- Email: [email protected]
41. Cryopreservation of greenlipped mussel (Perna canaliculus) trochophore larvae
Abstracts / Cryobiology 63 (2011) 306–342 with regard to characterization and documentation, and to share knowledge and expertise between national programmes, with regard to storage and use of germplasm and of conservation genetics. This work is funded by the Dutch Ministry of Economic Affairs, Agriculture and Innovation. Conflicts of interest: None declared. doi:10.1016/j.cryobiol.2011.09.042
40. Cryopreservation and recovery of avian genetic material. F.G. Silversides *, J. Liu, Y. Song, Agriculture and Agri-Food Canada, Agassiz Research Centre, Agassiz British Columbia, Canada Historically, storage of avian genetic material has been very difficult. Fertility obtained from cryopreserved avian semen is low and unpredictable and the structure of the avian egg prevents its cryopreservation altogether. Embryonic cells (blastodermal cells, embryonic stem cells and primordial germ cells) can be cryogencially stored and used to generate germline chimeras but this strategy requires complex procedures and results in very low efficiency. Until recently, the only effective method of conserving poultry germplasm has been in living animals, which is expensive and has led to a decimation of stocks kept in research institutions. At the same time, the efficiency of environmental control and the selection practiced by the poultry industry has resulted in consolidation of commercial breeding activities under two primary breeders responsible for more than 90% of all industrial layers, broilers, and turkeys. Cryogenic storage of germline genetic material requires that the material be preserved at subzero temperatures then warmed and recovered in a form that will produce viable individuals that can pass on the genetic material in a normal fashion. Successful development of techniques for cryopreservation and recovery of genetic material for birds will provide the genetic flexibility needed by the poultry industry to remain competitive and to adapt to change. In 2003, AAFC implemented a program of avian genetic resource conservation at the Agassiz Research Centre and accepted 18 lines of chickens and Japanese quail. At the same time, investigation began on methods of effective cryopreservation of avian genetic material. Cryogenic storage of ovarian tissue is a simple and effective method of conserving the female germline of some mammals but effective in vivo and/or in vitro recovery methods are required. Immunodeficient rodents are used as hosts for mammalian species to prevent tissue rejection when the cryopreserved/warmed tissue is recovered in vivo by transplantation, but these are not available for avian species. The in vitro approaches used in mammals such as in vitro maturation (IVM) and in vitro fertilization (IVF) of follicles retrieved from ovarian tissue are not applicable for avian species because of their special reproductive physiology. At Agassiz we have developed techniques of gonadal transplantation along with a commercially available immunosupressant and combined these techniques with cryopreservation to store and recover testicular tissue to a reproductive state. Based on this technology development, gonads from 18 chicken populations kept at Nova Scotia Agricultural College, the University of Saskatchewan, the University of Alberta, and the Agassiz Research Centre have been cryopreserved. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.043
Papers submitted for the Crystal Awards II
41. Cryopreservation of greenlipped mussel (Perna canaliculus) trochophore larvae. E. Paredes * 1, S.L. Adams 2, S.L. Gale 2, L.T. McGowan 3, J.F. Smith 2, H.R. Tervit 2, 1 Departamento de Ecoloxı´a e Bioloxı´a Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain, 2 Cawthron Institute, Private Bag 2, 98 Halifax Steet East, Nelson, New Zealand, 3 AgResearch, Private Bag 3123, Hamilton, New Zealand Cryopreservation is a powerful tool for aquaculture. The ability to cryopreserve gametes and/or larvae enables breeders to easily maintain a large number of family lines and allows for year-round juvenile production in hatcheries without the need to condition and maintain broodstock for out-of-season production. The aim of this study was to develop a method for cryopreserving trochophore larvae of the Greenlipped mussel. Mature mussels were induced to spawn using thermal cycling. For each experiment, at least three pools of trochophores were produced. The ability of trochophores to develop to D-larvae following treatment was assessed against a control. Exp 1: Five ethylene glycol (EG) concentrations in combination with three trehalose (TRE) concentrations were tested for toxicity. One-step vs. five-step cryoprotectant (CPA) addition and removal was also evaluated. Exp 2: 10% EG + 0.2 M TRE was used to evaluate two cooling regimes using 0.25 mL straws and to test whether addition of Bovine Serum Albumin (BSA) during thawing was beneficial. Exp 3: Evaluating the most promising CPAs from Expt 1 and cooling programme from
317
