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PubMed2004

Preliminary trial: motility comparisons of a unique freezing technology (UFT) to liquid nitrogen mist methodology for cryopreservation of porcine spermatozoa.

The motility outcomes of boar semen frozen with newly developed freezing techniques using a new unique freezing technology (UFT) compared with traditional liquid nitrogen methodology were investigated with the intent of improving current fertility outcomes using semen. The UFT is an electronically controlled cooling chamber that houses an organic fluid bath that can be maintained at temperatures below 0 degrees C without solidifying to freeze samples. Four ejaculates from four different boars were collected for this trial. Samples were handled consistently during the pre- and post-freeze processing. From each ejaculate, samples were separated into eight cryopreservation treatment groups, six UFT variations and two control liquid nitrogen groups, immediately before freezing, in replicates of two. After the initial cryopreservation was complete, all samples were stored in liquid nitrogen for at least 48 h. Post-thaw motilities and original motility return percentages were assessed on a random, individual-sample basis. After the initial evaluations, samples from two boars were recollected and frozen using the UFT for breeding purposes. Four sows were bred with the UFT frozen semen to confirm fertility capability. When assessing the individual UFT techniques, all of six UFT techniques had improved post-thaw motilities. However, treatments F (micro = 29%, return micro = 37%) and J (micro = 27%, return micro = 34%) showed the highest statistical improvement for post-thaw (p < 0.05) and original motility percent returns (p < 0.05) when compared with either the control cryo-tube (micro = 15%, return micro = 19%) or straw groups (micro = 12%, return micro = 16%). The UFT semen had a 50% conception rate, with an average of seven piglets from the sows that farrowed. Our preliminary data suggest a higher motility return with a slower pre-freeze phase below the freezing point before the acceleration to liquid nitrogen temperatures. The preliminary data suggest that the UFT could be utilized as a potential cryopreservation option for boar semen.

Animals↗
PubMed1988

Functional testing of hepatocytes following their recovery from cryopreservation.

Various tests of function have been suggested for assessing hepatocytes recovered from cryopreservation. In this study we have investigated hepatocyte attachment during tissue culture and cellular density in order to assess function and compared them with two classical dye exposure tests. The ability of hepatocytes to exclude trypan blue dye (TB) and metabolize fluorescein diacetate (FDA) was demonstrated. In populations of freshly prepared hepatocytes 88.07% were able to exclude TB and 87.31% were able to metabolize FDA. However in populations of hepatocytes recovered after cryopreservation using 1.5 M dimethyl sulfoxide as cryoprotectant only 33.44% were able to exclude TB and 31.59% able to metabolize FDA. Both of these tests gave the same estimate of functional ability. Density gradient centrifugation of hepatocytes on Percoll 400 (Pharmacia, Uppsala, Sweden) separated two populations of hepatocytes; one (density ca.1.07 g/ml Percoll) in which most of the cells were able to exclude TB and the second (density ca. 1.02 g/ml Percoll) in which they were stained blue. The dense population was highly enriched in dye-excluding hepatocytes: freshly prepared hepatocytes, 92.4%, and cryopreserved hepatocytes, 88.66%. When samples of these cells (2 x 10(6) dye-excluding cells per dish) were tested for their ability to attach to tissue culture dishes only 17.28% of the cryopreserved hepatocytes were able to attach compared to 55.28% of the freshly prepared cells. We conclude that cryopreservation of hepatocytes produces a population of cells which are not metabolically identical to a population of freshly prepared hepatocytes even though they appear to have the same buoyant density and dye-excluding capabilities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Assessment of pronuclear formation following in vitro fertilization with bovine spermatozoa obtained after thermal insulation of the testis.

