Collection, cryopreservation and thawing of stem cells for children weighing less than 25 Kg with high-risk neuroblastoma: A single center results in Morocco

ABSTRACT Introduction: An important component of the advances made in neuroblastoma treatment has been the use of peripheral blood stem cells to support high-dose chemotherapy. In this study, we report our experience on a series of small children who have undergone standard and large volume leukaphersis (LVL) procedures, provide an update on a single institution's experience with cryopreservation of autologous peripheral blood stem cells (PBSCs), using 10% dimethyl sulfoxide (DMSO) and applying post-thaw DMSO depletion and analyze a number of variables that may affect viability. Methods: A total of 36 aphereses were performed on 29 children weighing less than 25 kg between July 2016 and October 2019 at the Ibn Sina university hospital. Results: Seven females and twenty-two males, median bodyweight 14 kg (9 - 22). A single apheresis was sufficient to obtain at least 3 × 106/kg body weight (BW) of CD34+ cells in 82.8% of the cases. The LVL was performed in 22 aphereses. A median number of 5.9 × 106/ kg CD34 cells were collected per apheresis. A total of 60 PBSC samples were cryopreserved and 46 samples were infused. The mean cell viability percentage decreased from 94.75 ± 1.14% before freezing to 70.84 ± 8.6% after thawing (p < 0.001). No correlation was found between post-thaw viability and storage time (r = -0.233; p = 0.234) or number of total nucleated cells (r = 0.344; p = 0.073). Conclusion: Leukapheresis is safe and feasible in small pediatric patients if the appropriate measures are used. Cryopreservation poses numerous challenges, especially a decrease in cell viability after thawing.

Thawing rate predicts acute pulmonary vein isolation after second-generation cryoballoon ablation

OBJECTIVE: To evaluate whether thawing rate could be a novel predictor of acute pulmonary vein isolation (PVI) and explore the predictive value of thawing rate as a factor ensuring long-term PVI (vagus reflex). METHODS: A total of 151 patients who underwent cryoballoon ablation for atrial fibrillation (AF) were enrolled in this retrospective study between January 2017 and June 2018. The thawing rate was calculated using the thawing phase of the cryoablation curve. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of the thawing rate for acute PVI and vagus reflex. RESULTS: ROC curve analyses revealed that the interval thawing rate at 15°C (ITR15) was the most valuable predictor of PVI, with the highest area under curve (AUC) value of the ROC curve. The best cut-off value of ITR15 for PVI was ≤2.14°C/S and its sensitivity and specificity were 88.62% and 67.18%, respectively. In addition, the ITR15 of the successful PVI group after cryoballoon ablation was significantly slower than the failed PVI group. ITR15 was a predictor of vagus reflex and the occurrence of vagus reflex group had a slower ITR15 compared to the non-occurrence group. CONCLUSIONS: Thawing rate was a novel predictor of acute PVI and the ITR15 was the most valuable predictor of acute PVI. In addition, ITR15 was a predictive factor ensuring long-term PVI (vagus reflex). Our study showed that thawing rate may serve in the early identification of useless cryoballoon ablation.

Optimization of preparation of rat kidney decellularized scaffold by combining freeze-thawing with perfusion / 生物工程学报

We explored the improved method to prepare decellularized kidney scaffold and provide experimental basis for kidney tissue engineering and renal pathology and toxicology in vitro research. We perfused rat kidneys with PBS (group control) and prepared the decellularized kidney scaffolds with sodium dodecyl sulfate (SDS) (group S), Triton X-100 combined with SDS (group TS), and Triton X-100 combined with SDS after repeated freezing and thawing (group FTS) in different flow velocity. Meanwhile we measured their fluid distributions and vascular resistance. We examined the degree of decellularization of acellular scaffolds by HE, DAPI staining and DNA quantification. We examined the retention of main composition and structural integrity of decellularized scaffolds by Masson, PAS and immunohistochemical staining. We also detected the ultrastructure, cytotoxicity and the level of growth factor of the scaffolds by scanning electron microscope, MTT and ELISA, respectively. The results showed that the time of decellularization in group FTS was less than that in group S and TS. The vascular resistance of scaffolds decellularized at 10 mL/min flow velocity was lower. The fluid distribution in groups S, TS and FTS was different from that in control group. No residual cell was detected by HE and DAPI staining. DNA content was less than 50 ng/mg. Masson, PAS and immunohistochemical staining results showed that there was extracellular collagen, polysaccharide, type I collagen, type IV collagen, fibronectin and laminin in the decellularized scaffolds, and the scanning electron microscope result showed the scaffolds had the honeycomb structure. The cytotoxicity level of decellularized scaffolds was between grade 0 to 1. The level of VEGF, EGF, IGF-1 and PDGF-BB in group FTS were significantly higher than those in group S and TS. In concluding, combining freeze-thawing with perfusion can produce more ideal and effective whole organ decellularized scaffold of rat kidney, and make a foundation for the study of kidney tissue engineering and in vitro pathology and toxicology of kidney.

