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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 38 (1), 1-6 (2017) THE BENEFICIAL EFFECT OF CARBOXYLATED POLY-L-LYSINE ON CRYOSURVIVAL OF VITRIFIED EARLY STAGE EMBRYOS Yuta Tsuji1*, Toshiroh Iwasaki1, Hiromi Ogata1, Yukiko Matsumoto1, 1Hanabusa Women’s Clinic, 1-1-2 Sannomiyacho, Chuo-ku, Kobe city, Hyogo, 650-0021 Japan Abstract BACKGROUND: In the vitrification of embryos, dimethyl sulfoxide (DMSO) is one of the most effective cryoprotectant agents (CPAs), but cytotoxic effects of DMSO on embryos are well known. Carboxylated poly-L-lysine (CPLL) has been identified as an effective cryoprotectant of cultured cell lines and mammalian oocytes. OBJECTIVE: To evaluate the efficacy and safety of CPLL as a CPA for developmental stage embryos. MATERIALS AND METHODS: Mouse 8-cell embryos and blastocysts were vitrified with ethylene glycol (EG), DMSO/EG, or CPLL/EG and the developmental potency assessed in vitro. RESULTS: In 8-cell embryos, there were no differences between the levels of survival and developmental progress into the blastocyst stage in each solution. At the blastocyst stage, the proportion of dead cells was significantly higher in the EG compared with other solutions. In contrast, there were no differences between the DMSO/EG and CPLL/EG. CONCLUSION: These results indicate that CPLL can be used as a replacement for DMSO in the vitrification of mouse embryos. Keywords: vitrification, carboxylated poly-L-lysine, dimethyl sulfoxide, ethylene glycol, cryoprotection
CryoLetters 38 (1), 7-16 (2017) A SIMPLE AND EFFICIENT PROTOCOL FOR THE CRYOPRESERVATION OF ZYGOTIC EMBRYOS OF MACAW PALM [Acrocomia aculeata (JACQ.) LODD. EX MART.], A TROPICAL SPECIES WITH A CAPACITY FOR BIOFUEL PRODUCTION Zanderluce Gomes Luis1 and Jonny Everson Scherwinski-Pereira2* 1Department of Botany, University of Brasília (UnB), Campus Universitário Darcy Ribeiro, 70.910-900, Brasília, DF, Brazil. Abstract BACKGROUND: The development of cryopreservation protocols for zygotic embryos has the advantage of direct embryo regeneration into plantlets, an important factor when one´s purpose is to preserve genetic plant resources. OBJECTIVE: Current study developed an efficient and simplified cryopreservation protocol for zygotic embryos of the macaw palm, a type of palm tree with great capacity for biodiesel production. MATERIALS AND METHODS: Embryos were obtained from seeds and rehydrated for 15 h in a 3% sucrose medium. Zygotic embryos were subsequently desiccated in a laminar flow cabinet for 0, 2, 4, 6 and 8 h. After each period, some embryos was cultured on a germination medium and others were placed in sterile cryotubes and immersed directly in liquid nitrogen for up to 360 d. Cryotubes were rewarmed by immersion at 40°C during 90 s (quick thawing). RESULTS: Germinability reduction occurred in proportion to desiccation time, with averages of 97 and 75% for 0 h and 8 h, respectively. In the case of cryopreserved zygotic embryos, the highest germination (81%) was obtained after 6-h desiccation, when the embryos had approximately 10% water content (FW basis). CONCLUSION: The zygotic embryos cryopreservation protocol covered in this study represents another option for preserving Acrocomia aculeata genetic resources. Keywords: Arecaceae, Acrocomia aculeata, tropical crop, dehydration, preservation time, in vitro culture.