This study was conducted to follow the chronology of pronuclear formation in bovine zygotes after in vitro insemination with a population of spermatozoa having abnormal morphology. Semen samples were obtained and cryopreserved from four Holstein bulls before and after a scrotal insulation period of 48 h (Day 0). A pre-insult (Day 5) and a Day 20 post-insult semen sample were evaluated for morphology and used for IVF after standard swim-up sperm separation protocols. Pronuclear formation was scored on subpopulations of presumptive zygotes after they were fixed and stained at 3-h time intervals from 6 to 18 h post in vitro insemination (hpi). Post-thaw morphological evaluation of semen samples revealed a decrease in the percentages of normal spermatozoa in the post-insult samples compared with the pre-insult samples for Bull I (74-22%) and Bull III (68-1%). The sperm penetration rate decreased (P<0.05) between the pre- and post-insult samples for Bulls I (90-76%) and III (92-70%), but was not different for Bulls II (92-90%) and IV (78-85%). The pronuclear formation rates for post-insult zygotes for Bulls II and IV had comparable increases in development over time, whereas there was no increase in the pronuclear development for the zygotes from the post-insult samples for Bulls I and III, and generally a condensed sperm head was observed in the oolemma. At 18 hpi the fertilization rate between the pre- and post-insult samples for Bulls I (51-4%), II (88-75%) and III (94-2%) decreased (P<0.01), but there was no change for Bull IV (66%). In conclusion, we inferred that the failure in normal pronuclear formation was associated with an absence of normal decondensation of the penetrating spermatozoon; this suggested that the effect of morphologically abnormal spermatozoa occurred prior to cleavage, thus limiting early development.

Animals↗
PubMedDate not supplied

Pentoxifylline enhancement of post-thaw motility in cryopreserved semen of spinal cord-injured men.

OBJECTIVE: To investigate the effects of pentoxifylline on cryopreserved human semen of spinal cord injured men, in efforts to enhance post-thaw motility. DESIGN: Semen specimens were collected from: pregnancy-proven donors (n = 10), spinal cord injured (SCI) patients (n = 8), and infertility patients referred for white blood cell (WBC) screening of the semen (n = 18). Following at least 24 hours of freezing in 100% tes and tris-yolk buffer, 0.5-mL semen straws were thawed. Each sample was split into two equal aliquots, one of which was treated with 3.6 mM pentoxifylline. Following incubation, percent motility was recorded manually at 15 minutes, 1 hour, 2 hours, and 24 hours post-thaw. Percent original motility preserved (post-thaw motility/original motility X 100%) was calculated for each specimen. RESULTS: Statistically significant differences (P < .05) were noted in the SCI and WBC patients when samples with and without pentoxifylline were compared at 15 minutes, 1 hour, and 2 hours. No significant difference was noted in the donor population at any time interval, and no difference was seen in any group at 24 hours post-treatment with pentoxifylline. The greatest differential between treatment and control groups was noted at 1 hour in all three populations. CONCLUSIONS: Pentoxifylline enhances post-thaw motility of cryopreserved sperm in male infertility patients from 15 minutes to 2 hours post-treatment. Pentoxifylline appears to be most useful at 1 hour post-treatment in the cryopreserved samples of SCI patients obtained by electroejaculation. It does not appear to have any significant effect on the post-thaw motility of pregnancy- proven donors. Pentoxifylline loses its in vitro efficacy prior to 24 hours post-thaw.

Cryopreservation↗
PubMed2006

Recovery of human sperm motility and ability to interact with the human zona pellucida after more than 28 years of storage in liquid nitrogen.

Six donor semen samples were evaluated after 28 years cryopreservation in liquid nitrogen. The results showed that the samples retained good postthaw motility recovery and normal levels of binding to the human zona pellucida and that four of the five samples tested also gave normal levels of zona-induced acrosome reaction. In conclusion, human sperm can survive very long-term storage, which is pertinent information for clinicians referring boys and young men for sperm banking before chemotherapy.

Cells, Cultured↗
PubMed2004

Polyvinyl pyrrolidone: a novel cryoprotectant in islet cell cryopreservation.

The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37 degrees C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80 degrees C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean +/- SEM percent intact islet recovery was higher with PVP compared with DMSO (82 +/- 4.6 vs. 62.7 +/- 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.

