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PubMed2004

Cryopreservation of alginate-encapsulated recombinant cells for antiangiogenic therapy.

The potential benefit of continuous local administration of antiangiogenic proteins to CNS tumors in vivo has recently been demonstrated using endostatin-producing recombinant cells encapsulated in alginate beads. Due to the treatment potential of transplanted alginate-encapsulated cells producing therapeutic proteins, we describe a successful method of cryopreservation (CP) of such beads, in which cellular viability, alginate structure, and protein secretion were maintained. Alginate beads containing human embryonic kidney cells (HEK 293 cells) stably transfected with the gene encoding for endostatin were cryopreserved in dimethyl sulfoxide (DMSO) using a slow freezing procedure. Briefly, the DMSO concentration was gradually increased prior to the freezing procedure. The cryotubes were further supercooled to -7.5 degrees C and nucleated. Thereafter, the samples were cooled at a rate of 0.25 degrees C/min and stored in liquid nitrogen. The viability of the encapsulated cells was assessed using confocal microscopy quantification (CLSM) technique and a MTS assay. The cell cycle distribution inside the beads was assessed by DNA flow cytometry and endostatin production was determined by an endostatin-specific ELISA assay, both prior to and after CP. CLSM measurements showed sustained esterase activity in the beads after thawing, with only a slight transient decrease 24 h after CP. The MTS assay verified these findings by displaying similar variations of intracellular dehydrogenase activity. Flow cytometric analyses revealed no cryorelated disturbances in cellular ploidy. Furthermore, ELISA measurements showed a well-preserved endostatin production after CP. In conclusion, this work describes the successful CP of alginate-encapsulated recombinant cells secreting a therapeutic protein. Together with previous published reports, these results further substantiate the feasibility and potential of cell encapsulation therapy in the treatment of malignant tumors.

Alginates↗
PubMed1996

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells.

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.

Adult↗
PubMed2004

Proacrosin-acrosin activity in capacitated and acrosome reacted sperm from cryopreserved bovine semen.

Acrosin activity is associated with normal fertility in human and bovine spermatozoa. The aim of the study was to determine the variation of the enzyme activity in the proacrosin-acrosin system in capacitated and acrosome reacted cryopreserved bovine sperm. Enzyme activity was assessed spectrophotometrically using N-alpha-benzoyl-DL-arginine p-nitroanilide (BAPNA) as specific substrate for acrosin at pH 8. Capacitation with heparin and quercitin failed to induce conversion of proacrosin to acrosin. An increase in acrosin activity produced by the presence of progesterone, in a dose-dependent manner, was related with the induction of true acrosome reaction. The total level of acrosin activity registered showed that 96% of acrosin of capacitated sperm samples and control is present in the zymogen form. Moreover, progesterone is capable of duplicating the level of active enzyme, indicating that enzyme activity changes are related to acrosome reaction, suggesting that only a minor proportion of the total of proacrosin-acrosin system is required in the exocytotic process on cryopreserved bovine sperm.

Acrosin↗
PubMed1999

Improvements in the infectivity of cryopreserved third-stage larvae of Angiostrongylus cantonensis using a programmable freezer.

Although there have been some advances in the cryopreservation of Angiostrongylus cantonensis, the degrees of viability and infectivity of the cryopreserved developmental stages have not been high. A two-step freezing protocol using a programmable freezer was determined to be effective in improving the infectivity of the cryopreserved third-stage larvae of this parasite. After washing steps and suspension in 10% (v/v) dimethylsulfoxide and equilibrium at room temperature the larvae were placed into the freezer. The temperature was lowered first at 0.8 degrees C/min from room temperature to -40 degrees C and then at 10 degrees C/min to -70 degrees C. The samples were plunged into liquid nitrogen. After storage in liquid nitrogen for 7-15 days the larvae were thawed rapidly in 37 degrees C water and 27.6% were found to show vigorous "S-shape" movement without significant changes in appearance. These larvae (50/rodent) could develop to the fifth stage in mice (42.6%) and establish patent infection in rats (40.4%). Moreover, there was no significant difference in the recovery rates of cryopreserved worms and their unfrozen counterparts. These findings indicate that steady precooling conditions may decrease damage with regard to the infectivity of cryptopreserved third-stage larvae of A. cantonensis.

Angiostrongylus cantonensis↗
PubMed2002

Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates.