CryoLetters 38 (1), 17-28 (2017) COLD RESISTANCE OF AN ANT Camponotus herculeanus (HYMENOPTERA, FORMICIDAE) IN VARIOUS CLIMATES IN NORTH-EAST ASIA Daniil I. Berman, Zoya A. Zhigulskaya and Anna N. Leirikh* Institute of Biological Problems of the North, Far Eastern Branch, Russian Academy of Sciences, Magadan, 685000 Russia Abstract BACKGROUND: The relationship between cold resistance of widespread invertebrate species and overwintering temperatures has not been studied; however, this knowledge could have high prognostic value. OBJECTIVE: Using an ant species, Camponotus herculeanus, to investigate whether there is a relationship between cold resistance and overwintering temperatures in two climatically distinct regions, coastal and continental. MATERIALS AND METHODS: We determined seasonal changes in SCP and LТ100% for overwintering ant stages, as well as winter temperatures in the nests. RESULTS: In the watershed of Upper Kolyma River (Aborigen field station), minimal temperature in the nests dropped to 26°С; average SCP of the ants was 40°С; LТ100% – 37°С. At the coast of the Sea of Okhotsk (Magadan) the minimum in the nest was 14°С, SCP was 19 to 27°С and LТ100% was 32°С. CONCLUSION: We have established a relationship between cold tolerance and overwintering conditions for geographically separate populations in the two areas with different climatic regimes. Keywords: ants, Camponotus herculeanus, overwintering conditions, cold resistance, geographic variation
CryoLetters 38 (1), 29-36 (2017) EFFECTS OF TEMPOL AND QUERCETIN ON HUMAN SPERM FUNCTION AFTER CRYOPRESERVATION Leila Azadi1, Marziyeh Tavalaee1, Mohammad Reza Deemeh1, 1 Department of Reproductive Biotechnology. Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran; Abstract BACKGROUND: Quercetin is a flavonoid with high reactive oxygen species (ROS) scavenging and ion chelating activity. It also enhances the activity of antioxidant enzymes and reduces enzymatic activity such as NADPH oxidase and NADH-dependent oxido-reductase. Tempol, as a superoxide dismutase mimetic agent, converts superoxide to less toxic hydrogen peroxide (H2O2), but cannot reduce highly toxic hydroxyl radicals in Fenton or Haber-Weiss reactions mediated with free iron or cupper. OBJECTIVE: The study was to compare the effect of Quercetin and Tempol in an optimized commercial cryo-protective media on ROS induced cryoinjury for the first time. MATERIALS AND METHODS: Following administration of these compounds during freezing process, sperm motility, viability, ROS production and DNA integrity were assessed before and after freezing/thawing process. RESULTS: Data showed that 10 µM Quercetin and 5 µM Tempol significantly improved sperm motility and viability, but they together had no additive effect. Supplementation with Quercetin alone or combination of Quercetin with Tempol decrease the ROS concentration, but the reduction was not significant for Tempol alone compared to control group. Quercetin and Tempol significantly decrease DNA fragmentation. CONCLUSION: The supplementation of Quercetin or Tempol, but not their combination improves the quality of cryopreserved human semen. Keywords: Quercetin, Tempol, ROS, cryoinjury, DNA fragmentation, human sperm.
CryoLetters 38 (1), 37-42 (2017) NUMERICAL AND EXPERIMENTAL INVESTIGATION ON WATER-ME2SO MIXING IN MICROFLUIDIC DEVICE FOR OOCYTE CRYOPRESERVATION Yun Yang #, Xinli Zhou #*, Nanfeng Zhou, Wenqi Shao, and Leren Tao Institute of Biomedical Technology, University of Shanghai for Science and Technology, Shanghai,China.
Abstract BACKGROUND: Oocyte vitrification is a practical tool in assisted reproductive technology and fertility preservation. However, vitrification requires high concentration of cryoprotectants (CPAs), which may cause osmotic injury and is toxic to oocytes during the addition and removal process of CPAs. OBJECTIVE: To minimize osmotic and toxic injuries, a microfluidic device was designed to provide continuous concentration changes of CPAs to oocytes. METHODS: Numerical simulations were first used to investigate the effects of geometric parameters of microchannel on water-Me2SO mixing performance. A microfluidic device was then fabricated via soft lithography. Distilled water and 30% Me2SO containing 1mM fluorescence dye FITC were mixed using the microfluidic device to verify the CPA diffusion behavior. RESULTS: The mixing performance in the microfluidic device strongly depends on the channel geometry and the flow rates of the fluids. Linear CPA loading profiles with diverse slopes were obtained by controlling the flow rate and the loading time of CPA and the buffer solution in the microfluidic device. CONCUSION: The designed microfluidic device can yield linear CPA loading profiles with different slopes. Keywords: Osmotic injuries, microfluidic device, mixing efficiency, oocyte cryopreservation.
