Post-thaw Period Research Articles

Effect of Thawing Procedure and Thermo-Resistance Test on Sperm Motility and Kinematics Patterns in Two Bovine Breeds.

This investigation aimed to analyze the effect that thawing time and temperature in combination with a termo-resistance test had on straws from dairy bulls used for artificial insemination (AI) on semen motility and kinematic variables measured with CASA systems. Eight animals of Holstein and Jersey breeds were used, and nine frozen-thawed semen doses per animal were analyzed for each breed. Three temperatures (35, 37, and 40 °C) and three thawing times (35, 40, and 45 s) were evaluated using a factorial design. Motility and kinematic patterns were analyzed using CASA-mot (Computer-Assisted Semen Analysis of motility) technology at different post-thawing times (0.5, 1, and 2 h). Sperm motility in Jersey bulls was higher (p < 0.05) than in Holstein ones (64.52 ± 1.45% and 53.10 ± 1.40%, respectively). The same effect was seen with progressive motility among the two breeds (Jersey: 45.29 ± 1.00%; Holstein: 36.30 ± 0.98%, p < 0.05). The Jersey breed presented higher values (p < 0.05) of curvilinear velocity (VCL), rectilinear velocity (VSL), average velocity (VAP), linearity on forward progression (LIN), and wobble (WOB). The Holstein breed showed a lower mean value (p < 0.05) of the beat-cross frequency (BCF) compared to the Jersey breed, thus suggesting an effect on VCL and VAP. During the post-thaw period, a gradual increase in VCL was observed at 2 h. VSL and VAP showed a decrease (p < 0.05) as the post-thaw period was prolonged. The study showed differences in sperm quality between Holstein and Jersey breeds, influenced by cryopreservation, thawing, and post-thawing incubation. Thawing at 37 °C for 30 s was considered optimal in relation to sperm motility. In addition, a decrease in sperm quality was observed as post-thawing time increased.

Cryopreservation of Abies alba embryogenic tissues by slow-freezing method

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

Performance of bat-derived macrophages at different temperatures.

Heterothermy, as a temperature-dependent physiological continuum, may affect host-pathogen interactions through modulation of immune responses. Here, we evaluated proliferation and functional performance of a macrophage cell line established from the greater mouse-eared (Myotis myotis) bat at 8, 17.5, and 37°C to simulate body temperatures during hibernation, daily torpor and euthermia. Macrophages were also frozen to −20°C and then examined for their ability to proliferate in the immediate post-thaw period. We show that bat macrophages can proliferate at lower temperatures, though their growth rate is significantly slower than at 37°C. The cells differed in their shape, size and ability to attach to the plate surface at both lower temperatures, being spheroidal and free in suspension at 8°C and epithelial-like, spindle-shaped and/or spheroidal at 17.5°C. While phagocytosis at temperatures of 8 and 17.5°C amounted to 85.8 and 83.1% of the activity observed at 37°C, respectively, full phagocytic activity was restored within minutes of translocation into a higher temperature. Bat-derived macrophages were also able to withstand temperatures of −20°C in a cryoprotectant-free cultivation medium and, in the immediate post-thaw period, became viable and were able to proliferate. Our in vitro data enhance understanding of macrophage biology.

P-212 Impact of post-thaw blastocyst culture time prior to transfer on live birth rate in frozen-thawed embryo transfer cycles: a retrospective observational study