Spermatozoa collected from the caudae epididymides of 16 dogs of various breeds were suspended in an isotonic salt solution (DIMI medium) containing 0.6 M glycerol, frozen in liquid nitrogen, and their "survival" was measured after thawing. In the first experimental series, duplicate samples of spermatozoa from each of 11 dogs were cooled at rates of 0.5, 3, 11, 58, or 209 degrees C/min, stored in liquid nitrogen, and the frozen samples warmed at approximately 830 or at 33 degrees C/min. Sperm "survival" was judged by microscopic assessments of motility and of membrane integrity, the latter as assayed with Fertilight, a double fluorescent stain. Motility of frozen spermatozoa that were thawed rapidly, averaged for 11 dogs, was low at low rates, increased to a maximum at 11 degrees C/min, and then decreased significantly at higher rates (P<0.01). This inverted V-shaped curve was also observed with slow thawing, although the apparent optimum cooling rate ranged from 3 to 11 degrees C/min. The integrity of sperm plasma membranes showed a similar dependence on cooling rate, although the percentages of spermatozoa with intact plasma membranes were higher than the percentages of motile spermatozoa. Motility of spermatozoa, as a function of cooling rate, varied considerably from male to male (P<0.01), whereas membrane integrity was much more consistent among the 11 dogs. In the second experimental series with spermatozoa from 5 dogs, motility of spermatozoa frozen at 0.5 degrees C/min and warmed at 3.6, 33, 140, or 830 degrees C/min also exhibited an inverted V-shaped survival curve, in this case as a function of warming rate. In summary, high survival of frozen-thawed canine epididymal spermatozoa depended on both cooling and warming rates, but spermatozoa from each dog exhibited their own sensitivity to cooling and warming rates.

Animals↗
PubMed2002

Intracytoplasmic sperm injection with testicular spermatozoa in men with azoospermia.

PURPOSE: The aim of the study was to gain an insight into the optimal management of the infertile couple with the husband suffering from azoospermia. METHODS: One hundred and forty-two intracytoplasmic sperm injection (ICSI) cycles performed with testicular extracted spermatozoa were retrospectively analysed. The following factors were investigated for their possible influence on fertilization, cleavage, damage, pregnancy, and ongoing pregnancy rates: the use of fresh, cryopreserved, and preincubated (24 h) spermatozoa and the etiology of the husbands' azoospermia (obstructive and nonobstructive). All microinjections were performed with apparently normal spermatozoa--a head with a tail of normal length. In 116 cycles at least two embryos were available for transfer. RESULTS: The overall fertilization, clinical pregnancy, and ongoing pregnancy rates obtained for the 116 cycles were 65.0, 30.2, and 22.4% respectively. Similar outcomes were obtained for cycles using fresh testicular and cryopreserved testicular spermatozoa. Similarly, no significant differences were obtained between the cycles using spermatozoa from obstructive or nonobstructive azoospermic patients. An increase in motility after a 24-h preincubation was observed, and although this group was relatively small (n = 17), a significant improvement in fertilization (73.7%) and pregnancy (53.9%) rate was obtained when the testicular sample was preincubated for 24 h. This improvement prevailed in the obstructive azoospermic group, but was less pronounced in nonobstructive patients. CONCLUSIONS: This study shows that the outcome of fresh and frozen-thawed testicular spermatozoa in ICSI is comparable, obstructive and nonobstructive etiologies perform the same, and that preincubation of testicular spermatozoa results in increased fertilization and pregnancy rates. All testicular biopsies are therefore performed the day before oocyte retrieval, superfluous spermatozoa cryopreserved, and the remaining testicular homogenate preincubated for the 24 h prior to oocyte retrieval. With this regime, most azoospermic patients are treated successfully, irrespective of the use of fresh or frozen-thawed spermatozoa from obstructive or nonobstructive cases.

Cryopreservation↗
PubMed2005

Comparison by LC-MS and MALDI-MS of prostate-specific antigen from five commercial sources with certified reference material 613.

OBJECTIVE: Prostate-specific antigen (PSA) samples from five commercial sources were compared with PSA from the European Commission Community Bureau of Reference, known as CRM 613, using liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). DESIGN AND METHODS: The effects of storage at various temperatures, multiple freeze-thaw cycles and lyophilization vs. solution storage were investigated. RESULTS: : CRM 613 and the five commercially available samples varied in their heterogeneity and in the amount of PSA with a molecular mass of 28,430 kDa. Few changes were observed among any of the samples during the stability study or study of the effect of lyophilization and freeze-thaw cycles. CONCLUSIONS: Use of many of the commercial PSA sources as authentic PSA standards for calibrants for in vitro diagnostic tests could lead to significantly biased PSA measurements. Although all the PSA samples appear to be relatively stable for the time periods and conditions studied, some of the commercially available samples contain limited amounts of PSA and included other unidentified components.

