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ABSTRACT ARCHIVE |
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CryoLetters is a bimonthly, international journal for low temperature science and technology |
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CryoLetters 41 (1), 1-5 (2020) EFFECT OF AMIDE ON SEMEN CRYOPRESERVATION OF CURIMBA (Prochilodus lineatus) Izani Bonel Acosta1, Carine Dahl. Corcini1*, 1Nucleus of Teaching and Research in Animal Reproduction, Federal University of Pelotas, Pelotas, Brazil. Abstract BACKGROUND: The low molecular weight and high cellular permeability of amides make them suitable for use as penetrative cryoprotectants for sperm cells. OBJECTIVE: This study aims to evaluate the effect of dimethylformamide (DMF) and dimethylacetamide (DMA) on sperm cryopreservation of Curimba (Prochilodus lineatus). MATERIALS AND METHODS: Semen samples were diluted in media containing cryoprotectants [DMF, DMA and dimethyl sulfoxide (DMSO)]. Parameters of motility, membrane integrity, DNA integrity, mitochondrial functionality, viability and fertility were assessed upon thawing. RESULTS: As compared to the 10% DMSO, DMA at 5% and DMF at 2% obtained the best results for the integrity of membrane, DNA and mitochondria; the motility parameters were best in the 2% and 5% DMF treatments. The best fertilization rates were demonstrated in 2%, 5%, and 8% DMF treatment groups. CONCLUSION: DMF at 2%, 5%, and 8% provided the best results for both in vitro and in vivo assessments, and can efficiently cryopreserve semen of Prochilodus lineatus. Keywords: cryoprotectant, curimba, dimethylformamide, dimethylacetamide, spermatozoon.
CryoLetters 41 (1), 6-12 (2020) QUALITY IMPROVEMENT OF POST-THAWED STALLION EPIDIDYMAL SPERMATOZOA WITH SINGLE LAYER CENTRIFUGATION Fernanda Carlini Cunha dos Santos1*, Jane M. Morrell2, 1Programa de Pós Graduação Medicina Animal: Equinos, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Rio Grande do Sul, Brazil; Abstract BACKGROUND: Epididymal sperm cryopreservation represents the ultimate option to preserve spermatozoa of valuable stallions. OBJECTIVE: The study aims to evalute whether single layer centrifugation (SLC) prior to cryopreservation or after post-thawing improves the quality of stallion epididymal sperm. MATERIALS AND METHODS: Epididymal sperms of stallions were harvested (N=20). Sperm samples were subjected to treatments: conventional centrifugation, SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). All samples were cryopreserved, thawed and evaluated. SLC+ were thawed, single layer cenrifuged and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility, progressive motility, morphology, mitochondrial functionality, membrane integrity and DNA integrity were evaluated. RESULTS: SLC-PC and SLC+F yielded higher total motility, while SLC+F yielded the highest progressive motility. Mitochondrial functionality was significantly higher in all SLC groups. Membrane integrity was higher in SLC-PC. The percentage of morphologically normal spermatozoa was higher in SLC-PC and SLC+F. CONCLUSION: SLC prior to cryopreservation or post-thaw improves the quality of stallion epididymal spermatozoa. When SLC is performed post-thaw, freezing extender is the best medium to resuspend the pelleted semen. Keywords: colloid, cryopreservation, epididymides, sperm selection.