Breast Milk-Transmitted Cytomegalovirus Infection in Preterm Infants

PURPOSE: The purpose of this study is to describe the rate of cytomegalovirus (CMV) virolactia, and the prevalence of breast milk (BM)-transmitted postnatal CMV infection among premature infants after freeze-thawing (FT) and Holder pasteurization (HP) of breast milk. METHODS: This is a single-center, retrospective study of 312 infants born at less than 32 weeks of gestation, or with a birth weight less than 1,500 g from January 2013 to June 2017. All infants were screened for CMV-specific immunoglobulin (Ig) G and IgM at birth. Initial CMV specific polymerase chain reaction (PCR) and CMV culture were performed on mothers' BM and babies' urine within the first 21 days of life. FT and HP of BM was used to prevent the transmission of CMV. For the surveillance of postnatal CMV infection, CMV culture and CMV specific PCR of urine from babies were repeated one to two months after the initial screening. Screening for viremia and viruria was performed if postnatal CMV infection was suspected. RESULTS: Among 178 BM samples obtained from mothers of CMV-IgG-seropositive infants, 80 (44.9%) were CMV PCR positive. CMV deoxyribonucleic acid (DNA) was detected in five of the 22 BM samples (22.7%) obtained from the mothers of CMV-IgG seronegative infants. When CMV DNA load in BM was measured before and after HP, various results were shown. Sixty-three infants out of 232 (27.2%) were evaluated for postnatal CMV infection and four infants out of 63 (6.3%) were infected. CONCLUSION: Interventions to prevent BM-transmitted CMV infection can reduce the chance of postnatal CMV infection, but not completely eliminate it.

Adição de antioxidantes ao sêmen de carneiros e seus efeitos após a descongelação / Addition of antioxidants in ram semen and the effects after thawing

Objetivou-se avaliar o efeito da adição dos antioxidantes ácido ascórbico, melatonina e Trolox C, associados ao sêmen diluído de carneiros sobre o estresse oxidativo e o potencial fecundante após criopreservação. Foram coletados 10 ejaculados de 3 carneiros (n=30) e diluídos em Tris-Gema de ovo até a concentração final de 200x106 sptz/mL e, mantidos em banho maria a 32°C. Os antioxidantes foram adicionados da seguinte forma: controle (sem adição de antioxidantes); 100µM de melatonina (MEL) + 0,05% de ácido ascórbico (AA); 100µM de MEL + 90µL de Trolox C (TRO); 90µL de TRO + 0,05% de AA; e 100µM de MEL + 0,05%AA + 90µL de TRO. Depois, o sêmen foi resfriado em câmara fria a 5°C por duas horas, após esse período, envasado e lacrado em palhetas de 0,5mL, e então acondicionado sob vapor de nitrogênio liquido (N2L), a 8cm da lâmina líquida por 15 minutos, e depois imersos no N2L. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática e da membrana acrossomal, atividade mitocondrial, teste de ligação e a quantificação do estresse oxidativo. As variáveis foram submetidas à análise de variância e medias comparadas pelo teste de Tukey a 5% de probabilidade. A motilidade (total e progressiva) foi maior (P<0,05) quando adicionado à associação MEL+AA+TRO (67% e 49,89%), MEL+AA (64,37% e 45,61%) e MEL+TRO (61,65% e 41,15%) comparado ao tratamento controle (55,52% e 36,54%) e TRO+AA (57,07% e 38,40%). A adição de MEL+AA+TRO ao sêmen diluído manteve (P<0,05) a integridade da membrana plasmática (30,75%) e acrossomal (84,53%) dos espermatozoides quando comparado ao tratamento controle (15,60 e 68,16%, respectivamente), além de ter promovido maior (P<0,05) atividade mitocondrial (96,43%) quando comparado aos demais tratamentos. O número de espermatozoides que apresentaram à capacidade de ligação a membrana perivitelina da gema de ovo foi maior (P<0,05) no sêmen tratado com as diferentes associações de antioxidante quando comparado ao controle, sendo a associação MEL+AA+TRO (178,36%) superior (P<0,05) aos demais tratamentos. Não houve diferença (P>0,05) entre os tratamentos quanto a quantidade de espécies reativas ao ácido tiobarbitúrico produzidos. Conclui-se que a adição de MEL+AA+TRO ao sêmen diluído de carneiros, nas doses avaliadas, melhora a qualidade espermática após descongelação.(AU)