Animals↗
PubMed1983

GCT-conditioned medium: an unsuitable stimulus for monitoring granulocyte--macrophage colony-forming cells in cryopreserved bone marrow.

Assays of granulocyte-macrophage colony-forming cells provide a means of testing the viability of cryopreserved bone marrow cells intended for autologous transplantation. We have compared two different sources of granulocyte-macrophage colony-stimulating activity, giant cell tumor-conditioned medium (GCT-CM) and peripheral blood leukocyte feeder layers, to determine whether the former is a suitable substitute for leukocyte feeder layers in the assay. The results show that GCT-CM, while providing a comparable stimulus to that provided by leukocyte feeder layers for colony formation by fresh bone marrow samples, is an inadequate stimulus when cryopreserved bone marrow samples are cultured. GCT-CM is not therefore suitable for use in monitoring cryopreserved bone marrow, since there is gross underestimation of the number of colony-forming cells present when this stimulus is used. The results suggest that great care should be taken when selecting alternative sources of granulocyte-macrophage colony-stimulating activity for culture of cryopreserved material.

Adult↗
PubMed2004

Lung fibroblasts from patients with emphysema show a reduced proliferation rate in culture.

Emphysema is characterised by a loss of alveolar structure, as reflected in elastic recoil and gas exchange. As fibroblasts play a key role in the maintenance of structure, the current authors hypothesised that their proliferation might be constitutively impaired in lung emphysema. Using explant cultures, lung fibroblasts were obtained from resected lungs of 10 patients with emphysema (median forced expiratory volume in one second (FEV1) 40% predicted) and 10 control patients (FEV1, 95% pred). The doubling time (DT) was measured over 4 days under standard conditions (10% foetal calf serum) prior and after cryopreservation. Additionally, in seven samples per group the total population doubling level (PDL) was determined. In emphysema, mean+/-sem DT was 33.6+/-2.8 h compared with 24.8+/-1.4 h in controls. The differences in DT were preserved after cryopreservation. Groups also differed in the initial slope of the PDL plot during long-term culture (up to 35 days). However, the median (range) maximum PDL did not differ significantly between groups (13.8 (7.4-22.6) versus 20.2 (11.2-25.5)). The current authors, therefore, suggest that the reduced proliferation rate in vitro of lung fibroblasts from patients with emphysema reflects a persistent, intrinsic failure of cellular replacement and maintenance in this disease, possibly in relation to pre-term aging.

Aged↗
PubMedDate not supplied

Storage and preservation of whole blood samples for use in detection of human immunodeficiency virus type-1 by the polymerase chain reaction.

Methods used in the diagnosis of human immunodeficiency virus type-1 (HIV-1) infection by the polymerase chain reaction (PCR) usually require the separation of lymphocytes from a whole-blood sample within 24 hours of patient sampling. A method is described in which blood samples are mixed with a cryopreservative ('Glycigel'), stored frozen, and DNA suitable for use in an HIV PCR recovered. Samples can be stored at -20 degrees C for up to 3 months and still give positive results with all samples from infected patients; storage at -80 degrees C for at least 3 months shows no loss of titre. The method shows no loss of sensitivity compared to previously described sample preparation methods. Deglycerolised Glycigel supernatants were found to be suitable for conventional anti-HIV-1 serological studies and loss of sensitivity only represented the dilution effect due to sample preparation. Application of the method as a means of storing samples frozen at the point of sampling and transporting them to a central laboratory for processing is demonstrated using samples taken from HIV-1-infected mothers and their babies.

Adult↗
PubMed1995

The effect of amphotericin B on the viability of cryopreserved human skin.