Abstract Study question Does post-thaw blastocyst culture time prior to transfer influence the live birth rate in frozen-thawed embryo transfer cycles? Summary answer Our study revealed that different culture times of frozen-thawed blastocysts prior to transfer does not influence live birth rate. What is known already Several studies demonstrated a significantly increased clinical pregnancy rate after a frozen-thawed embryo transfer (FET) compared to a fresh embryo transfer (ET). FET reduces the risk of ovarian hyperstimulation syndrome without facing adverse effects of supraphysiological hormonal levels over endometrial receptivity. However, little is known on the effect of post-thaw embryo culture duration prior to transfer on live birth rate in FET cycles. Recent findings described an improved blastocyst morphological grade after a 20-22h post-thaw culture. Of note, the overnight culture of non-ideal morphology thawed blastocysts prior to transfer resulted in an enhanced live birth rate. Study design, size, duration We compared the live birth rate of patients undergoing FET after a short blastocyst culture time (2-4h) prior to transfer to that of patients in whom thawed blastocysts were transferred after a long embryo culture time (20-22h). Frozen-thawed transfers of blastocysts vitrified at Day 5 and subsequently thawed were evaluated. Only transfers of single blastocyst were included. Participants/materials, setting, methods The study was conducted at the Infertility Center – Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan. We included couples with indications for IVF cycles who underwent frozen-thawed blastocyst transfer procedure on the following natural cycles for freeze-all strategy or to transfer supernumerary embryos from 2014-2021. Data were analyzed using SPSS Statistics software. We evaluated statistically significant differences using various tests including Fisher's test, chi-squared test and Wilcoxon's test. Main results and the role of chance A total of 2012 frozen-thawed cycles were included in the analysis; in 977 of them, blastocysts were transferred after a 2-4h post-thaw period and in 1035, blastocysts were transferred after a 20-22h post thaw culture period. The age of the women was comparable (35.4 ± 4.2 and 35.3 ± 4.1; p = 0.5, respectively in the short and long culture period). According to statistical analysis, no significant difference in pregnancy outcomes was found between the two groups. Live birth rate was respectively 40% and 42% ( p = 0.2) in the short and long post-thaw blastocyst culture period prior to transfer. Limitations, reasons for caution Our study was limited by the retrospective nature and was performed in a single fertility center. Moreover, only blastocysts vitrified on day 5 were evaluated and findings might not be generalized to day 6 and 7 blastocysts. Wider implications of the findings These findings might be useful in the context of the organization of the laboratory workflow. Indeed, the possibility to thaw blastocysts on the day before transfer may represent an advantage in order to avoid an overload of the laboratory procedures during very busy days. Trial registration number 28_2022

(Re)shaping Literary Canon in the Soviet Indigenous North

In this article I demonstrate how in the post-Thaw period—the period of “soft” socialist realism—the northern indigenous minorities began to (re)invent literary writing and manifest their own version of the canon. Due to the lack of a pre-Soviet written literary tradition, “young” literatures were born as a symbiosis of folklore, beliefs, indigenous-Christian customs and the surrogate literary tradition of the Russian-European center: the Soviet “master plot.” Having graduated from universities in Moscow or Leningrad, the first generations of writers “(re)invented” a view of themselves as simultaneously native and Other. A consequence of the fact that the authors internalized the role of the youngest “brother” was, among others, the amalgamation of children's and adults' narrative and pedagogical zeal, which combined folklore ethics with socialist realist moralism. The study is of a transitional time: before the local authors had experienced a cardinal reevaluation of their values during perestroika and afterwards.

Bez Ludwika i panny służącej. Projekty nowoczesnych kuchni jako zwierciadło przemian roli kobiet w poodwilżowej Polsce

The development of a post-Stalinist modern kitchen in Poland was informed by the activities of different individual and institutional actors: experts in ‘professional’ home engineering, architects and designers and modernist taste-makers. The image of the model kitchen is surprisingly coherent: a rational laboratory kitchen, where the housewife’s work is orchestrated according to Taylorism-inspired rules that aim at reducing the burden of domestic chores and introducing modern and hygienic equipmentand attitudes. The discourse, inspired by similar discussions in Europe and United States, mainly by the works of Swedish Research Institute, reflects the prewar ideas of kitchen-laboratories and ‘home engineering’. What’s new and different is the temporal (limited to a short post-Thaw period) enthusiasm for open-plan kitchens presented as spaces where a housewife can seamlessly perform two duties at the same time: housework and care work. This phenomenon mirrors changing attitudes towards women’s roles in society which, in the post-Stalinist period, were marked by ongoing conservatism. Drawing on the concept of a ‘mediation junction’ and the historical production-consumption-mediation paradigm in design, the article traces changing attitudes towards women’s roles in society, reflected both in popular and professional discourses on kitchen design.

