• Reproductive and Developmental Biology
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• 제35권3호
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• pp.203-207
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• 2011

The objective of this study was to determine the effect of semen extenders on the motility, viability and fertility in vitro of spermatozoa during storage of fresh boar semen diluted in different commercial extenders used for pig artificial insemination (AI). In this experiment, semen were diluted in Androhep plus, Beltsville Thawing Solution (BTS), Modena, Seminark and Vitasem LD. Five ejaculates were collected from three Duroc boars and sub-samples were diluted ($30{\times}10^6$ spermatozoa/ml) in different extenders. Semen was stored at $170^{\circ}C$ for 10 days. Sperm motility and viability was assessed using Computer-Assisted Semen Analysis (CASA) and flow-cytometry on 1, 3, 5 and 10 day post collection The motility of spermatozoa stored in different extenders was gradually decreased by increasing the duration of storage of semen. However, there was not significant1y different in the sperm motility and viability among other extenders. On the other hand, the in vitro-matured oocytes were fertilized and cultured in vitro to assess the fertility of boar spermatozoa stored for 3 and 10 days in different extenders. The percentage of morula and blastocyst were taken as indicators of fertility in vitro of spermatozoa. Therefore, there were no differences in the rate of embryos developed to the molular and blastocyst stage. There were no differences in the motility and fertility in vitro among 5 kinds of commercial boar semen extenders.

• 한국수정란이식학회지
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• 제23권3호
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• pp.161-166
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• 2008

The techniques for the collection, cooling and freezing of semen and artificial insemination of horses are not fully understood in Korea. We investigated the percentages of total motile (TM) and progressively motile (PM) sperms after the collection, cooling and freezing of stallion semen. The average volume of semen was 167 ml in Thoroughbred and 68 ml in Arab. The average numbers of spermatozoa in Thoroughbred and Arab were $104\times10^6/ml$ and $86\times10^6/ml$ respectively. The average percentages of TM and PM were 82.3% and 88.6% in Thoroughbred, and 61.4% and 82.6% in Arab, respectively. The average percentage of TM at 4 hr after cooling at $5^{\circ}C$ was significantly lower than that at 0 hr ($30.0\pm4.1%\;vs.\;78.0\pm2.5%,\;p<0.05$), but the percentage of PM was similar between 66.5 and 73.2% at 0, 1, and 4hr. The average percentage of frozen-thawed Thoroughbred semen frozen in MFR5 extender was 56.2%, which was significantly higher than that of the semen frozen in LE extender (average 32.9%, p<0.05). The percentage of TM in Arab was similar for semen frozen in MFR5 extender and LE extender (18.2% and 21.2%, respectively), but the percentage of PM was significantly higher in sperm frozen in MFR5 extender than in sperm frozen in LE extender (69.0% vs. 36.4%, p<0.05). Four mares were artificially inseminated by Thoroughbred frozen-thawed semen and one of them fertilized at 11 day after artificial insemination. In this study, the collection, cooling and freezing of equine semen were possible under domestic conditions.

• 한국가축번식학회지
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• 제10권1호
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• pp.19-26
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• 1986

This experiment was carried out to investigate the effect of bovine serum ablumin (BSA), sugars, glycerol equilibration time, straw size and thawing method on the survival index and the morphology of frozen boar spermatozoa. The results obtained were summarized as follow: 1. When the semen frozen in BF5 dilutor as pellet form was thawed in BTS at 37$^{\circ}$and 50$^{\circ}C$, BF5 dilutor with fructose showed higher sperm survival index than that with dextrose, however, when the semen was thawed on dry test tube at 37$^{\circ}C$, BF5 dilutor with sucrose showed higher sperm survival index than with other sugars. 2 When the semen forzen in BF5 dilutor with straw and thawed at 37$^{\circ}C$, BF5 dilutor with dextrose showed higher sperm survival index than those with other sugars, and there was no difference in sperm survival index between 0.5 and 1.0 ml straws. 3. The sperm survival index of frozen sperm was significantly (P<0.05) improved due to addition of BSA (0.05%) to BF5 dilutor. 4. When the extended semen with BF5 dilutor contatining 0.01 to 0.05% of BSA was frozen in the straw, the semen without glycerol equilibration showed significantly (P<0.05) higher sperm survival index than those with 2, 4 and 6 hrs glycerol equilibration time. 5. The sperm frozen in BF5 dilutor with dextrose or fructose, sucrose and raffinose showed 77 to 88% in normal acrosome rate and no difference among sugars. 6. The frozen semen showed lower normal acrosome rate than the first and second diluted semen, whereas the frozen semen showed higher swollen, damaged and missing acrosome rate than the first and second diluted semen. 7. Damaged and missing acrosome rate of sperm head due to freezing was somewhat inhibited by addition of BSA (0.01 to 0.05) to the BF5 dilutor.

