• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.55-60
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• 2011

The objective of this study was carried out to evaluate the efficiency of sperm collection methods on the post-thaw viability of cat semen. The cat semen was collected by artificial virginal (AV) and electronic ejaculate (EE) methods. The composition of semen extender was consisted of Tris-buffer supplemented with 20% egg yolk and 1% P/S antibiotics in Ext I, and more added 8% glycerol, 1.0% Equex STM paste of total volume in Ext II. The collected semen was adjusted the concentration and then diluted in Ext I for optimal concentration. The diluted semen was cooling to $5^{\circ}C$ temperature in refrigerator for at least 2 hrs and then diluted stepwise with Ext II for at least 1 hrs. After an equilibration for 1 hrs, the cooled semen was packaged in 0.5 ml straw and then freezing on the $LN_2$ vapor over 5 cm above from $LN_2$ and then immersed directly in $LN_2$ for cryopreservation. The frozen semen was thawed in $38^{\circ}C$ water for 15 sec and then evaluated the motility, viability, and morphology. Post-thaw semen were calculated the motility by SMI (sperm motility index). The live-dead sperm was evaluated by Eosin-B and morphological evaluation was by Diff-quik kit staining. The post-thaw concentration ($89{\times}10^6$ /ml vs. $128{\times}10^6$ /ml), viability ($22.6{\pm}10.6%$ vs. $37.1{\pm}26.1%$), morphological normality ($27.0{\pm}50.2%$ vs. $45.6{\pm}123.0%$) of EE and AV groups were not significant different, but the post-thaw motility was significant lower in EE than that in AV group ($53.1{\pm}3.6$ vs. $73.6{\pm}5.7$) (p<0.05). In conclusion, semen collection methods did not significant different between EE and AV groups except of post-thaw motility and so both semen collection methods could be applied in feline semen collection methods.

• Clinical and Experimental Reproductive Medicine
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• v.49 no.2
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• pp.87-92
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• 2022

Objective: The aim of this study was to determine the effects of melatonin and selenium in freezing extenders on frozen-thawed rat sperm. Methods: Semen samples were collected from 20 adult male Wistar albino rats. Following dilution, the samples were divided into six groups: four cryopreserved groups with 1 mM and 0.5 mM melatonin and selenium supplements, and two fresh and cryopreserved control groups. The rapid freezing technique was used to freeze the samples. Flow cytometry was used to assess plasma membrane integrity, mitochondrial membrane potential, and DNA damage, while computer-assisted sperm analysis was used to assess motility. Results: Total motility was higher in the 1 mM melatonin supplementation group than in the cryopreserved control group (mean±standard error of the mean, 69.89±3.05 vs. 59.21±1.31; p≤0.05). The group with 1 mM selenium had the highest plasma membrane integrity (42.35%±1.01%). The cryopreserved group with 0.5 mM selenium had the highest mitochondrial membrane potential, whereas the cryopreserved control group had the lowest (45.92%±4.53% and 39.45%±3.52%, respectively). Conclusion: Cryopreservation of rat semen supplemented with 1 mM melatonin increased sperm motility after freeze-thawing, while supplementation with 0.5 mM selenium increased mitochondrial activity.

• Korean Journal of Poultry Science
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• v.44 no.1
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• pp.59-65
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• 2017

This study examined factors affecting the analysis of motility of chicken semen. The viability of spermatozoa was estimated using varying dilution ratios and supplementation with BSA or fatty acid free (FAF)-BSA as protein sources in semen diluent. Fresh semen was examined after preparing dilutions in beltsvile poultry semen extender (BPSE) of 1/8, 1/16 and 1/32 at $25^{\circ}C$. The motility of incubated semen at each dilution was observed at 3 min (89.9%, 69.9% and 53.2%), 30 min (86.7%, 71.4% and 51.7%), 1 h (89.5%, 74.0% and 53.5%) and 3 h (78.5%, 66.5% and 45.7%), respectively. The addition of BSA or FAF-BSA to BPSE diluent significantly increased the viability of semen in 1/32 dilution with results of 53.2% (control), 84.8% (BSA) and 92.9% (FAF-BSA) (p<0.05). This phenomenon was also observed in the dilution of frozen semen, where FAF-BSA treatment increased the viability of thawed semen from 17.6% to 34.0% in a 1/8 dilution (p<0.05). When the protein sources were used in the dilution, the survival rates of diluted chicken semen were also increased with time lapse. These results show that FAF-BSA may act to protect chicken semen and is suitable as a basic component of chicken semen diluent for the method of analyzing rooster semen after freezing.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.363-367
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• 2011

The purpose of this study was to improve of frozen-thawed sperm using magnetized water in Korean native cattle. Before cryopreservation, without egg-yolk Triladyl$^{(R)}$ solution was flowed though magnet [0, 2000, 4000 and 6000 Gauss(G)] for S min. The freezing of dilluted semen added with Triladyl containing 20% egg-yolk. Analysis of frozen-thawed sperm was estimated viability with SYBR14/PI double stain, membrane intact with hypoosmotic swelling test (HOST), acrosome reaction with FITC-PNA, mitochondria membrane function with Rhodamin 123 by flow- cytometry. Sperm viability was significantly higher in 4000G group than other groups (p<0.05). However, the Hypoosmotic Swelling Test(HOST) was significantly higher in fresh, 4000 and 6000G than 0 and 2000G (p<0.05). In addition, mitochondria membrane damage and acrosome damage were significantly lower in 6000G group than other groups (p<0.05). in conclusion we suggest that magnetized water could be improve ability of sperm on cryopreservation in Korean native cattle.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.1-5
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• 2006