Human serum albumin and chromatin condensation rescue ex vivo expanded γδ T cells from the effects of cryopreservation

Clinical applications of gamma delta (γδ) T cells have advanced from initial interest in expanding γδ T cells in vivo to the development of a manufacturing process for the ex vivo expansion. To develop an “off-the-shelf” allogeneic γδ T cell product, the cell manufacturing process must be optimized to include cryopreservation. It is known that cryopreservation can dramatically reduce viability of primary cells and other cell types after thawing, although the exact effects of cryopreservation on γδ T cell health and functionality have not yet been characterized. Our aim was to characterize the effects of a freeze/thaw cycle on γδ T cells and to develop an optimized protocol for cryopreservation. γδ T cells were expanded under serum-free conditions, using a good manufacturing practice (GMP) compliant protocol developed by our lab. We observed that cryopreservation reduced cell survival and increased the percentage of apoptotic cells, two measures that could not be improved through the use of 5 GMP compliant freezing media. The choice of thawing medium, specifically human albumin (HSA), improved γδ T cell viability and in addition, chromatin condensation prior to freezing increased cell viability after thawing, which could not be further improved with the use of a general caspase inhibitor. Finally, we found that cryopreserved cells had depolarized mitochondrial membranes and reduced cytotoxicity when tested against a range of leukemia cell lines. These studies provide a detailed analysis of the effects of cryopreservation on γδ T cells and provide methods for improving viability in the post-thaw period.

Improvement of bovine in vitro embryo production by fetal calf serum and cysteamine supplementation and investigation of freezability

The aim of this study was to investigate the effects of Cysteamine (Cys) and Fetal Calf Serum (FCS) in Synthetic Oviduct Fluid (SOF) and Charles and Rosenkrans (CR1aa) on the bovine in vitro embryo production and its ability of freeze. The oocytes were divided into two groups and allowed to mature in TCM-199, with and without cysteamine. They were divided into 4 subgroups according to whether they contain Cys and FCS in 2 different culture media (SOF and CR1aa). Accordingly, 8 groups were formed as SOF+FCS+Cys, SOF+Cys, SOF+FCS, S Dulbeco's OF, CR1aa+FCS+Cys, CR1aa+Cys, CR1aa+FCS and CR1aa. Embryos were cultured for 7-9 days and were frozen using vitrification method. Development of embryos was observed during the first 24 hr post-thaw period. The highest rate of compact morula was 20% in SOF+FCS+Cys and 19.4% in CR1aa+FCS+Cys. The rate of blastocyst in SOF+FCS and CR1aa+FCS were found 17.9% and 15.4% respectively and the difference between groups was statistically significant (P&amp;lt;0.05). During the 12 hr vitality assessment, the highest rate was determined in SOF+FCS (47%). The results indicate that FCS has a positive effect in reaching compact morula and blastocyst regardless of the presence of the culture medium or antioxidant used. There was no statistically difference between the values of post-thaw embryos.

Somatic Embryogenesis in Selected Conifer Trees Pinus nigra Arn. and Abies Hybrids.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

CRYOPRESERVATION OF SHEEP OOCYTES AND EMBRYOS USING LOCAL, MODIFIEID VITRIFICATION TOOL

The present study was aimed to cryopreserve mature, immature oocytes and in vitro produced embryos in Iraqi sheep using vitrification technique by local, simple and cost effective vitrification tool. This tool is an innovative straw called vitripeace invented, designed and used for the first time. Immature oocytes were aspirated from ovaries of slaughtered ewes and subjected to in vitro maturation and in vitro fertilization programs.The immature, mature oocytes and embryos were vitrified using Vitripeace tools, then thawed and assessed for the morphology and viability. The results revealed non-significant effect of time on viability (%) and normal morphology (%) of vitrified immature and mature oocytes for post-thawing and 2 hours post-thawing. The results showed significant (P&lt;0.05) reduction in the viability (%) of 2 cell embryo namely 88.89% and 77.78 % for post-thawing and two hours post-thawing respectively. The results revealed a significant (P&lt;0.05) reduction on normal morphology of 1 cell embryo namely 88.24 % and 76.47 % for post-thawing and two hours post-thawing respectively. Significant (P&lt;0.05) differences in the percentage of normal morphology were found at post-thawing period for all stages of embryo development which were 90.74%, 88.31, 88.24 and 83.33 for immature , mature oocytes, 1 cell and 2 cell embryos, respectively while no significant differences in the viability at post-thawing period among all stages of embryo development. It was concluded that, successful vitrification of oocytes and embryos was resulted using Vitripeace which was novel, simple and cost effective vitrification tool.