CryoLetters 38 (1), 43-50 (2017) CRYOPRESERVATION OF NILI-RAVI BUFFALO BULL SPERM IN CRYODILUENT SUPPLEMENTED WITH Lolium perenne PROTEIN PREPARATIONS S. Qadeer1, M.A. Khan1, M.S. Ansari1, B.A. Rakha1, R. Ejaz1, 1Animal Physiology Laboratory, Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi-46300, Pakistan Abstract BACKGROUND: Semen from the Nili-Ravi buffalo bull, Bubalus bubalis, shows poor survival after freeze storage compared to bovine (Bos taurus and Bos indicus) semen. Freeze-susceptibility distinctions in these two genera have been attributed to differences in sperm membranes. MATERIALS AND METHODS: We measured the impact of protein preparations derived from a frost-resistant perennial grass, Lolium perenne, with ice recrystallization inhibition activity on the low temperature storage of B. bubalis semen. RESULTS: When the L. perenne preparations (0.1, 1, 10 µg/mL) were added to buffalo semen [2 ejaculates per bull (N=3) per replicate (r=3)] in Tris-citrate extender (50×106sperm mL-1), there was no impact on semen quality, as measured by sperm motility and plasma membrane integrity, after storage at 4ºC (P>0.05). However, when semen supplemented with the grass proteins (0.1 and 1 µg mL-1) was evaluated after freezing and storage in liquid nitrogen for 24 h, post-thaw sperm progressive motility and plasma membrane integrity was higher (P<0.05) than in control samples. Post-thaw sperm viability and sperm acrosome integrity was similar (P > 0.05) to controls. CONCLUSION: The improvement in cryopreserved buffalo sperm progressive motility and plasma membrane integrity suggests that the use of these easily-made preparations may improve fertility after cryopreservation and offers the prospect of improved conception rates after artificial insemination with cryopreservation. Keywords: Buffalo bull sperm, antifreeze proteins, cryopreservation, Lolium perenne, ice recrystallization inhibition
CryoLetters 38 (1), 51-57 (2017) EFFECT OF MONOSACCHARIDES IN GLYCEROL-FREE TRIS EXTENDER ON REACTIVE OXYGEN SPECIES AND APOPTOSIS IN DOG SPERM CRYOPRESERVATION Md. Ataur Rahman, Sang-Hyun Park and Il-Jeoung Yu* Laboratory of Theriogenology and Reproductive Biotechnology, College of Veterinary Medicine and Bio-safety Research Institute, Chonbuk National University, Iksan 54596,
Republic of Korea. Abstract BACKGROUND OBJECTIVE: The present study investigated the effect of monosaccharides in a glycerol-free tris (GFT) extender on reactive oxygen species (ROS) levels and apoptosis in cryopreserved dog spermatozoa. MATERIALS AND METHODS: The sperm pellets were resuspended (5 × 107 mL-1) with GFT containing 172 mM glucose (G), 86 mM glucose + 86 mM fructose (GF), 86 mM glucose + 86 mM galactose (Gg), 172 mM fructose (F), 172 mM galactose (g) or 86 mM fructose + 86 mM galactose (Fg). The sperm (500 µL) were loaded in 0.5 mL straws and cooled for 50 min at 4°C. The straws were then frozen 7 cm from the surface of liquid nitrogen (LN2) for 20 min and were finally plunged into LN2. After freezing-thawing, the sperm motility and viability were evaluated. The ROS level (H2O2) and apoptosis index were assessed by using flow cytometry. RESULTS: GFT supplemented with GF resulted in significantly higher (P< 0.05) progressive sperm motility and viability followed by only G, which had greater ROS reducing capacity. However, sperm cell apoptosis had no significant differences among all the experimental groups. CONCLUSION: Cryopreservation of dog sperm in GFT containing GF yields more motile and viable sperm followed by only G, which has greater ROS reducing efficiency. Keywords: cryopreservation, dog sperm, glycerol-free tris, monosaccharides, ROS
CryoLetters 38 (1), 58-64 (2017) Viability and infectivity of Tuber borchii after cryopreservation Federica Piattoni1, Pamela Leonardi1, Boutahir Siham1, Mirco Iotti2* 1Department of Agricultural Sciences, Bologna University, Viale Fanin 46, 40127, Bologna, Italy Abstract BACKGROUND: Truffles (Tuber spp.) are the most precious ectomycorrhizal edible mushrooms whose biodiversity is seriously endangered. OBJECTIVE: To develop a protocol for cryopreservation of Tuber spp. mycelia using T. borchii as a model species, verifying whether conservation in liquid nitrogen may affect viability, growth rate, hyphal morphology and infectivity. MATERIALS AND METHODS: Cryopreservation was performed using sorbitol, sucrose and DMSO as cryoprotectants. The morphological