• Title, Summary, Keyword: Liquid Semen

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Effect of Ethylene Glycol Concentration and Freezing Speed on Post-thawed Semen Viability and Acrosome Integrity in Korean Jeju Black Bull (제주흑우 동결정액 제조시 Ethylene Glycol의 농도와 예비 동결 조건이 정자의 생존율 및 첨체양상에 미치는 영향)

  • Choi, Sun-Ho;Ko, Min-Hee;Kang, Tae-Young;Cho, Sang-Rae;Park, Yong-Sang;Oh, Shin-Ae
    • Reproductive and Developmental Biology
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    • v.35 no.3
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    • pp.377-383
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    • 2011
  • The objective of this study was to examine effect of ethylene glycol for semen cryopreservation in Korean Jeju Black Bull. The semen was cryopreserved with extenders containing cryoprotectants (7% glycerol and 3%, 5%, 7% ethylene glycol) and packed to 0.5 ml straws. The semen straws were located above 3 cm of liquid nitrogen for 5 min, 5 cm for 10 min and 8 cm for 10 min. And then frozen straw was plunged into $LN_2$. Post-thawed sperm motility, viability and membrane integrity were significantly higher in 5% ethylene glycol ($72.5{\pm}5.00%$, $54.88{\pm}0.66%$ and $46.00{\pm}2.40%$; p<0.05). Motility and viability were similar between 7% glycerol and 5% ethylene glycol. However, the membrane integrity was significantly higher in 5% ethylene glycol ($34.69{\pm}4.64%$ vs $46.00{\pm}2.40%$; p<0.05). The viability and membrane integrity were significantly higher in 5 cm for 10 min and 8 cm for 10 min than 3 cm for 5 min (viability: $55.81{\pm}2.94$, $55.19{\pm}3.34$ vs $47.94{\pm}3.48%$; p<0.05 and membrane integrity: $44.94{\pm}3.51$, $46.06{\pm}2.25$ vs $40.38{\pm}1.03%$; p<0.05). The percentage of capacitated sperm assessed by CTC staining, percentage of F pattern was higher in 7% glycerol, 5% and 7% ethylene glycerol, and AR pattern was significantly higher in 3% ethylene glycol. F pattern was significantly increased in 5 cm for 10 min and 8 cm for 10 min (p<0.05), but AR pattern was significantly increased in 3 cm for 5 min (p<0.05).

Quantitative Analysis of the Marker Constituents in Yongdamsagan-Tang using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (LC-ESI-MS/MS를 이용한 용담사간탕의 주요 성분 분석)

  • Seo, Chang-Seob;Ha, Hyekyung
    • Korean Journal of Pharmacognosy
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    • v.48 no.4
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    • pp.320-328
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    • 2017
  • Yongdamsagan-tang has been used to treat the urinary disorders, acute- and chronic-urethritis, and cystitis in Korea. In this study, an ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS/MS) method was established for simultaneous analysis of the 20 bioactive marker compounds, geniposidic acid, chlorogenic acid, geniposide, liquiritin apioside, acteoside, calceolarioside B, liquiritin, nodakenin, baicalin, liquiritigenin, wogonoside, baicalein, glycyrrhizin, wogonin, glycyrrhizin, wogonin, saikosaponin A, decursin, decursinol angelate, alisol B, alisol B acetate, and pachymic acid in traditional herbal formula, Yongdamsagan-tang. Chromatographic separations of all marker compounds were conducted using a Waters Acquity UPLC BEH $C_{18}$ analytical column ($2.1{\times}100mm$, $1.7{\mu}m$) at $45^{\circ}C$ using a mobile phase of 0.1% (v/v) formic acid in water and acetonitrile with gradient elution. The MS analysis was performed using a Waters ACQUITY TQD LC-MS/MS coupled with an electrospray ionization source in the positive and negative modes. The flow rate was 0.3 mL/min and injection volume was $2.0{\mu}L$. The correlation coefficient of 20 marker compounds in the test ranges was 0.9943-1.0000. The limits of detection and quantification values of the all marker components were 0.11-6.66 and 0.34-19.99 ng/mL, respectively. As a result of the analysis using the optimized LC-ESI-MS/MS method, three compounds, geniposidic acid (from Plantaginis Semen), alisol B (from Alismatis Rhizoma), and pachymic acid (from Poria Sclerotium), were not detected in this sample. While the amounts of the 17 compounds except for the geniposidic acid, alisol B, and pachymic acid were $0.04-548.13{\mu}g/g$ in Yongdamsagan-tang sample. Among these compounds, baicalin, bioactive marker compound of Scutellariae Radix, was detected at the highest amount as a $548.13{\mu}g/g$.

