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PubMed1985

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

Animals↗

PubMed1982

The survivability of Mycoplasma meleagridis in frozen-thawed turkey semen.

Semen from 15 turkeys was pooled and divided into two groups and diluted with glycerated freezing medium. One group contained naturally occurring levels of Mycoplasma meleagridis while the other was inoculated with a 24-hr culture of the organism. Both groups were frozen under identical conditions. Semen was evaluated for motility and life-dead analysis during various stages of cryopreservation. The number of viable mycoplasmas in both groups was determined prior to and at 1-, 2-, and 6-months frozen storage. The experiment was repeated twice. Semen motility and live-dead evaluation showed no apparent trends outside of the normal decline seen during various stages of cryopreservation. There was no significant decline in M. meleagridis levels in either treatment group in either trial when samples were tested at intervals up to 6 months. The naturally infected semen contained approximately 10(3) cfu/ml, while the inoculated semen had 10(5) cfu/ml. It was concluded that viable numbers of M. meleagridis do not substantially decline in turkey semen during cryopreservation and subsequent thawing. Consideration must be given to potential pathogens in turkey semen cryopreserved for long-term storage.

Animals↗

PubMed1991

Concurrent infection with Leishmania donovani and Leishmania major in a Kenyan patient: clinical description and parasite characterization.

Leishmania isolates aspirated a few months apart from the spleen of an indigenous adult male kala-azar patient from Baringo District, Kenya, were biochemically characterized and compared. The patient lived within a dual focus of L. donovani kalazar and L. major cutaneous leishmaniasis. A primary Leishmania isolate from splenic aspirates was cryopreserved (NLB-294). The patient was treated with sodium stibogluconate for kala-azar and discharged. Three months later, he had clinical relapse and returned for retreatment. During his second visit, the patient participated in a diagnostic study in which urine and nasopharyngeal samples were cultured for leishmaniasis. Urine, nasopharyngeal, and splenic samples were positive for Leishmania. Secondary isolates from splenic (NLB-294-I) and urine (NLB-318) cultures were cryopreserved and characterized by cellulose acetate electrophoresis (CAE) using 20 enzymes. Whereas the urine isolate was typed as L. donovani, the splenic aspirate culture revealed a mixed infection with L. donovani and L. major. The primary isolate (NLB-294) was then characterized and also showed a mixed infection. To exclude the possibility of protein post-translational modifications in electrophoretic assays, the primary and secondary isolates were grown and processed under identical cultural and lysis conditions, and compared using CAE. The results were identical to the first electrophoretic assays showing mixed promastigote banding patterns. Stationary-phase promastigotes of the secondary splenic isolate (NLB-294-I) inoculated subcutaneously, intraperitoneally, and intracardially into Syrian hamsters and BALB/c mice produced both kala-azar and cutaneous leishmaniasis within 6.5 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1998

Controlled-rate versus uncontrolled-rate cryopreservation of peripheral blood progenitor cells: a prospective multicenter study. Group for Cryobiology and Biology of Bone Marrow Transplantation (CBTMO), Spain.

BACKGROUND AND OBJECTIVE: Cryopreservation of hemopoietic progenitors for transplantation has been traditionally performed by the use of a controlled-rate freezer. Several groups have reported successful cryopreservation of progenitor cells at -80 degrees C without a controlled-rate freezer. In an attempt to elucidate whether both methods are equally efficient, we compared controlled-rate versus uncontrolled cryopreservation of peripheral blood progenitor cells (PBPC) in a prospective, multicenter study. DESIGN AND METHODS: Apheresis products from patients undergoing PBPC mobilization were split into two aliquots, and cryopreserved simultaneously by both methods, in autologous plasma plus 10% dimethylsulfoxide. Controlled-rate samples were placed into a programmable freezer with a cooling rate of 1-2 degrees C/min. Uncontrolled-rate samples were directly introduced into a -80 degrees C mechanical freezer. After thawing, cell counts, assays for viability, clonogenic cultures and CD34+ cell enumeration were performed. RESULTS: A total of 105 cases were included. No significant differences were found in viability (mean 88.8 +/- 13% in the controlled-rate group vs. 89.7 +/- 12% in the uncontrolled-rate group), nucleated cell loss (23.5 +/- 23% vs. 23 +/- 22%), mononuclear cell loss (19 +/- 23% vs. 19.1 +/- 22%), and loss of CD34+ cells (34.3 +/- 33% vs. 28.6 +/- 34%). On the other hand, recovery of granulomonocytic colony-forming units (CFU-GM), was significantly better with the controlled-rate technique, than with the non-controlled-rate method (104.3 +/- 95 vs. 86.5 +/- 80, respectively; p = 0.048). INTERPRETATION AND CONCLUSIONS: Our results indicate that both techniques are suitable for cryopreservation of PBPC, although a better recovery of committed progenitors is achieved by the controlled-rate method. Therefore, the use of controlled-rate freezer should probably be recommended.

