• Title, Summary, Keyword: Liquid Semen

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Effects of Skimmed Goat Milk as a Semen Extender on Preservation of Bull Spermatozoa (탈지산양유(脫脂山羊乳)가 우정자보존(牛精子保存)에 미치는 영향(影響))

  • Lee, Hyo Jong;Oh, Soo Kak
    • Korean Journal of Veterinary Research
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    • v.15 no.2
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    • pp.207-213
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    • 1975
  • Skimmed goat milk heated at $92^{\circ}C$ for 10 minutes was used as a basal extender for bull semen. The extenders for liquid semen were prepared by adding simultaneously at various ratio of 5% dextrose solution and egg yolk to skimmed goat milk. After bull seven was diluted with the extenders at the rate of 20 million spermatozoa per ml of the extenders. The extenders were stored at $5^{\circ}C$ and the survival rates of spermatozoa were examined at 4 and 24 hours, and 3, 5 and 7 days after dilution. The extenders for frozen semen were prepared by adding various ratlo of glycerol to skimmed goat milk containing 20 parts of 5% dextrose solution and 3 parts of egg folk to 77 parts of skimmed goat milk. After bull semen was diluted with the extenders at the rate of 40 million spermatozoa per ml of the extenders, the extenders were frozen in liquid nitrogen tank. The frozen extenders were thawed at $40^{\circ}C$ for 2 minutes, and the revival rates of the spermatozoa in the extenders were examined. These thawed extenders were stored at $5^{\circ}C$ and the survival rates of the spermatozoa were examined at 10 minutes and 24 hours and 3 and 5 days after thawing. The results obtained were as follows: 1. Among the extenders stored at $5^{\circ}C$, the survival rate of the sperm was the highest in the extender including 20 parts of 5% dextrose solution and 3 parts of egg yolk to 77 parts of skimmed goat milk, and the survival rate was significantly higher that of the spermatozoa in egg folk-2.9% sodium citrate (1 : 4) extender. (P<0.05) 2.Among the extenders frozen in liquid nitrogen tank, the revival rate of the spermatozoa was the highest in the extender containing 7ml of glycerol per 100ml of the extender with consisted of 77 parts of skimmed goat milk, 20hparts of 5% dextrose solution and 3 parts of egg yolk, and the revival rate was significantly higher than that of the spermatozoa in egg yolk-2.9% sodium citrate (1 : 4) extender containing 8ml of glycerol per 100ml of the extender (p<0.01). 3. Among the extenders stored at $5^{\circ}C$ after thawing, the survival rate of the spermatozoa was the highest in the extender containing 7ml of glycerol per 100ml of extender which consisted of 77 parts of skimmed goat milk, 20 parts of 5% dextrose solution and 3 parts of egg yolk, and the survival rate was significantly higher than that of the spermatozoa in egg yolk -2.9% sodium citrate (1 : 4) extender containing 8ml of glycerol per 100ml of the extender (p<0.01).

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Prevention of Epimerization and Quantitative Determination of Amygdalin in Armeniacae Semen with Schizandrae Fructus Solution

  • Joo, Woo-Sang;Jeong, Ji-Seon;Kim, Hyo-Geun;Lee, Yong-Moon;Lee, Je-Hyun;Hong, Seon-Pyo
    • Archives of Pharmacal Research
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    • v.29 no.12
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    • pp.1096-1101
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    • 2006
  • Armeniacae Semen not only contains amygdalin, but emulsin also, which is an enzyme that hydrolyzes amygdalin. The extraction yield of amygdalin from Armeniacae Semen was low, due to the presence of emulsin, when extracted with water. When Schizandrae Fructus solution was used as the extractant; however, amygdalin was almost completely extracted, regardless of the cutting size, due to the absence of the influence of emulsin. In addition, when the crude powder or small piece forms were used with Schizandrae Fructus solution, on epimerization of the D-amygdalin into neoamygdalin occurred. D-amygdalin and its conversion product, neoamygdalin, were quantitatively analyzed by reverse-phase, high-performance liquid chromatography (HPLC), with an optimized eluent of 10 mM sodium phosphate buffer (pH 2.3), containing 11.5% acetonitrile. The concentration and detector response were linearly correlated over the range 0.05 to 2 mM. The detection limits for both D-amygdalin and neoamygdalin were approximately $5\;{\mu}M$ for the amount injected.