Aimed to evaluate the effect of adding antioxidants as ascorbic acid, melatonin and Trolox C to diluted semen of ram with oxidative stress to potenciate fertilization after cryopreservation. Ten samples collected were diluted in Tris-egg yolk to a final concentration of 200x106 sperm/mL and kept in a water bath at 32°C. Antioxidants were added as follows: 100µM melatonin (MEL) +.05% ascorbic acid (AA); 100µM of MEL + 90µL of Trolox C (TRO); 90µL of TRO + 0.05% AA; and 100µM of MEL0.05% AA + 90µL of TRO. Semen was cooled in a cold chamber at 5°C for two hours and packaged, sealed in 0.5mL straws, packaged under liquid nitrogen vapor (N2L), 8cm of water depth for 15 minutes, and then immersed in N2L. Samples were assayed for motility, integrity of the plasma membrane and acrosomal membrane, mitochondrial activity, binding assay and oxidative stress spermatozoa. The variables were analyzed by ANOVA and means compared by Tukey test (P<0.05). Percentage of total and progressive motility was higher for sperm treated with MEL+AA+TRO (67% and 49.89%), MEL+AA (64.37% and 45.61%) and MEL+TRO (61.65% and 41.15%) compared with the other treatments (P<0.05). The integrity of the plasma membrane and acrosome was higher for all semen treated with antioxidant associations compared with control (P<0.05). Mitochondrial activity was higher in sperm treated with MEL+AA+TRO compared all treatments (P<0.05). The number of sperm binding to perivitelline membrane was higher for semen treated with antioxidant associations compared with control; also sperm treated with MEL+AA+TRO demonstrated higher effect of all (P<0.05). No difference was observed between the treatments by oxidative stress sperm (P>0.05). The addition of melatonin, ascorbic acid and Trolox C in diluted semen of ram improves sperm quality after thawing.(AU)

Application value of frozen-thawed mouse round spermatids in in vitro fertilization / 中华男科学杂志

Objective@#To investigate the application value of the frozen-thawed round spermatids of the mouse in in vitro fertilization (IVF).@*METHODS@#Haploid spermatids of the mouse obtained in vitro were divided into a frozen-thawed and a fresh group and oocytes were collected from 6－8 weeks old female mice. After diamidino-phenyl-indole (DAPI) staining, the oocytes were subjected to intracytoplasmic round spermatid injection (ROSI), 259 in the frozen-thawed and 238 in the fresh group. Comparisons were made between the two groups in the capacities of fertilization and embryonic development.@*RESULTS@#The survival rate of the frozen-thawed haploid spermatids was (75.9 ± 2.3) %. No statistically significant differences were observed between the frozen-thawed and fresh groups after ROSI in the rates of fertilization (51.9 vs 55.7%, P >0.05), 2-cell embryos (51.0 vs 62.2%, P >0.05), 4－8-cell embryos (41.8 vs 42.9%, P >0.05), or morula-blastocysts (12.2 vs 21.4%, P >0.05).@*CONCLUSIONS@#Frozen-thawed round spermatids of the mouse are similar to fresh ones in their capacities of fertilization and embryonic development.