Chromatography, High Pressure Liquid↗
PubMed1996

Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA.

BACKGROUND/AIMS: Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay. METHODS: We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months. RESULTS: A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%. CONCLUSIONS: These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.

Blood Preservation↗
PubMed1998

Effect of storage on protein concentration of tear samples.

PURPOSE: To determine the effect of storage on protein concentration of tear samples stored at room temperature (RT), 4 degrees C, -20 degrees C and -70 degrees C. METHODS: Total protein concentration of closed, open (basal tears) and reflex tears (stimulated tears) was measured by modified Bradford's method. SDS-PAGE gel electrophoresis was used to determine the intensity of protein bands. Quantity of various tear proteins was determined by HPLC analysis. RESULTS: Compared to control samples (0 h) protein concentrations decreased significantly in tear samples stored beyond 4 h (for closed) and 8 h (for open and reflex) at RT. However, no significant change in protein concentrations was observed in closed and reflex tears when stored up to 1 week at 4 degrees C, up to 2 months at -20 degrees C; and up to 4 months at -70 degrees C. Multiple freeze-thaw procedures (6 x per day at 2 h intervals) resulted in 8% decrease (at -20 degrees C) and 10% decrease (at -70 degrees C) of protein concentrations in closed eye tears. SDS-PAGE analysis of tear samples stored at -20 degrees C and -70 degrees C for 4 months greatly affected the intensity of the protein bands. HPLC analysis of tear samples stored at these conditions showed the significant reduction in the peak corresponding to secretory IgA in closed eye tears and a split in the peak corresponding to lysozyme in case of reflex tears. CONCLUSIONS: Reduction in the total protein concentration, intensity of the protein bands as well as changes in the quantity of tear proteins were observed in the tear samples stored for longer duration of time at various temperatures.

Chromatography, High Pressure Liquid↗
PubMed1996

Methodological aspects of flow cytometry DNA analysis in endometrial carcinoma, with special reference to sampling and reproducibility.

Two adjacent paraffin-embedded sections manifested concordance in ploidy status in 96% of cases (45/47), and the standard deviation (SD) for SPF was 2.7%. Analysis of 'micro-heterogeneity', within a distance of < or = 700 microm, yielded results for concordant ploidy status in 94% of cases, and the SD for SPF was 1.9% (n = 17). Frozen and paraffin-embedded material yielded concordant results for ploidy status in 87% (39/45) of cases, and SPF values were significantly lower (mean difference 1.5%) in the frozen samples. Diagnostic and repeat curettage material yielded concordant results for DNA ploidy status in 85% (40/47) of cases, and no significant difference in mean SPF (12% vs. 11%) was found. Discordant DNA ploidy results were attributable to small differences in the DNA histograms influencing the interpretation of near-diploid, near-tetraploid and small non-diploid cell populations, and the influence of debris on SPF estimation. On the basis of our findings and the practical advantage we recommend paraffin-embedded material from diagnostic curettage for FCM DNA analysis; the results are available sooner and the handling and transportation of tumor samples is more convenient.

Cryopreservation↗
PubMed2004

[Comparison of sperm motion parameters in pre-freeze and post-thaw semen samples using computer-assisted sperm analysis].

OBJECTIVE: To observe sperm motion parameters in pre-freeze and post-thaw semen samples using computer-assisted sperm analysis (CASA) system. METHODS: Semen analyses of 238 samples before freezing and after thawing were separately performed by Hamilton-Thorne Sperm Analyzer. RESULTS: Sperm motility in post-thaw samples was significantly decreased. There was significant correlation and difference between pre-freeze and post-thaw samples in sperm motion parameters, including average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), straightness (STR) and linearity (LIN), except heat cross frequency (BCF). The percentage of sperm movement velocity parameters (VAP, VSL and VCL) and moving pattern parameters (ALH) significantly decreased, while that of LIN and STR significantly increased in post-thaw samples. CONCLUSION: CASA system is of clinically applied value and is a useful tool for evaluating sperm motion parameters in pre-freeze and post-thaw semen samples.

Cryopreservation↗
PubMed2004

Cost analysis of singleton versus twin pregnancies after in vitro fertilization.

