• 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).

• Archives of Pharmacal Research
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• v.25 no.4
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• pp.453-456
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• 2002

Tonin(Persicae Semen) is the herb medicine that contains amygdalin as a major ingredient. Amygdalin in water is decomposed into benzaldehyde, HCN, and glucose by emulsin, a hydrolysis enzyme in tonin. A useful and practical method for the optimum extraction condition of amygdalin without enzymatic hydrolysis is required. The extraction yield of amygdalin of natural formula to nin was 0.1 % from crude powders, 1.4% from small pieces, 3.5% from half pieces and 2.4% from whole pieces. The extraction yield of amygdalin of outer shell-eliminated to nin was 0.3% from crude powders, 1.4% from small pieces, and 3.5% from half pieces and whole pieces respectively. The extraction yield of amygdalin was most high when using the size larger than half.

• 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.

• 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 $25{\times}10^6 cells/ml$

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.92-92
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• 2002

Considerable attention has been focused on the cryopreservation of semen and estrus induction in dog, as consequence of poor productivity caused by long anestrus period, in order to enhance the productivity of youngs and to preserve the breeds. The objectives of this study were to evaluate semen quality after cryopreservation and to evaluate the Pregnancy rate after insemination (AI). Fifty infertilie dogs (age 2∼3 years) were selected for the study and divided into three different estrus induction treatment groups. Group 1: dogs (n=15) were given clomifene (0.1 mg/kg) orally for five days at 12 hr intervals. Group 2: dogs (n=15) were given bromocriptine (50 $\mu\textrm{g}$/kg) orally for five days at 12 hr intervals, followed by single injection intravenously of 500 IU GnRH (Group 3, n=20) when pro-estrus occurred. The rates of pregnancy in estrus inducted dogs mated naturally compared to those inseminated artificially with ejaculated fresh semen and frozen-thawed semen. Estrus detection was performed using the method of vaginal smear and confirmed by the plasma progesterone assay. The ejaculated semen to freeze was exposed to a mixture of Tris extender with cryoprotectant (Trisma, 81 mM: TES, 209 mM: citric acid, 6 mM; glucose, 5 mM; glycerol, 8%) and cryopreserved gradually by slow-cooling at 17 cm above the surface of liquid nitrogen (LN$_2$) for 23 min. The motility of frozen-thawed spermatozoa was assessed by phase-contrast microscopy. To assess their viability and acrosome content, spermatozoa were stained with a vital stain and Fluorescence conjugated lectin Pisum Savitum Agglutinin (FITC/PAS), respectively. Pregnancy was confirmed by ultrasonograpy on day 25, 35 and 55 post insemination. The use of fresh semen, the pregnancy rates were observed 66.6, 66.6, 75.0 and 83.3% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. The use of frozen-thawed semen, the pregnancy rates were observed 66.6, 33.3, 50.0 and 60.0% in natural estrus, clomifene induced, bromocriptine induced and a combination of GnRH and bromocriptine, respectively. No difference was observed in the number of offspring produced among natural estrus and treated groups inseminated with fresh or frozen-thawed semen. In conclusion, the pregnancy rate of dogs treated with a combination of GnRH and bromocriptine was more effective than use of clomifene or bromocriptine only. In addition, frozen-thawed semen can be used successfully far artificial insemination in dog.

• 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.

• 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.

• 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.

• 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.

• 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.