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Cryopreservation
Kinds of Cryopreservation Terms modified by Cryopreservation Selected AbstractsCRYOPRESERVATION OF THE GAMETOPHYTIC CELLS OF LAMINARIALES (PHAEOPHYTA) IN LIQUID NITROGEN,JOURNAL OF PHYCOLOGY, Issue 3 2004Kazuyoshi Kuwano The gametophytic cells of six species of Laminariales, Laminaria japonica Areschoug, L. longissima Miyabe, Kjellmaniella crassifolia Miyabe, Ecklonia stolonifera Okamura, E. kurome Okamura, and Undaria pinnatifida (Harvey) Suringar, were subjected to cryopreservation in liquid nitrogen. The cells were suspended in various cryoprotective solutions and slowly cooled to ,40°C over a period of 4 h. After this slow cooling step, the suspensions were immediately immersed in liquid nitrogen. All the species of Laminariaceae used in the present study survived maximally in a mixture of ethylene glycol and proline. On the other hand, the gametophytic cells of Undaria pinnatifida, a member of the Alariaceae, survived maximally in the mixture of glycerol and proline. The viability of the thawed gametophytic cells decreased during postthawing incubation. The decrease in viability continued for 4,6 days, and the minimum levels ranged from 36.2% to 67.2%. After 4,6 days of incubation, the percentage viability of all strains began to increase due to the renewal of cell division. [source] Cryopreservation of semen from a stallion with seminal vesiculitisEQUINE VETERINARY EDUCATION, Issue 5 2010L. C. Fennell Summary A 6-year-old Warmblood stallion was admitted for semen collection and cryopreservation. On the seventh and subsequent collection days semen samples were contaminated with purulent debris. A diagnosis of seminal vesiculitis was made following ultrasonography and endoscopy of the seminal vesicles. The stallion was treated with systemic and topical antimicrobial therapy and, although this did not cure the condition, subsequent ejaculates were suitable for cryopreservation. [source] Cryopreservation of fish sperm: applications and perspectivesJOURNAL OF APPLIED ICHTHYOLOGY, Issue 5 2010E. Cabrita Summary Cryopreservation is of interest not only for fish farming but also for the conservation and genetic improvement of resources. This technique has been well established in some freshwater fish species mainly, salmonid, sturgeons and carps, however, only in the last decade research was focused in marine fish species. The benefits of sperm cryopreservation include: (i) synchronization of gamete availability of both sexes, (ii) sperm economy; (iii) simplification of broodstock management, (iv) transport of gametes from different fish farms, and (v) germplasm storage for genetic selection programs or conservation of species. These issues would certainly benefit the aquaculture industry. The tremendous impact that biotechnology is having in aquaculture has been particularly obvious in recent years. Several species are being used as research models not only for aquaculture development applications but also for medical research. Sperm cryopreservation can give an important contribution in the germ storage of all transgenic lines. However, in all applications in fish sperm, cryopreservation needs to overcome a lack in standardization of methodologies and procedures, a correct assay of seminal quality and the development of tools to characterize cryoinjury. Many efforts have recently been made in the study of DNA using different approaches such as the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labelling), SCSA (sperm chromatin structure assay) and the analysis of specific DNA sequences using RT-PCR, since DNA damage may impair fertility or embryo development. Cryopreservation of gametes would certainly benefit from a higher concern on male improvement, basically through nutrition or selection of resistant stocks (e.g. stress resistant individuals or highly adapted to captivity) producing gametes of higher quality. There is a huge window of opportunities for improve the resistance of cells to cryopreservation through diet supplementation of certain compounds such as amino acids (taurine and hypotaurine), vitamins (Vit. E and C) and lipids or through a direct supplementation of the extender media. An equilibrium of those compounds will improve spermatozoa and seminal plasma composition protecting cells against oxidative stress (lipid peroxidation, protein oxidation, DNA fragmentation, enzyme protection) that is gaining each day more importance in cryodamage research. [source] Improved Cryopreservation of Sperm of Paddlefish (Polyodon spathula)JOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 4 2006ÁKos Horváth Experiments were performed to improve protocols for sperm cryopreservation of paddlefish (Polyodon spathula), a species for which there has been limited study. The first experiment was conducted to investigate the effects of two extenders (modified Tsvetkova's extender: mT and modified Hanks' balanced salt solution: mHBSS) in combination with methanol (MeOH) and dimethyl sulfoxide in two concentrations (5 and 10%) on the postthaw motility and fertilization rates of cryopreserved sperm. The highest postthaw motility (85 ± 5%) was observed