Thawing Modes on Stability of Coagulation Control Products after Frozen / 现代检验医学杂志

Objective To investigate the thawing modes on stability of coagulation control products after frozen,looking for a new theoretical basis for cost control and the quality and safety of laboratory.Methods Using ACL TOP 700 automated co-agulation analyzer and supporting the same batch of reagents and quality control materials conduct of the study:after daily QC,recycled the remaining control materials immediately and dispensed into two EP tubes and frozen at-40℃,respectively thawed by room-temperature and 37℃water bath after 24 hours,and examined together with the date of quality control ma-terial,got 20 pairs of data for analysis the financial impact of two alternate ways on coagulation QC parameters.Results For the room-temperature thawing group,FIB high value increasedby an average 0.23 g/L (t=4.026 9,P0.05).For the 37℃water bath group,both normal and high value of APTT,PT,FIB,TT and D-Dimer were no significant difference after tha-wing (P>0.05).Conclusion The commercialization of coagulation control materials can be for the second QC,just follow the principle of rapid after melting and timely detection,other laboratories can be used as a reference.

Changes of coagulation factors stored for different lengths of time after fresh frozen plasma melting / 军事医学

Objective To study the changes in coagulation factors stored for different lengths of time after fresh frozen plasma(FFP)thawing.Methods Thirty-two samples of thawed FFP were detected by an automated coagulation analyzer SYSMEX CA-1500 to observe the changes in coagulation function and coagulation factors stored for different lengths of time at 4℃.Results Activated partial thromboplastin time (APTT) was significantly prolonged 12 hours after thawing.However, no significant changes were observed in prothrombin time (PT), thrombin time (TT) or fibrinogen (FIB).Coagulation factors showed varying degrees of attenuation in 24 hours.FⅤ, FⅦ and FⅧ were attenuated significantly in 6 hours by 17.2%, 9.47% and 12.5%, respectively.FⅨ began to attenuate in 12 hours and reached, the lowest rate of 21.1%, while FⅧ had the highest attenuation rate (52.0%) and showed the lowest stability.Conclusion The activity of coagulation factors is decreased with time after FFP thawing ,so it should be transfused as soon as possible to ensure the curative effect and clinical application.

Physical, Mechanical and Antimicrobial Evaluations of Physically Crosslinked PVA/Chitosan Hydrogels Containing Nanoparticles.

Chitosan/montmorillonite (MMT) and Poly vinyl Alcohol (PVA) / Silver nanoparticles (AgNPs) nanocomposites were prepared and formed hydrogel membranes using freeze thawing technique. The properties of the prepared hydrogels were investigated and compared to hydrogel membranes in presence and absence of nanometals. The physical behavior, mechanical properties and antibacterial activity was examined. Also the surface morphology monitored using scanning electron microscope. Antimicrobial activity against bacteria and yeast was also examined. The obtained results showed positive effect of nanometals especially AgNPs on swelling percent on the other hand tensile strength were combined by presence of MMT nanoparticles. The surface morphology showed homogenous images for all samples except samples containing MMT. All prepared samples containing nanoparticles showed antibacterial activity especially hydrogel membranes containing AgNPs.

Modern Trends: The Cryopreservation of Human Oocytes.

Oocyte freezing has been considered as an experimental procedure for decade while it would be an important tool to use in IVF routine. In fact, a safe and efficient program would be of substantial benefit for infertile patients and also for women at risk of loosing their fertility due to radio or chemio therapy treatments. Moreover, egg cryopreservation could replace embryo freezing which instead involves important legal and ethical drawbacks. After the initial disappointment due to the low survival rate diverse methods have been developed raising the post thaw recovery. The fertilization and cleavage performance of frozen eggs is now similar to that of fresh sibling oocytes, even though not a great deal is known about the early implantation potential. There is an increased global interest in analyzing intrinsic factors connected to the ability of these embryos to give a live birth. What is clear, up to now is that certain oocyte cryopreservation protocols may affect cell division and thus being associated with low implantation rates. Other freezing methods, instead, seem not to affect the post implantation development and are going to be used in laboratories as an alternative mode to embryo freezing. It would be of immense importance that further randomized studies are carried out which may underline any possible differences between various technical approaches and also to further generate more analytical data on the cumulative pregnancy rates along with long term follow up of the babies born as associated with oocyte cryopreservation.