parameters analyzed were: hyphal diameter, septal distance and hyphal growth unit. Cryopreserved mycelium infectivity was assessed by inoculating Quercus robur seedlings. RESULTS: In T. borchii cryopreserved mycelium, the lag-phase lasted for 6-42 days but no differences in growth curve evolution, growth rate and hyphal morphology were observed except for hyphal growth unit. No differences in mycorrhizal colonization were observed between the seedlings inoculated with non-cryopreserved and cryopreserved mycelium. CONCLUSION: The established protocol is suitable for long-term conservation of Tuber mycelium and opens up the possibility of creating a Tuber spp. germplasm bank to preserve truffle diversity. Keywords: cryopreservation, Tuber borchii, vitality, mycelium infectivity, mycorrhization
CryoLetters 38 (1), 65-74 (2017) CHARACTERIZATION AND EXPRESSION ANALYSIS OF ATTACINS, ANTIMICROBIAL PEPTIDE-ENCODING GENES, FROM THE DESERT BEETLE Microdera punctipennis IN RESPONSE TO LOW TEMPERATURES Jieqiong Li, Xueying Lu and Ji Ma* Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi, China Abstract BACKGROUND: Antimicrobial peptides (AMPs) constitute the insect innate immune defense system. AMP production is usually induced by microbes. However, mounting evidence links insect immune reaction to cold hardiness. OBJECTIVE: We aimed at characterizing two attacins, MpAtt1 and MpAtt2, from the desert beetle Microdera punctipennis in Coleoptera and investigating the expression profiles of the two genes in response to cold stress. RESULTS: Full lengths MpAtt1 and MpAtt2 cDNAs were obtained from low temperature transcriptomic data. The newly identified attacins show characteristics different from those previously reported in other insects. MpAtt1 and MpAtt2 encode precursor proteins of 151 and 166 amino acid residues, respectively. These two attacins show 28.1% identity at amino acid level, and 38.3% identity at nucleotide level. Phylogenetic tree shows that the Mp attacins and the other Coleoptera attacins diverge earlier than Diptera and Lepidoptera attacins in evolution. The results of real-time quantitative PCR showed that MpAtt1 and MpAtt2 are highly expressed in hindgut plus Malpighian tube, and MpAtt1 also highly expressed in fat body. The mRNA levels of MpAtt1 and MpAtt2 were increased when the insects were treated at 4ºC and 4ºC, but the responsiveness to -4ºC was lower than to 4ºC. CONCLUSION: The cold inducible expression of attacin genes from the desert beetle M. punctipennis from the aspect of antimicrobial peptide synthesis supports the hypothesis that the insect immune system is stimulated during cold exposure. Keywords: Microdera punctipennis, cold-responsive genes, attacin, antimicrobial peptide, cold stress
CryoLetters 38 (1), 75-79 (2017) COMPARISON OF MITOCHONDRIAL FUNCTION IN BOAR AND BULL SPERMATOZOA THROUGHOUT CRYOPRESERVATION BASED ON JC-1 STAINING Chuanhuo Hu, Xinjie Zhuang, Yingming Wei, Ming Zhang,Shengsheng Lu, College of Animal Science and Technology, Guangxi University, Nanning, 530004, China. Abstract BACKGROUND: Poor reproductivity hampers the commercialization of cryopreserved boar semen. OBJECTIVE: This study was to determine the differences in the sperm mitochondrial function between boar and bull semen at different cryopreservation stages. MATERIALS AND METHODS: Boar and bull fresh, equilibrated, and frozen-thawed spermatozoa were evaluated for mitochondrial function using JC-1 under a fluorescent microscope. RESULTS: Bull and boar percentage of spermatozoa staining green (PSSG) showed no difference between fresh and equilibrated semen (P > 0.05). However, frozen-thawed bull and boar semen demonstrated significantly higher PSSG (P < 0.01) than fresh and equilibrated semen. Frozen-thawed boar semen represented a significantly higher PSSG (P < 0.01) than bull semen. CONCLUSION: Negative cryopreservation influence on boar and bull spermatozoa was not significantly produced by pre-freezing procedures, but rather by freezing and thawing. Cryopreservation has more pronounced negative effects on boar than on bull spermatozoa, which partly explains lagged commercialization of frozen boar semen. Keywords: Boar, bull, sperm, mitochondrial membrane potential, cryopreservation, JC-1 staining. |
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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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