Expt 2 using 0.25 mL straws. Exp 4: Four volumes were tested with the best CPAs from Experiment 3 (10% EG + 0.4 M TRE and 15% EG + 0.2 M TRE). Exp 5: Development beyond the D-larval stage was investigated. Cryopreserved trochophores were reared through to the D-larval stage, and then transferred to the Cawthron Ultra-Density Larval System (CUDLS) for on-growing and reared to the pediveliger stage. A protocol to cryopreserve Greenlipped mussel trochophores was developed and consists on: CPA addition of 10% EG + 0.4 M TRE in one step, allowing 15 min equilibration time. Then trochophores are loaded into 0.25 mL straws and introduced into a Freeze Control System. The protocol chosen starts with a hold at 0 °C for 5 min, then cool to 10 °C at 1 °C min 1, hold for 10 min, then cool at 0.5 °C min 1 to 35 °C, then plunge into liquid nitrogen. Straws were thawed at 28 °C for 6 s and then allowed 15 min equilibration in sea water with 0.1% BSA (w/v). Thawed trochophores were incubated at 20 °C until D-larve stage was reached, obtaining around 40–60% normal D-larvae. Development beyond the D-larval stage was investigated. Frozen trochophores mean survival at day 4 was 60% followed by a significant decrease at day 8. From day 9 onwards, survival is relatively constant under 10%. This attempt to cryopreserve Greenlipped mussell trochophores for the aquaculture industry is encouraging. Future work will investigate the long-term viability of resulting D-larvae by further examining their ability to develop to the eyed-larval stage and scale-up issues for application in selective breeding and hatcheries. This research was funded by projects from Cawthron Institute (New Zealand) and Universidade de Vigo (Spain). Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.044
42. Fluorescence as a better approach to gate cells for cryobiological studies with flow cytometry. Anthony J. Reardon * 1, JanetA.W. Elliott 2, Locksley E. McGann 1, 1 Departments of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, 2 Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada Flow cytometry is an important tool in biology and medicine. There is a generalization that light scattered at small angles is proportional to cell volume, leading to the acceptance of forward light scatter as an appropriate estimation of cell size [Millaney et al., Rev. Sci. Instrum. 40(8) (1968) 1029–1032], and commonly used to identify cells from debris. However, in addition to size, other cell characteristics also affect forward scatter measurements [H.M. Shapiro, Practical Flow Cytometry 4th Ed. 2003, p. 275]. Cells with compromised membranes, such as those damaged by freeze–thaw stress produce smaller forward scatter signals, implying that strategies to analyze flow cytometric data developed for other areas of biology may not be appropriate for cryobiological studies. In this study, human umbilical vein endothelial cells (HUVEC) were used to demonstrate the use of fluorescence as an alternative to forward scatter gates for identification of cells in cryopreservation studies. Samples were exposed to two conditions: (i) control cells in suspension at room temperature, and (ii) cells plunged directly into liquid nitrogen and then thawed. Assessment of HUVEC under these conditions was conducted using flow cytometry with JC-1. JC-1 demonstrates cells with functional, polarized mitochondria as a high red/green fluorescence intensity ratio, whereas cells with depolarized mitochondria have a low red/green ratio [Smiley et al., PNAS, 88 (1991) 3671–3675]. A parallel assessment of membrane integrity to verify the results of JC-1 was conducted using a combination of Syto13 and ethidium bromide (SytoEB). Control cells displayed the highest ratio of red/green (JC-1) fluorescence, indicative of the formation of J-aggregates in polarized mitochondria, whereas plunged cells showed a shift to a lower ratio of red/green fluorescence indicating depolarized mitochondria. In addition to these findings the JC-1 (green) fluorescence under these conditions exhibited a distinctive intensity of cells separated from lower intensity events, allowing a threshold to be established to identify cells from debris. These results were verified with a parallel SytoEB study that identified the viable control and non-viable plunged cells in samples based on membrane integrity. In cryobiological studies, isolating cell populations for analysis is more effectively accomplished by gating on fluorescence rather than light scatter, as dyes such as JC-1 allow for identification of cells regardless of the integrity of their plasma membrane. Although forward scatter gates are an effective method of discriminating debris in scatterplots for healthy cell populations, they are ineffective for cell populations with compromised membranes. This is due to the possibility of damaged cells falling below the threshold of the established gate and excluded from the final assessment, leading to an inflated percentage of surviving cells. Green fluorescence emission then makes for an ideal threshold to differentiate cells from debris without the use of forward scatter gating. This research was funded by the Canadian Institutes of Health Research (MOP 86492), and the University of Alberta. J.A.W. Elliott held a Canada Research Chair in Interfacial Thermodynamics during this research. doi:10.1016/j.cryobiol.2011.09.045