CryoLetters 41 (1), 13-18 (2020) EXOGENOUS ATP IN THE CRYOPRESERVATION OF Brycon orbignyanus SPERMATOZOA Carolina Trindade Perry,1 Carine Dahl Corcini,2 Izani Bonel Acosta,2 1Federal University of Rio Grande, Biology of Continental Aquatic Environments. Rio Grande, 96207 900, Brazil. Abstract BACKGROUND: ATP exogenous (ATPe) has been used successfully in improving motility and fertility for many animal species. However this has not yet been tested on Brycon orbignyamus. OBJECTIVE: The objective of this study was to evaluate the use of ATPe for the cryopreservation of sperm from B. orbignyamus. MATERIALS AND METHODS: The ATPe concentrations tested were 1.0 µM, 5.0 µM and 10 µM combined with Beltsville Thawing Solution™ extender and dimethylformamide at 7.5%. The sperm were frozen in a nitrogen vapour vessel and stored in liquid nitrogen at -196 ºC. The parameters of viability post-thawing were evaluated using CASA, and flow cytometer. RESULTS: The ATPe did not promote improvements in spermatic kinetics, and in the higher concentrations caused a worsening in these parameters. Also there was loss of mitochondrial functionality and greater cellular disruption with the concentration of 10 μM. CONCLUSION: We do not recommend the addition of ATP for cryopreserving B. orbignyamus. Keywords: ATPe, fish, cryopreservation, motility.
CryoLetters 41 (1), 19-25 (2020) THE EFFECT OF VITAMIN E AND POLY ETHYLENE GLYCOL (PEG) ASSOCIATION ON CHILLED RABBIT SPERM: IMPACT ON SPERM MOTILITY AND OXIDATIVE STRESS STATUS A. Amokrane1, R. Kaidi2 and M. Iguer-Ouada1 1Associated Laboratory in Marine Ecosystems and Aquaculture, Department of Biological Sciences of the Environment, Faculty of Nature and Life Sciences,
Abderrahmane-Mira-University, Route de Targua Ouzemmour, 06000 Bejaia, Algeria. Abstract BACKGROUND: Vitamin E is the major lipid-soluble antioxidant, however, its potent effect is limited by its poor solubility. OBJECTIVE: The present study aimed to investigate whether polyethylene glycol 6000 (PEG) can enhance Vitamin E solubility and help protect sperm motility and against oxidative status. MATERIALS AND METHODS: Sperm groups consisted of the control aliquot diluted with Tris buffer and aliquots treated with Tris buffer containing polyethylene glycol (PEG), vitamin E (Vit E) or vitamin E- PEG complex (PEG/Vit E). Sperm motility was measured using a Computer Aided Semen Analysis at 0, 1, 3, 6 hours of cooling at 4°C. The oxidative stress status was measured at 4 hours using ABTS radical scavenging capacity. RESULTS: Sperm motility and oxidative status were significantly protected when using PEG and Vit E individually; however the most potent effects were observed in PEG/Vit E treatment. CONCLUSION: The present study demonstrated that treating rabbit semen with vitamin E complexed to PEG 6000 (PEG/Vit E) is effective in protecting sperm cells during chilling at 4°C. Keywords: rabbit sperm, chilling at 4°C, PEG 6000, vitamin E, oxidative stress.
CryoLetters 41 (1), 26-30 (2020) MICROWAVE-WATER BATH HYBRID WARMING FOR FROZEN CRYOPROTECTANT SOLUTION USING A HELICAL ANTENNA Hailing Ruan 1,2, Tao Wang 1* and Cai Gao 1* 1 School of Automotive and Traffic Engineering, School of Electronic Science and Applied Physics, Hefei University of Technology, Hefei, P.R. China. Abstract BACKGROUND: Successful cryopreservation of organs and/or tissues of large dimension is challenging due to damages by solute concentration and thermal stress caused by crystallization during cooling and devitrification/recrystallization during rewarming. The key to reduce thermal stresses in cryopreserved biomaterials during rewarming is fast and uniform heating. OBJECTIVE: To explore a hybrid warming process using two heat sources (microwave and water bath) simultaneously to achieve faster and more uniform heating. MATERIALS AND METHODS: Rewarming of frozen cryoprotectants (CPA) using microwave and 37 °C water bath at the same time was experimentally studied. A helical antenna was installed at the center of a 1.8 mL cryovial. Microwave (2.4 GHz) was generated, amplified and transported to the helical antenna through the matched coaxial cables. Frozen CPA solution in the cryovial at an initial temperature of -196°C was rewarmed by microwave and water bath. The temperature of two selected points in the sample with the maximum temperature difference was measured by thermocouples during rewarming. RESULTS: During rewarming of the frozen sample in 37° C water bath without microwave, the warming rate was 70.2°C min-1 with the maximum temperature gradient of 1.07°C mm-1 in the sample. With microwave added to form a hybrid warming process, the warming rate was increased to be 100.5 °C min-1 with a smaller temperature gradient of 0.68°C mm-1. CONCLUSION: The study indicated that warming rate and temperature uniformity increased with the microwave-water bath hybrid heating process. Keywords: microwave-water bath hybrid heating, warming rate, temperature uniformity.