when sperm were frozen using mT extender with 10% MeOH as cryoprotectant. Extenders (P = 0.0018) and cryoprotectants (P = 0.0040) each had a significant effect on the postthaw motility of paddlefish sperm. The highest fertilization (80 ± 3%) was found when eggs were fertilized with sperm frozen with mT extender in combination with 10% MeOH. However, there was no significant difference among fertilization rates when MeOH was used as a cryoprotectant in either concentration or in combination with either mT or mHBSS extenders. In the second experiment, 4000 eggs were fertilized with the pooled contents of five straws of thawed sperm (total volume of 1.25 mL) using mT extender in combination with 5% MeOH, and hatch rates as high as 79 ± 5% were observed. A third experiment was also conducted to clarify the role of MeOH concentration; however, no significant difference was found among fertilization and hatch rates when either 5 or 10% MeOH was used as a cryoprotectant. These results suggest that MeOH is a safe and reliable cryoprotectant for freezing of paddlefish sperm and obtaining viable postthaw sperm for consistent fertilization and hatch rates. Further, this experimental protocol is relatively simple and applicable for commercial hatchery production of paddlefish. [source] Cryopreservation of YamúBrycon siebenthalae MiltJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 4 2004Pablo E. Cruz-Casallas The yamúBrycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher (P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control (P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO (P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 109 spermatozoa per g of eggs was higher (P <0.05) than with 1.6 × 109 spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 109 and 6.4 × 109 spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 109 and 6 × 109 spermatozoa per g of fresh eggs. [source] Cryopreservation of primary human hepatocytes: The benefit of trehalose as an additional cryoprotective agentLIVER TRANSPLANTATION, Issue 1 2007Ekaterina Katenz Problems with the limited availability of human hepatocytes for cell transplantation may be overcome by efficient cryopreservation techniques and formation of appropriate cell banking. In this study we investigated the effect of the disaccharide trehalose on the cryopreservation of human hepatocytes. For analysis, liver cells were frozen in culture medium containing 10% dimethyl sulfoxide (DMSO) that was supplemented with varying concentrations of trehalose. During the postthawing culture period, viability, plating efficiency, total protein, cell proliferation, enzyme leakage, albumin and urea formation, as well as phase I and II metabolism were analyzed. In the pilot study, among the concentrations investigated, 0.2 M trehalose showed the best overall outcome. Compared to the use of DMSO alone, we found significant improvement in postthaw cell viability (62.9 ± 13 vs. 46.9 ± 11%, P < 0.01) and plating efficiency (41.5 ± 18 vs. 17.6 ± 13%, P < 0.01) in the trehalose group. The use of trehalose as an additive for cryopreserving human hepatocytes resulted in a significantly increased total protein level in the attached cells, higher secretion of albumin and a lower aspartate aminotransferase (AST) level after thawing. In conclusion, the use of trehalose as cryoprotective agent significantly improves the outcome of human hepatocyte cryopreservation. Liver Transpl, 2007. © 2006 AASLD. [source] Morphological changes in mouse embryos cryopreserved by different techniquesMICROSCOPY RESEARCH AND TECHNIQUE, Issue 4 2007A.R.S. Coutinho Abstract Cryopreservation of mammalian embryos is an important tool for the application of reproductive biotechnologies. Subjective evaluation to determine embryo viability is often used. The determination of the best cryopreservation protocol depends on morphological and molecular analysis of cellular injuries. The main objective of this study was to compare two methods of cryopreservation by assessing morphological alterations of frozen embryos using light, fluorescence, and transmission electron microscope. Fresh (control), slow frozen, and vitrified mouse embryos were composed. To evaluate the viability of the embryos, the cell membrane integrity was assessed using Hoechst33342 and propidium iodide (H/PI) staining. Morphological analyses using hematoxylin and eosin (HE) staining were performed to test different techniques (in situ, paraffin, and historesin) by both light and fluorescence microscopy. Transmission electron microscope was used to detect ultrastructural alterations in Spurr- and Araldite-embedded samples. H/PI staining detected more membrane permeability in the vitrification (69.8%) than in the slow freezing (48.4%) or control (13.8%) groups (P < 0.001). Historesin-embedded samples showed to be more suitable for morphological analyses because cellular structures were better identified. Nuclear evaluation in historesin sections showed the induction of pycnosis in slow freezing and vitrification groups. Cytoplasm evaluation revealed a condensation and an increase in eosinophilic intensity (indicating apoptosis) in the slow freezing