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PubMed2006

Strategies to improve the fertility of frozen-thawed boar semen for artificial insemination.

Although cryopreservation of boar semen for artificial insemination (AI) was developed 35 years ago, cryopreservation conditions and AI strategies are still considered sub-optimal. AI with excessive numbers of frozen-thawed sperm (5-6 x 10(9) cells), still does not achieve fertility levels similar to AI using liquid semen because of reduced sperm survival. Frozen-thawed (FT) spermatozoa have therefore not been the preferred option for commercial breeding programmes. However, substantial progress has been made regarding boar sperm cryopreservation. Adjustment of cooling and re-warming rates to biophysical properties of boar spermatozoa, new sperm package systems and the achievement of accurately consistent freezing of large numbers of samples using programmable freezers have contributed to post-thaw survival rates above 50%, a threshold similar to that used for bull AI-semen. Moreover, these post-thaw sperm survival rates are consistent within a large population of boars selected for sperm freezability potential, as occurs with AI-bull sires. When such post-thaw boar semen is deposited intra-utero, acceptable fertility (in terms of farrowing rates and litter size) is obtained. Currently, the most effective application of FT-semen for AI is achieved using deep uterine-AI (DUI) which allows placement of a minimal semen dose (in volume 0.5 to 10 mL and sperm number 0.5 to 1 x 10(9) total spermatozoa) into the anterior 1/3 of one uterine horn, with levels of fertility close to AI with liquid semen. However, owing to their shorter life span, FT-boar spermatozoa require an AI-to-ovulation interval not longer than 4-6 h, making peri-ovulatory AI a pre-requisite to obtain the highest possible fertility. Spontaneous ovulation most often occurs when two-thirds of oestrus has passed. Estimation of the duration of oestrus, taking into account the weaning-to-oestrus interval, is helpful when establishing appropriate AI-schedules. However, as the length of oestrus varies within and between farms, different AI strategies should be established a priori. The development of bio-sensors for spontaneous ovulation will widen the use of AI with frozen-thawed frozen semen.

Animals↗
PubMedDate not supplied

Somatic gene mutations in vivo as indicated by the 6-thioguanine-resistant T-lymphocytes in human blood.

The clonal and the autoradiographic assays for 6-thioguanine-resistant (TGr) T-lymphocytes (T-Lys) in human blood are reviewed. Studies of TGr colonies recovered from clonal assays show that the mutant T-Lys (i) are either helper (T4) or suppressor (T8) cells, (ii) possess stable TGr phenotypes, (iii) are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT), and (iv) have structural alterations in the hprt gene. TGr T-Ly mutant frequencies (Mfs) determined by clonal assays are of the order of 10(-6)-10(-5) for normal adults. Autoradiographically determined variant frequencies (Vfs) are also in this range for normal adults when lymphocytes are cryopreserved before study to remove 'phenocopies'. Cancer exposed to potentially mutagenic treatments have elevated TGr T-Ly Vfs. Comparative clonal and autoradiographic assays of the same blood samples give generally similar results when allowances are made for potential sources of error in each assay. The TGr T-Ly system is presented for human specific-locus mutagenicity monitoring.

Adult↗
PubMed1983

T-cell subset analysis of cryopreserved human peripheral blood mononuclear cells.

A criticism of current techniques for monitoring changes in T-cell subset numbers over extended periods in individuals with disease states in which such changes might provide insight in the fact that serial samples taken are usually analysed fresh and therefore not in the same assay. To try to overcome this problem we have stored peripheral blood mononuclear cells frozen in liquid nitrogen and thence examined their ability to form sheep red blood cell (E) rosettes and to label with OK monoclonal antibodies. Results obtained show that cell viabilities following freezing and T-cell subset analysis of E-rosette positive cells are no different when fresh or frozen and subsequently thawed peripheral blood mononuclear cells are used.

Antibodies, Monoclonal↗
PubMed1996

Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation.

