• Korean Journal of Animal Reproduction
• /
• v.12 no.3
• /
• pp.141-147
• /
• 1988

These experiments were carried out to develop new techniques for in vitro separation of x-and Y-bearing spermatozoa. The results obtained in these experiments were summarized as follows: 1. Following centrifugation of discontinuous percoll density gradient, populatin of spermatozoa increased progressively from low to high density. The highest concentration of spermatozoa was observed at the 4th fraction which included 36.6% of spermatozoa. 2. As increasing percoll concentration, the higher motility index was obtained and the highest motility index(74.2) was obtained at the 5th fraction. 3. The percentage of B-body bearing spermatozoa following percoll density gradient centrifugation was decreased from 39.7% to 25.6%. 4. The sperm population following chromatography by sephadex gel and percoll density gradient centrifugation was decreased in 1st, 5th and 6th fractions but the reverse was turn for 2nd, 3rd and 7th fractions, and the highest sperm concentration was observed at the 7th fraction which included 37.4% of spermatozoa. 5. Motility index of spermatozoa was increased from 77.6 to 79.4 after the sephadex gel filtration, however it was decreased at all fractions after percoll density gradient centrifugation. The lowest motility index(33.2) was obtained from the 7th fraction. 6. The rate of B-body bearing spermatozoa was shown the trend to decrease by the sephadex gel filtration and the trend was accelerated by the percoll density gradient centrifugation. The lowest percentage of B-body bearing spermatozoa, 12.0% was obtained from the 5th fraction.

• Asian-Australasian Journal of Animal Sciences
• /
• v.12 no.5
• /
• pp.702-707
• /
• 1999

A percoll density gradient technique was developed for producing high quality turkey semen and improving the fertility by removing deleterious cellular components, including spermiophages, bacteria, abnormal or dead spermatozoa, and other cellular debris. The combination of three different percoll densities, 1.05, 1.07, and 1.08 showed the best resolution and was selected to prepare a discontinuous percoll density gradient to obtain healthy spermatozoa from semen smples. Bacteria, spermiophages, and abnormal or dead spermatozoa were detected from the density range from 1.05, 1.05 to 1.07, and 1.07 to 1.08, respectively. Healthy spermatozoa were collected from the density greater than 1.08. Spermatozoa obtained from percoll density gradient centrifugation showed better sperm motility than those from unprocessed pooled semen. Bacteria including Escherichia coli, Staphylococcus aureus, and Proteus spp., were predominant contaminants in turkey semen, and the numbers of cells were approximately $5{\times}10^5$ to $1{\times}10^9cfu/ml$. The overall fertility rates in hens inseminated with processed percoll density gradient were higher than those in hens with unprocessed semen especially for unhealthy sperm. In conclusion, semen quality can be improved by percoll density gradient centrifugation, which augmented the fertility of turkey breeders.

• The Korean Journal of Food And Nutrition
• /
• v.8 no.4
• /
• pp.307-317
• /
• 1995

The purity of mtDNAs isolated from Archicitrus and Metacitrus leaves was higher in percoll density gradient centrifugation than differential and sucrose density gradient centrifugation. The most clear mtDNAs were obtained from mitochondria included in the Interface band of between 21% and 45% under isomotic, low viscosity conditions in the three step discontinuous percoll density gradient centrifugation. DNase treatment to the crude mitochondrlal suspension still more increased purity of mtDNA by the effective removal of the nuclear and chloroplast DNA and mtDNAs were appeared as a single band at middle position of tube by EtBr /cscl density gradient centrifugation. Agarose gel electrophoresis of mtDNAs resolved a single, broad band containing high molecular weight DNAs In all preparation. Yield of mtDNAs was about 110 and 2 ug Per 2009 in mature and immature leaves respectively. The mtDNA fragment patterns showed by EcoR I treatment were indistinguishable with respect to nom bet and position of bands in Archicitrus and Metacitrus. In the pattern of Hind E restriction, the Metacitrus displayed the unique band between 5.0 and 4.0kb, in addition to four fragments about 5.0, 2.4, 2.15, and 2.0kb, respectively, different from Archicitrus. Also the pattern of total mtDNAs fragment by the treatment of Pst I showed that the distinguishable fragment pat tern was not appeared in Archicitrus(C. iyo Tanaka), but about 6.0, 5.5, 5.0 and 2.Bkb fragments were appeared only in Metacitrus(C. junos Sieb). Therefore it was indicated that two species in intra-subgenus were identical each other, whereas considerable difference was revealed for inter-subgenus.