CryoLetters 41 (1), 31-37 (2020) THE PI3K/Akt/mTOR SIGNALING PATHWAY PLAYS A ROLE IN REGULATING AUTOPHAGY IN MOUSE OOCYTES DURING VITRIFICATION-WARMING AND IN VITRO MATURATION Jian-Min Zhanga*, Ran Guoa, Yong-Zhi Caob aSchool of Nursing, Shandong Xiehe University, Jinan, P.R. China. Abstract BACKGROUND: It has been shown that vitrified-warmed oocytes exhibit autophagic activation. However, its regulatory molecular mechanism of autophagy needs to be clarified. OBJECTIVE: To determine the role of signaling pathway of PI3K/Akt/mTOR in the regulation of autophagy in oocytes during vitrification-warming and IVM. MATERIALS AND METHODS: Oocytes from mice were vitrified-warmed and IVM. The expressions of LC3-II, Beclin-1, PI3K, Akt, and mTOR protein were determined. Moreover, the ATP level, viability of vitrified-warmed oocytes, and their developmental potential were measured. RESULTS: At 6 h of incubation of warmed oocytes, the LC3-II and Beclin-1 expressions were markedly up-regulated, whereas PI3K, Akt, and mTOR proteins expressions were significantly down-regulated. In addition, autophagy inhibition significantly decreased ATP level, viability of oocytes, and their developmental potential. CONCLUSION: Autophagy plays a protective role in the oocytes during vitrification-warming and IVM. The PI3K/Akt/mTOR pathway participated in regulating autophagy activity in oocyte during vitrification-warming and IVM. Keywords: oocyte, cryopreservation, autophagy, PI3K/Akt/mTOR.
CryoLetters 41 (1), 38-43 (2020) INHIBITION OF mTORC1 SIGNALING PATHWAY IS A VALID THERAPEUTIC STRATEGY IN TRANSPLANTATION OF CRYOPRESERVED MOUSE OVARIAN TISSUE Jian-Min Zhang1*, Xi-Lan Lu2, Hong-Xia Wang2 and Xi-Lan Lu2 1Weifang Nursing Vocational College, Qingzhou City, P.R. China. Abstract BACKGROUND: Blockage of mTOR1 can inhibit the transformation of primordial follicles into growing follicles in the ovaries. OBJECTIVE: The aim of this study was to investigate the role of mTORC1 inhibition in the cryopreservation and transplantation of mouse ovarian tissues. MATERIALS AND METHODS: ICR (Institute of Cancer Research) mice were randomly divided into control group (autograft), cryopreservation group (cryopreservation + autograft), and mTORC1 inhibition group (cryopreservation + autograft + mTOR inhibitor). After 30 days of auto-transplantation, the follicle number of graft and kit ligand (KL) immunostaining in grafts were quantified. In addition, serum concentration of anti-Müllerian hormone (AMH) was examined by ELISA. RESULTS: The graft in mTORC1 inhibition group showed a significantly higher proportion of primordial follicles and a significantly lower proportion of growing follicles compared with cryopreservation group. Furthermore, a significant decrease in expression of KL (a marker gene related to follicular development) was observed in mTORC1 inhibition group in contrast to cryopreservation group. The follicle number of graft and serum AMH concentration in mTORC1 inhibition group were significantly higher than that in cryopreservation group. CONCLUSION: Inhibition of mTORC1 signaling pathway is a valid therapeutic strategy in transplantation of cryopreserved mouse ovarian tissue via suppression of primordial follicle activation. Keywords: mTOR inhibition, ovarian tissue, cryopreservation, transplantation, follicle activation.