group, and weakly eosinophilic structures and degenerated cells (indicating oncosis) in the vitrification group (P < 0.05). Ultrastructural analyses confirmed HE morphological findings. It was concluded that both cryopreservation techniques resulted in oncosis and apoptosis injuries. However, vitrification caused more severe cellular alterations and reduced embryonic viability compared to slow freezing. Microsc. Res. Tech., 2007. © 2006 Wiley-Liss, Inc. [source] Cryopreservation of vascularized ovary: An evaluation of histology and function in ratsMICROSURGERY, Issue 5 2008Shijie Qi M.D. Cryopreservation of organs has been investigated to sustain the reproductive function of patients undergoing sterilizing chemotherapy and radiotherapy or reproductive surgery. A modified protocol for whole organ cryopreservation was described and the outcome of cryopreservative ovaries was evaluated, and apoptosis of cryopreservative cells stored for different time period and the viability of cryopreserved cells stored at different temperature was examined in rats. Lewis rat ovarian grafts were perfused for 30 min at 0.35 ml/min with M2 medium containing 0.1M fructose and increasing concentrations of 0,1.5M dimethylsulfoxide, cooled to ,140°C controlled by a computerized program, and stored in liquid nitrogen (,196°C) for 24 hours. After being thawed, ovaries were transplanted to syngeneic recipients after bilateral oophorectomy. Graft functions were monitored postoperatively. The major findings were that: 1) A 100% survival rate of rat ovaries was achieved in this study. Ovarian hormone secretion recovered in 80% rats which had received cryopreservative ovarian grafts. Postoperative serum estradiol levels in the cryopreservative graft group were lower than in the sham surgery control, but much higher than in the bilateral oophorectomy group. 2) Histological examination of cryopreservative ovarian grafts showed preantral and antral follicles. Two gestations were obtained. 3) Estradiol levels remained low in ovariectomized rats while in the oophorectomized rats given cryopreservative ovarian grafts levels started to rise after 14 ± 3 days. 4) The average viability in the cells from cryopreservative ovary organ (,196°C) was about 71 ± 18% compared to 90 ± 9% of fresh cells. This success should encourage further improvement of cryopreservative techniques for large organs. © 2008 Wiley-Liss, Inc. Microsurgery, 2008. [source] Technical Aspects of Laparoscopic Ovarian Autograft in Ewes After Cryopreservation by Slow-Cooling ProtocolREPRODUCTION IN DOMESTIC ANIMALS, Issue 1 2010J Massardier Contents Iatrogenic ovarian failure and infertility are long term-term side effects of anticancerous gonadotoxic treatments in children or women of reproductive year. Ovarian cortex cryopreservation can be a solution to preserve immature germinal cells before gonadotoxic treatment, for later transplantation. The aim of our study was to prove the efficiency of a laparoscopic technique for orthotopic graft after a slow-freezing/thawing protocol, and to evaluate the effect of ovarian cryopreservation and autograft on the primordial follicle survival rate. Experimental surgical study was performed on 6- to 12-month-old ewes. The study was approved by the ethic committee of the Lyon-veterinary-school. The left ovary was removed by laparoscopy and cut in half, and medulla was excised. In group 1 (n = 6), autograft was performed immediately on the right ovary, and in group 2 (n = 6), graft was performed after a slow-freezing/thawing protocol. The second hemi-ovary served as an ungrafted control fragment. A polypropylene/polyglactin mesh was included between graft and base to separate the two structures, to help histological analysis. The mean graft performance time was 71 ± 8 min in the first group and 57 ± 10 min in the second. Freezing did not affect the number of primordial follicles. In the ungraft control fragments, the global anomaly rate (cytoplasm plus nuclear anomaly) increased after freezing (p < 0.05). Others results did not reach signification. Pelvic adhesion occurred only once. The post-graft primordial follicle survival rate was 5.1 ± 2.8% in the non-frozen group vs. 6.3 ± 2.3% after freezing/thawing. Kruskal,Wallis and Wilkoxon non-parametric tests were used for statistical analysis. Laparoscopy seems to be a well-adapted technique for ovarian tissue orthotopic autograft. The main follicle loss occurs before graft revascularization. Our orthotopic graft's procedure has to be improved to obtain a better graft's neovascularization, and to have a better long-term post-graft primordial follicle survival rate. [source] Sperm Cryopreservation in Brown Bear (Ursus arctos): Preliminary AspectsREPRODUCTION IN DOMESTIC ANIMALS, Issue 2008L Anel Contents The development of sperm cryopreservation procedures in brown bear is the basis for establishing a specific genetic resource bank aimed at the preservation of a Cantabric brown bear population, which is seriously threatened. Several issues complicate the development of these cryopreservation procedures: lack of previous specific studies, a high incidence of urospermia and spermagglutination observed in bear