Blood Preservation↗

PubMed1993

Cryopreservation of bovine buffy coat leukocytes for use in immunologic studies.

A simple cryogenic technique for preserving bovine buffy coat leukocytes was developed. This was coupled with a variation of the standard discontinuous gradient technique to purify mononuclear cells that retained immunologic function. The total number of mononuclear cells recovered from cyropreserved samples were only 87 to 42% of those recovered from freshly obtained blood samples. However, the functional capabilities of mononuclear cells from cyopreserved buffy coat preparations were retained. Polyclonal proliferative responses to 3 mitogens were measured, using a titration of mitogen concentrations, and were found to be normal, compared with those of cells isolated from fresh blood. Blood samples collected after vaccination with Brucella abortus contained leukocytes that responded to irradiated B abortus. These antigen-specific responses were also retained through cyopreservation. Cell surface expression of T-lymphocyte antigens, CD2, CD4, and CD8, and cell-surface IgM on B lymphocytes was also evaluated. Flow cytometric analysis of fresh and cryopreserved mononuclear cell preparations indicated that the relative proportions of different subpopulations were not altered. The technical simplicity of our cryopreservation system will allow processing of large numbers of samples with the ability to assay various immune functions at a later time.

Animals↗

PubMedDate not supplied

[Expression of the membrane antigens in native and cryopreserved embryonic precursor cells].

Native and cryoconservated human embryonic liver cells at different stages of gestation (5, 6 and 7 weeks) have been studied. Tissue desagregation was performed by trypsinisation technique. A part of the samples was studied after their trypsinisation, another part - after 3-week storage. Cell viability of all samples was higher than 90%. CD34 antigen expression was highest in 5-week gestation samples. The tendency of the deacrease of CD34 antigen expression was seen in 7-week gestation samples. CD34 antigen expression was higher in the samples after their cryoconservation than in native ones. The CD34 antigen expression afer their cryoconservation was accompanied by an increase in numbers of cells expressing CD38, CD45RA, CD33, CD13 and CD14 antigens as well as markers of erythroid precursor cells -CD71, CD36 and CD45RO. Percentage of alpha-actin positive cells was higher in 6- and 7-week samples. An increase in alpha-actin positive cell fraction has been seen also in samples after their cryogenic conservation.

Antigens, CD↗

PubMed2004

Cryopreservation of human whole blood for pyrogenicity testing.

Human whole blood assays are increasingly employed to test immune function or detect pyrogenic contamination, since they offer advantages, such as ease of performance, few preparation artifacts and a physiological cell environment. However, the approach is often limited by the availability of freshly drawn blood, putative safety concerns in the case of infected donors and interindividual donor differences. To overcome these limitations, a method was developed and optimized to produce batches of cryopreserved blood that can be used directly after thawing without any washing steps. Mononuclear cells remained intact as shown by FACS analysis. Cytokine release could be induced by a variety of immunological stimuli. The cell preparation released higher amounts of interleukin-1beta (IL-1beta) and IL-6 compared to fresh blood, but no TNF. These differences could be attributed to the presence of the cryoprotectant dimethylsulfoxide (DMSO) alone by addition of DMSO to fresh blood. Large batches of cryopreserved blood could be produced by mixing blood donations of up to 10 donors, independent of differing blood groups. The detection limit for the World Health Organization (WHO) lipopolysaccharides (endotoxin, LPS) reference preparation (EC-6) with regard to the induction of IL-1beta release was at least 0.5 endotoxin equivalent units (EU)/ml. Endotoxin spikes at the limit concentrations prescribed in the European Pharmacopoeia could be detected in a series of drugs, showing that the In vitro Pyrogen Test (IPT) can also be run with cryopreserved blood. Further possible applications include high-throughput screening for immunomodulators or toxins as well as preservation of patient samples for later analysis of cell functions.