• 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.

• 한국임상수의학회지
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• 제17권2호
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• pp.431-437
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• 2000

Evaluation of viability and capacitation of canine sperm is of great importance to deter- mine good condition for freezing canine semen and consequently to improve conception rate by arti-ficial insemination. Semen were collected from nine male dots which had been proved to be fertile in the post and the semen were treaded for freezing procedure. Semen were thawed at 37$^{\circ}C$ for 30 seconds. In this study, hypoosmotic swelling(HOS) test and chlortetracycline(CTC) test were per- formed to evaluate post-thaw viability and capacitated status of sperm, respectively. In HOS test far canine sperm, the highest percentage of curled sperm was shown at 60 mOsm. In HOS test for canine semen, there were considerably significant correlation between HOS values and sperm motil- ity(r=0.9064, p<0.01) and converse correlation between HOS values and sperm abnormality(r=- 0.6905, p<0.05). The sperm viability and HOS-values for chilled extended semen were significantly decreased from 0 to 72 hours during storage at 5$^{\circ}C$ (p<0.05). Of the media added to canine semen after thawing, the most capacitated sperm were shown in CCM(p<0.01), and then This Fructose Cit- rate(TFC) medium with calcium from 3 hours after incubation with media. It was concluded that HOS test is of great value to determine the viability and motility of canine sperm, whereas CTC test is usable to determine the capacitated status. Consequently, both tests were thought to be useful as the additional tests to standard semen analysis.

• 한국수정란이식학회지
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• 제32권3호
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• pp.221-226
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• 2017

Spermatozoa viability can be assessed by microscopy, flow cytometry, and other methods using fluorescent stain. Flow cytometry can be used to examine the morphological and functional characteristics of spermatozoa in a short time. The purpose of this study was to compare the viability of cryopreserved spermatozoa in Jeju black cattle by two dual fluorescent stain methods. Semen of Jeju black cattle raised in Subtropical Livestock Research Institute, National Institute of Animal Science, RDA were collected with artificial vaginal technique. Sperm was diluted with $Triladyl^{(R)}$-egg yolk diluent and then was performed cryopreservation. There was no significant difference in viability of spermatozoa according to the two dual fluorescent stain methods. However, when the distribution of spermatozoa according to the staining method was compared, the spermatozoa group stained with 6-CFDA/PI was more clearly distinguished than the spermatozoa group stained with calcein AM/PI.

• 한국수정란이식학회지
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• 제21권4호
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• pp.339-344
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• 2006

본 연구는 염소 액상 정액의 활용에 적합한 희석액을 검토하여, 국내 염소 인공수정 실용화를 위한 기초 자료를 확보하기 위하여 실시되었다. 두 마리의 흑염소 수컷으로부터 전기 자극법으로 정액을 채취하였다. 채취된 정액은 BTS로 희석하여 500g에서 5분간 원심분리하여 정장을 제거하고, BTS, Modena, Triladyl$^{(R)}$로 희석하여 $4^{\circ}C$ 냉장고나 $17^{\circ}C$ 온장고에서 8일간 보관되었다. BTS와 Modena로 희석된 정자의 운동성은 하루 만에 소멸되었으며, BTS로 희석된 정액을 $17^{\circ}C$에 보관하였을 때 2일째에 30$\sim$45%로 떨어졌으며, Modena에서는 3일째까지 인공수정에 가능한 활력을 유지하였다. 한편 Triladyl$^{(R)}$로 희석하여 $4^{\circ}C$나 $17^{\circ}C$에서 보관한 경우에는 좀더 좋은 생존성을 나타내었으며, 4일까지도 인공수정이 가능한 운동성을 보여주었다.

• 한국가축번식학회지
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• 제20권2호
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• pp.101-109
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• 1996