ejaculates. Moreover, the availability of individuals for research from these threatened populations is problematic. In the case of the Cantabric brown bear, we have used males from other populations, but of the same species, as surrogates, to carry out a direct extrapolation of the results. Urospermia , Moreover, 70% of the ejaculates are urine contaminated and spermagglutination have a detrimental effect on post-thawing cell quality recovery in this species. Considering the high value of these samples (autochthonous population with few individuals), a pre-selection of the ejaculates is not a viable alternative. Preventive methods reducing the mentioned detrimental effects need to be developed. On the basis of previous data, we can suppose that bear spermatozoa resist freezing injuries well. Nevertheless, because of the scarcity of this information, it is necessary to conduct further research on bear semen freezing under field conditions. Epidydimal spermatozoa can be important for genetic resource banking of threatened populations and thus specific cryobiological protocols need to be assayed. To date, 168 brown bear ejaculates have been frozen by the ITRA-ULE group at the University of León (Spain) in the development of methodologies for the preservation of brown bear sperm. [source] Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct SubpopulationsREPRODUCTION IN DOMESTIC ANIMALS, Issue 4 2007J Rubio-Guillén Contents Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30°C in a skim milk,egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser® (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure. [source] OC1 Effects of Cryopreservation on the Expression of Glut-3, Glut-5 and As-A Proteins in Iberian Boar Sperm MembranesREPRODUCTION IN DOMESTIC ANIMALS, Issue 2006S Sancho In order to determine the injure produced in boar spermatozoa through cryopreservation process, we analyzed the expression of the hexose transporters Glut-3 and Glut-5 and the zona pellucida binding protein As-A (P68) in three different steps of the freezing-thawed protocol: at 17°C (fresh BTS-diluted semen, 1 : 2 v/v, step 1), at 5°C (after glycerol addition; step 2), and post-thawing (step 3). All sperm analyses were carried out with immunogold techniques under electronic microscopy. For this study eight healthy post-pubertal Iberian boars were submitted to a collection of twice per week through 3 months, evaluating two ejaculates from each boar. Glut-3 maintains the expression in the acrosome region post-thawing but not along the tail where is reduced. The expression of Glut-5 and As-A is majority located at the post-acrosome region of the spermatozoa at step 1, but in step 2 and step 3 this expression is relocated to sperm tail area. In conclusion, while cryopreservation affects the localization and the expression of Glut-3 and Glut-5, its fertilizing capacity is not significantly reduced. The stabilization of boar semen at 5°C was found to be the most crucial step for sperm survival. [source] Cryopreservation of Black Bengal buck semen: Effects of diluents and freezing on sperm motility and morphologyANIMAL SCIENCE JOURNAL, Issue 5 2008Sadia AFROZ ABSTRACT Semen from Black Bengal bucks was collected to establish a cooling protocol (to ,196°C) for buck semen preservation, and to study the effect of freezing on sperm motility and morphology. Semen was diluted with diluents (Triladyl & Tris) and cryoprotectants, filled into straws, sealed, cooled (to 5°C) and equilibrated. After dilution, motility ranged from 75.00% to 76.67% and from 73.33% to 80.00% in Triladyl and Tris diluents, respectively. Motility of sperm after cooling to 5°C in Triladyl and Tris diluents ranged from 65.00% to 66.67% and from 63.33% to 70.00%, respectively. After equilibration in straws, the semen was subjected to a freezing protocol in a computer-controlled biofreezer CL-3000 (cooling at 10°C per minute, from 5°C to ,80°C) and plunged into liquid nitrogen. Sperm motility of re-thawed semen varied from 38.33% to 43.33% and from 6.00% to,6.67% in Triladyl and Tris diluents, respectively. Sperm morphology of re-thawed semen was studied and head damage or cryoinjury was found in 2,3% of sperm in Triladyl diluents and 3,6% in Tris diluents. Whether the differences of sperm motility and head damage reflect fertility after artificial insemination is yet unknown and needs to be studied further. [source] Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservationANNALS OF APPLIED BIOLOGY, Issue 3 2009Q.C. Wang Abstract Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (,196°C), and is considered as an ideal means for long-term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana (Musa spp.), Citrus spp., grapevine (Vitis vinifera), Prunus spp., raspberry (Rubus idaeus), potato (Solanum tuberosum) and sweet potato (Ipomoea batatas) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing ,citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy. [source] Cryopreservation of isolated blastomeres and embryonic stem-like cells of Leopard danio, Brachydanio frankeiAQUACULTURE RESEARCH, Issue 4 2010Padmanav Routray Abstract This study aimed at developing a suitable cryopreservation