Influence factors of macronutrients and energy content of donor human milk / 实用医学杂志

Objectives To explore the effect of holder pasteurization, frozen storage time and thawing methods on macronutrients and energy content of donor human milk, and to provide theoretical basis for the rational use of breast milk. Methods Thirty-three samples of donor human milk were collected and an aliquot of each sample was analyzed before and after pasteurization. The remaining milk after pasteurization was split into 9 aliquot , and frozen at -20 ℃. After 30, 60, and 90 days, the milk was thawed by three different methods of room tempe-rature, 4 ℃ refrigeration, and 37 ℃ water bath, respectively. The nutrient components of each aliquot were analyzed and compared. Results We observed a mild reduction in fat and energy content after pasteurization (P <0.05). A significant decrease of fat, protein and energy content with the prolonged storage time was observed (P <0.01), and during the whole process (pasteurization + frozen storage), the decrease of fat, protein and energy content was 36.6%, 32.6%and 22.6%, respectively. The protein was influenced mostly by different thawing methods and the content of protein reached highest while thawed at 4 ℃ refrigeration. Conclusions Holder pasteurization and frozen storage at-20℃significantly reduce fat, protein and energy content of donor human milk. The donor milk should be used as quickly as possible when applied for preterm infants and thawing at 4 ℃ refrigeration is recommended before delivery to newborn infants.

The Expression Profile of Angiotensin System on Thawed Murine Ovaries

Freezing and thawing is one of the most widely used tissue engineering techniques for the preservation of ovaries. Many cells and tissues demonstrate changes in functional gene expression after thawing. Several studies have reported the important roles of angiotensin (AT) system during the ovarian follicular growth. AT system consists of ATII, and ATII receptors type I (ATII-RI) and type II (ATII-RII). However, little is known whether frozen-thawed ovaries show any alteration of AT system member gene expression when treated with survival-enhancing factors. We aimed to investigate whether mass freezing and thawing with or without the use of Rho-associated kinase (ROCK) inhibitors up- or down-regulate the expression of ATII, ATII-RI, and ATII-RII genes on frozen-thawed ovarian tissues. Significant changes in the expression of ATII, ATII-RI, and ATII-RII genes were observed on thawed ovaries when compared to fresh control. The treatment with ROCK inhibitors did not significantly alter their expression. In conclusion, freezing and thawing of ovarian tissue may affect the mRNA expression levels of intra-ovarian AT system genes, and modulation of ROCK inhibitor activity may not regulate AT system on the frozenthawed ovarian tissue.

Application of high hydrostatic pressure in development of melanoma vaccine / 中国免疫学杂志

Objective: To explore the application of high hydrostatic pressure in the development of melanoma vaccine.Methods:The high hydrostatic pressure,liquid nitrogen freezing and thawing were used to break the murine B16 melanoma cells and then the cell suspension was mixed with Freund's adjuvant to prepare vaccine for immunizing the mice.Results:After immu-nization,the murine B16 melanoma cells were injected intravenously and subcutaneously.The immune results of the vaccines were evaluated,by comparing survival time of the mice, subcutaneous tumor volume, DTH experiments and the fluorescence imaging of tumors.Conclusion:Compared with the method of liquid nitrogen freeze-thaw broken,high hydrostatic pressure crushing cells has more advantages in the development of tumor vaccine.