본 연구는 돼지 액상정액을 인공수정용 100ml 플라스틱 병에 보존하면서 Lactose-Egg yolk 희석액과 B tschwiler 희석액 간에 보존 온도별 차이를 조사하고, Lactose-Egg yolk 희석액에서의 글리세롤 농도의 효과를 조사하여 돼지 액상정액을 좀더 장기간 사용할 수 있는 방법을 찾고자 실시하였다. 돼지 액상정액을 5$^{\circ}C$ 냉장고에 보존하면서조사한 바에 의하면, 37$^{\circ}C$에서 0.5 및 2시간 배양 후의 정자 운동성은 전체 보존기간동안 Lactose-Egg yolk 희석액이 B tschwiler 희석액보다 유의하게 (P,0.05) 높게 나타났고, 정상첨체비율은 두 희석액간에 차이가 없었다. 돼지 액상정액을 15$^{\circ}C$에 보존하면서 조사한 바에 의하면, 3일부터 7일 보존시까지 정자 운동성과 정상첨체비율에 있어서 B tschwiler 희석액이 Lactose-Egg yolk 희석액보다 유의하데 높게 나타났다. Lactose-Egg yolk 희석액을 이용한 돼지 액상정액의 글리세롤 농도의 효과에 있어서는 최종 글리세롤 농도가 0, 1, 3 및 5%보다 2%일 때 가장 높은 정자 운동성과 정상첨체비유을 나타내었다. 분만율, 복당 생존자돈수 그리고 출생시 평균 생시체중은 Lactose-Egg yolk 희석액과 B tschwiler 희석액간에 차이가 없었다. 이상의 연구 결과를 종합해 볼 때 Lactose-Egg yolk 희석액은 5$^{\circ}C$에서 B tschwiler 희석액은 15$^{\circ}C$에서 6~7일 동안 돼지 액상정액을 보존할 수 있었다.

• 한국수정란이식학회지
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• 제18권2호
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• pp.91-96
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• 2003

본 연구는 개의 동결정액제조 시 동결보호 희석액 내에 첨가되는 당의 종류와 조합이 동결융해 후 정자 두부의 첨체의 손상과 생존율 및 운동성 등에 미치는 영향에 대하여 조사하고자 실시하였다. 당의 종류에 따른 정자의 첨체손상 정도는 정상 정자비 율은 Fru+Tre, 처리구가 Fructose, Trehalose, Fru+Tre+Xyl, Fru+Xyl, Tre+Xyl, Xylose구에 비해 가장 높았다(83.0$\pm$5.6%, 82.3 $\pm$3.1%, 81.7$\pm$2.1%, 81.0$\pm$5.6%, 80.3$\pm$4.5%, 76.7$\pm$3.8%, 72.0$\pm$2.0%;(P<0.05). Fru+Tre처리구에서 CASA 분석 후 운동성이 Fru +Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose구에 비해 가장 높았다. (79$\pm$6 vs 75$\pm$3, 74$\pm$8, 71$\pm$11, 70$\pm$4, 66$\pm$15, 63$\pm$12%; P< 0.05). 전진운동율은 Fru+Tre 처리구가 Fru+Tre+Xyl, Tre+Xyl, Fru+Xyl, Xylose, Trehalose, Fructose구에 비해 가장 높았다(67$\pm$7, 64$\pm$3, 62$\pm$6, 61$\pm$8, 60$\pm$2, 57$\pm$13, 53$\pm$10%; P<0.05). 본 연구결과 70 mM Fru+Tre (two combination) 가 첨체 손상정도가 낮았으며 가장 높은 운동성 및 전진 운동율의 결과를 얻을 수 있었다.

• 생명과학회지
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• 제24권11호
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• pp.1252-1257
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• 2014

DMSO는 배양포유류세포 동결보존의 동결보호제로써 일반적으로 사용 되어져 왔지만, DNA 메틸화 및 히스톤의 수식에 의해 일부 세포에서는 분화를 일으키는 것으로도 알려져 있다. 동결보존시의 배양세포의 안정된 분화형질유지에는 메틸화를 일으키는 DMSO 이외의 동결보호제의 사용이 필요하다. 세포독성이 낮고, 동물정자동결보존에 효과적인 것으로 알려진 아미도 화합물이 동일하게 포유류의 배양세포의 동결보존에서 동결보호작용이 있는지를(8종류의 아미드 화합물) 배양 마우스 혈관내피세포를 이용해 조사했다. 조사한 아미드 화합물 중에 아세트아미드와 락트아미드의 2종류가 배양세포에 대해서 동결보호작용이 있고, 가장 효과적인 것은 농도가 1.5 M의 락트아미드이다. 배양세포의 동결보존에 관해서는 삼투압 스트레스를 받지 않을 필요가 있기 때문에, 1.5 M 락트아미드 용액을 제작 시, 용매를 각 희석율의 PBS로 하고, 삼투압을 바꾼 동결 보존액에 동결세포의 생존율을 조사했다. 그 결과, 0.4배 농도의 PBS가 삼투압 스트레스를 가장 낮고 생존율이 가장 높음을 확인했다. 동결보존배지에 고 분자량재료를 첨가하면 세포생존율이 개선되는 것이 알려져 있기 때문에 BSA, HES, 데키스트란의 효과를 조사했다. 그 결과, 락트아미드를 이용한 동결보존배지는 $0.4{\times}PBS$를 이용한 1.5 M 락트아미드용액에 1%의 BSA를 첨가한 경우, DMSO의 동결보호작용에 필적하는 동결보호작용을 나타내는 것을 확인했다.