protocol for embryonic stem (ES)-like cells of a tiny freshwater fish Leopard danio (Brachydanio frankei). Embryonic stem (ES)-like cells derived from blastomeres of the early blastulae stage of the developing embryo were cultured in vitro in a medium containing Leibowitz-15 supplemented with 10% foetal bovine serum, leopard danio embryo extract, sodium bicarbonate, sodium selenite, basic fibroblast growth factor, epidermal growth factor and leukaemia inhibitory factor. The ES-like cells showed properties similar to ES cells in other species. They were morphologically small, round to polygonal and present in patches and extensively expressed alkaline phosphatase and stage-specific embryonic antigen. The toxicity and chilling sensitivity of these cells were determined using ethylene glycol (EG), propylene glycol (PG) and glycerol as cryoprotective agents at molar concentrations of 0.6, 1.0, 1.4, 1.8 and 2.0. Among them, 1.8 M EG showed 70% significant viable ES-like cells (P<0.05). The post-thawed cells retained similar properties of non-cryopreserved ES-like cells with a viability rate of 65%. Similarly, blastomeres cryopreserved following the slow cooling rate with EG and PG yielded a viability of more than 70%. [source] Cryopreservation of silver barb Puntius gonionotus (Bleeker) spermatozoa: effect of extender composition, cryoprotective agents and freezing rate on their postthawing fertilization abilityAQUACULTURE RESEARCH, Issue 15 2008Padmanav Routray Abstract The effects of extender composition, cryoprotectant concentration and freezing and thawing on the fertilization efficiency of cryopreserved spermatozoa of Puntius gonionotus were evaluated. Computer-aided motility analysis of semen was conducted to check the suitability of spermatozoa for cryopreservation after mixing with different extenders and cryoprotective agents (CPAs). Extender-4 with an osmolality 260 mOsmol kg,1and pH 7.6 was used for the cryopreservation study. Among the CPAs, dimethyl sulphoxide (DMSO) was least toxic and more than 60% fertilization was achieved when used at 1.4 M at 0 °C for 10 and 30 min, whereas the toxicity of all CPAs to spermatozoa was evident when tested at 30 °C. Semen frozen at ,16 °C min,1 with 1.4 M DMSO showed 70% fertilization, which was significantly higher (P<0.05) than other freezing rates. Samples thawed at 35 °C water showed a fertilization rate comparable with that of fresh semen. Computer-assisted semen analysis of fresh and frozen semen after thawing showed variations in different types of motility in spermatozoa and in their class. There was no significant difference in motility before or after cryopreservation; however, significant differences could be observed in the average path velocity (VAP), straight line velocity (VSL) and curve linear velocity (VCL). Semen of silver barb could be cryopreserved with extender-4 by addition of 1.4 M DMSO to a final cryopreservation medium (MED 2) cooled at a rate of ,16 °C min,1, stored in liquid nitrogen (,196 °C) and utilized after thawing at 35±2 °C. [source] Cryopreservation of black ear catfish, Pangasius larnaudii, (Bocourt) spermAQUACULTURE RESEARCH, Issue 9 2006Samorn Kwantong No abstract is available for this article. [source] Cryopreservation of sperm in marine fishAQUACULTURE RESEARCH, Issue 3 2000M. Suquet Since the first work of Blaxter in 1953, fish sperm cryopreservation has been attempted on about 30 marine species. The present paper reviews the techniques used and the results published in these species. Particular attention is paid to the handling procedure of sperm before freezing, the problems of semen ageing and semen contamination with urine. The quality of frozen,thawed semen was evaluated using previously standardized biotests, such as a two-step motility activation technique adapted for the different species and fertilization assays using a discriminating insemination technique. Most extenders used in marine fish are saline or sugar solutions. From the investigated cryoprotectants, dimethyl sulphoxide (DMSO) generally leads to the best results. Cooling rates range from 8 °C to 99 °C min,1; the thawing rate is generally high. Compared with freshwater species, a high percentage of spermatozoa survives cryopreservation. Therefore, and because of the simplicity of the techniques, the cryopreservation of marine fish sperm is suited for application in aquaculture. [source] Cryopreservation of Fibroblasts Immobilized Within a Porous Scaffold: Effects of Preculture and Collagen Coating of Scaffold on Performance of Three-Dimensional CryopreservationARTIFICIAL ORGANS, Issue 7 2010Hirotoshi Miyoshi Abstract As a preliminary investigation to establish a cryopreservation method suited for bioartificial livers (BALs), three-dimensional (3-D) cryopreservation experiments with fibroblasts were performed, in which the cells were firstly seeded into a porous scaffold, and the scaffold containing the cells was then cryopreserved. After thawing, 65% of the initially applied cells were still attached to the scaffold, and this efficiency was significantly higher than that in the control experiments (39%), in which fibroblasts cryopreserved in a suspension were seeded