- Email: [email protected]
145 Preliminary attempts to cryopreserve sperm of the Dredge Oyster, Ostrea chilensis
Abstracts / Cryobiology 67 (2013) 398–442 IBPs for the basal planes of ice. A 3D simulation of a melting process using a velocity profile that reflects melting perpendicular to the basal plane confirmed our findings. Our results show a clear difference in the ice shaping mechanisms of moderate and hyperactive IBPs. This study implies that basal plane affinity may be predicted by simple observation of an ice crystal. Understanding the differences between the interactions of the various IBPs and ice is important for the future utilization of these proteins in industry and medicine. Source of funding: M.B.D was supported by The Lady Davis Fellowship thrust and The Valazzi-Pikovsky Fellowship Fund. The research was funded by the National Science Foundation (NSF), the Israel Science Foundation (ISF), the European Research Council (ERC). Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.149
144 Kinetics of hyperactive and moderate antifreeze proteins. Ran Drori 1, Yeliz Celik 2, Peter L. Davies 3, Ido Braslavsky 1,2, 1 Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel, 2 Department of Physics and Astronomy, Ohio University, Athens, OH, USA, 3 Department of Biochemistry, Queen’s University, ON, Canada Ice binding proteins (IBPs) protect cold-adapted organisms from freeze injuries by inhibiting the growth of endogenous ice crystals. IBPs have been found in fish, insects, plants, fungi and bacteria, and hold great potential in different fields such as the food industry, medicine and agriculture. IBPs include antifreeze proteins (AFPs), which adsorb to the surface of ice crystals and lower the temperature at which ice crystals grow, thereby creating a gap (thermal hysteresis [TH]) between the melting point and the non-equilibrium freezing point, within which ice growth is arrested. The accepted model explaining the mechanism of action of AFPs is the adsorption–inhibition model (Raymond and DeVries, 1977). Although this model requires irreversible binding of AFP to ice, there is still an ongoing debate over whether AFPs adsorb to ice irreversibly; furthermore, the time-dependence of this process is still unclear. Our results on the kinetics of AFP binding show that for a hyperactive AFP from Tenebrio molitor (TmAFP), TH activity increased with time by 3- to 10-fold for all of the concentrations measured (1–40 lM). In an AFP from spruce budworm (Choristoneura fumiferana) (sbwAFP) the increase of TH activity with time was the highest, reaching a 43-fold increase. Using a novel microfluidic system (Celik, Drori et al., 2013) to investigate the binding mechanism of AFPs to ice, we exposed an ice crystal to AFP molecules, and then exchanged the bathing solution with AFP-free buffer. Assuming irreversible binding, upon exchange of the AFP solution, the crystal must remain covered with AFPs. Our results with a TmAFP-GFP solution show that after the removal of the AFP solution, TH activity was similar to that before the exchange, and the crystal remains covered with TmAFP-GFP (Celik, Drori et al., 2013). Similar to the exchange of TmAFP-GFP solution, after the removal of an AFPIII-GFP solution, bound AFP were sufficient to inhibit ice growth in a supercooled solution (0.11–0.23 °C). However, the ice growth inhibition of AFPIII (which do not bind to the basal plane of the crystal) is dependent on the crystal shape; if the bipyramidal shape is complete, the basal plane is minimal and ice growth is suppressed. Our microfluidic system also enables us to measure and calculate the spacing between AFP molecules on the ice surface. We found that depending on solution concentration and exposure time of the crystal, the distance between AFPs is 5–30 nm (note that the dimensions of AFPs are about 3 nm2), and this value has a direct correlation to the TH activity. Our results suggest that AFPs accumulate on the ice surface, and that any desorption rate must be much slower than the adsorption rate. These findings reveal new insights about the mechanism of action of AFPs, and assist in better understanding how to manipulate these proteins for our benefit. Source of funding: This research was funded by Israel Science Foundation, The European Research Council, and the Canadian Institutes of Health Research. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.150
145 Preliminary attempts to cryopreserve sperm of the Dredge Oyster, Ostrea chilensis. Serean L. Adams 1, John F. Smith 1, Jolene Taylor 1, H. Robin Tervit 1, Lindsay T. McGowan 2, 1 Cawthron Institute, Nelson, New Zealand, 2 AgResearch, Ruakura Research Centre, Hamilton, New Zealand The dredge oyster, Ostrea chilensis, is native to New Zealand and Chile. Although this species has been commercially fished for decades, it is only recently that farm-
439