Blood↗

PubMed1994

Human plasma LDL cryopreserved with sucrose maintains in vivo kinetics indistinguishable from freshly isolated human LDL in cynomolgus monkeys.

We previously reported that freeze-thawing of LDL causes marked alterations in its structure and in vitro biological behavior, and that such changes can be completely abolished by the addition of sucrose to the LDL solution prior to freezing. (Rumsey, S. C. et al., J. Lipid Res. 1992. 33: 1551-1561). We now questioned whether the cryopreservative action of sucrose would be equally effective in maintaining the in vivo metabolic characteristics of LDL. Two dual-label LDL turnover studies were performed in cynomolgus monkeys (n = 8) comparing freshly isolated human LDL with human LDL that was frozen in sucrose (10% w/v) for a short (20 h) or long period (6 months). The same sucrose-cryopreserved LDL was used for both the short- and long-term studies; different fresh LDL preparations were used in each study. Absorption spectrophotometry, gel filtration, and electron microscopy of LDL samples frozen with sucrose showed no evidence of physical alterations or aggregation, and there was no evidence of very rapid clearance of cryopreserved LDL from monkey plasma after injection. Fractional catabolic rates (FCR) of fresh and frozen LDL were very similar in either the short-term or long-term experiments: 2.09 +/- 0.86 versus 2.16 +/- 0.88, short-term and 3.03 +/- 2.28 versus 3.08 +/- 2.29, long-term (pools per day; mean +/- SD). The difference between FCR of fresh and frozen LDL for each animal averaged -0.076 +/- 0.074 and 0.01 +/- 0.22 (mean +/- SD), for short-term and long-term freezing, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Effect of dialysis before storage or cryopreservation on fertilizing ability of fowl semen.

Semen of broiler breeder males was collected and used for artificial insemination. Semen was used undiluted and diluted and both inseminated immediately after collection. Only diluted semen was used after storage. Half of the semen samples to be stored were first dialyzed. Storage of fresh, diluted semen was for 4 or 24 h at 5 C or by cryopreservation. The Beltsville Poultry Semen Extender was used for dilution and dimethylsulfoxide (DMSO) was used as cryoprotectant. Fertilizing ability was determined as the percentage fertilized eggs of eggs collected from Day 2 to 17 after a single insemination. Dialysis had a small positive effect on fertilizing ability of semen stored for 24 h. Dialysis before cryopreservation did not improve fertilizing ability. The effects of storage for 4 h were only apparent in eggs collected 9 or more d after insemination. Semen stored for 24 h gave reduced fertilizing ability in the 1st wk after insemination, especially when no dialysis was applied.

Animals↗

PubMed1993

The use of a liquid-phase controlled rate freezing system for the successful cryopreservation of mouse pre-embryos.