Efecto del medio extensor y de las curvas de enfriamiento y congelamiento en la célula espermática en caballos criollos colombianos

El objetivo fue analizar los parámetros de motilidad, vigor, morfología e integridad de membrana plasmática para cada uno de los crioprotectores. Cada eyaculado se dividió en dos alícuotas iguales al azar con variaciones en la adición del crioprotector (dimetilformamida 5% o glicerol 5%) y se congelo por el protocolo de criopreservación, con una curva de enfriamiento de 5ºC durante 120 minutos y exposición a vapor de nitrógeno durante 15 minutos antes de ser sumergido al nitrógeno líquido. La viabilidad espermática (motilidad) fue evaluada mediante el test de termorresistencia, la morfología de las células se evalúo fijando el semen en formol salino y la integridad de la membrana plasmática de la cola del espermatozoide mediante el test hiposmótico. Al evaluar el semen pos descongelación no se observó diferencia significativa (p&gt; 0.05) entre los dos crioprotectores respecto a los parámetros evaluados. Los resultados nos permiten concluir que la utilización del medio extensor INRA 82 modificado con azúcares asociado a leche descremada con adición del 2% de yema de huevo, y a una curva de enfriamiento moderada de 120 minutos a 5ºC muestran efectos benéficos en los parámetros espermáticos pos descongelamiento de forma independiente al crioprotector utilizado en los caballos criollos colombianos.

The 70-kDa Heat-shock Protein as a Potential Biomarker of Quality of the Parapenaeus longirostris Shrimp Flesh

ABSTRACTThe deep-water rose shrimp, Parapenaeus longirostris, is a target species of the Mediterranean fisheries mostly caught by trawlers offshore, processed, and frozen on board. The effects of thawing on shrimp muscle exudate collected at 0, 1, 2, 3 days after thawing were investigated. In total, 70-kDa heat-shock protein (Hsp70), alpha (α)-enolase, and manganese superoxide dismutase (Mn-SOD) were selected as metabolic and stress-related proteins and analyzed by immunoblotting on exudates. Data were compared for the amount of exudates collected and the pH values. Among the investigated proteins, only the Hsp70 levels showed a decrease related to the post-thawing period and correlated with both the significant increase of the exudate amount and the pH values. These data strongly suggest the potential use of Hsp70 as an early predictive biomarker for quality of the P. longirostris shrimp after thawing.

Transcriptome profiling of fully open flowers in a frost-tolerant almond genotype in response to freezing stress.

Spring frost is a major limiting abiotic stress for the cultivation of almonds [Prunus dulcis (Mill.)] in Mediterranean areas or the Middle East. Spring frost, in particular, damages almond fully open flowers, resulting to significant reduction in yield. Little is known about the genetic factors expressed after frost stress in Prunus spp. as well as in almond fully open flowers. Here, we provide the molecular signature of pistils of fully open flowers from a frost-tolerant almond genotype. The level of frost tolerance in this genotype was determined for all three flowering stages and was confirmed by comparing it to two other cultivars using several physiological analyses. Afterwards, comprehensive expression profiling of genes expressed in fully open flowers was performed after being exposed to frost temperatures (during post-thaw period). Clean reads, 27,104,070 and 32,730,772, were obtained for non-frost-treated and frost-treated (FT) libraries, respectively. A total of 62.24Mb was assembled, generating 50,896 unigenes and 66,906 transcripts. Therefore, 863 upregulated genes and 555 downregulated genes were identified in the FT library. Functional annotation showed that most of the upregulated genes were related to various biological processes involved in responding to abiotic stress. For the first time, a highly expressed cold-shock protein was identified in the reproductive organ of fruit trees. The expression of six genes was validated by RT-PCR. As the first comprehensive analysis of open flowers in a frost-tolerant almond genotype, this study represents a key step toward the molecular breeding of fruit tree species for frost tolerance.