A Comparison of Spinosin Content in Zizyphi Semen and Its Processed Products by Roasting (산조인의 수치에 따른 Spinosin 함량 비교)

  • Seo, Chang-Seob;Kim, Jung-Hoon;Shin, Hyeun-Kyoo;Kim, Byoung-Soo
    • Korean Journal of Pharmacognosy
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    • v.47 no.4
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    • pp.360-365
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    • 2016
  • The aim of this study was to compare the amount of spinosin in the 70% ethanol extracts of non-processed Zizyphi Semem (ZS) and processed ZS by roasting using a high-performance liquid chromatography equipped with photodiode array detector. Separation of the spinosin was used $SunFire^{TM}$ $C_{18}$ analytical column ($5{\mu}m$, $4.6{\times}150mm$) using two mobile phase consisting of distilled water and acetonitrile, both with 1.0% (v/v) acetic acid. The flow rate was 1.0 mL/min and injection volume was $10{\mu}L$. Calibration curve of the spinosin was y = 22339.45x+483.99 in tested concentration range ($1.28-20.00{\mu}g/mL$) and correlation coefficient was 1.0000. In non-processed ZS sample, the amount of the spinosin was 0.94 m/g, while, the amount of the marker compound in processed ZS samples were 0.66-1.10 mg/g.

Acute Oral Toxicity Study of Standardized Herbal Preparations(Gami-Samhwang-San, SH-21-B) in Rats (HPLC로 표준화한 가미삼황산(加味三黃散) 분획물(SH-21-B)의 랫드에 대한 단회경구투여독성시험)

  • Yu, Young-Beob;Kim, Seon-Hyeong;Yoon, Yoo-Sik
    • Toxicological Research
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    • v.21 no.3
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    • pp.255-261
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    • 2005
  • Gami-Samhwang-San, a herbal prescription for obesity treatment, is composed of seven crude herbs such as Rehmanniae Radix Preparata, Ephedrae Herba, Scutellariae Radix, Acori Gramineri Rhizoma, Polygalae Radix, Typhae Pollen, Armeniacae Semen, Menthae Herba. In this study, marker substances in n-butanol fraction (SH-21-B) from Gami-Samhwang-San were analyzed by high performance liquid chromatography (HPLC) and acute toxicity of standardized SH-21-B was evaluated by good laboratory practices (GLP) guideline of Korea Food and Drug Administration. Therefore we confirmed that there were baicalin of 15.92%, amygdalin of 6.57% and ephedrine of 2.49% in SH-21-B. SH-21-B was administered in rats at dose of 0 mg/kg, 2,000 mg/kg, and 5,000mg/kg. Clinical signs of both sexes of rats were observed daily for 14 days after single oral administration. Two female rats one administered at 2,000 mg/kg and the other administered at 5,000 mg/kg, died, but no dead animal was observed among male rats. Therefore $LD_{50}$ in the female rat is observed to be 8,710 mg/kg, and MLD (Minimun Lethal Dose) of the male rat is observed to be more than 5,000 mg/kg.

HPLC analysis of Gami-Samhwang-San and prediction of active compounds using QSAR (가미삼황산(加味三黃散) 분획물(SH-21-B)의 지표성분 정량과 구조활성상관(QSAR) 예측)