ing trials have been initiated. The ‘‘wild” commercial fishery was decimated in the 1980s by the parasite, Bonamia and so breeding for disease resistance is imperative for the progress of the aquaculture industry. Cryopreservation can provide a powerful tool in selective breeding programmes enabling breeders to bank germplasm from valuable genetic lines. The reproductive strategy of dredge oysters is challenging for both selective breeding and cryopreservation as they are protandric hermaphrodite brooders: sperm are held as spermatozeugmata in the gonad and then released into the water column but eggs are fertilized and retained within the mantle cavity where they are reared until shortly before settlement. Mature broodstock was obtained from marine farms in the Marlborough Sounds, New Zealand during the reproductive season (November 2011 and 2012). Oysters were shucked and stripped for sperm which was then pooled and stored at 4 °C until used in experiments. Two pools were collected each season. Motility was evaluated visually using the 0–5 scale developed by Fabbrocini et al. 2011 (Cryobiology 40:46–53) and viability was assessed using SYBR-14 and propidium iodide staining in combination with fluorescent microscopy. Cryoprotective agents (CPAs: ethylene glycol, dimethyl sulphoxide (Me2SO) and propylene glycol) were made up in Milli-Q water at twice the desired final concentration and were evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. Three other combinations (8% Me2SO + 0.25 M trehalose, 5% Me2SO + 0.625 or 0.5 M trehalose, final) were also tested. Sperm was diluted 1:1 with CPA then frozen in 0.5 mL straws on a rack 3 cm above the liquid nitrogen surface for 10 min before plunging into liquid nitrogen. Straws were thawed in an ambient temperature (17–18 °C) water bath. Sperm collected during the 2011 season had lower fresh motility than that collected during the 2012 season and only one of the two pools from the 2011 season was frozen. The post-thaw motility of the pool was poor (0–0.5). However, some treatments (viz: 10% and 15% ethylene glycol with or without trehalose, 8% Me2SO + 0.25 M trehalose, 5% Me2SO + 0.625 or 0.5 M trehalose) had equivalent viability (up to 100% live) to that obtained with fresh sperm. For the 2012 pools, post-thaw sperm motility remained high for many combinations (e.g. 2.5–3.5 for 10% Me2SO) but was less than that of fresh sperm (3.0–4.5). Again, post-thaw viability (about 90% live for most treatments) was equivalent to that of fresh sperm. This is a preliminary step towards developing a cryopreservation method for dredge oyster sperm. Further work will develop methods to assess fertilization in-vitro and for application of sperm cryopreservation in selective breeding. Source of funding: This work was funded by the New Zealand Ministry of Business Innovation and Employment(CAWX0802). Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.151
146 Evaluation of various storage conditions of laboratory testing samples. Koh Furuta 1, Teruto Hashiguchi 2, Yoh Hidaka 3, Dongchon Kang 4, Katsuyoshi Ikeda 5, Masato Maekawa 6, Hiroyuki Matsumoto 7, Kazuyuki Matsushita 8, Shigeo Okubo 9, Tatsuyuki Tsuchiya 10, The Japanese Society of Laboratory Medicine (JSLM), The Committee for Standardization, Japan1 Division of Clinical Laboratories, National Cancer Center Hospital, Japan, 2 Laboratory and Vascular Medicine, Kagoshima University Graduate, School of Medical and Dental Sciences, Japan, 3 Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Japan, 4 Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Japan, 5 Department of Clinical Laboratory, Kumamoto University Hospital, Japan, 6 Department of Laboratory Medicine, Hamamatsu University School of Medicine, Japan, 7 Department of Medical Technique, Nagoya University Hospital, Japan, 8 Department of Molecular Diagnosis & Division of Clinical Genetics and Proteomics, Graduate School of Medicine, Chiba University, Japan, 9 Department of Clinical Laboratory, The University of Tokyo Hospital, Japan, 10 Nihon University School of Medicine, Department of Pathology and Microbiology Division of Laboratory Medicine, Nihon, Japan Background: One of the current big concerns not only for laboratory medicine communities but also for research communities is the quality of human materials. Despite these concerns, few investigations focus on the degradation mechanism surrounding human materials, especially liquid samples. This situation inspires us to attempt the investigation of the human liquid samples and their stabilities in various temperatures for short or long storage. Purpose: The aim of this multi-center study is to clarify the appropriate handling conditions, such as temperature and duration,of human liquid samples.By this clarification we can standardize the handling procedures and minimize the artificial effects to the samples. Materials: We utilized sera, plasmas, and urines of post clinical test samples which were submitted to each clinical laboratory as a routine testing. For this purpose, at least 20 samples were analyzed per each clinical laboratory. By using these samples, the evaluation of various storage temperature and durations based on 38 routine testings were done. Methods: After the routine analytical procedure, submitted each sample was divided into 300 lL. Each 300 lL sample was stored at room temperature (23 °C), 4 °C, 20 °C, and 80 °C without light exposure. Plasmas and urines