into the scaffold. This higher efficiency was mainly caused by higher immobilization efficiency at the time of cell seeding (83%) than in the controls (54%). Collagen coating of the scaffold in the 3-D cryopreservation enhanced immobilization efficiency at the time of cell seeding, and 1-day precultures before the 3-D cryopreservation considerably improved cell growth after thawing. From these favorable results, this 3-D cryopreservation method may become useful for developing BALs. [source] Effects of Cryopreservation and Hypothermic Storage on Cell Viability and Enzyme Activity in Recombinant Encapsulated Cells Overexpressing Alpha-L-IduronidaseARTIFICIAL ORGANS, Issue 5 2010Fabiana Quoos Mayer Abstract Here, we show the effects of cryopreservation and hypothermic storage upon cell viability and enzyme release in alginate beads containing baby hamster kidney cells overexpressing alpha-L-iduronidase (IDUA), the enzyme deficient in mucopolysaccharidosis type I. In addition, we compared two different concentrations of alginate gel (1% and 1.5%) in respect to enzyme release from the beads and their shape and integrity. Our results indicate that in both alginate concentrations, the enzyme is released in lower amounts compared with nonencapsulated cells. Alginate 1% beads presented increased levels of IDUA release, although this group presented more deformities when compared with alginate 1.5% beads. Importantly, both encapsulated groups presented higher cell viability after long cryopreservation period and hypothermic storage. In addition, alginate 1.5% beads presented higher enzyme release after freezing protocols. Taken together, our findings suggest a benefic effect of alginate upon cell viability and functionality. These results may have important application for treatment of both genetic and nongenetic diseases using microencapsulation-based artificial organs. [source] Cryopreservation and in Vitro Expansion of Chondroprogenitor Cells Isolated from the Superficial Zone of Articular CartilageBIOTECHNOLOGY PROGRESS, Issue 1 2005Juan M. Melero Martin Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 ,L FCS cm,2 h,1 was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 × 105 cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 × 105 cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-,1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time. [source] Feasibility of commercial interferon-,-based methods for the diagnosis of latent Mycobacterium tuberculosis infection in Finland, a country of low incidence and high bacille Calmette,Guérin vaccination coverageCLINICAL MICROBIOLOGY AND INFECTION, Issue 8 2007T. Tuuminen Abstract The performances of the QuantiFERON-TB Gold in Tubes (QFGT), T SPOT-TB (ELISPOT) and the Mantoux test were compared for the diagnosis of latent tuberculosis infection in Finland, a country of low tuberculosis incidence. In Cohort A (16 students), freshly isolated peripheral blood mononuclear cells (PBMCs), and in Cohort B (21 school children), cryopreserved PBMCs, were used for the ELISPOT assay. Cryopreservation of cells in fetal calf serum, but not in serum-free medium, produced false-positive results. Discrepancies between the results of the assays were observed. It was concluded that the accuracy of these ex-vivo methods needs additional evaluation. [source] DNA damage and repair measurements from cryopreserved lymphocytes without cell culture,A reproducible assay for intervention studiesENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 7 2006Jyh-Lurn Chang Abstract Single-cell gel electrophoresis (the Comet assay) can be used to measure DNA damage and DNA repair capacity (DRC). However, to test DRC of cryopreserved lymphocytes, published methods include steps for cell culturing and phytohemagglutinin stimulation, which may limit use of this assay in intervention studies. We developed a modified Comet assay protocol that allows us to measure DRC from cryopreserved lymphocytes without these in vitro manipulations. Assay reproducibility was evaluated by performing the assay six times on different dates using six aliquots from one blood draw of one individual. The interindividual variation was assessed by performing the assay using one aliquot from six individuals. When ,-irradiation was used as the mutagen, intra-assay coefficients of variation (CVs.) for baseline DNA damage, damage after ,-irradiation exposure, and DRC,measured as tail moment,were 8, 31, and 10%, respectively. Interindividual CVs. were higher. When H2O2 was used as the mutagen, intra-assay CVs. for damage measurements were lower for a protocol modification that included damage and repair at 37°C (CVs. ranging from 8 to 35%) than for the more standard 4°C protocol. Analyzing moment arm,the average distance of DNA migration within the tail,yielded similar results. DNA repair was successfully detected in each experiment. Comparing freshly isolated lymphocytes to cryopreserved lymphocytes from the same individuals' blood draw indicated that DRC was highly correlated when determined using moment arm values. This modified protocol extends the use of the Comet assay to measuring DRC in intervention studies (e.g., dietary