PURPOSE: The present study evaluates the clinical significance of detection of cytokeratin 19 (K19) in the bone marrow of patients with breast cancer undergoing high-dose chemotherapy (HDCT) and autologous bone marrow transplantation (ABMT). PATIENTS AND METHODS: We studied retrospectively cryopreserved bone marrow aspirates from 83 patients with high-risk stage II, III, and IV breast cancer obtained before bone marrow harvest but after induction chemotherapy. All samples were histologically negative for metastases. Polymerase chain reaction (PCR) for K19 was performed according to methods described previously and results were correlated with the probability of relapse following HDCT and ABMT. RESULTS: The incidence of occult metastases as defined by PCR for K19 message was 52% for 19 stage II, 57% for 14 stage III, and 82% for 50 stage IV patients (two-tailed P = .0075, chi 2 test). The probability of relapse at 3 years after ABMT was 32% and 94% for K19-positive stage II/III and stage IV patients, respectively, versus 10% and 14% for K19-negative stage II/III and stage IV patients, respectively. The difference was significant for stage IV patients (two-tailed P = .0002). CONCLUSION: It has been shown that PCR is a highly sensitive method to detect K19 message in the bone marrow. The incidence of K19 positivity in bone marrow increases significantly with advancing stage. In patients with breast cancer, especially metastatic breast cancer, undergoing HDCT and ABMT, the presence of K19 is associated with a poor prognosis.

Adult↗
PubMed2002

Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulfoxide concentration.

UNLABELLED: The development of cryopreservation (CP) strategies has traditionally focused on the cellular chemo-osmometric characteristics attendant to the freeze-thaw process. This approach coupled with a limited understanding of cellular physiological and biochemical responses to the CP process often yields sub-optimal cell survival. Recently, we have reported on the benefits of the utilization of an intracellular-like preservation solution, HypoThermosol (HTS), as well as incorporating a molecular approach to improving CP outcome [In Vitro Cell. Dev. Biol. Anim. 36(4) (2000) 262]. We now report on the elucidation of a cryoprotective agent (CPA)-dependent survival limit (cap) associated with standard CP methodologies. We further demonstrate an elevation and shift in the CP cap through the utilization of HTS coupled with a reduction in CPA levels necessary to achieve "successful" cell preservation. METHODS: Human fibroblasts, keratinocytes, hepatic, and renal cells were cryopreserved in a standard fashion (approximately 1 degrees C min-1 cooling and storage in LN2) in culture media (serum-free) or HTS with varying levels of dimethyl sulfoxide (Me2SO). Samples were allowed to recover for 24-h prior to survival assessment. Survival was assessed using alamarBlue (metabolic activity indicator) and calcien-AM (membrane integrity stain) in comparison with non-frozen controls. RESULTS: (1) A limit in cell survival was identified following CP in media-based CP solutions yielding a cell-type specific CPA-dependent survival limit, (2) peak cell survival resulted in the identification of "optimal" Me2SO concentrations for CP of each cell type, (3) incorporation of HTS as the carrier medium at typical Me2SO concentrations substantially elevated survival, and (4) utilization of HTS allowed for the successful preservation of all systems examined at significantly reduced Me2SO levels. CONCLUSION: The data presented in this study illustrate that the utilization of HTS as the carrier medium during CP facilitated a significant improvement in efficacy at reduced Me2SO levels. Further, the utilization of HTS offers the potential for successful Me2SO-free CP. These findings may prove significant to the advancement in the development of cell-based clinical therapies by providing an improved biocompatible CP methodology.

Animals↗
PubMedDate not supplied

Impact of cryopreservation on spermatozoa from infertile men: implications for artificial insemination.

The objective of this study was to evaluate the effect of cryopreservation-thawing on the quality of sperm from men with subfertile semen parameters. Twenty-seven patients with teratozoospermia, six of whom also had asthenozoospermia, were studied and their sperm parameters were compared to those of fertile donors (n = 71) in their fresh, post-thaw, and washed samples. After thawing, the percentage decrease in motility was significantly greater in patients than in donors, but the motility yield (post-thaw motility/prethaw motility) reached an average of 58% in the patient group vs. 68% in the donors (p = .02). No single characteristic of the fresh samples from patients or donors could significantly predict post-thaw outcome. For the patient group, however, multiple regression analysis provided a cutoff sperm concentration (50 x 10(6)/mL) and motility (40%) below which a very poor post-thaw recovery was obtained. The frozen-thawed-washed specimens had significantly higher velocity than the frozen-thawed samples, both in patients and donors. The results suggest that some patients with teratoasthenozoospermia yield acceptable sperm parameters after freezing-thawing-washing, and therefore these ejaculates could be used individually (or pooled) in artificial insemination.

Cryopreservation↗
PubMed2002

Quantitative, functional, morphological and ultrastructural recovery of platelets as predictor for cryopreservation.