144 Kinetics of hyperactive and moderate antifreeze proteins. Ran Drori 1, Yeliz Celik 2, Peter L. Davies 3, Ido Braslavsky 1,2, 1 Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel, 2 Department of Physics and Astronomy, Ohio University, Athens, OH, USA, 3 Department of Biochemistry, Queen’s University, ON, Canada Ice binding proteins (IBPs) protect cold-adapted organisms from freeze injuries by inhibiting the growth of endogenous ice crystals. IBPs have been found in fish, insects, plants, fungi and bacteria, and hold great potential in different fields such as the food industry, medicine and agriculture. IBPs include antifreeze proteins (AFPs), which adsorb to the surface of ice crystals and lower the temperature at which ice crystals grow, thereby creating a gap (thermal hysteresis [TH]) between the melting point and the non-equilibrium freezing point, within which ice growth is arrested. The accepted model explaining the mechanism of action of AFPs is the adsorption–inhibition model (Raymond and DeVries, 1977). Although this model requires irreversible binding of AFP to ice, there is still an ongoing debate over whether AFPs adsorb to ice irreversibly; furthermore, the time-dependence of this process is still unclear. Our results on the kinetics of AFP binding show that for a hyperactive AFP from Tenebrio molitor (TmAFP), TH activity increased with time by 3- to 10-fold for all of the concentrations measured (1–40 lM). In an AFP from spruce budworm (Choristoneura fumiferana) (sbwAFP) the increase of TH activity with time was the highest, reaching a 43-fold increase. Using a novel microfluidic system (Celik, Drori et al., 2013) to investigate the binding mechanism of AFPs to ice, we exposed an ice crystal to AFP molecules, and then exchanged the bathing solution with AFP-free buffer. Assuming irreversible binding, upon exchange of the AFP solution, the crystal must remain covered with AFPs. Our results with a TmAFP-GFP solution show that after the removal of the AFP solution, TH activity was similar to that before the exchange, and the crystal remains covered with TmAFP-GFP (Celik, Drori et al., 2013). Similar to the exchange of TmAFP-GFP solution, after the removal of an AFPIII-GFP solution, bound AFP were sufficient to inhibit ice growth in a supercooled solution (0.11–0.23 °C). However, the ice growth inhibition of AFPIII (which do not bind to the basal plane of the crystal) is dependent on the crystal shape; if the bipyramidal shape is complete, the basal plane is minimal and ice growth is suppressed. Our microfluidic system also enables us to measure and calculate the spacing between AFP molecules on the ice surface. We found that depending on solution concentration and exposure time of the crystal, the distance between AFPs is 5–30 nm (note that the dimensions of AFPs are about 3 nm2), and this value has a direct correlation to the TH activity. Our results suggest that AFPs accumulate on the ice surface, and that any desorption rate must be much slower than the adsorption rate. These findings reveal new insights about the mechanism of action of AFPs, and assist in better understanding how to manipulate these proteins for our benefit. Source of funding: This research was funded by Israel Science Foundation, The European Research Council, and the Canadian Institutes of Health Research. Conflict of interest: None declared. Email address: [email protected] http://dx.doi.org/10.1016/j.cryobiol.2013.09.150
145 Preliminary attempts to cryopreserve sperm of the Dredge Oyster, Ostrea chilensis. Serean L. Adams 1, John F. Smith 1, Jolene Taylor 1, H. Robin Tervit 1, Lindsay T. McGowan 2, 1 Cawthron Institute, Nelson, New Zealand, 2 AgResearch, Ruakura Research Centre, Hamilton, New Zealand The dredge oyster, Ostrea chilensis, is native to New Zealand and Chile. Although this species has been commercially fished for decades, it is only recently that farm-
439