To evaluate whether amphotericin B has a detrimental effect on the viability of cryopreserved skin, human skin samples were incubated during 7 and 24 h in three different media: 0.9% phosphate-buffered saline and Medium 199 with and without 10 micrograms/ml amphotericin B. The viability of the tissue was assessed by measuring the tetrazolium reductase activity before and after cryopreservation. A significant decrease in cryopreserved human skin viability was observed after 7 and 24 h incubation with amphotericin B. These results indicate that in the processing of skin preservation, the beneficial effect of using amphotericin B as an antifungal agent may be reduced by its negative effect on tissue viability.

Amphotericin B↗
PubMed1994

Growth of primary human acute lymphoblastic and myeloblastic leukemia in SCID mice.

The in vivo growth of 8 human primary acute lymphoblastic (ALL) and 17 primary acute myeloblastic (AML) cell populations was investigated in severe combined immunodeficient (SCID) mice. Bone marrow (BM) or peripheral blood (PB) samples, either fresh or cryopreserved, were implanted i.v. into irradiated SCID mice. The cells from 5/8 patients with ALL resulted in engraftment with systemic proliferation and dissemination leading to morbidity and mortality of the animals within 12-18 weeks from implantation. In contrast, none of the 17 AML samples resulted in sustained engraftment. Four of the 5 engrafted ALL populations have been successfully passaged into fresh recipients and phenotypic and karyotypic characteristics remain unaltered. The data presented indicate that the SCID mouse may be a useful model for studying the pathogenesis of ALL and may also enable the development and investigation of new therapies for this disease.

Adult↗
PubMed1993

Rapid freezing of whole blood or buffy coat samples for polymerase chain reaction and cell culture analysis: application to detection of human immunodeficiency virus in blood donor and recipient repositories. The Transfusion Safety Study Group.

Storage of lymphocytes for later use in prospective epidemiologic studies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When the Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain reaction, a simple thawing and pelleting technique for recovering hemoglobin-free total white cells (WBCs) was developed. To validate the technique, parallel analysis was conducted of BCs, whole blood (WB), and PBMC samples from human immunodeficiency virus type 1 (HIV-1)-seropositive subjects. Immediate postthaw cell courts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 and 75 percent, respectively, which is as sensitive as that obtained with freshly separated PBMC lysates. Quantitative HIV-1 proviral load analysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express viral load relative to total WBCs, PBMCs, or CD4+ cells. These results establish the basis for simplified virologic analysis of cryopreserved BC or WB specimens.

Blood Donors↗
PubMed1980

Improvement of spermatozoal motility characteristics.

In this study were collected 34 normal semen samples, and samples from 8 men showing prostatic hypofunction of unknown origin and from 5 men with agglutinating autoantibodies in their sera. Among the various biological solutions tested, Tyrode's solution, with or without glucose, bovine serum albumin, and sperm-Ringer solution containing fructose best maintained the motility of normal spermatozoa, which was nearly the same as with semen samples. The ATP content of the spermatozoa was highest in the Tyrode's solution. When seminal plasma of the samples with low acid phosphatase and poor sperm motility was replaced with normal donor seminal plasma, the motility was increased almost to the normal level. All other biological solutions tested were weaker in this respect. 7.2 mmol/l caffeine had neither clear improvement on the motility of normally motile sperm cells nor on the spermatozoal ATP content. After cryopreservation and thawing, the samples containing caffeine showed the best motility, which was, however, surprisingly low. Spermatozoa coated by autoantibodies showed very abnormal cervical mucus penetration. After washing with Tyrode's solution the penetration improved, but did not reach normal penetration level.

Antibodies↗
PubMed1996

Analysis of stallion semen and its relation to fertility.

This article describes some of the basic methodology for conducting stallion semen evaluations. Not all of these assays will likely be conducted on every semen sample collected. Routine evaluations should include determination of semen volume, sperm concentration, and an estimation of the percentage of progressively motile sperm, at a minimum. Other assays can be used if a seminal problem is discerned at the beginning of the breeding season or after a stallion has recovered from an illness or injury. Additionally, laboratory assays are particularly important to conduct on cryopreserved spermatozoa, because these cells have been exposed to substantial temperature and osmotic insults (see Cryopreservation of Stallion Spermatozoa, this issue). The percentage of fully functional sperm is reduced during cryopreservation and the longevity of the cells surviving freezing is shortened. Therefore, to maintain high fertilization rates using cryopreserved sperm, only sperm samples containing sufficient numbers of high-quality cells should be used. Unfortunately, no single laboratory assay can estimate the fertilizing potential of a semen sample. What these analyses can do is discern semen samples that are likely to have poor fertilizing capacity.