CryoLetters 41 (1), 44-49 (2020) IMPACT OF OSCILLATING MAGNETIC FIELD ASSISTED FREEZING ON Lactobacillus plantarum VIABILITY: EFFECTS OF FROZEN STORAGE TIME AND FREEZE-THAW REPETITIONS Lucía Cartagena, Eduardo Puértolas * and Iñigo Martínez de Marañón AZTI, Food Research Division. Parque Tecnológico de Bizkaia. Astondo Bidea, Edificio 609, 48160 Derio (Bizkaia), Spain. Abstract BACKGROUND: Oscillating magnetic field (OMF) assisted freezing has been proposed for improving the frozen preservation of biological materials. However, contradictory results have been reported on foods, tissues and cells, and so doubts exist in its actual benefit. OBJECTIVE: To study the effect of OMF-assisted freezing on the viability of fermentation starter Lactobacillus plantarum. MATERIALS AND METHODS: L. plantarum bacteria were frozen in a batch air blast freezer (-20°C, 5 m s-1) by OMF-assisted freezing (0.57 mT). Bacterial viability was examined after 60 days of frozen storage and 5 freeze-thaw cycles. RESULTS: OMF showed no statistically significant effects (P > 0.05) in key freezing curve characteristics, refuting Cell Alive System (CAS) technology claims. OMF-assisted freezing did not improve bacterial preservation. CONCLUSION: There was no effect of the application of the maximum OMF (0.57 mT) on cell viability independently of the frozen storage time (up to 60 days) and freeze-thaw repetitions (up to 5 cycles). Keywords: Lactobacillus plantarum, oscillating magnetic field, bacteria, Cell Alive System.
CryoLetters 41 (1), 50-56 (2020) NEW CRYOPRESERVATION TECHNOLOGY OF HMSCS: FIRST PRECLINICAL RESULTS USING DMSO-CONTAINING MEDIUM Jandová Miroslava1, 2*, Šponer Pavel3, Vokurková Doris4, 1Tissue Bank, University Hospital Hradec Králové, Hradec Králové 500 05, Czech Republic. Abstract BACKGROUND: Human mesenchymal stem cells (hMSCs) have tremendous potential in regenerative medicine, making it desirable to cryopreserve and bank them to increase their access and availability. OBJECTIVE: This research is part of a clinical trial performed on six patients that aimed to use advanced therapy medicinal products (ATMPs) based on hMSCs in patients undergoing repeated total hip replacement. MATERIALS AND METHODS: To compare the characteristics of fresh and frozen hMSCs, we used the trypan blue exclusion test (cell viability), flow cytometry (cell viability and phenotyping), sterility determinations and the clonogenic assay of cell proliferation. RESULTS: Cryopreserved hMSCs showed good quality parameters after thawing in comparison with fresh hMSCs in suspension. When using a medium containing dimethyl sulfoxide (DMSO), the viability was higher than 90% in all cases. The cell purity determined by flow cytometry was also acceptable. CONCLUSION: These initial results show that the prepared cryopreserved ATMP exhibited good viability and phenotype characteristics. Keywords: mesenchymal stem cells, advanced therapy medicinal product, dimethyl sulfoxide, cryopreservation. |
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For Abstracts published from meetings, such as SLTB meetings, go to the
relevant Volume Year of the journal (above). For Full text Free Access Content (from 2000 onwards) go to CryoLetters at Ingenta and look for the blue symbol. |
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