8.2 and 80.0$\pm$6.3). However, Ext II-RJ containing 0.1% RJ yielded higher viability than Ext II, Ext II-RJ containing 0.01% at or 1% 1 h after thawing (69.5$\pm$8.1 vs. 55.0$\pm$12.9, 57.5$\pm$9.6 and 41.5$\pm$12.6; P<0.05). At 1 h after thawing, the viability of spermatozoa thawed in 7$0^{\circ}C$ (68.8$\pm$12.5) was significantly higher than that of spermatozoa thawed in 38$^{\circ}C$ (48.8$\pm$16.3), although there was no difference in the viability between both groups immediately after thawing (77.5$\pm$9.6 and 81.3$\pm$8.1). Post-thaw viability and longevity of post-thaw spermatozoa in Ext II-RJ containing 0.1% RJ was higher in those in Ext II at 1 h (65.0$\pm$12.9 vs. 42.5$\pm$12.6), 2 h (52.5$\pm$12.6 vs. 27.5$\pm$17.1) and 3 h (40.0$\pm$14.1 vs. 20.0$\pm$12.1) 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.

• 한국수정란이식학회지
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• 제10권3호
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• pp.203-208
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• 1995

This study was carried out to efficiently use the ultrarapid freezing method in the cryopreservation of mouse ova. For this, the effects of dehydration method, oval vigour and $0^{\circ}C$ controlling method on post-thawing viability were investigated. Fresh mouse ova were dehydrated in mPBS with 3.5M DMSO and /or 0.25M sucrose, and directly immersed in L$N_2$ for ultrarapidly freezing. The frozen ova were thawed at 37$^{\circ}C$, rehydrated in mPBS with 0.25M sucrose, and then repeatedly washed in HAM's Fl0 before evaluating the morphological normality of frozen-thawed ova. The results obtained showed that there was difference between treatments in a experiment. 1) The post-thawing viability of ova dehydrated in multi-step (48.4$\pm$13.8%) was higher than that of ova in two-step (40.9$\pm$14.0%). 2) The post-thawing viability of fertilized ova (87$\pm$14.0%) was significantly(p<0.0l) higher than that of unfertilized ova (5.4$\pm$5.4%). 3) The post-thawing viability of ova dehydrated and rehydrated using a cooling machine (95.8$\pm$4.2%) was significantly(p<0.05) higher than that on ice(84.1$\pm$9.9). In conclusion, in order to efficiently cryopreserve ova in vitro with ultrarapidly freezing method, highly viable embryos should be selected, heavy osmotic shock to the dehydrating ova should be avoided, and embryos in high osmotic condition were dehydrated and rehydrated in a constantly low temperature.

• 한국임상수의학회지
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• 제10권1호
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• pp.11-18
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• 1993

Four extenders such as tris-fructose-citrate, Tris-glucose-citrate, glycine-glucose-citrate and lactose that the more frequently utilized types of semen extenders used for freezing dog semen evaluated with sperm motility, viability and acrosomal score in the processing procedures to prior freezing and after frozen-thawing respectively. Each extender contained 4% glycerol and 20% egg yolk were treated by same methods in dilution, freezing, storage and thawing. The results were obtained as follows; 1. The sperm motility and viability in procedure from dilution to frozen-thawing appeared superiorly with recovery rate of 53.2%, 54.8% in tris-fructose-citrate but appeared inferiorly with recover rate of 8.4%, 8.3% in lactose to others. 2. In the processing procedure course to prior-freezing, glycine-glucose-citrate appeared superiorly with decrease rate of 5.4% in motility, and lactose with decrease rate of 4.6% in viability, but tris-glucose-citrate appeared inferiorly with decrease rate of 12.2%, 11.4% in the sperm motility and viability. 3. During frozen-thawing, tris-fructose-citrate appeared superiorly with decrease rate of 35.2% in motility and 30.7% in viability but lactose appeared inferiorly with decrease rate of 76.7% in motility and 75.7% in viability. 4. The variation of acrosome morphology in the total processing procedures appeared that glycine-glucose citrate were superior with acrosome score of 0.1191$\pm$0.029, that tris-fructose-citrate were inferior with acrosome score of 0.1941$\pm$0.045 to others.