40. Cryopreservation and recovery of avian genetic material. F.G. Silversides *, J. Liu, Y. Song, Agriculture and Agri-Food Canada, Agassiz Research Centre, Agassiz British Columbia, Canada Historically, storage of avian genetic material has been very difficult. Fertility obtained from cryopreserved avian semen is low and unpredictable and the structure of the avian egg prevents its cryopreservation altogether. Embryonic cells (blastodermal cells, embryonic stem cells and primordial germ cells) can be cryogencially stored and used to generate germline chimeras but this strategy requires complex procedures and results in very low efficiency. Until recently, the only effective method of conserving poultry germplasm has been in living animals, which is expensive and has led to a decimation of stocks kept in research institutions. At the same time, the efficiency of environmental control and the selection practiced by the poultry industry has resulted in consolidation of commercial breeding activities under two primary breeders responsible for more than 90% of all industrial layers, broilers, and turkeys. Cryogenic storage of germline genetic material requires that the material be preserved at subzero temperatures then warmed and recovered in a form that will produce viable individuals that can pass on the genetic material in a normal fashion. Successful development of techniques for cryopreservation and recovery of genetic material for birds will provide the genetic flexibility needed by the poultry industry to remain competitive and to adapt to change. In 2003, AAFC implemented a program of avian genetic resource conservation at the Agassiz Research Centre and accepted 18 lines of chickens and Japanese quail. At the same time, investigation began on methods of effective cryopreservation of avian genetic material. Cryogenic storage of ovarian tissue is a simple and effective method of conserving the female germline of some mammals but effective in vivo and/or in vitro recovery methods are required. Immunodeficient rodents are used as hosts for mammalian species to prevent tissue rejection when the cryopreserved/warmed tissue is recovered in vivo by transplantation, but these are not available for avian species. The in vitro approaches used in mammals such as in vitro maturation (IVM) and in vitro fertilization (IVF) of follicles retrieved from ovarian tissue are not applicable for avian species because of their special reproductive physiology. At Agassiz we have developed techniques of gonadal transplantation along with a commercially available immunosupressant and combined these techniques with cryopreservation to store and recover testicular tissue to a reproductive state. Based on this technology development, gonads from 18 chicken populations kept at Nova Scotia Agricultural College, the University of Saskatchewan, the University of Alberta, and the Agassiz Research Centre have been cryopreserved. Conflict of interest: None declared. Source of funding: None declared. doi:10.1016/j.cryobiol.2011.09.043
Papers submitted for the Crystal Awards II
41. Cryopreservation of greenlipped mussel (Perna canaliculus) trochophore larvae. E. Paredes * 1, S.L. Adams 2, S.L. Gale 2, L.T. McGowan 3, J.F. Smith 2, H.R. Tervit 2, 1 Departamento de Ecoloxı´a e Bioloxı´a Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain, 2 Cawthron Institute, Private Bag 2, 98 Halifax Steet East, Nelson, New Zealand, 3 AgResearch, Private Bag 3123, Hamilton, New Zealand Cryopreservation is a powerful tool for aquaculture. The ability to cryopreserve gametes and/or larvae enables breeders to easily maintain a large number of family lines and allows for year-round juvenile production in hatcheries without the need to condition and maintain broodstock for out-of-season production. The aim of this study was to develop a method for cryopreserving trochophore larvae of the Greenlipped mussel. Mature mussels were induced to spawn using thermal cycling. For each experiment, at least three pools of trochophores were produced. The ability of trochophores to develop to D-larvae following treatment was assessed against a control. Exp 1: Five ethylene glycol (EG) concentrations in combination with three trehalose (TRE) concentrations were tested for toxicity. One-step vs. five-step cryoprotectant (CPA) addition and removal was also evaluated. Exp 2: 10% EG + 0.2 M TRE was used to evaluate two cooling regimes using 0.25 mL straws and to test whether addition of Bovine Serum Albumin (BSA) during thawing was beneficial. Exp 3: Evaluating the most promising CPAs from Expt 1 and cooling programme from
317