Production of Pups Following Artificial Insemination by Canine Intrauterine Inseminator (개 자궁내 인공수정기에 의한 인공수정 후 산자생산)

  • 공일근;조성균;임용택;이상인;위성하
    • Journal of Veterinary Clinics
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    • v.16 no.2
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    • pp.375-380
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    • 1999
  • This study was conducted to develop an intrauterine inseminator (IUI) to deposit of frozen semen into uterus and to evaluate the results obtained after artificial insemination by IUI. Two Japanese spitzs (2 to 4 years of age) were used as semen donors. Semen was collected by manual masturbation into sterile glass collection tubes and separated into 3 fractions with only the sperm-rich fractions retained for further examination. Sperm motility >70%, sperm concentration of 200 to $400{\times}10^6 cells/ml$$\times$g for 5 min and poured out the suspended solution, and then diluted with 2 ml Tris-buffer which was consisted of 2.4 g Tris, 1.4 g citric acid, 0.8 g glucose, 0.1 $\mu\textrm{g}$/ml streptomycin, 100 IU/ml penicillin, 20 ml egg yolk to 100 ml mili-Q water (Ext I) or supplemented with 8 ml glycerol and 1 ml Equex STM paste to 100 rnl (Ext II). The diluted semen was cooled to 5$^{\circ}C$ in cold room, where the temperature in the sample reached 5$^{\circ}C$. Two h after beginning the cooling procedure, 2 ml of Ext II, also at 5$^{\circ}C$, was added and mixed by gently reversing the tubes several times during 1 h. The final sperm concentration for freezing was approximately $50{\times}10^6 cells/ml$. After equilibration, the semen was loaded into 0.5 ml straw and frozen on the liquid nitrogen vapour in styrofoam box. The straws were thawed at 7$0^{\circ}C$ for precisely 6 sec. After thawing of each straw, the frozen semen can survived over 50% motility. All the females were inseminated twice with 1 ml of $25{\times}10^6 cells/ml$

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Evaluation of Boar Sperm Viability by MTT Reduction Assay in Beltsville Thawing Solution Extender

  • Byuna, J.W.;Choo, S.H.;Kim, H.H.;Kim, Y.J.;Hwang, Y.J.;Kim, D.Y.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.4
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    • pp.494-498
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    • 2008
  • MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored water-soluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay. Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the fresh BTS extended semen from a local A.I center. Semen sample was diluted to $3.0{\times}10^7$ sperms/ml in BTS. The rates of formazan production were measured in 96-well microtiter plates immediately and 1h after incubation at $17^{\circ}C$ using a spectrophotometer at wave length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory.

Establishment of Cryopreservation of Leopard Cat Semen Collected by Electro-ejaculation Method

  • Ha, A-Na;Jo, A-Ra;Kim, Yu-Gon;Yoon, Jin-Ho;Bang, Jae-Il;Deb, Gautam K.;Fakruzzaman, M.;Lim, Yang-Mook;Yong, Hwan-Yul;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.26 no.4
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    • pp.245-250
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    • 2011
  • The aim of this study was to evaluate the post-thawed characteristics of leopard cat semen. In this experiment, semen was collected from two leopard cats (A and B) at wild animal center in Seoul Grand Park in Korea. After collection, the sperms were washed with D-PBS and diluted by the freezing medium (Irvine science, USA) and stored in liquid nitrogen. The post-thawed concentration was $357{\times}10^6sperms/ml$ for A and $97{\times}10^6sperms/ml$ for B. The viability of post-thawed sperm from A and B individual was 24.0% and 19.0%, respectively. Pre-freezing motility of A and B individual semen was 68.54% and 56.65. Leopard cat A had more normal sperm than that of B (69.5% vs. 54.5%). Acrosome integrity analysis detected live (14.5% vs. 9.0%), damage (39.0% vs. 44.0%) and dead (46.0% vs. 47.0%) in leopard cat A and B, respectively. The present results concluded that leopard cat semen can be collected successfully by electro-ejaculation method and cryopreserved successfullyfor future use in different assisted reproductive technologies. The cryopreservation protocol needs to be modified for increasing post-thawed viability of leopard cat spermatozoa.