OBJECTIVE: To determine the difference in costs between singleton and twin pregnancies after IVF treatment from pregnancy to 6 weeks after delivery from a health care perspective. DESIGN: Retrospective cost analysis. SETTING: IVF department at the University Medical Center Nijmegen, The Netherlands. PATIENT(S): A representative sample of singleton and twin pregnancies after IVF treatment between 1995 and 2001 at the University Medical Center Nijmegen. INTERVENTION(S): IVF with or without intracytoplasmic sperm injection and with or without cryopreservation. MAIN OUTCOME MEASURE(S): Medical costs per singleton and twin pregnancy after IVF. RESULT(S): In patients pregnant with twins, the incidence of hospital antenatal care, complicated vaginal deliveries, and cesarean sections was higher and was associated with more frequent and longer maternal and neonatal hospital admissions. Maternal and neonatal hospital admissions were the major cost drivers. The medical cost per twin pregnancy was found to be more than five times higher than per singleton pregnancy, 13,469 and 2,550, respectively. CONCLUSION(S): The medical cost per twin pregnancy was more than 10,000 higher than per singleton pregnancy. A reduction in the number of twin pregnancies by elective single ET will save substantial amounts of money. This money might be used for the additional IVF cycles that will probably be needed to achieve similar success rates between single ET and two-embryo transfer.

Adult↗
PubMedDate not supplied

Fetal calf serum versus human serum: ultrastructural evaluation of protein support influence on human ovarian tissue cryopreservation.

The aim of this study was to examine the effect of the different protein supports in the cryopreservation solution on improving human ovarian tissue preservation after frozen-thawed procedures. Biopsies of ovarian cortical tissue were obtained from 14 subjects. All specimens were cryopreserved using a slow freezing/rapid thawing method in a solution consisting of propanediol and sucrose in different proportions of 3 protein supports: 30% human serum (HS) (solution A), 20% HS (solution B), or 20% fetal calf serum (solution C). After thawing, 191 follicles and a total of 70 samples were analyzed using transmission electron microscopy (TEM). The post-thaw preservation rate of the follicles in solution A was significantly higher with respect to solution C (p < 0.05). Unlike the follicles, the stromal cell morphology was not affected by any of the solutions investigated. By comparing stromal morphology and the patient age, it was found that HS better preserved the tissue in patients over 20 years of age with respect to younger ones, which showed a wider variability in ovarian preservation. TEM evaluation showed that 30% HS is more suitable for human ovarian tissue cryopreservation, and research should be focused on defining cryopreservation protocols specific to young patients.

Adolescent↗
PubMed2003

Cytogenetic and molecular study of a premature male infant with 46,XX derived from ICSI: case report.

We investigated the aetiology of the male phenotype in a premature infant derived from ICSI with a 46,XX karyotype. A karyotypically normal couple underwent ICSI because of obstructive azoospermia in the male partner. Sperm were retrieved by testicular sperm extraction (TESE), cryopreserved, and later used for ICSI. The pregnancy after ICSI ended at 20 weeks. A normal-appearing male was delivered but he did not survive. Umbilical cord blood and placenta were sampled and used for molecular and cytogenetic investigation. The 46,XX karyotype from G-banding in this male infant correlated to a balanced female comparative genomic hybridization (CGH) profile in placental tissue. No PCR amplification of SRY on the p arm of the Y chromosome was observed while fluorescence in-situ hybridization (FISH) with the SRY probe also could not detect the gene in cord blood or placental tissues. CGH and FISH, with X and Y centromeric probes, failed to detect mosaicism in the trophoblast, stroma and amnion. Skewed X-chromosome inactivation (81%) was found in the chorionic villi. The molecular and cytogenetic studies indicated a 46,XX male infant without the SRY gene or 46,XX/XY mosaicism. The possible mechanism in this SRY-negative XX male by ICSI is discussed.

Adult↗
PubMed1982

A simplified liquid nitrogen vapour method for the cryopreservation of human semen.

A simple manually operated liquid nitrogen vapour freezing apparatus has been developed for the cryopreservation of human semen. A test method using this apparatus was evaluated by comparison with a conventional freezing method using an ultra cold (-70 degrees C) freezer. Twenty different unselected fresh semen samples were analysed, diluted with cryoprotective medium and then divided into equal numbers of straws prior to freezing by the two methods. A comparison of the average sperm motility before freezing and after thawing using these two methods demonstrated a statistically significant superiority of the simplified test method.

Freezing↗
PubMed1995

Quality control of platelet concentrates by the Thrombostat 4000.