Cryopreservation of platelets is of great interest, since it could extend the shelf life of therapeutic platelet concentrates and facilitate stockpiling and inventory control in blood banking. Despite the use of many cryopreservation procedures the optimal cryopreservation procedure is not defined yet. We have compared the cryopreservation of human platelets by various protocols employing controlled-rate and non-controlled-rate freezing procedures in combination with different concentrations of DMSO (6% and 10%) or 5% DMSO + 6% HES combination. After storage for 1 to 3 months, samples were thawed and analyzed. Measurements included cell recovery, platelet viability according to hypotonic shock response (HSR), platelet aggregation with ADP, morphological and ultrastructural properties of defrozen platelets. Our findings show that the application of our original procedure for controlled-rate freezing consisting of six cooling steps (cooling rate 1 degree C/min) with compensation of released heat of fusion (cooling rate 2 degrees C/min) has significantly influenced the quality of thawed platelets. At the same time, a concentration of 6% DMSO proved to be the most effective. In summary, cryopreservation of human platelets using controlled-rate freezing procedure in combination with lower (6%) DMSO concentration resulted in less damage from freezing and higher recovered function of platelets.

Blood Platelets↗
PubMed2002

Cryopreservation of the semen collected by electroejaculation from the Hokkaido brown bear (Ursus arctos yesoensis).

Four adult Hokkaido brown bears were used as semen donors, and semen characteristics were examined before freezing and after thawing. A total of 10 electroejaculates were diluted with Tris-egg yolk extender and cooled to 4 degrees C over 90 min. Spermatozoa were equilibrated with 4.7% glycerol for 80 min. Semen packed in 0.25 ml plastic straws were frozen with liquid nitrogen vapor. Percentages (mean +/- SD) of motile and live sperm were 96+/-2 and 86.5+/-7.2% before freezing, and 43+/-5 and 67.4+/-3.9% after thawing, respectively. Although the number of progressively motile sperm after thawing varied among samples (1.8+/-1.2 x 10(8) cells/ejaculate), frozen semen in the present study might serve for artificial insemination.

Animals↗
PubMed2006

Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of egg yolk, glycerol and cooling rate.

Two experiments were conducted to determine the effects of egg yolk (EY), glycerol, and cooling rate on the cryosurvival of red deer epididymal spermatozoa. The aim of Experiment 1 was to examine the effects of two EY types (clarified EY, CE, prepared by centrifugation, and whole EY, WE), and four EY concentrations (0, 5, 10 and 20%) on cryosurvival of red deer epididymal spermatozoa. Sperm samples were diluted to a final sperm concentration of approximately 200 x 10(6)spermatozoa/ml with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility, viability and of plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Cryopreservation of red deer epididymal spermatozoa frozen in a clarified EY extender, and with a 20% EY resulted in more vigorous post-thaw and post-incubation motilities (P<0.0001). Moreover, our results showed that regardless of the egg yolk concentration tested, the best sperm quality was obtained with the use of CE. Therefore, the objective of Experiment 2 was to explore the post-thaw effects of four clarified egg yolk concentrations (0, 5, 10 and 20%), two final glycerol concentrations (3 and 6%), and two cooling rates from 22 to 5 degrees C (slow: 0.23 degrees C/min; rapid: 4.2 degrees C/min) on red deer epididymal spermatozoa. At thawing, the effects of CE and glycerol concentrations, and cooling rate, all independently affected post-thaw sperm quality, while there were no effects of interactions on post-thawing sperm quality. Therefore, we studied each variable separately. Differences (P<0.05) for most of the semen parameters evaluated were found between the two final glycerol concentrations tested, with the high values after thawing found with the use of 6% glycerol (58.8+/-1.4 versus 46.2+/-1.4, for sperm motility). Moreover, the cooling rate did not have an effect on the semen characteristics, except for NAR (P<0.05), with the high values after thawing found with the use of the rapid protocol (64.5+/-1.4 versus 59.9+/-1.4). In conclusion, the use of 20% CE and 6% glycerol in combination with a rapid cooling rate, significantly improved red deer epididymal spermatozoa freezability.

Acrosome↗
PubMed2006

Development of sperm sexing and associated assisted reproductive technology for sex preselection of captive bottlenose dolphins (Tursiops truncatus).