  • Yu, Young-Beob
    • Journal of Korean Traditional Oncology
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    • v.11 no.1
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    • pp.95-103
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    • 2006
  • Objective: Gami-Samhwang-San, a herbal prescription for obesity treatment, is composed of seven crude herbs such as Ephedrae Herba, Scutellariae Radix, Acori Gramineri Rhizoma, Polygalae Radix, Typhae Pollen, Armeniacae Semen, Nelumbo Folium. This study was aimed to evaluate marker substances in n-butanol fraction (SH-21-B) from Gami-Samhwang-San by high performance liquid chromatography (HPLC). And we predicted inhibition activity of major compounds of Gami-Samhwang-San using Quantitative Structure Activity Relationships (QSAR) Methods: The separation was performed on a YMC J,sphere-H80 CI8(250${\times}$4.6 mm I.D) column by gradient elution with $H_3PO_4$ buffers in acetonitrile as the moblie phase at a flow-rate of 1.0ml/min. Results: HPLC was employed to determine the quantities and the qualities of several marker substances such as ephedrine, pseudoephedirne, baicalin, ${\beta}-asarone$, tenuifoliside, naringenin, amygdalin and hyperoside in the SH-21-B. Conclusion: We suggest this results could be a useful evidence for quality control of SH-21-B.

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Studies on Correlation Among Sperm Characteristics, Farrowing Rates by AI and Chromatin Structure in Boars (돼지에서 정액 성상 및 인공수정 분만율과 염색질 구조 분석(SCSA)과의 상관관계에 관한 연구)

  • 유재원;김인철;이장희;조규호;지달영;이주형;김일;이종완;윤희진;방명걸;류범용;정영채;김창근
    • Journal of Animal Science and Technology
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    • v.48 no.6
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    • pp.777-784
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    • 2006
  • This study was designed to investigate between the semen characteristics and sperm chromatin structure in boar with different farrowing rates and relationship between fertility by AI and results of sperm chromatin structure assay (SCSA). The CASA (computer-aided sperm analysis) and SCSA were performed with liquid semen in boars. The all SCSA parameters based on the farrowing rates by AI were significantly differ (P<0.05). The significant negative correlations (P<0.05) were observed between all SCSA parameters and farrowing rate obtained by AI in the field. In conclusion, these results suggest that the sperm parameters evaluated in these studies may be useful indicators to predict the fertility by AI.

Studies on Genetics and Breeding in Rainbow Trout(Oncorhynchus mykiss) VII. Fertilization of Fresh Egg with Co-Preserved Sperm and Ultrastructural Changes (무지개 송어의 유전 육종학적 연구 VII. 동결보존시킨 정자와 신선한 난모세포의 수정 및 미세구조적 변화)

  • PARK Hong-Yang;YOON Jong-Man
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.25 no.2
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    • pp.79-92
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    • 1992
  • This study was carried out to develop new techniques useful for cryopreservation, thawing and artificial insemination, and ultrastructural changes of cryopreserved spermatozoa in rainbow trout(Oncorhynchus mykiss) . Two extenders, such as Tyrode solution and Whittingham's $T_6$ solution, were used to preserve rainbow trout sperm in refrigerator $(-20,\;-40\;and\;-70^{\circ}C)$ or liquid nitrogen $%(-196^{\circ})$. Hand-stripped semen was diluted to 1:16 with two extenders, an then the semen were frozen after mixing semen and each extender containing 1M or 1.5M DMSO solution to 1:1. After 60 days cryopreserved semen was thawed in a $13^{\circ}$ water bath, and subsequently centrifugated. After centrifugation at 1,000 rpm for 5 min thawed semen was washed with extenders, and then fertilized with fresh eggs. The results obtained in these experiments were summarized as follows: After cryopreservation, over 75% of spermatozoa were appeared motile and the survival rate was high. Following cryopreservation by the addition of cryoprotectant such as DMSO, methanol and glycerol, the fertilization rate of the thawed spermatozoa appeared over $99\%$ compared with the control having $99\%$ of fertilization rate. There was no difference between the control and experimental groups such as $(-20^{\circ}C\;-40^{\circ}C\;and\;-70^{\circ}C)$ and $-196^{\circ}$ in fertilization rate. Following cryopreservation at $-196^{\circ}$ by the addition of 1M DMSO of cryoprotectant, each fertilization rate following 24 hours and hatching rate following 24 days showed $96\%$ and $8\%$ by the addition of BSA, but showed $98\%\;and\;10%$ by no addition of BSA. Following 2 months of cryopreservation by the addition of 1M DMSO of cryoprotectant, there were $10%$ of hatching rate at $-196^{\circ}\;and\;10\%\;and\;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1M methanol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C,\;and\;28\%,\;at\;-70^{\circ}C$ Following 2 months of cryopreservation by the addition of 1M glycerol of cryoprotectant, there were $22\%$ of fertilization rate at $-20^{\circ}C$, and $33\%,\;at\;-70^{\circ}C$. pollowing 2 months of cryopreservation by the addition of 1.5M DMSO of cryoprotectant, there were $27\%$ of fertilization rate at $-20^{\circ}C,\;an\;36\%\;and \;35\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C$. Following 2 months of cryopreservation by the addition of 1.5M glycerol of cryoprotectant, there were $34\% \;of\;fertilization\;rate\;at\;-20^{\circ}C, \;and\;31\%\;and\;31\%,\;respectively,\;at \;-40^{\circ}C\;and\;-70^{\circ}$. Following 2 months of cryopreservation by the addition of 1.5M methanol of cryoprotectant, there were $28\%$ of fertilization rate at $-20^{\circ}C,\;and\;29\%\;and\;28\%,\;respectively,\;at\;-40^{\circ}C\;and\;-70^{\circ}C.$ From 10 days and 15 days following fertilization at $13^{\circ}C\;and\;10^{\circ}C$, respectively, the mortality rate of fertilized ova was markedly increased. The middle piece of spermatozoa had two set of central doublets, nine set of outer coarse fibres, and mitochondrial sheath. Spermatozoa went through morphological changes during storage, e.g. winding of flagella, detachment of the nuclear envelope and the plasma membrane from the nucleus of the sperm head. There were $1\%$ abnormal spermatozoa in fresh sperm and about $15\%$ during storage.