Cellular immunity of influenza vaccine lyophilized liposome produced by freeze-thawing / 中国药科大学学报

@#To investigate the cellular immunogenicity of influenza vaccine liposomes dry powder using the film-dispersion and freeze-thawing. Mice were divided into the non-liposomal influenza vaccine group, film-dispersion prepared liposomal influenza vaccine group, freeze-thawing lyophilized influenza vaccine liposome group, positive control group and negative control group(n=5). 6 μg of hemagglutinin of H1N1 subtype per mouse was delivered tracheally to the mice of lyophilized liposomes groups, with the same dose for non-liposomes intraperitoneally delivered groups as the positive control, and PBS intraperotoneal injection group as the negative control. After 28-day of immunization, the proliferationofsplenic lymphocytes was detected by MTT assay; CD4+cell and CD8+cell were analyzed by flow cytometry. The dry powder of influenza vaccine liposomes prepared by the above two methods, both induce cellular immunity, with no significant difference in the induced on immunogenicity between the prepared products(P< 0. 05). The results showed that freeze-thawing method is feasible in the preparation of vaccine liposomes, leading to the attenuated live vaccine liposome preparation.

Cryostorage of sunflower seeds / Crioarmazenamento de sementes de girassol

Sunflower seeds present a great economic importance due to the high oil content. The aim of this study to evaluate the effect of storage sub-zero temperatures and the quick and slow thawing methods on the physiologic quality of seeds of Helianthus annuus, cultivars BRS 122 and BRS 324. The experiment was set up in a randomized complete design, in a factorial 2x6 (cultivars x procedures) with four replications; means were compared by the Tukey test at 5%. Were evaluated: first count and percentage of germination, mean time of germination, germination speed index, dry mass seedling, root and shoot length and stability of membranes, through electrical conductivity test solution soaking of seed. Sunflower seeds tolerate storage in sub zero temperatures up to six months; the seeds of cultivars BRS and BRS 324 122 present different answers in relation to the type of freezing and thawing.

As sementes de girassol apresentam grande importância econômica devido ao grande conteúdo de óleo. Objetivou-se neste trabalho avaliar o efeito do armazenamento em temperaturas sub-zero e os métodos de descongelamento rápido e lento sob a qualidade fisiológica de sementes de Helianthus annuus cultivares BRS 122 e BRS 324. Utilizou-se o delineamento experimental inteiramente casualizado, num fatorial 2x6 (cultivares x procedimentos), com quatro repetições; as médias foram comparadas pelo teste de Tukey em nível de 5%. Foram avaliados: primeira contagem e porcentagem de germinação, tempo médio de germinação, índice de velocidade de germinação, massa seca de plântulas, comprimento de raiz e parte aérea e estabilidade das membranas, através de teste de condutividade elétrica da solução de embebição das sementes. As sementes de girassol toleram o armazenamento em temperaturas sub-zero até seis meses; as sementes dos cultivares BRS 122 e BRS 324 apresentam respostas diferentes em relação ao tipo de congelamento e descongelamento.

Ultrastructural observation of dormant mouse embryos cultured in vitro after freezing-thawing / 中国实验动物学报

Objective The aim of this study was to investigate the differences of the cell ultrastucture of normal mouse hatched blastocysts and their dormant ones cultured in vitro after freezing-thawing, and to explore whether the dor-mant embryos have a better anti-freezing shock property than the normal hatched mouse embryos .Methods By transmis-sion electron microscopy , the ultrastructure of these two types of mouse embryos was observed and analyzed .Results By comparative analysis of their ultrastructure , the results showed that the dormant embryos before freezing are being austerity and with lower energy metabolism at a ‘ground state ’ .After freezing-thawing and culture , their cellular structure seemed to be similar to that of the normal embryos cultured in vitro before freezing.However, after freezing-thawing and culture, the number of mitochondria decreased , the nuclei were loose , and their heterochromatin also increased .Conclusions From the ultrastructural observation , compared with the normal mouse hatched embryos , the cellular state of dormant mouse em-bryos after freezing-thawing is more favorable for material storage and energy metabolism , thus, indicating that they have a better anti-freezing property than normal hatched embryos .