Research was conducted to develop sperm sorting and novel sperm preservation methodologies for sex predetermination in the bottlenose dolphin (Tursiops truncatus) using artificial insemination. In Study 1, the effect of seminal plasma (SP), sperm concentration and freezing rate (FR) on in vitro sperm quality of liquid-stored, non-sorted spermatozoa was examined. There was no effect (P > 0.05) of prefreeze SP addition on post-thaw quality (progressive motility, kinetic rating, sperm motility index (SMI), viability and acrosome integrity). Post-thaw motility parameters and viability were higher (P < 0.05) for slow FR than fast FR samples. In Study 2 investigating the effects of liquid storage and sorting on sperm quality, motility and SMI after sorting and centrifugation were lower (P < 0.05) than those of the initial ejaculate. The sort rate for enrichment (91 +/- 4% purity) of X- and Y-bearing spermatozoa was 3400 +/- 850 spermatozoa sex(-1) s(-1). In Study 3, compared with a modified straw method, directional freezing resulted in enhanced in vitro quality of sorted and non-sorted spermatozoa derived from liquid-stored semen (P < 0.05). In Study 4, endoscopic insemination of three dolphins with sorted, frozen-thawed X-bearing spermatozoa resulted in one conception and the birth of a female calf. High-purity sorting of dolphin spermatozoa, derived from liquid-stored semen, can be achieved with minimal loss of in vitro sperm quality and samples are functional in vivo.

Animals↗
PubMed1996

OCT embedded sections of pathological specimens as a source of high quality RNA for reverse transcriptase/polymerase chain reaction.

OCT embedded cryostat sections of stored pathological specimens of non-Hodgkin's lymphoma were used to provide RNA. After reverse transcription to produce cDNA, the polymerase chain reaction was performed with primers for standard and variant forms of the CD44 molecule. Using Southern transfer and hybridisation with a probe specific for exon 4 of the CD44 gene, both standard and variant forms were visualised by autoradiography. This method was shown to be applicable to other gene products by using primers specific for the abl and bcr genes. This technique permits retrospective analysis of RNA from small amounts of stored pathological samples.

Biomarkers, Tumor↗
PubMed2005

Freezability of equine semen after glass beads column separation.

REASONS FOR PERFORMING STUDY: The success rate of artificial insemination following the freezing of stallion semen is limited; therefore, improving the stallion semen quality after the freezing and thawing process is a necessary objective. OBJECTIVES: To investigate the influence of glass bead column separation on the freezability of stallion semen. HYPOTHESIS: Glass beads in a column separator remove damaged and dead spermatozoa in the ejaculate during centrifugation. METHODS: In total, 50 ejaculates from 6 Lipizzaner stallions were studied. Each ejaculate was divided into 2 parts, one half processed following standard procedure and the second half used for the column separation procedure. After freezing, semen quality was evaluated using standard tests for motility, morphology and viability of semen. RESULTS: Motility and progressive motility of the column-separated (CS) semen were significantly higher (P < 0.001) before freezing and immediately, 24 and 48 h after thawing. A significant increase (P < 0.001) in the percentage of hypoosmotic positive spermatozoa was observed in CS samples. The percentage of total morphological changes in the separated samples before and after freezing was significantly lower (P < 0.001) compared with samples prepared using the standard procedure. A substantial decrease (P < 0.001) was found in the percentage of spermatozoa with damaged acrosomes. However, the percentage of spermatozoa with coiled tails was increased in the separated samples (P < 0.001). CONCLUSIONS: Column separation before freezing has a positive effect on the quality of thawed equine semen. POTENTIAL RELEVANCE: The quality of CS frozen/thawed samples indicates their potential use for increasing insemination success in mares.

Animals↗
PubMed2004

Effect of post-thaw incubation on sperm kinematics and acrosomal integrity of ram spermatozoa cryopreserved in medium-sized French straws.

The objectives were to assess the effect of post-thaw in vitro incubation on motion characteristics and acrosomal integrity of ram spermatozoa of native Malpura and Bharat Merino breeds maintained under a semi-arid tropical environment. Good quality semen samples of both breeds were diluted, packaged in medium-sized straws, and frozen under controlled conditions. Straws were thawed at 60 degrees C for 10s and thawed samples were incubated at 37 degrees C for 4h. Post-thaw motion characteristics and acrosomal integrity of incubated spermatozoa were assessed (by computer-aided semen analysis and Giemsa staining, respectively) just prior to incubation and at hourly intervals thereafter. There was a significant effect of incubation time on motility characteristics and the proportion of spermatozoa with normal acrosomes; 81.4% (arcsin transformed value, 65.2) of spermatozoa were motile at the start of incubation, with 47.9% (arcsin transformed value, 44.4) motile after 4h. At the corresponding times, there were normal acrosomes in 65.8 (arcsin transformed value, 54.8) and 55.7% (arcsin transformed value, 48.9) of spermatozoa, respectively. The percentage straightness of spermatozoa varied during incubation (P < 0.01). However, there was no significant change in percentage linearity, curvilinear velocity, average path velocity, straight line velocity, lateral head displacement, and beat cross frequency of spermatozoa during incubation. There were no breed variations in any motility parameters during incubation, except percentage straightness (P < 0.05), lateral head displacement (P < 0.05) and beat cross frequency (P < 0.01). That sperm motility and acrosomal morphology were very acceptable immediately post-thaw and after 4h of incubation indicated the efficacy of cryopreserving ram spermatozoa under controlled conditions in medium-sized straws.