OBJECTIVES: To investigate the ability of a liquid-phase, controlled-rate freezing machine to generate reproducible freezing gradients with a constant rate of change; temperature fluctuations and heat dissipation during seeding; to assess the viability of mouse pre-embryos exposed to the silicone liquid cooling phase and the rates of survival and viability of mouse pre-embryos cryopreserved using this system. DESIGN: Freezing gradients were generated from the bath or a sample and compared for reproducibility and slope. Temperature fluctuations and gradients around the freezing chamber and the temperature rises and dissipation during seeding were measured. The toxicity of the silicone polymer freezing-phase was tested with mouse pronuclear pre-embryos. Different developmental stages of mouse pre-embryos were frozen and thawed and survival recorded in vitro and in vivo. SETTING: Research Laboratories, Sinai Hospital of Baltimore, Baltimore, Maryland. MAIN OUTCOME MEASURES: The reproducibility of freezing gradients; temperature fluctuations during seeding; and the in vitro and in vivo viability of mouse pre-embryos exposed to the silicone polymer or frozen and thawed. RESULTS: The freezing gradients generated were constant and reproducible with a constant rate of change; no temperature differences were recorded around the freezing chamber; temperature changes at seeding are minimal and rapidly dispersed. The silicone polymer was nontoxic to mouse pre-embryos and mouse pre-embryos frozen with the system and subsequently thawed are viable both in vitro and in vivo. CONCLUSIONS: This liquid-phase cryopreservation system is an attractive option for assisted reproductive technologies laboratories because liquid nitrogen is not required for operation, it is reliable, there are small temperature fluctuations during seeding, and it can be kept constantly running.

Animals↗

PubMed2000

Apoptosis: a method for evaluating the cryopreservation of whole blood and peripheral blood mononuclear cells.

We sought to compare the effect of cryopreservation and storage at -30 degrees C, -70 degrees C and -150 degrees C of human whole blood versus matched peripheral blood mononuclear cell (PBMC) samples using apoptosis as an indicator of cell fitness. Following 10 weeks of storage the samples were thawed and assessed for viability (trypan blue exclusion), levels of apoptosis (using the nuclear stain bis-benzimide) and cell function (ability to be transformed by Epstein-Barr virus, EBV). When comparing storage temperatures, the levels of apoptosis in whole blood and PBMC samples stored at -30 degrees C were significantly higher than the values for samples stored at -70 degrees C or -150 degrees C (P<0.004). Whole blood samples stored at -150 degrees C had significantly less apoptosis than those stored at -70 degrees C (P<0.03). A comparison of the cell preparations showed that at all three storage temperatures there was significant sample deterioration (viability, apoptosis, and function) in whole blood relative to PBMC samples. This study indicates that careful consideration should be given to storage conditions and that apoptosis can be used as a sensitive measure of cell fitness following cryopreservation.

Apoptosis↗

PubMed2005

Methods for etiologic and early marker investigations in the PLCO trial.

With the rapid development of biomarkers and new technologies, large-scale biologically-based cohort studies present expanding opportunities for population-based research on disease etiology and early detection markers. The prostate, lung, colorectal and ovarian cancer (PLCO) screening trial is a large randomized trial designed to determine if screening for these cancers leads to mortality reduction for these diseases. Within the Trial, the PLCO etiology and early marker study (EEMS) identifies risk factors for cancer and other diseases and evaluates biologic markers for the early detection of disease. EEMS includes 155,000 volunteers who provide basic risk factor information. Serial blood samples are collected at each of six screening rounds (including one collection for cryopreserved whole blood) from screening arm participants (77,000 subjects) and buccal cells are collected from those in the control arm of the trial. Etiologic studies consider environmental (e.g., diet), biochemical, and genetic factors. Early detection studies focus on blood-based biologic markers of early disease. Clinical epidemiology is also an important component of the PLCO trial.

Biomarkers, Tumor↗

PubMed2005

Semen collection in the dog.

This review will discuss semen collection in the dog. Semen samples may be collected from male dogs for the purposes of artificial insemination, cryopreservation or diagnosis. The materials needed for semen collection depend on which method is used and the collector's level of expertise with this procedure. At minimum, two sterile centrifuge tubes or specimen cups can be used to collect semen as it is ejaculated (for the combined first and second fractions and for the third fraction). The most common method for semen collection in the dog is by digital stimulation. Under ideal conditions, this procedure is performed in the presence of an estrous bitch. Initially, the dog's penis is vigorously massaged through the prepuce at the level of the bulbus glandis (caudal-most aspect of the prepuce) until a partial erection develops (initial engorgement of the bulbus glandis). The prepuce is quickly retracted past the bulbus glandis and firm constant pressure is applied to the penis behind the bulbus glandis by squeezing the penis between index finger and thumb. Pelvic thrusting may occur following application of pressure behind the bulbus glandis during the development a "full" erection. The ejaculate is composed of three fractions: first (sperm-poor), second (sperm-rich) and third (prostatic fluid). In addition to digital stimulation of the penis, spermatozoa have been collected from dogs using electroejaculation and pharmacologic methods.