ing trials have been initiated. The ‘‘wild” commercial fishery was decimated in the 1980s by the parasite, Bonamia and so breeding for disease resistance is imperative for the progress of the aquaculture industry. Cryopreservation can provide a powerful tool in selective breeding programmes enabling breeders to bank germplasm from valuable genetic lines. The reproductive strategy of dredge oysters is challenging for both selective breeding and cryopreservation as they are protandric hermaphrodite brooders: sperm are held as spermatozeugmata in the gonad and then released into the water column but eggs are fertilized and retained within the mantle cavity where they are reared until shortly before settlement. Mature broodstock was obtained from marine farms in the Marlborough Sounds, New Zealand during the reproductive season (November 2011 and 2012). Oysters were shucked and stripped for sperm which was then pooled and stored at 4 °C until used in experiments. Two pools were collected each season. Motility was evaluated visually using the 0–5 scale developed by Fabbrocini et al. 2011 (Cryobiology 40:46–53) and viability was assessed using SYBR-14 and propidium iodide staining in combination with fluorescent microscopy. Cryoprotective agents (CPAs: ethylene glycol, dimethyl sulphoxide (Me2SO) and propylene glycol) were made up in Milli-Q water at twice the desired final concentration and were evaluated at final concentrations of 10% and 15% with or without 0.2 M trehalose. Three other combinations (8% Me2SO + 0.25 M trehalose, 5% Me2SO + 0.625 or 0.5 M trehalose, final) were also tested. Sperm was diluted 1:1 with CPA then frozen in 0.5 mL straws on a rack 3 cm above the liquid nitrogen surface for 10 min before plunging into liquid nitrogen. Straws were thawed in an ambient temperature (17–18 °C) water bath. Sperm collected during the 2011 season had lower fresh motility than that collected during the 2012 season and only one of the two pools from the 2011 season was frozen. The post-thaw motility of the pool was poor (0–0.5). However, some treatments (viz: 10% and 15% ethylene glycol with or without trehalose, 8% Me2SO + 0.25 M trehalose, 5% Me2SO + 0.625 or 0.5 M trehalose) had equivalent viability (up to 100% live) to that obtained with fresh sperm. For the 2012 pools, post-thaw sperm motility remained high for many combinations (e.g. 2.5–3.5 for 10% Me2SO) but was less than that of fresh sperm (3.0–4.5). Again, post-thaw viability (about 90% live for most treatments) was equivalent to that of fresh sperm. This is a preliminary step towards developing a cryopreservation method for dredge oyster sperm. Further work will develop methods to assess fertilization in-vitro and for application of sperm cryopreservation in selective breeding. Source of funding: This work was funded by the New Zealand Ministry of Business Innovation and Employment(CAWX0802). Conflict of interest: None declared. Email address: [email protected]
http://dx.doi.org/10.1016/j.cryobiol.2013.09.151
146 Evaluation of various storage conditions of laboratory testing samples. Koh Furuta 1, Teruto Hashiguchi 2, Yoh Hidaka 3, Dongchon Kang 4, Katsuyoshi Ikeda 5, Masato Maekawa 6, Hiroyuki Matsumoto 7, Kazuyuki Matsushita 8, Shigeo Okubo 9, Tatsuyuki Tsuchiya 10, The Japanese Society of Laboratory Medicine (JSLM), The Committee for Standardization, Japan1 Division of Clinical Laboratories, National Cancer Center Hospital, Japan, 2 Laboratory and Vascular Medicine, Kagoshima University Graduate, School of Medical and Dental Sciences, Japan, 3 Department of Laboratory Medicine, Osaka University Graduate School of Medicine, Japan, 4 Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Japan, 5 Department of Clinical Laboratory, Kumamoto University Hospital, Japan, 6 Department of Laboratory Medicine, Hamamatsu University School of Medicine, Japan, 7 Department of Medical Technique, Nagoya University Hospital, Japan, 8 Department of Molecular Diagnosis & Division of Clinical Genetics and Proteomics, Graduate School of Medicine, Chiba University, Japan, 9 Department of Clinical Laboratory, The University of Tokyo Hospital, Japan, 10 Nihon University School of Medicine, Department of Pathology and Microbiology Division of Laboratory Medicine, Nihon, Japan Background: One of the current big concerns not only for laboratory medicine communities but also for research communities is the quality of human materials. Despite these concerns, few investigations focus on the degradation mechanism surrounding human materials, especially liquid samples. This situation inspires us to attempt the investigation of the human liquid samples and their stabilities in various temperatures for short or long storage. Purpose: The aim of this multi-center study is to clarify the appropriate handling conditions, such as temperature and duration,of human liquid samples.By this clarification we can standardize the handling procedures and minimize the artificial effects to the samples. Materials: We utilized sera, plasmas, and urines of post clinical test samples which were submitted to each clinical laboratory as a routine testing. For this purpose, at least 20 samples were analyzed per each clinical laboratory. By using these samples, the evaluation of various storage temperature and durations based on 38 routine testings were done. Methods: After the routine analytical procedure, submitted each sample was divided into 300 lL. Each 300 lL sample was stored at room temperature (23 °C), 4 °C, 20 °C, and 80 °C without light exposure. Plasmas and urines