• Clinical and Experimental Reproductive Medicine
• /
• v.46 no.1
• /
• pp.8-13
• /
• 2019

Objective: Density gradient centrifugation (DGC) is frequently used to isolate high-motility fractions of spermatozoa. We compared the efficacy of four DGC media in terms of the percentage of morphologically normal spermatozoa, DNA fragmentation level, and hyaluronic acid (HA) binding ability. Methods: Thirty men with a total motile spermatozoa count > 80 million participated. Semen samples were divided into four aliquots, which were processed using PureSperm, PureCeption, Sidney, and SpermGrad media, respectively. The DNA fragmentation level was measured using the Halosperm assay kit and HA binding ability was measured using the HBA assay kit. Results: The mean percentage of morphologically normal spermatozoa was significantly enhanced after DGC using all four media (10.3%, 9.9%, 9.8%, and 10.7%, respectively; p< 0.05 for each when compared with 6.9% in raw semen). The DNA fragmentation level was significantly reduced after DGC using PureSperm, PureCeption, and SpermGrad media (6.0%, 6.5%, and 4.9%, respectively; p< 0.05 for each when compared with 11.2% in raw semen), but not after DGC using Sidney media (8.5%, p> 0.05). HA binding ability did not change after DGC using any of the four media. Conclusion: The four media were equally effective for obtaining a sperm fraction with highly motile, morphologically normal sperm. PureSperm, PureCeption, and SpermGrad media were equally effective for acquiring a sperm fraction with less DNA fragmentation.

• Journal of Life Science
• /
• v.19 no.6
• /
• pp.728-734
• /
• 2009

In this work, a simple, inexpensive and reproducible technique of flotation density gradient centrifugation was developed to isolate monocytes with high purity and yield from peripheral blood mononuclear cells (PBMC) using Histopaque solution, density and osmolarity of which were modified to 1.072 g/ml and 335 mOsm with phosphate-buffered saline (PBS) and sodium chloride (NaCl) solution, respectively. The average purity of monocytes was 74.75${\pm}$3.84%, with the individual purity ranging from 71.44% to 82.38%. The average yield of monocytes was 32.62${\pm}$11.16%, with the individual yields ranging from 21.02 to 53.63%. The monocytes isolated by floatation density gradient centrifugation could be successfully cultured into morphologically, phenotypically and functionally dendritic cells in vitro. In conclusion, the entire procedure seemed to be faster and more convenient, simple and cost-effective than other monocyte isolation methods, including plastic adherence and density gradient methods, and has the potential to be developed as a closed system for clinical scale generation of dendritic cells.

• Korean Journal of Microbiology
• /
• v.30 no.6
• /
• pp.524-527
• /
• 1992

Synechococcus sp. cyanophage was isolated from Baekwoon reservoir located in KyonggiDo. The cyanophage was purified by employing ultrafiltration. differential centrifugation. and sucrose density gradient centrifugation. Electron microscopic observation indicated that the sizes of its isometric head and contractile tail are 89 nm and] II nm. respectively. which means that the isolated cyanophage is included in the group. Myoviridae. The cyanophage maintained the stability of more than 50 percent from $20^{\circ}C$ to $40^{\circ}C$ and from pH 5 to 8. and had the maximal infectivity at $30^{\circ}C$ and pH 9 implying its ecological significance.

• The Journal of the Korean Society for Microbiology
• /
• v.22 no.3
• /
• pp.219-224
• /
• 1987

A strain of Leptospira interrogans(UM-19) was isolated in 1984 from a patient with epidemic pulmonary hemorrhagic fever. The bacteria has been characterized morphologically by a polymorphism showing spiral forms, short and long rods in culture. In order to study the biological relatedness among these forms of the bacteria, the short rods were separated from others by means of continuous sucrose density gradient centrifugation, and cultured at various temperatures such as $10^{\circ}C$, $15^{\circ}C$, $30^{\circ}C$ and $37^{\circ}C$. It was revealed that cultivation or subcultivation of the short rods has resulted in morphological changes observed by darkfield examination, silver stain, and scanning electron microscope showing spiral forms as well as short and long rods.