Characterization of Bacteria and Their Antibiotic Sensitivities in Porcine Liquid Semen (돼지 액상정액 내 세균오염과 항생제 감수성에 관한 연구)

  • Ryu, Jae-Weon;Cho, Kyu-Ho;Hong, Joon-Ki;Kim, Myung-Jick;Park, Jun-Chul;Jung, Il-Byung;Kim, In-Cheul
    • Journal of Animal Science and Technology
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    • v.50 no.6
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    • pp.793-798
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    • 2008
  • The semen collection process in the porcine is far from being a sterile procedure. Consequently, porcine ejaculates commonly contain bacterial contaminants. The aim of this study is to identify the bacteria in porcine semen and to find the antibiotics resistance of bacteria. Twelve porcine originating from four AI center were used to collect semen. Bacteria were identified by automated instrument for rapid organism identification system and bacterial sensitivities of 8 antibiotics were tested. The Bacterial contaminants of Staphylococcus genus(37.8%), Proteus genus(7.0%), Bacillus genus (6.1%), Pasteulla genus(5.7%), Acinetobacte genus(5.2%), Serratia genus(4.3%) and others(33.9%) were frequently isolated. However, amikacin showed higher antibiotic sensitivity than other antibiotics. General sanitation protocols can contribute partly to inhibit the bacterial contamination, with monitoring boar housing, semen collection areas and the extended semen. But, proper selection of preservative antibiotics by microbial sensitivities can minimize the influence of bacteria.

Effects of Seeding during Freezing Procedure on Post-Thaw Viability and Acrosome Integrity of Boar Spermatozoa (돼지정액 동결중 식빙처리가 융해후 정자생존율 및 침체형태에 미치는 영향)

  • Kim Yong-jun;Kim Yong-hwan;Lee Young-jun;Kim Sue-hee;Ji Dong-beom
    • Journal of Veterinary Clinics
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    • v.21 no.4
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    • pp.363-368
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    • 2004
  • To investigate the effects of seeding during freezing procedure on post-thaw viability, motility, and acrosome integrity of boar spermatozoa, semen from 5 Yorkshire boars were collected for this experiment. Raw semen were diluted with Merck I, subsequently added with cooling diluent containing lactose and egg yolk and with freezing diluent containing glycerol. The diluted semen were frozen on the rack in the styrofoam box filled with liquid nitrogen at the distance of 5 cm or I cm above LN2 level. Seeding was performed to only a group of straws frozen at 5 cm away on the surface of LN2. The frozen semen were thawed in $50^{\circ}C$C water and the viability and local motility were analyzed by sperm analysis imaging system. A part of thawed semen was taken for the examination of morphology of apical ridge of the acrosome to compare with the effect of seeding between the seeding-treated and non treated groups. I. Post-thaw viability was considerably higher in seeding-treated sperm than non-seeding group (p<0.01), however, no difference of local motility was obtained among the groups. 2. At three hours after thawing, viability was also higher in seeding-treated group than non-treated group (p<0.05), along with no difference of motility among the groups. 3. Higher normal acrosome integrity was obtained in the seeding-treated sperm than non-treated groups (p<0.01). 4. Between non-seeded groups, higher normal acrosome integrity was obtained in the sperm group frozen at 5cm upper on the surface of LN2 than that frozen at 1cm away (p<0.01). These results indicated that seeding treatment during freezing boar spermatozoa was beneficial to post-thaw viability and normal acrosome integrity.