interventions) in that it assesses cellular response after cryopreservation without cell culture or other extensive manipulation. Environ. Mol. Mutagen., 2006. © 2006 Wiley-Liss, Inc. [source] Cryopreservation of semen from a stallion with seminal vesiculitisEQUINE VETERINARY EDUCATION, Issue 5 2010L. C. Fennell Summary A 6-year-old Warmblood stallion was admitted for semen collection and cryopreservation. On the seventh and subsequent collection days semen samples were contaminated with purulent debris. A diagnosis of seminal vesiculitis was made following ultrasonography and endoscopy of the seminal vesicles. The stallion was treated with systemic and topical antimicrobial therapy and, although this did not cure the condition, subsequent ejaculates were suitable for cryopreservation. [source] Gonadal dysfunction in male cancer patients before cytotoxic treatmentINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 1 2010Niels J. Van Casteren Summary Male patients diagnosed with cancer are often referred for semen cryopreservation before gonadotoxic treatment but often have low semen quality. The aim of this study was to evaluate which type of cancer affects gonadal function and proposes a risk factor for low pre-treatment semen quality. Between January 1983 and August 2006, 764 male cancer patients were referred for semen cryopreservation prior to chemotherapy and radiotherapy. We compared semen characteristics and reproductive hormones between different groups of cancer patients. In addition, we evaluated the role of tumour markers in patients with testicular germ-cell tumours (TGCT) on fertility. Abnormal semen parameters were found in 489 men (64%) before cancer treatment. Patients with TGCT and extragonadal germ-cell tumours had significantly lower sperm concentrations and inhibin B levels than all other patient groups. No semen could be banked in 93 patients (12.2%). Eight hundred and thirty-nine of 927 (90%) produced semen samples were adequate for cryopreservation. Inhibin B in all groups showed to be the best predictor of semen quality. Although pre-treatment raised tumour markers were associated with a decrease in inhibin B and increased follicle stimulating hormone, both predictive for low semen quality; no direct linear association could be found between raised beta-HCG, alfa-fetoprotein and semen quality. Only 1/3 of cancer patients had normal semen parameters prior to cancer treatment. Patients with TGCT and extragonadal GCT have the highest risk for impaired semen quality and gonadal dysfunction at the time of semen cryopreservation. [source] Penile vibratory stimulation and electroejaculation in the treatment of ejaculatory dysfunction,INTERNATIONAL JOURNAL OF ANDROLOGY, Issue 6 2002JENS SØNKSEN Summary The purpose of this review is to present the current understanding of penile vibratory stimulation (PVS) and electroejaculation (EEJ) procedures and its clinical use in men with ejaculatory dysfunction. Unfortunately, the record of treating such individuals has been quite poor, but within recent years development and refinement of PVS and EEJ in men with spinal cord injury (SCI) has significantly enhanced the prospects for treatment of ejaculatory dysfunction. The majority of spinal cord injured men are not able to produce antegrade ejaculation by masturbation or sexual stimulation. However, approximately 80% of all spinal cord injured men with an intact ejaculatory reflex arc (above T10) can obtain antegrade ejaculation with PVS. Electroejaculation may be successful in obtaining ejaculate from men with all types of SCI, including men who do not have major components of the ejaculatory reflex arc. Because vibratory stimulation is very simple in use, non-invasive, it does not require anaesthesia and is preferred by the patients when compared with EEJ, PVS is recommended to be the first choice of treatment in spinal cord injured men. Furthermore, EEJ has been successfully used to induce ejaculation in men with multiple sclerosis and diabetic neuropathy. Any other conditions which affect the ejaculatory mechanism of the central and/or peripheral nervous system including surgical nerve injury may be treated successfully with EEJ. Finally, for sperm retrieval and sperm cryopreservation before intensive anticancer therapy in pubertal boys, PVS and EEJ have been successfully performed in patients who failed to obtain ejaculation by masturbation. Nearly all data concerning semen characteristics in men with ejaculatory dysfuntion originate from spinal cord injured men. Semen analyses demonstrate low sperm motility rates in the majority of spinal cord injured men. The data give evidence of a decline in spermatogenesis and motility of ejaculated spermatozoa shortly after (few weeks) an acute SCI. Furthermore, it is suggested that some factors in the seminal plasma and/or disordered storage of spermatozoa in the seminal vesicles are mainly responsible for the impaired semen profiles in men with chronic SCI. Home insemination with semen obtained by penile vibratory and introduced intravaginally in order to achieve successful pregnancies may be an option for some spinal cord injured men and their partners. The majority