Tissue regeneration of Abies embryogenic cell lines after 1 year storage in liquid nitrogen

AbstractEmbryogenic tissues of hybrid firs (

COMPARAÇÃO ENTRE OS SISTEMAS AUTOMATIZADO E CONVENCIONAL DE CRIOPRESERVAÇÃO DE SÊMEN BOVINO

The objective of this study was to compare the efficiency of bovine semen cryopreservation using the controlled-rate freezing machine versus the conventional method (uncontrolled curve) by the parameters of sperm quality and viability in post-thaw period. Semen from four adult crossbreed bulls was cryopreserved in Tris-yolk-glycerol medium. The computer assisted analysis of thawed semen detected the following results: PM 56.50±22.25%; VAP 34.77±4.25?m/s; VSL 28.17±4.25?m/s; VCL 58.45±6.85?m/s; STR 82.00±2.31%; LIN 49.50±3.32%, to automated system and PM 57.00±13.11%; VAP 25.75±1.66?m/s; VSL 23.32±1.99?m/s; VCL 63.32±1.79?m/s; STR 82.25±3.59?m/s; LIN 50.00±4.97?m/s to conventional cryopreservation system. The results of plasma membrane and acrosome integrity evaluation were 54.7±12.55% and 36.13±22.20% for the automated system and 53.22±13.22% and 47.26±5.74% for the conventional system, respectively. The parameters evaluated demonstrated that there was no statistical difference between the cryopreservation systems. Thus, the choice of the bovine semen cryopreservation method to be used on a farm is a responsibility of the technician, and should be based on the reality of each farm. Therefore, it is always necessary to consider that the conventional system of bovine semen cryopreservation can vary more than the automated system, which, despite the cost of the equipment, can ensure repeatability of the results and consequent quality of cryopreserved bovine semen. KEYWORDS: artificial insemination; biotechnology; bovine sperm; cryopreservation; cryopreservation curve .

Cryogenic Freezing of Silver Barb (&lt;i&gt;Barbonymus gonionotus&lt;/i&gt;) Spermatozoa for Gene Pool Conservation

Experiments were conducted to develop and standardize the protocols of cryopreservation of sperm of Barbonymus gonionotus. Three extenders Alsever’s solution, urea-egg-yolk and egg-yolk citrate and four cryoprotectants methanol, ethanol, DMSO and DMA were used. Cryodiluents were prepared by adding 10% cryoprotectant to 90% extender (% v/v). Milt and cryodiluents were mixed at a ratio of 1 : 9 for Alsever’s solution and 1 : 4 for urea egg-yolk and egg-yolk citrate solutions. Alsever’s solution with 10% DMSO showed the best performance and produced 78 ± 2.55% sperm motility at the post-thaw period. Urea egg-yolk and egg-yolk citrate with 10% DMSO produced 76 ± 1.87% and 74 ± 1.87% post-thaw motility respectively. When cryopreserved sperm was stored in liquid nitrogen for 130 days, a gradual reduction in motility ranging from 31.25 to 37.50% was observed. This could be happened due to frequent opening of the nitrogen dewar that could cause thawing of the sample. In breeding trials, sperm preserved with Alsever’s solution and urea egg-yolk plus 10% DMSO produced 14.28 ± 7.06% and 15.46 ± 5.50% hatching respectively. In contrast, sperm preserved with egg-yolk citrate and 10% DMSO produced poor hatching as 8.01 ± 2.15%.DOI: http://dx.doi.org/10.3329/pa.v20i1-2.16863 Progress. Agric. 20(1 &amp; 2): 117 – 124, 2009