Effect of freezing and thawing rates on sperm motility in Bocachico Prochilodus magdalenae (Pisces, Characiformes) / Efecto de la congelación y descongelación sobre movilidad espermática en Bocachico Prochilodus magdalenae (Pisces, Characiformes)

Objective. To determine the freezing and thawing rates necessary to maintain sperm viability during cryopreservation of Bocachico semen. Materials and methods. Four interactional treatments were implemented between two freezing (rapid and slow) and two thawing (rapid and slow) curves, in a 2x2 factorial as follows: rapid freezing-rapid thawing, rapid freezing-slow thawing, slow freezing-rapid thawing, and slow freezing-slow thawing. After thawing by Sperm Class Analyzer (SCA) curvilinear velocity (VCL) and straight-line (VSL) (μm sec-1) were analyzed; total, rapid, medium, and slow motility, were compared among treatments. Results. The rapid freezing-slow thawing treatment was lethal for all variables of velocity and motility, causing a significant (p<0.01) post-thaw inmotility of 100%. The slow freezing-rapid thawing interaction had a significantly higher effect than the other treatments (p<0.05), particularly on variables such as rapid motility (10.1 ± 1.1%), medium motility (30.16 ± 4.1%), and curvilinear velocity (51.5 ± 4.75 μm sec.-1) also decreased the percentage of sperm with slow motility (41.7 ± 4.45%). Independently of the applied thawing rate, the freezing rate generated the main significant effect on total motility. Conclusions. It is possible to conclude that the interaction effect between freezing and thawing rates is nil (except for slow motility) during cryopreservation process. However, the independent effects of these factors (main effects) on remaining motility variables are positively significant and decisive to the maintenance of these features, especially the freeze factor (when it is slow). This becomes the first successful report of sperm cryopreservation from Bocachico Prochilodus magdalenae in the world and may be used in conservation programs for this endangered species.

Objetivo. Determinar la tasa de congelación y descongelación para mantener la viabilidad espermática durante la crioconservación seminal de Bocachico. Materiales y métodos. Fueron implementados cuatro tratamientos de interacción entre dos curvas de congelación (rápida, lenta) y dos curvas de descongelación (rápida, lenta) en un factorial 2x2, así: congelación rápida-descongelación rápida, congelación rápida-descongelación lenta, congelación lenta-descongelación rápida y congelación lenta-descongelación lenta. Posterior a la descongelación espermática y mediante el software Sperm-Class-Analyzer, fueron analizadas la velocidad curvilínea (VCL) y lineal (VSL) (µm sec-1); movilidad total, rápida, media y lenta, y fueron comparadas entre tratamientos. Resultados. El tratamiento congelación rápida-descongelación lenta fue letal para las variables de velocidad y movilidad espermática, causando una significativa (p<0.05) inmovilidad pos-descongelación (100%). La interacción congelación lenta-descongelación rápida tuvo un efecto significativamente mejor (p<0.05) que los demás tratamientos, particularmente sobre variables como movilidad rápida (10.1 ± 1.1%), movilidad media (30.16 ± 4.1%) y velocidad curvilínea (51.5 ± 4.75 µm sec.-1), así mismo, generó una disminución del porcentaje de espermatozoides con movilidad lenta (41.7 ± 4.45%). La tasa de congelación posee un efecto principal significativo sobre la movilidad total. Conclusiones. El efecto de la interacción entre las tasas de congelación y la descongelación es nulo (excepto para la movilidad lenta), sin embargo, los efectos independientes de estos factores (efectos principales) sobre el resto de variables de movilidad son positivamente significativos y determinantes para el mantenimiento de aquellas características, especialmente el factor congelación (cuando es lento). Este es el primer reporte exitoso de crioconservación espermática de Bocachico P. magdalenae en el mundo, pudiendo ser usado en programas de conservación de esta especie en riesgo de extinción.

Fat Loss in Thawed Breast Milk: Comparison Between Refrigerator and Warm Water.

Objective: To compare the fat loss between refrigerator and warm water thawed breast milk. Design: Experimental. Setting: Tertiary-care pediatric university hospital. Participants: Ninety samples of expressed breast milk were collected from mothers with singleton babies of a gestational age 32-42 weeks. Main Outcome Measures: Fat content in fresh breast milk (FM); thawed breast milk by refrigeration (RM); and thawed breast milk by warm water (WM). R E S E A R C H P A P E R INDIAN PEDIATRICS 877 VOLUME 49__NOVEMBER 16, 2012 Results: The mean (SD) total fat content in FM, RM and WM were 2.98 (0.97), 2.76 (0.99) and 2.66 (0.88) g/100 mL, respectively. The mean difference (SD) of the total fat in FM declined significantly after the frozen milk was thawed by refrigeration or warm water at -0.22 (0.50) g/100 mL (P=0.0001) and -0.32 (0.45) g/100 mL (P<0.0001), respectively. The mean (SD) total fat loss of frozen breast milk thawed by refrigeration was less than thawing in warm water at 0.094 (0.38) g/100 mL (P=0.02). Conclusion: The fat loss of thawed breast milk by refrigeration was significantly less than placing it in warm water.