Quality control of platelet concentrates (PC) is an important prerequisite for good transfusion praxis. However, direct measurement of platelet function is complex, since available methods (e.g. aggregometry, serotonin release) are time consuming and require special equipment. Therefore a test system is needed, which is easy to handle, fast, and achieves reliable results. The present paper compares the results of conventional platelet function tests with those of a modified in-vitro bleeding test (IVBT) (Thrombostat 4000) in liquid-stored and cryopreserved PCs. A high correlation between aggregometry, serotonin release, GMP 140 expression upon stimulation, and IVBT was demonstrated. Therefore IVBT seems to be a good alternative to the conventional platelet function tests for quality control of PCs. In addition, a good correlation between the results of IVBT of patients' blood after PC transfusion and IVBT of patients blood before transfusion supplemented with platelets of the respective PC could be found. Therefore IVBT seems to be able to predict PC transfusion success. However, since these data were obtained in a small sample undergoing bone marrow transplantation, further studies are needed to verify this hypothesis.

Bleeding Time↗
PubMed1996

Factors affecting motion characteristics of frozen-thawed stallion spermatozoa.

Five experiments were conducted to evaluate damage incurred in each processing step for cryopreservation of stallion spermatozoa. In Experiment 1, semen was centrifuged for 9 centrifugation times and the percentage of spermatozoa recovered after each treatment was calculated and spermatozoal motion characteristics analysed. Recovery of spermatozoa was > or = 80% when spermatozoa were centrifuged for > or = 10 min. Experiment 2 evaluated spermatozoa cryopreserved at 5 different concentrations in each of 2 extenders (skim milk-egg yolk-glycerol, SM-EYG; and lactose-EDTA, LAC). In SM-EYG, TMOT and PMOT were higher at spermatozoal concentrations of 20, 200 and 400 x 10(6)/ml (51%/41%, 52%/44%, 50%/43%, respectively) than for samples frozen at > or = 800 x 10(6) spermatozoa/ml (41%/35%, 32%/27%; P < 0.05). Spermatozoa frozen in LAC at a concentration of 20 x 10(6)/ml resulted in the highest TMOT and PMOT (43% and 30%, respectively, P < 0.05). The effect of freezing rate on motion characteristics of spermatozoa was evaluated in Experiment 3. The VCL of spermatozoa frozen in SM-EYG was the only parameter affected by freezing rate (P < 0.05). Experiment 4 evaluated motion characteristics after cryopreservation of spermatozoa in different sized straws (0.5 or 2.5 ml) in each of 2 extenders (SM-EYG and LAC). In SM-EYG, PMOT (38%) and VCL (109 microns/s) were highest when spermatozoa were frozen in 0.5 ml straws (P < 0.05). In Experiment 5, spermatozoa thawed immediately after cryopreservation or thawed after storage in liquid nitrogen for 24-48 h were evaluated. There was no effect of length of storage in liquid nitrogen on spermatozoal motion characteristics (P < 0.05). Experiment 6 evaluated the effects of cooling time to 5 degrees C (0, 2.5 and 5 h) on motion characteristics of spermatozoa cryopreserved in 2 extenders (SM-EYG and LAC). TMOT and PMOT were effected by cooling time, and there was a cooling-time-by-extender interaction (P < 0.05). In SM-EYG, TMOT and PMOT were higher if spermatozoa were cooled to 5 degrees C prior to initiation of freezing than if freezing was initiated at 20 degrees C (P < 0.05). A suggested protocol for cryopreservation of stallion spermatozoa would include: 1) centrifugation at 400 g for 14 to 16 min; 2) extension at 23 degrees C with SM-EYG to 400 x 10(6) spermatozoa/ml; 3) cool to 5 degrees C for 2.5 h; 4) package in 0.5 ml straws at 5 degrees C; 5) freeze in liquid nitrogen vapour at -160 degrees C; and 6) thaw for 30 s in 37 degrees C water.

Animals↗
PubMedDate not supplied

Effect of fluctuations in temperature encountered during handling and shipment of human cryopreserved semen.

The effect of temperature fluctuations which cryopreserved spermatozoa may undergo during routine shipping and handling was evaluated in sperm frozen with two cryoprotectants. Sperm frozen in TEST-yolk buffer maintained motility better than those frozen in glycerol solution in all studies. Sperm motility was significantly compromised in samples stored more than one day in dry ice, regardless of the cryoprotectant, and more than two days in a liquid nitrogen shipping dewar if frozen in glycerol solution. Sperm motility was not compromised following exposure to room temperature for up to 3 min if TEST-yolk buffer was used as the cryoprotectant, but was compromised following 1 min exposure using glycerol cryoprotectant. This study describes the limits of non-ideal conditions that spermatozoa may undergo during shipping and handling, and demonstrates the effects of the cryoprotectant on those limits.

Cryopreservation↗