of men will further enhance their fertility potential when using either penile vibratory or EEJ combined with assisted reproduction techniques such as intrauterine insemination or in-vitro fertilization with or without intracytoplasmic sperm injection. [source] Effect of pentoxifylline on motility and membrane integrity of cryopreserved human spermatozoaINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2002P. Stanic The purpose of this study was to examine the effects of pentoxifylline used before and after semen cryopreservation,thawing on sperm motility and membrane integrity. Twenty-four semen samples were split into four equal aliquots. Aliquots were incubated at 37 °C for 30 min, followed by cryopreservation with TEST-yolk freezing medium using slow programmable freezing protocol. After 2 weeks the sperm samples were thawed, washed twice in Quinn's Sperm Washing Medium (modified HTF with 5.0 mg/mL Human Albumin) and incubated at 37 °C for 30 min. Aliquots were treated by adding 3 mmol/L pentoxifylline to: (1) fresh sperm samples during incubation period prior to cryopreservation, (2) sperm samples as a supplement to the cryoprotectant prior to cryopreservation, and (3) thawed sperm samples during incubation period. One aliquot received no treatment (control group). The addition of 3 mmol/L pentoxifylline to fresh semen during incubation period prior to cryopreservation significantly decreased progressive and total motility compared with controls. However, the addition of 3 mmol/L pentoxifylline to cryopreserved semen after thawing significantly increased progressive and total motility compared with controls. After post-thaw, no differences in motion characteristics between sperm samples treated by adding 3 mmol/L pentoxifylline as a supplement to the cryoprotectant and control groups were observed. Post-thaw hypoosmotic swelling (HOS) test scores did not improve with the addition of pentoxifylline compared with the control group. It is concluded that pentoxifylline enhanced post-thaw motility of cryopreserved human spermatozoa when added after thawing. No improvement was found by freezing sperm with pentoxifylline. [source] Low cost autologous peripheral blood stem cell transplantation performed in a municipal hospital for a patient with plasma cell leukaemiaINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 3 2002K. Ghosh Autologous peripheral blood stem cell transplantation (PBSCT) is a costly procedure. In India, the cost varies from US$20 000 to 25 000 and most patients cannot afford it. Using several cost-cutting measures, we were able to treat a patient with plasma cell leukaemia by autologous PBSCT. A 42-year-old-male presented with plasma cell leukaemia. He was treated with VAD therapy, followed by high-dose cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) for mobilization of peripheral blood stem cells. The patient was conditioned with high dose melphalan, followed by autologous PBSCT. The procedure was performed in a municipal hospital in which there was no prior experience with stem cell transplantation. Costs were reduced by: (i) using oral medication whenever possible; (ii) having a relative of the patient prepare his food under medical guidance; (iii) starting G,CSF on day 7 rather than on day 1; (iv) short-term storage of the PBSC in an ordinary refrigerator at 4 °C without cryopreservation; (v) infusing a large number of CD34+ cells, which shortened the time to engraftment; (vi) delegating many of the functions of a marrow transplant nurse to a resident physician. The cost of transplantation was thereby reduced to about US$ 6000, with successful engraftment by day +13. The patient remained in remission for 7 months, after which he relapsed and was treated with chemotherapy and electron beam radiation to the skin. [source] Microsurgical vasoepididymostomy with sperm cryopreservation for future assisted reproductionINTERNATIONAL JOURNAL OF UROLOGY, Issue 12 2000Hatsuki Hibi Abstract Background Although obstructive azoospermia is treatable with microscopic seminal reconstruction, the number of patients who choose to undergo vasoepididymostomy is limited because of recent advances in assisted reproductive technology (ART). We attempted to define the outcome of surgical reconstruction in patients with suspected epididymal obstruction and no previous history of vasectomy. Methods We described 40 eligible end-to-side vasoepididymostomy procedures performed on 24 azoospermic patients who had either bilateral or unilateral epididymal obstruction. Results The overall patency rate following surgery was 54% (13/24) and for four patients (17%), natural intercourse resulted in pregnancy. Two pregnancies were initiated with intracytoplasmic sperm injections using frozen sperm collected during vasoepididymostomy. Conclusions In the era of modern ART, microsurgical vasoepididymostomy with cryopreservation of sperm collected during the operation is recommended for patients with epididymal obstructions. [source] Commercial considerations in tissue engineeringJOURNAL OF ANATOMY, Issue 4 2006Jonathan Mansbridge Abstract Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well. [source] | |||||||||||||||||||