Effects of Kinds of Cryoprotectants on the Characteristics of Frozen Fowl Semen (닭 정액 동결 시 동결 보호제가 정액 성상에 미치는 영향)

  • Choi, Jin Seok;Shin, Dan-Bi;Ko, Yeoung-Gyu;Do, Yoon-Jung;Byun, Mijeong;Park, Soo-Bong;Seong, Hwan-Hoo;Kim, Hyun;Kong, Il-Keun;Kim, Sung Woo
    • Korean Journal of Poultry Science
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    • v.40 no.3
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    • pp.171-178
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    • 2013
  • The purpose of this study was to evaluate the sperm viability, normal acrosome and mitochondrial activity in the frozen-thawed fowl semen by different cryoprotectants. The experiment was carried out on 10 sexually adult roosters of Ogye. The semen was collected twice a week and pooled semen was diluted 1:1 EK extender containing no cryoprotectant at $5^{\circ}C$. After equilibration for 30 minutes, diluted chicken semen was diluted 1:1 extender containing either 7% dimethylacetamide (DMA), 7% dimethylformamide (DMF) or 7.5% methylacetamide (MA) at final concentration and was put in 0.5 mL plastic straws and frozen for 30 minutes by exposure to liquid nitrogen vapor 4 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen. Frozen semen was thawed in water bath at $5^{\circ}C$ for 2 minutes. For cytometric analysis, the frozen-thawed semen was diluted with EK extender to a final concentration of 90 million spermatozoa per mL. Sperm membrane integrity was evaluated as SYBR-14 and propidium iodide (PI). Acrosome integrity was assessed with fluorescein isothiocyanate-labeled PSA and PI. The percentage of mitochondrial function was estimated by using Rhodamine123 (R123) and PI. In conclusion, freezing rooster semen by using 7% DMF as cryoprotectant was significantly highest in rates of survival and mitochondrial function while its rate of damage of acrosome was significantly lowest. As a result, DMF is the cryoprotectant that has the lowest influences on sperm membranes and acrosome integrity. Therefore it could be used for freezing method of animal genetic conservation method for poultry diversity.

Rhei Rhizoma Mainly Blended Prescriptions According to the Fomula, Manipulation, Related Co-herb in Dongeuibogam (『동의보감(東醫寶鑑)』 중 대황(大黃)이 주약(主藥)으로 배오(配伍)된 방제(方劑)의 제형(劑形), 포제(?製), 약대구성(藥對構成)에 따른 활용(活用))