본 연구는 개 채취 정액의 동결후의 생존성과 신선 및 동결 정자의 capacitation, acrosome reaction과 생존성을 조사하고, 아울러 신선 및 동결 정액을 자연 발정 또는 발정 유기 암캐 에게 인공수정 후 임신율을 조사하였다. 개 채취 정액의 동결 융해 후의 생존성은 $64.5{\pm}2.30%$로서 신선 정액의 생존성에 비해 유의하게 낮게 나타났다(p<0.05). 신선 및 동결 정액의 capacitation, acrosome reaction 및 생존성은 각각 $52.5{\pm}4.5%,\;9.5{\pm}0.6%,\;68.8{\pm}4.5%$ 및 $16.2{\pm}3.2%,\;3.2{\pm}0.5%,\;24.5{\pm}2.5%$로 나타났다. 신선 및 동결 정액을 자연 발정 또는 발정을 유기한 암캐에 인공수정했을 때 임신율은 각각 50.0% 및 33.3%로서 동결 정액을 이용했을 때 임신율이 신선 정액에 비해 낮은 임신율을 나타냈다.

• Journal of Embryo Transfer
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• v.33 no.4
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• pp.321-326
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• 2018

In this study, two epididymal spermatozoa recovery methods in relation to sperm number, motility, viability and acrosome reaction were examined. Seven bulls were castrated and 7 testicles with epididymides were transferred to the laboratoy. Epididymis in each bull was randomly used for flushing and mincing methods with semen extender (Optixcell, IMV, France). The recovered spermatozoa with adjusted sperm concentration to $40{\times}10^6cells/mL$ was diluted with optixcell and cryopreserved. In experiment 1, the difference in the total number of spermatozoa using flushing and mincing methods was insignificant (2570.0 and $2505.2{\times}10^6cells/mL$, respectively). For experiment 2, the percentage of motile spermatozoa and motility parameters between flushing and mincing methods were studied through the use of sperm class analyzer after frozen-thawing. The percentage of total motile sperm between flushing and mincing methods was almost the same with $89.5{\pm}12.8$ and $91.4{\pm}7.9%$, respectively. The same is the case with experiment 3 wherein the viability and acrosomal integrity of frozen-thawed epididymal spermatozoa by flushing and mincing was insignificantly different. The results from the study showed that both flushing and mincing methods can be used for epididymal spermatozoa recovery in bull.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.157-162
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• 2005

To find out the possible inefficiencies of artificial inseminators at rectovaginal insemination in cows, inseminators' skill were evaluated by controlling the semen thawing procedure adopted and by using the technique of dye deposition in the genital tract of slaughtered cows. This was followed by refreshment training for the inseminators. Thirty seven artificial insemination technicians regularly working in the government, cooperative and NGO (Non Government Organization) artificial insemination programmes at different places of Bangladesh were included in the study. Individual technicians were asked to thaw a semen straw and deposit dye in the genital tract of slaughtered cows following the procedures they would have adopted in their actual practices of insemination. The time and water temperature adopted by technicians were recorded and genital tract after sham artificial insemination was dissected to determine the site of dye deposition. Then, the inseminators took part in a three days intensive training program. The training program was ended up with the same tests for thawing frozen semen straw and dye deposition in the genital tract of slaughtered cows. At pre training evaluation, only $25\%\;and\;72\%\;(n=36)$ inseminators adopted co..ect thawing time and temperature, respectively. At post training evaluation, all inseminators thawed semen straws for proper time and temperature. At pretraining evaluation, $21(57\%),\;11 (30\%)\;and\;3(8\%)$ inseminators deposited dye at the body of uterus, in the vagina or in cervix, and into the horn of uterus, respectively. In $2(5\%)$ cases dye did not pass into the genital tract, instead back flowed through the space between the barrel of insemination gun and sheath. At post training evaluation, all inseminators successfully deposited dye in the body of uterus. Frequent evaluation of inseminators' skill and subsequent training would help improvement of the artificial insemination technicians' skill.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.139-143
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• 2009

Most captive canids and felids at Zoos in advanced countries have been examined enough to apply artificial reproductive techniques to them. We investigated reproductive hormones and vaginal epithelial cells of a 6-year-old, female coyote, hoping these data could eventually be extended to artificial insemination with frozen-thawed conspecific semen at Seoul Zoo. As a relative of pet dogs, coyote exhibited a similar appearance with only minor differences. In vaginal smear, an increase in the number of superficial cells suggests that the bitch has reached a state close to estrus. A sudden decrease of estradiol and increase of progesterone is considered as a preovulatory event. Vaginal epithelial cells and hormones might be useful for determining the optimal time of artificial insemination in coyotes' breeding.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.277-277
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• 2004

The purpose of this study was to investigate the effect of different conditions (osmolity, solution, incubation times, comparison of fresh and frozen/thawed semen and storage times) on the swelling of canine spermatozoa. Employing the hypoosmotic swelling test (HOST), the membrane integrity of spermatozoa in different solutions (sucrose, fructose, latose, Na-citrate, Na-citrate plus sucrose, Na-citrate plus fructose and Na-citrate plus lactose were 61.4%, 66.2%, 62.5%, 68.1%, 62.0%, 68.5% and 60.2%, respectively. (omitted)

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.143-152
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• 1992

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.