Animals↗
PubMedDate not supplied

Probability of lethal damages of cryopreserved biological objects during storage.

This study demonstrated that with a cyclic change of the temperature from -196 degree C up to -130 degree C or -100 degree C part of cryopreserved microorganisms, tumor and human embryo cells died. This should be taken into account when moving and transporting the cryopreserved objects. Storage of samples under the above-mentioned stable temperatures did not result in additional death of cells.

Animals↗
PubMed2005

The incidence of apoptosis after IVF with morphologically abnormal bovine spermatozoa.

Normal embryonic development depends on the maintenance of a population of normal healthy cells within each embryo. The aim of this study was to use a combination of apoptotic measures to assess differences in embryo quality after IVF with semen samples with high percentages of abnormal spermatozoa. Semen samples were obtained and cryopreserved from four Holstein bulls before (5 day prior) and after (2 week-post-insult; 2 week-PI and day 20; 3 week-PI) a scrotal insulation period of 48 h (day 0). The swim-up sperm separation method was used. The post-thaw morphology revealed a decrease (P < 0.01) in the percentages of normal spermatozoa in the 3 week-PI samples in comparison with the pre-insult samples for Bulls I and III (74-22.3 and 67.7-0.5%, respectively). The percentage of vacuolated spermatozoa increased significantly for Bull II. After 18 h of sperm-oocyte co-incubation, zygotes were cultured and subpopulations were removed from culture at day 8 and subjected to either the TUNEL or caspase assay. On day 8, caspase intensity increased significantly for both Bull I (217+/-147) and Bull III (229+/-98) for the 3 week-PI embryo groups compared to the equivalent embryo groups for Bull II (98+/-115) and Bull IV (90+/-111). In conclusion, the inability to consistently measure apoptosis with TUNEL alone complicated the assessment of differences in embryo quality. Thus, it is uncertain exactly when during early pre-implantation development the differences in embryo quality are first manifest. Despite discrepancies, our results clearly indicated a difference in the embryo quality between embryos obtained after IVF with semen samples from bulls that had an intense response to scrotal insulation.

Animals↗
PubMed2000

Endothelial and smooth muscle changes of the thoracic and abdominal aorta with various types of cryopreservation.

BACKGROUND: There is a need for cryopreserved arterial allografts in vascular surgery. Vitrification was examined as an alternative to slow cooling. Function of endothelial and adjacent cells was evaluated in the thoracic and abdominal regions of the porcine aorta. MATERIAL AND METHODS: Aortas from young pigs were harvested and cryopreserved with several treatments: slow cooling at 1 degrees C/min with 10% dimethyl sulfoxide (DMSO) and vitrification with a vitrification solution (VS), performed either in the presence (medium) or in the absence (air) of the cryoprotective medium surrounding the sample. Tetrazolium salt reduction and oxygen consumption were used to assess the mitochondrial activity of the endothelial cells and smooth muscle cells from the aorta. RESULTS: Fresh aorta showed an increased function from below the aortic arch to the iliac branch. Exposure to cryoprotective solutions reduced significantly the oxygen consumption of the abdominal sections. Samples from the thoracic region had a better response to cryopreservation than those from the abdominal region. There was a significant reduction in tetrazolium salt reduction and oxygen consumption when the samples were cryopreserved immersed in the medium. Significant flaking of the endothelium was observed after vitrification and contributes generally to lower the function. CONCLUSION: The thoracic region of the aortic wall was more tolerant to the preservation injury, and freezing with DMSO in air offered a better choice of preservation.

Animals↗