• Clinical and Experimental Reproductive Medicine
• /
• v.44 no.4
• /
• pp.201-206
• /
• 2017

Objective: The aim of this study was to compare the efficacy of swim-up and density gradient centrifugation (DGC) for reducing the amount of sperm with fragmented DNA, sex chromosome aneuploidy, and abnormal chromatin structure. Methods: Semen samples were obtained from 18 healthy male partners who attended infertility clinics for infertility investigations and were processed with swim-up and DGC. The percentages of sperm cells with fragmented DNA measured by the sperm chromatin dispersion test, normal sex chromosomes assessed by fluorescence in situ hybridization, and abnormal chromatin structure identified by toluidine blue staining were examined. Results: The percentage of sperm cells with fragmented DNA was significantly lower in the swim-up fraction (9.7%, p= 0.001) than in the unprocessed fraction (27.0%), but not in the DGC fraction (27.8%, p= 0.098). The percentage of sperm cells with normal X or Y chromosomes was comparable in the three fractions. The percentage of sperm cells with abnormal chromatin structure significantly decreased after DGC (from 15.7% to 10.3%, p= 0.002). The swim-up method also tended to reduce the percentage of sperm cells with abnormal chromatin structure, but the difference was not significant (from 15.7% to 11.6%, p= 0.316). Conclusion: The swim-up method is superior for enriching genetically competent sperm.

• Clinical and Experimental Reproductive Medicine
• /
• v.42 no.1
• /
• pp.22-29
• /
• 2015

Objective: Sperm must be properly prepared in in vitro fertilization (IVF)-embryo transfer (ET) programs in order to control the fertilization rate and ensure that embryos are of high quality and have appropriate developmental abilities. The objective of this study was to determine the most optimal sperm preparation method for IVF. Methods: Patients less than 40 years of age who participated in a fresh IVF-ET cycle from November 2012 to March 2013 were included in this study. Poor responders with less than three mature oocytes were excluded. Ham's F-10 medium or sperm-washing medium (SWM) was used in combination with the density-gradient centrifugation/swim-up (DGC-SUP) or SUP methods for sperm preparation. A total of 429 fresh IVF-ET cycles were grouped according to the media and methods used for sperm preparation and retrospectively analyzed (DGC-SUP/Ham's F-10, n=82; DGC-SUP/SWM, n=43; SUP/Ham's F-10, n=181; SUP/SWM, n=123). Results: There were no significant differences among these four groups with respect to the mean age of the female partners, duration of infertility, number of previous IVF cycles, and retrieved oocytes. We determined that both the DGC-SUP and SUP methods for sperm preparation from whole semen, using either Ham's F-10 or SWM media, result in comparable clinical outcomes, including fertilization and pregnancy rates. Conclusion: We suggest that both media and both methods for sperm preparation can be used for selecting high-quality sperm for assistive reproductive technology programs.

• Journal of the Korean Society of Tobacco Science
• /
• v.11 no.2
• /
• pp.233-240
• /
• 1989

This experiment was carried out to select of heterokaryon based on the different buoyant densities of protoplasts. Protoplats were isolated from cultured cells (calli) of Nicotiana tobacum(cv. BY4) and from mesophyll cells of N. glauca. The two types of protoplats were fractionated by centrifugation in an iso-osmotic (770 mOs/kg. H2O) density gradients condition. Major difference in the buoyant density exists between two types of protoplasts isolated from different cells. The mesophyll protoplasts were fractionated in the higher gradient interphases than that of callus protoplasts. The two types of fractionated protoplasts were fused with 40% polyethylene glycol (PEG), and the protoplasts treated with PEG were separated by centrifugation in the same density gradients condition. The heterokaryons were fractionated in the intermediate density gradients.