• 한국임상수의학회지
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• 제17권2호
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• pp.420-430
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• 2000

These studies were preformed to investigate the freezing conditions to achieve good post-thaw viability of spend and the practical methods of artificial insemination frozen canine semen. Semen were collected from nine male dogs which had been proved to be fertile in the past and the semen were treated for freezing procedure. Post-thaw motility and viability of canine sperm were evaluated to investigate individual tolerance of freezing, difference among freezing extenders, dif-ference among freezing equipments and freezing conditions, difference between fast and slow cooling rate, difference according to different glycerol concentration, effect of seeding on post-thaw viability, difference according to cutting part of straw, difference according to thawing temperatures, and dif-ference according to media added to thawed semen. Thawed semen for insemination were added with equal volnme of canine capacitation medium (CCM) and the volume of semen and the number per insemination were adjusted as 2-3 ml and $20-30 {\times}10^7,$ respectively. The semen were inseminated in vagina using balloon catheter and en17ryos were cellected from 9 to 11 days after the second Al to d determine fertilization.

• Reproductive and Developmental Biology
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• 제31권3호
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• pp.175-179
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• 2007

본 연구는 0.5ml straw를 이용한 정자 동결융해 시 응해 온도가 동결정자의 성상에 미치는 영향을 파악하고 미니 돼지의 동결정액에 최적화된 적정 응해 조건을 찾기 위하여 실시하였다. 정액동결 straw를 37, 50 및 $70^{\circ}C$에서 각각 5초, 10초 및 45초간 융해하여 생존율(SYBR-14/PI staining), 정자원형질막기능검사 (Hypoosmotic Swelling Test) 및 첨체반응율(CTC : chlorotetracycline staining)을 검사 한 결과, $70^{\circ}C$에서 5초간 융해한 정자의 생존율과 CTC 결과가 $37^{\circ}C$와 $50^{\circ}C$에서 10초 및 45초간 융해한 처리구보다 유의적(p<0.05)으로 높은 생존율과 낮은 비율의 첨체 반응율을 얻었다. 따라서 미니돼지의 동결 정액은 고온에서 단시간 융해를 하는 것이 정자의 성상에 유리한 것으로 사료된다.

• 한국임상수의학회지
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• 제19권1호
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• pp.80-85
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• 2002

This study was performed to investigate the freezomg condition especially focused on extender composition to achieve good post-thaw viability and motility of canine sperm. Semen were collected from 6 male dogs which had been proved to be fertile in the past and were treated for freezing. Equex-STM paste was contained in both the 1st(3%) and the 2nd(7%) diluent and the 2nd diluent was added to the 1st diluent following glycerol equilibration for an hour and a half. To investigate the effect of Equex-STM paste in the extender on post-thaw canine sperm characteristics, the post-thaw viability, motility, and HOS(Hypoosmotic swelling) values were evaluated according to the different composition of extender with or without Equex-STM paste, thawing conditions, and different thawing media added to thawed semen. 1. Canine sperm removed from seminal plasma and frozen )n Sweden extender containing Equex showed higher post-thaw viability, motility, and HOS values than those frozen in the extender containing Equex-STM paste with seminal plasma and those frozen in the extender without Equex and seminal plasma. 2. Canine sperm frozen in Sweden extender containing Equex-STM paste with 5% glycerol showed higher post-thaw viability, motility, and HOS values than those frozen with 3%, 8% glycerol or 5% DMSO. 3. The canine semen frozen in Sweden extender with 5% glycerol and Equex-STM paste showed higher viability, motility, and HOS values when thawed at $70^{\circ}C$ for 8 seconds than when thawed at $37.5^{\circ}C$ for 1 min and at $18-20^{\circ}C$ for 5 min. 4. TFC (tris -fructose-citrate) and PB S (phosphate buffered saline) medium added immediately to thawed canine semen brought better viability, motility, and HOS values for the sperm than those semen added with TGC(tris-glucose-citrate) and no medium. These results indicated that Equex-STM paste in Sweden extender for freezing the canine sperm which were removed from seminal plasma brought good post-thaw viability and motility of canine sperm. Also of the freezing conditions of canine sperm with the same extender containing Equex, the concentration of 5% glycerol, the thawing condition at $70^{\circ}C$ for 8 sec, and TFC and PBS medium added to the thawed semen brought better post-thaw viability and motility of canine sperm than the other conditions used in this study.