Acrosome↗
PubMed2005

Cryopreservation of human endothelial cells for vascular tissue engineering.

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to -80 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 +/- 5% and a viability of 79 +/- 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.

Cell Adhesion Molecules↗
PubMed1998

Improvement of ram sperm cryopreservation protocols assessed by sperm quality parameters and heterogeneity analysis.

Semen cryopreservation processes generally decrease sperm quality and fertility rate, but this is highly dependent on the specific susceptibility of sperm cells to low temperatures. In order to study the effect of cooling, freezing, and thawing on ram spermatozoa, sperm viability (plasma membrane integrity), HOS test (hyposmotic swelling test), and individual motility were assessed in fresh, cooled, and frozen-thawed samples. The cryoprotective ability of four extenders [Triladyl-yolk (20% Triladyl, 20% egg yolk), Salamon (9.9% raffinose, 2% sodium citrate, 15% egg yolk, 5% glycerol), I.N. I.A. (6.31% Tes, 1.51% Tris, 6% egg yolk, 3% glycerol), and Fiser (F1: 3.25% Tris, 9.3% fructose, 1.7% citric acid, 25% egg yolk, 2% glycerol; F2: 0.68% sodium citrate, 0.15% TES, 0.36% glycine, 10.18% lactose, 1.18% raffinose, 0.5% fructose, 3.95% dextran (150,000-200, 000 MW); 12% of the obtained solution was replaced by the same volume of glycerol)] was tested, as well as the effect of adding various compounds (bovine lactalbumin and serum albumin, vitamin E, dithiothreitol, bull seminal plasma, glycine betaine, proline, phosphatidylcholine, and cholesterol) to Fiser's medium. Simultaneously, centrifugal counter-current distribution in an aqueous two-phase system was carried out to analyze the effect of the four extenders on sperm surface heterogeneity. The results showed that ram spermatozoa undergo a dramatic loss of heterogeneity, viability, motility, and positive response to the HOS test during freezing and thawing. These changes were partially prevented by diluting samples in Fiser's extender containing egg yolk and glycerol. A considerable increase in sperm quality with respect to that obtained in Fiser's control sample was achieved by the addition of bovine lactalbumin, vitamin E, bovine serum albumin, and bovine and ram seminal plasma.

Animals↗
PubMed2002

[Treatment of fungal keratitis with partial lamellar keratoplasty].

OBJECTIVE: To study the indication and clinical effect of treatment of fungal keratitis with partial lamellar keratoplasty (LKP). METHODS: Sixty-eight cases of fungal keratitis who could not be controlled by routine antifungal medicine were treated with partial LKP with the cornea donors cryopreserved in glycerin. All patients were followed up for 6 months to 3 years after surgery. The ratio of transparent grafts and surgical complications were recorded. The disease corneal sample was divided into 2 equal parts, one for fungal culture and the other for histopathologic examination. RESULTS: Sixty-four cases (94%) resulted in successful resolution of fungal infection. The corrected visual acuities were 0.2 approximately 1.0; 4 cases (6%) recurred; positive fungal culture were found in 59 cases, and were identified as Fusarium in 41, Aspergillus in 8, Candida in 4, Penicillium in 2 and other Hyphomycetes in 4 cases. Hyphae in histologic sections of corneal buttons were detected in 61 cases. In most cases hyphae could not be found in the deeper layer of the sections except the 4 cases with recurrence. CONCLUSIONS: Partial LKP is one of the effective operations that can succeed curing fungal infection which can not be controlled by antifungal medicines. It is effective for fungal keratitis whose infected lesion lies in the superficial or middle layer, especially when the corneal ulcer is widespread or not at the central area.

Adolescent↗