Cryofreezing of Grass Carp (&lt;i&gt;Ctenopharyngodon idella&lt;/i&gt;) Spermatozoa for &lt;i&gt;Ex Situ&lt;/i&gt; Conservation

Experiments were conducted to develop and standardize the protocols for cryopreservation of sperm of grass carp (Ctenopharyngodon idella). Seven extenders such as Alsever’s solution, Urea egg-yolk, Egg-yolk citrate, Kurokura- 1, Kurokura-2, 0.9% NaCl and 0.6% Glucose and five cryoprotectants i.e. DMSO, methanol, ethanol, DMA and glycerol were employed for finding suitable combinations. Cryodiluents were prepared by mixing the cryoprotectants at 10% concentration of the extenders (v/v). Milt and cryodiluents were mixed at a ratio of 1:9 for Alsever’s solution, Kurokura-1, Kurokura-2, 0.9% NaCl and 0.6% glucose, and 1:4 for urea egg-yolk and egg-yolk citrate. Among the 35 combinations of extenders and cryoprotectants, Alsever’s solution with ethanol and methanol, urea egg-yolk and egg-yolk citrate with DMSO found suitable for preservation and it produced 74 ± 2.44%, 72 ± 2.54%, 76 ± 2.44% and 75 ± 2.23% spermatozoan motility at the post-thaw period respectively. Rest of the combinations, on the other hand, produced &lt;60% motility at the post-thaw period and glycerol was found to be clotted after freezing. The dilution of milt with cryodiluent has been tested using six dilution ratios (1:2, 1:4, 1:7, 1:9, 1:15, 1:20) and found that 1:9 dilution ratio produced the highest post-thaw spermatozoan motility with Alsever’s solution (&gt;75%) and 1:4 with urea eggyolk and egg-yolk citrate (&gt;70%). As an optimum level of cryoprotectant, 10% concentration was found effective to produce significantly highest (P&lt;0.05) percentage of spermatozoan motility compared to those of other four concentrations (5, 15, 20 and 30%).DOI: http://dx.doi.org/10.3329/pa.v21i1-2.16763 Progress. Agric. 21(1 &amp; 2): 141 - 150, 2010

Risk factors for microbial contamination of peripheral blood stem cell products

Despite the well-known contamination rates and presence of microbial agents in stem cell products, the risk factors affecting microbial contamination have not been well described. In a 12-year period, we retrospectively reviewed culture results of peripheral blood stem cell products with the intent of identifying risk factors for microbial contamination. Microbial contamination was detected in 28 (5.7%) products of the postprocessing period and in 18 (3.66%) products of the postthawing period. Large-volume leukapheresis (LVL; odds ratio [OR], 5.85; 95% confidence interval [CI], 1.52-22.49; p = 0.01) and high numbers of stem cell culture sampling (OR, 1.4; 95% CI, 1.03-1.91; p = 0.03) were found to be risk factors for postprocessing bacterial contamination. The presence of postprocessing bacterial contamination was a risk factor for postthawing (OR, 28.89; 95% CI, 6.67-125.15; p < 0.001) and posttransplant (OR, 3.25; 95% CI, 1.24-8.50; p = 0.01) microbial growth. In transplants that were performed using contaminated products, the same pathogen was detected in 20% of patients and different pathogens were found in 35% of patients. Cultures should be carefully monitored in LVL products and in samples with high numbers of cultures performed. Growth of different bacterial pathogens must be considered in transplants that are performed with contaminated products.

Monitoring Study of Shaft Lining Concrete Strain in Freezing Water-Bearing Soft Rock During Mine Shaft Construction Period in West China