Cryopreservation of Agaricus blazei in liquid nitrogen using DMSO as cryoprotectant / Criopreservação de Agaricus blazei em nitrogênio líquido usando DMSO como crioprotetor

The preservation of Agaricus blazei is generally done using successive subcultivations that are laborious and are subject to contaminations or genetic degenerations, resulting in loss of biotechnological interest characteristics. An alternative process would be cryopreservation, but there are no reports of methodologies for this basidiomycete in liquid nitrogen. Thus, the objective of this study was to evaluate mycelial viability of A. blazei strains after cryopreservation in liquid nitrogen in order to establish the initial parameters of species preservation. Five strains grown on malt extract agar (MEA) were used. Disks of MEA containing A. blazei mycelium were transferred for screwcap cryovials containing the cryoprotectant, 10% dimethyl sulfoxide. Then, they were cooled at 8 ºC for 30 min and kept at -196 ºC with liquid nitrogen. After 1.5 year of cryopreservation, the cryovials were thawed in water bath at 30 ºC for 15 min. The disks with mycelia were transferred to MEA culture media without cryoprotectant and kept at 28 ºC for 30 days. A. blazei strains respond differently to the cryopreservation method at -196 ºC by varying mycelial viability recovery. Cryopreservation with liquid nitrogen, using dimethyl sulfoxide as cryoprotectant, is not the most appropriate one for A. blazei preservation.

A preservação de Agaricus blazei ocorre usualmente por subculturas sucessivas que são laboriosas e estão sujeitas a contaminações ou degenerações genéticas, com eventual perda das características de interesse biotecnológico. Uma alternativa a este processo é a criopreservação, no entanto não existem relatos de metodologias para este basidiomiceto com uso de nitrogênio líquido. Desta forma, o objetivo deste trabalho foi avaliar a criopreservação em nitrogênio líquido de linhagens de A. blazei, visando estabelecer os parâmetros iniciais de criopreservação para a espécie. Foram utilizadas cinco linhagens do fungo crescidas em meio de ágar-extrato de malte (MEA). Os discos de MEA contendo o micélio crescido foram transferidos em temperatura ambiente (25 ºC) para criotubos com rosca contendo o agente crioprotetor dimetilsulfóxido a 10%. Em seguida foram resfriados a 8 ºC por 30 min e mantidos a -196 ºC com nitrogênio líquido. Após 1,5 anos de criopreservação os criotubos foram descongelados por imersão em água a 30 ºC por 15 min. Os discos com micélio foram transferidos para meio de cultura MEA, sem o agente crioprotetor, e mantidos por 30 dias a 28 ºC. As linhagens de A. blazei respondem de forma diferente ao processo de criopreservação a -196 ºC com variação na recuperação da viabilidade do micélio. A criopreservação em nitrogênio líquido de A. blazei, com dimetilsulfóxido como crioprotetor, não é a forma mais adequada para a preservação desta espécie.

Rapid yeast DNA extraction by boiling and freeze-thawing without using chemical reagents and DNA purification

The purpose of this work was to study a rapid yeast DNA extraction by boiling and freeze-thawing processes without using chemical reagents or any purification procedures, to obtain a high grade PCR-product. A specific DNA fragment of the 18S region of Dekkera bruxellensis and Saccharomyces cerevisiae was chosen. The described boiling and freeze-thawing protocols generated the PCR-grade product preparations and could be used to process many samples. The amplification of the fragments could be observed after 30 and 35 cycles. These processes of extraction without using any kind of chemical reagents, especial water, and purification procedures proved to be efficient, reproducible, simple, fast, and inexpensive.