Expt 2 using 0.25 mL straws. Exp 4: Four volumes were tested with the best CPAs from Experiment 3 (10% EG + 0.4 M TRE and 15% EG + 0.2 M TRE). Exp 5: Development beyond the D-larval stage was investigated. Cryopreserved trochophores were reared through to the D-larval stage, and then transferred to the Cawthron Ultra-Density Larval System (CUDLS) for on-growing and reared to the pediveliger stage. A protocol to cryopreserve Greenlipped mussel trochophores was developed and consists on: CPA addition of 10% EG + 0.4 M TRE in one step, allowing 15 min equilibration time. Then trochophores are loaded into 0.25 mL straws and introduced into a Freeze Control System. The protocol chosen starts with a hold at 0 °C for 5 min, then cool to 10 °C at 1 °C min 1, hold for 10 min, then cool at 0.5 °C min 1 to 35 °C, then plunge into liquid nitrogen. Straws were thawed at 28 °C for 6 s and then allowed 15 min equilibration in sea water with 0.1% BSA (w/v). Thawed trochophores were incubated at 20 °C until D-larve stage was reached, obtaining around 40–60% normal D-larvae. Development beyond the D-larval stage was investigated. Frozen trochophores mean survival at day 4 was 60% followed by a significant decrease at day 8. From day 9 onwards, survival is relatively constant under 10%. This attempt to cryopreserve Greenlipped mussell trochophores for the aquaculture industry is encouraging. Future work will investigate the long-term viability of resulting D-larvae by further examining their ability to develop to the eyed-larval stage and scale-up issues for application in selective breeding and hatcheries. This research was funded by projects from Cawthron Institute (New Zealand) and Universidade de Vigo (Spain). Conflict of interest: None declared. doi:10.1016/j.cryobiol.2011.09.044
42. Fluorescence as a better approach to gate cells for cryobiological studies with flow cytometry. Anthony J. Reardon * 1, JanetA.W. Elliott 2, Locksley E. McGann 1, 1 Departments of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada, 2 Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta, Canada Flow cytometry is an important tool in biology and medicine. There is a generalization that light scattered at small angles is proportional to cell volume, leading to the acceptance of forward light scatter as an appropriate estimation of cell size [Millaney et al., Rev. Sci. Instrum. 40(8) (1968) 1029–1032], and commonly used to identify cells from debris. However, in addition to size, other cell characteristics also affect forward scatter measurements [H.M. Shapiro, Practical Flow Cytometry 4th Ed. 2003, p. 275]. Cells with compromised membranes, such as those damaged by freeze–thaw stress produce smaller forward scatter signals, implying that strategies to analyze flow cytometric data developed for other areas of biology may not be appropriate for cryobiological studies. In this study, human umbilical vein endothelial cells (HUVEC) were used to demonstrate the use of fluorescence as an alternative to forward scatter gates for identification of cells in cryopreservation studies. Samples were exposed to two conditions: (i) control cells in suspension at room temperature, and (ii) cells plunged directly into liquid nitrogen and then thawed. Assessment of HUVEC under these conditions was conducted using flow cytometry with JC-1. JC-1 demonstrates cells with functional, polarized mitochondria as a high red/green fluorescence intensity ratio, whereas cells with depolarized mitochondria have a low red/green ratio [Smiley et al., PNAS, 88 (1991) 3671–3675]. A parallel assessment of membrane integrity to verify the results of JC-1 was conducted using a combination of Syto13 and ethidium bromide (SytoEB). Control cells displayed the highest ratio of red/green (JC-1) fluorescence, indicative of the formation of J-aggregates in polarized mitochondria, whereas plunged cells showed a shift to a lower ratio of red/green fluorescence indicating depolarized mitochondria. In addition to these findings the JC-1 (green) fluorescence under these conditions exhibited a distinctive intensity of cells separated from lower intensity events, allowing a threshold to be established to identify cells from debris. These results were verified with a parallel SytoEB study that identified the viable control and non-viable plunged cells in samples based on membrane integrity. In cryobiological studies, isolating cell populations for analysis is more effectively accomplished by gating on fluorescence rather than light scatter, as dyes such as JC-1 allow for identification of cells regardless of the integrity of their plasma membrane. Although forward scatter gates are an effective method of discriminating debris in scatterplots for healthy cell populations, they are ineffective for cell populations with compromised membranes. This is due to the possibility of damaged cells falling below the threshold of the established gate and excluded from the final assessment, leading to an inflated percentage of surviving cells. Green fluorescence emission then makes for an ideal threshold to differentiate cells from debris without the use of forward scatter gating. This research was funded by the Canadian Institutes of Health Research (MOP 86492), and the University of Alberta. J.A.W. Elliott held a Canada Research Chair in Interfacial Thermodynamics during this research. doi:10.1016/j.cryobiol.2011.09.045