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Effects of Fertilization Time and Culture Medium of Pig Oocytes Matured In Vitro by liquid Boar Sperm Stored at $4^{\circ}C$ (체외성숙된 돼지난포란을 $4^{\circ}C$ 보존 액상정액으로 체외수정시 수정시간과 배양배지의 영향)

  • Park, C. S.;Y. J. Yi;Kim, M. Y.;Y. J. Chang;Lee, S. H.;D. I. Jin
    • Korean Journal of Animal Reproduction
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    • v.27 no.3
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    • pp.215-223
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    • 2003
  • This study was to investigate the effects of fertilization time and culture medium of pig oocytes matured in-vitro by liquid boar sperm. The sperm rich fraction (30∼60 ml) was slowly cooled to room temperature (20∼23$^{\circ}C$) by 2 h after collection. Semen was transferred into 15 ml tubes, centrifuged at room temperature for 10 min 800 ${\times}$ g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 ml of the LEN diluent to provide 1.0${\times}$10$^{9}$ sperm/ml at room temperature. The resuspended semen was cooled in a refrigerator to 4$^{\circ}C$. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin B$_{12}$ , 25 mM HEPES, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Oocytes were inseminated with liquid boar sperm stored at 4$^{\circ}C$ for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 ${mu}ell$ mTBM fertilization media with 1.0${\times}$10$^{6}$ sperm/ml concentration, respectively. Thereafter, oocytes were transferred into 500 ${mu}ell$ NCSU-23, HEPES buffered NCSU-23, PZM-3 and PZM-4 culture media, respectively, for further culture of 6, 48 and 144 h. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times for 6 and 9 h than in those for 1 and 3 h. The rates of cleaved oocytes were higher in the fertilization times for 6 and 9 h (85.0 and 84.6%) than in those for 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time for 6 h (33.6%) than in that for 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9, 27.6, 26.3 and 24.4 in the fertilization times for 6, 9, 3 and 1 h, respectively. The rate of blastocyst from the cleaved oocytes and the number of cells per blastocyst were higher in HEPES buffered NCSU-23 culture medium than in NCSU-23, PZM-3 and PZM-4 culture media. In conclusion, we found out that liquid boar sperm stored at 4$^{\circ}C$ could be used for in-vitro fertilization of pig oocytes matured in-vitro. Also, we recommend the coincubation time of 6 h in 500 ${mu}ell$ TBM fertilization medium with 1${\times}$10$^{6}$ sperm/ml concentration and the HEPES buffered NCSU-23 culture medium for in-vitro fertilization of pig oocytes matured in-vitro.