Animals↗

PubMed1995

Cord blood as an alternative source for stem and progenitor cell transplantation.

Blood collected from the umbilical cord and placenta at the birth of a child is a rich source of immature blood cell elements and has been used clinically as an alternative source of transplantable stem and progenitor cells. Studies on the proliferative and replating capacities of cord blood stem and progenitor cells have documented their extensive capacity for division and self renewal. Studies on the immune cells in cord blood have shown them to be less immunologically reactive in a number of situations. These characteristics are consistent with the experience in children receiving HLA-matched sibling cord blood cells, in which these cells have been transplantable in a large number of clinical disorders with low or absent graft-versus-host disease. Stem and progenitor cells from cord blood are efficiently transduced with new genetic material by retroviral and adeno-associated viral vectors and may be of efficacy in the future for autologous gene therapy approaches to treat disease. Efforts in banking of cryopreserved cord blood cells have been undertaken, and a number of such stored samples have been used for fully and partially HLA-matched unrelated transplantation. Efforts to better understand the cells in cord blood and their clinical utility are continuing.

Animals↗

PubMed1994

Biochemical and cellular characterization of cardiac valve tissue after cryopreservation or antibiotic preservation.

It has been reported that aortic homografts that have been cryopreserved before transplantation remain viable longer as an allograft than tissue stored at 4 degrees C in an antibiotic solution. In the present study, we tested the hypothesis that storage of cardiac valve tissue by cryopreservation or by antibiotic preservation may alter the metabolic status of the tissue. Initially, we collected aortic valves composed of cardiac tissue, aortic root, and valvular tissue from cadaver donors. These specimens were divided into three equal portions, and one portion was analyzed before storage while the other two parts were stored for 3 weeks at either 4 degrees C in an antibiotic solution or at -196 degrees C in liquid nitrogen. All specimens were examined with regard to the following parameters: tissue structure, tissue viability, cell proliferative capacity, metabolic function, and identification of cell-specific antigens. We found no significant alterations in the structure of any of the three tissue components after antibiotic preservation or cryopreservation; however, cell viability and cell number were decreased in all three groups. All tissue samples grew in culture before storage. When we compared activities of the following organellar marker enzymes--lysosomal acid lipase, plasma membrane 5' nucleotidase, mitochondrial cytochrome oxidase, and microsomal neutral alpha-glucosidase--we observed no major differences between tissues stored by either technique. In addition, we observed no loss of enzymic activity as a result of storage. Finally, when cell lines isolated from each tissue specimen were incubated with monoclonal antibodies against cell-specific antigens in an immunoperoxidase assay, all the cell cultures proved to be endothelial cells. These results suggest that although cardiac valve tissue stored by cryopreservation or by antibiotic preservation retained its normal structure and metabolic capabilities, both storage techniques produced significant decreases in cell numbers and viability. However, only endothelial cells from tissue stored by cryopreservation retained the capacity to proliferate in vitro. These findings have important implications for the function of aortic homografts transplanted after storage.

5'-Nucleotidase↗

PubMed1996

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws.

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.

Journal Article↗

PubMed1994

Assessment of acrosomal integrity of vervet monkey spermatozoa after cryopreservation.

Cryopreservation of Vervet monkey semen caused a highly significant reduction in the percentage of normal acrosomes and a highly significant increase in the percentage of mildly damaged, severely damaged, and lost acrosomes. This was demonstrated by staining ten post-thaw semen samples, which enabled a visualization of the acrosomal damage. The incidence of post-thaw intact acrosomes in this cryopreservation method was found to be similar to that reported for man and cynomolgus monkey semen.

Acrosome↗

PubMed1997

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa.

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.

Journal Article↗