Abstract In the water-bearing soft rock strata of West China, a field measurement is carried out on the shaft lining in bedrock section. The shaft lining is built with freezing method. From the measurement, some changes of regularity in construction, thawing and post thawing period are obtained, including the temperature, freezing pressure and the vertical and hoop strain of the shaft lining. Measurement studies have shown that: during the plastic stage of shaft construction period, with the release of hydration heat, the thermal expansion occurs in the concrete and the strain gauge, leading to tensile strain. In the earlier stage, the elastic modulus and strength of concrete increases rapidly. When the shaft lining temperature reaches its maximum, the cooling and growth in the freezing pressure make the hoop strain increase sharply. However, the hoop strain is at lower tensile or compressive value. In the early stage and mature stage, the strain change is mainly affected by temperature. In the thawing and post thawing period, the concrete strain is also primarily influenced by temperature change. During the measurement, when the maximum compressive strain is less than ultimate value, the shaft lining is in a safe state. To ensure the security of the shaft lining, the concrete strength growth and sidewall temperature should be controlled in the similar shaft condition. In addition, the reinforcement ratio should not be too small for the shaft lining in frozen bedrock. And further, the fiber reinforced concrete, shrinkage-compensating concrete and low hydration heat concrete technology can also be adopted to reduce the cracks as much as possible and to avoid penetrating cracks. For the soft aquifers in West China, the further field measurement is still needed to obtain the stress and deformation regularities of the shaft lining, which can not only provide a basis for security assessment of the shaft, but also guide its design and optimization to improve the design theory.

284 IN VITRO FUNCTION OF FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM UNDERGOING SORTING AND RECRYOPRESERVATION

Artificial insemination (AI) using sex-selected sperm of bottlenose dolphins is currently used for the reproductive and social management of captive populations, but distance of males to the sorting facility represents a limitation of the procedure. Sorting and recryopreservation of previously frozen–thawed (FSF) sperm would facilitate the global application of this technology. Although a calf has been produced using FSF sperm (O’Brien et al. 2009 Theriogenology 71, 98–107), a comprehensive examination of the in vitro quality of such samples is needed. The objective was to compare the in vitro quality of nonsorted (CNTR) and sorted (FSF) dolphin sperm before and after recryopreservation using straw (STR) and directional freezing (DF) methods. At all assessment intervals, sperm were evaluated for 1) motility parameters with computer-assisted sperm analysis (CASA); 2) plasma membrane integrity (viability) and acrosome integrity using propidium iodide/fluorescein isothiocyanate-labeled peanut agglutinin (PI/FITC-PNA) staining and 3) DNA denaturation using the sperm chromatin structure assay (SCSA). Semen from 3 ejaculates × 3 males was cryopreserved by DF. After thawing, samples were divided into CNTR and FSF. The CNTR sperm were recryopreserved using STR and DF methods with assessments performed after the first thaw (PT1) and before recryopreservation (PF2). The FSF sperm were prepared for sorting using a density gradient centrifugation (DGC) method, stained with Hoechst 33342, sorted (SX MoFlo®, Dako, Fort Collins, CO, USA), then recryopreserved using STR and DF methods. The FSF sperm were assessed post-PT1, post-DGC, post-stain, post-sort, and at PF2. After the second thaw (PT2), CNTR and FSF samples were diluted (1:0.1, vol/vol) with Androhep Enduraguard™ (AE; Minitube of America, Verona, WI, USA), incubated at room temperature, and assessed at 0, 6, 12, 18, and 24 h PT2. The PT1 samples retained high proportions of their PF1 total motility (TM) and progressive motility (PM) (mean ± SD; 87.9 ± 7.3% and 92.2 ± 5.9%, respectively). The FSF sperm had improved (ANOVA; P &lt; 0.05) motility (TM, PM, VAP, VCL, VSL) and viability at PF2 compared with PF1. The FSF sperm recryopreserved using DF had higher (P &lt; 0.05) motility over the 24-h post-thaw incubation period compared with STR. The CNTR sperm DNA fragmentation remained unchanged throughout the process. The DNA fragmentation of FSF samples increased after staining (P &lt; 0.05), then decreased during the PT2 incubation period, stabilising at lower values (P &lt; 0.05) than CNTR from 6 to 24 h PT2. This unusual pattern indicates a possible interaction between Hoechst 33342 and acridine orange. After recryopreservation, the viability of FSF sperm was higher (P &lt; 0.05) than that of CNTR sperm. Results indicate that bottlenose dolphin sperm undergoing cryopreservation, sorting, and recryopreservation are of adequate quality for use in AI.