Viability Assessment of Fresh and Frozen-thawed Dog Spermatozoa by Flow Cytometry (Flow Cytometry에 의한 개 신선정액과 동결정액의 생존성 분석)

  • Hong Y. M.;Kim Y. J.;Yu I.;Ji D. B.;Kim M. S.
    • Reproductive and Developmental Biology
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    • v.28 no.3
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    • pp.167-172
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    • 2004
  • This study was performed to examine the correlations among dog sperm viabilities evaluated by flow cytometry, by microscopic evaluation (ME), by carbo-xifluorescein diacetate and propidium iodide (CFDA/PI) and by hypoosmotic swelling (HOS) test. Semen were collected from 5 dogs ranging in age from 2 to 4 years. Each ejaculate was divided into 3 aliquots and different proportions of freeze-killed cells were added to each aliquot (1:0, 1:1 and 1:3). In the other experiment, semen was extended with Sweden extender containing 5% glycerol and equex STM paste, and frozen using liquid nitrogen vapor. Fresh and frozen-thawed dog sperm viability were assessed by flow cytometry using PI staining method. The accuracy of flow cytometry was evaluated by comparing with other classic assessments, microscopic evaluation, epifluorescence microscopic analysis using CFDA/PI, and HOS test. High correlations of sperm viabilities were found among flow cytometry, epifluorescence evaluation, HOS test (p<0.01) in fresh semen. Especially, sperm viability assessed by HOS test was highly correlated with viability by flow cytometry in all the ratios of live and dead spermatozoa, 1:0, 1:1 and 1:3 (p<0.01). The viability evaluated by ME were significantly correlated with that by flow cytometry in ratios of 1:0 and 1:3 (p<0.05) however, there was no significance in ratio of 1:1. The viability evaluated by C/p were highly correlated with that by flow cytometry in ratio of 1:0 and 1:1 (p<0.01) and significantly correlated in ratio of 1:3 (p<0.05). In frozen-thawed spermatozoa, the viability determined by HOS test was considerably correlated with that by flow cytometry (p<0.01). There was significant correlation between the viabilities by ME and by flow cytometry (p<0.05). But the viability evaluated by CFDA/PI was not correlated with viability by flow cytometry. The result from this study validate the use of flow cytometry as a precise method for assessing the viability of fresh and frozen-thawed dog spermatozoa.

Development of Quality Control Method for a Novel Herbal Medicine, HPL-1 using UHPLC (UHPLC를 이용한 새로운 한약제제 HPL-1의 품질관리법 개발)

  • Kim, Se-Gun;Lamichhane, Ramakanta;Lee, Kyung-Hee;Jung, Hyun-Ju
    • The Korea Journal of Herbology
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    • v.30 no.3
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    • pp.19-24
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    • 2015
  • Objectives : HPL-1, a novel herbal medicine which is composed of five herbs such as Kalopanacis Cortex, Chaenomelis Fructus, Raphani Semen, Atractylodis Rhizoma and Pulvis Aconiti Tuberis Purificatum, was developed for treatment of osteoarthritis. This study is aimed to develop analytical method for consistent quality control of HPL-1 and validate chromatographic method. Methods : Chromatographic analysis was performed using ultra-high performance liquid chromatography - diode array detector (UHPLC-DAD) equipped with RP-amide column, column oven, and auto sampler. Marker compounds [protocatechuic acid, chlorogenic acid, liriodendrin, 3,5-dicaffeoylquinic acid, ${\beta}$-D-(3-O-sinapoyl)-fructofuranosyl-$\alpha$-D-(6-O-sinapoyl)glucopyranoside and benzoylmesaconine] were separated by step gradient elution of acetonitrile and 0.1% phosphoric acid/water. The method validation was evaluated by quantitative validation parameters of linearity, accuracy, precision, limit of detection (LOD) and limit of quantification (LOQ) according to KFDA guideline.Results : An optimized method for six marker compounds in HPL-1 was established by UHPLC-DAD. The correlation coefficient (R2) with each calibration curve was greater than 0.99. The LOD and LOQ were within the range of 0.008-0.090 and $0.023-0.274{\mu}g/mL$, respectively. The relative standard deviation (RSD) of intra- and inter-day variability were less than 4.0%. The result of recovery test was range from 93.3-106.3% with RSD < 4.0%.Conclusions : These results suggest that the quantitative UHPLC method is precise, accurate, effective for quality evaluation of HPL-1. The method may also contribute to improve quality of crude drug preparations used for treatment of various diseases.