  • Joh, Hae-In;Kook, Yoon-Bum
    • Herbal Formula Science
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    • v.25 no.4
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    • pp.553-574
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    • 2017
  • The purpose of this study is to find out effects of prescriptions according to the formula, manipulation of Rhei Rhizoma, configuration. The following results were reached through investigations on the prescriptions using Rhei Rhizoma as a main component. Objectives : Analysis of prescriptions According to the formula : Liquid Extract Prescriptions were used widely on three parts to treat fever and damp heat in the interior organs. Powder Prescriptions were taken with hot water, thin porridge, tea etc. to treat damp heat, congestion of phlegm, acute episodes. Liquid Mixed Pill treat congestion of QI, damp heat, phlegm. Honey Mixed Pill treat accumulated fever, distension, acute excessive fever. Paste Pill treat blood stagnation, excessive toxic-fever, epidemic diseases. External Application treat inflammation by injury, swelling due to severe fever by internal damage. Methods : Analysis of prescriptions According to the manipulation of Rhei Rhizoma : Prescriptions including Liquor processed Rhei Rhizoma treat excessive toxic-fever, congestion of phlegm, blocking orifices on the upper side. Steamed Rhei Rhizoma strengthen effects of making evacuate and cooling of heat. Processed Rhei Rhizoma with vinegar strengthen effects of removing blood stagnation by activating blood movement, releasing gathering. Results : Analysis of prescriptions According to the Composition of Rhei Rhizoma : 41% of the total prescriptions were on the area of less than 20%. In case of lower groups show increased frequency of combination with Pharbitidis Semen, Persicae Semen, Scutellariae Radix and manipulation of baking, steaming, roasting. In case of higher groups show increased frequency of treating excess syndrome, critical illness, acute severe illness, and using proccesed Rhei Rhizoma with vinegar. Treatment of damp heat on the liver and gallbladder, disorder of the spleen and stomach is done mostly by prescriptions on the area of less than 30%. Conclusions : Rhei Rhizoma-Coptidis Rhizoma pair treat damp heat, heat toxins in blood, and Constipation caused by excessive heat. Rhei Rhizoma-Glycyrrhizae Radix pair relieve effects of Rhei Rhizoma passing blocked feces, removing the poison, activating blood movement, releasing gathering with the effects of Glycyrrhizae Radix relaxing tension by harmonizing Middle. Rhei Rhizoma-Magnoliae Cortex pair are used to treat damp heat in middle area, excessive heat in the stomach and intestine. Rhei Rhizoma-Pharbitidis Semen pair act on both blood system and QI system treating edema, damp, stagnation, heat toxins, feces. Rhei Rhizoma-Persicae Semen pair treat blood stagnation with fever on blood system.

Seminal Attributes and Semen Cryo-banking of Nepalese Indigenous Achhami (Bos indicus) Bull under Ex-situ Conservation

  • Jha, Pankaj Kumar;Sapkota, Saroj;Gorkhali, Neena Amatya;Pokharel, Bhoj Raj;Jha, Ajeet Kumar;Bhandari, Shishir;Shrestha, Bhola Shankar
    • Journal of Animal Reproduction and Biotechnology
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    • v.34 no.4
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    • pp.272-279
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    • 2019
  • The study was conducted to evaluate the seminal attributes and cryobanking of Achhami (Bos indicus) bull semen. Of two Achhami bulls, 8 ejaculates from each bull were evaluated for seminal attributes. For semen freezing and cryo-banking, 4 ejaculates (having ≥2 mL semen volume, ≥75% of sperm motility and ≥1,000 × 106 cells/mL of sperm concentration) from each bull were used. Semen samples were diluted in egg-yolk-tris-citrate extender using a two-step dilution protocol, and were frozen in liquid nitrogen (LN2) vapour in a styrofoam box. The mean semen volume, colour, sperm mass activity, motility, viability, concentration, abnormal acrosome, midpiece and tail and, abnormal head of two Achhami bulls were 4.4 ± 0.5 mL vs. 2.5 ± 0.2 mL, 2.5 ± 0.1 vs. 2.4 ± 0.1, 3.5 ± 0.1 vs. 3.5 ± 0.1, 77.0 ± 1.1% vs. 78.3 ± 1.3%, 94.4 ± 0.5% vs. 91.0 ± 0.6%, 1137.7 ± 73.7 × 106 cells/mL vs. 1060.0 ± 44.3 × 106 cells/mL, 10.2 ± 0.5% vs. 10.3 ± 0.5% and 6.7 ± 0.5% vs. 8.2 ± 0.3%, respectively. The post-thawed sperm motility and viability were 53.0 ± 2.0% vs. 50.0 ± 0.0% and 80.2 ± 0.4% vs. 73.2 ± 0.7%, while evaluating by computer-assisted sperm analysis (CASA) system, the percentage of the progressive motility, fast motility, slow motility, local motility and immotile sperm were 75%, 68%, 7.4%, 16.6% and 8.6%, respectively. A total number of 620 doses semen straw were cryo-banked. Due to the acceptable post-thawed sperm motility and viability recorded, cryopreservation of Achhami semen is hereby recommended so as to preserve the Achhami breed. For further validation, the fertility will be observed from the produced frozen semen.