• 한국수정란이식학회지
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• 제27권1호
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• pp.45-50
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• 2012

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 ($37^{\circ}C$ for 20 sec, 45 sec and $75^{\circ}C$ 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 : $60.3{\pm}2.4$, NAI : $58.6{\pm}2.2%$), TLE ($61.3{\pm}2.4$, $62.2{\pm}2.2%$) extender significantly(p<0.05) increased than that in LEY ($50.2{\pm}2.4$, $54.5{\pm}2.2%$) extender thawed at $75^{\circ}C$ for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE ($66.1{\pm}3.2$, $66.2{\pm}1.0%$) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE ($43.3{\pm}0.5%$) while that in LEY ($63.5{\pm}2.3%$) is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

• 한국동물생명공학회지
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• 제36권2호
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• pp.69-75
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• 2021

Cryopreservation is a widely-used efficient means of long-term sperm preservation. However, unlike other types of semen, cryopreserved boar semen has reduced fertility and the efforts continue to optimize post-thawing sperm recovery. In this study, we evaluated the effects of various washing solutions (Hulsen solution, lab-made DPBS and commercial DPBS) on post-thawing porcine sperm kinematics (CASA system), viability (SYBR-14/PI) and acrosome integrity (PSA/FITC). We also examined the effect of washing-centrifugation on frozen-thawed semen kinematics. The results indicate that type of washing solution and post-thawing centrifugation alters parameters linked to sperm quality (total motility, progressive motility, viability and acrosome integrity). Significantly higher (p < 0.05) motility and progressive motility were obtained when cryopreserved semen was processed with Hulsen solution. The post-thaw percentage of live and intact acrosomal sperm was significantly higher in group 1 (Hulsen solution) as compared to other groups. Following thawing-centrifugation, the results showed significantly higher motility and progressive motility in group 1 than other groups. However, the latter two DPBS groups did not differ statistically. Taken together, Frozen-thawed spermatozoa motility, acrosome integrity and viability can be affected by the type of washing solution used. Moreover, centrifugation of frozen-thawed semen has an unfavorable effect on total motility and progressive motility.

• 한국임상수의학회지
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• 제8권2호
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• pp.183-190
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• 1991

Pellet and straw methods in canine semen freezing are compared with respect to motility, viability and acrosome demage of sperm during each of the two major processing steps, to prior-freezing and to frozen-thawing. Senen was extended with a tris-buffered egg yolk contained 4% glycero1 Pellet freezing in the hole of dry ice and straw freezing on the surface of liquid nitrogen were carried out, respectively. The frozen semen 10 days after storage in liquid nitrogen container. wao thawed. In the comparison of two freezing methods, the straw freezing method with 42.7% in motility. 49.2% in viability and 0.186 acrosome score after thawing seems to be superior to the pellet freezing method with 31.2%, 34.5% and 0.314%, respectively. Sperm motility of processing step to frozen-thawing against decrease rate 12.67% to Prior freezing appeared of 33.84% and 49.37% in straw and pellet freezing and increase of 0.02 in acrsomal score to prior freezing appeared of 0.08 and 0.21 in straw and pellet freezing method to frozen-thawing