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83. Non-equilibrium ice formation affects the survival of bull sperm at rapid cooling rates
Abstracts / Cryobiology 61 (2010) 362–408 tunica entirely removed were less successful. Of three potential VSs introduced to the tissues in a stepwise fashion and removed by stepwise reduction of non-penetrating cryoprotectant, only 40% EG/0.6M sucrose gave consistently high survivals (>90%). Short exposure and removal (10/15 min) was no better than longer exposure time (15/20 min), and processing at ambient temperature was better than at either 37 or 4 °C. By comparison with conventional freezing, vitrification of these tissue pieces was considerably superior. Testosterone production fell over 9 days in culture while inhibin B production was maintained, but in both cases the cryopreserved tissues were not significantly different from controls, suggesting that the culture conditions were not optimal for Leydig cells but may have been suitable for Sertoli cells. Using magnetic-activated cell sorting and fluorescent activated cell sorting, we showed that after sorting, the cryopreserved putative SSC fraction was 87% viable VS 92% of control. This preliminary study demonstrates the potential for this approach to optimise the cryopreservation and recovery of viable SSCs for future use. Conflict of interest: None declared. Source of funding: Grant NMRC/EDG/0029/2008. doi:10.1016/j.cryobiol.2010.10.086
83. Non-equilibrium ice formation affects the survival of bull sperm at rapid cooling rates. Jon E. Green 1, Idoia Celorrio Fernandez 2, Brian W.W. Grout * 3, Fernanda Fonseca 4, 1 Sustainability Research Institute, University of East London, 4-6 University Way, London E16 2RD, UK, 2 ABEREKIN, S.A., Centro de Inseminación, Parque Tecnológico, Edificio n ° 600, 48160, Derio, Bizkaia, Spain, 3 Faculty of Life Sciences, University of Copenhagen, Hoejbakkegaard Alle 21, 2630 Taastrup, Denmark, 4 Chargée de Recherche, UMR 782 Génie et Microbiologie des Procédés Alimentaires INRA, AgroParisTech, F- 78850 Thiverval-Grignon, France A pooled semen population was prepared from three bulls of different breeds, each recorded as routinely producing semen samples with high viability following cryopreservation. The semen was prepared for cryopreservation using a glycerol-based extender (BioxcellÒ, IMV Technologies) and samples were frozen in 0.25 ml straws at rates of 2, 5, 10, 20, 40, 60, 80, 300 and 500 °C min 1 and by direct immersion in liquid nitrogen. Samples were thawed by plunging into a water bath at 36 °C and recovery estimated by recording progressive motility and viability. Progressive motility and viability were highest following cooling at 20 °C min 1 and decreased markedly and progressively between 80 °C min 1 and the faster cooling rates. This reduction in performance at faster cooling rates coincided with nonequilibrium development of the ice fraction in the extender, visualised by Differential Scanning Calorimetry using comparable cooling rates. This pattern of sperm cell survival and ice fraction formation mirrors the previous study with equine material [1], and comparable ice fraction development has also been recorded in glycerol/NaCl solutions [2]. The data adds support to the argument that, at rapid and ultra-rapid cooling rates, ice formation in glycerol-based solutions is significantly influenced by increased viscosity, leading to variable ice formation and deviation of the concentration of the extracellular matrix from equilibrium values. Thawing from conditions where the magnitude of deviation is high i.e. very rapid rates and ultra-low temperatures, will impose severe osmotic shock on the spermatozoa, leading to the loss of viability. Conflict of interest: None declared. Source of funding: Funding for the study was provided proportionately by the University of Copenhagen, INRA and ABEREKIN, S.A.
References [1] Morris GJ, Goodrich M, Acton E, Fonseca F. The high viscosity encountered during freezing in glycerol solutions: effects on cryopreservation. Cryobiology 2007;52:323–34. [2] Morris GJ, Faszer K, Green JE, Draper D, Grout BWW, Fonseca F. Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology 2006;68:804–12. doi:10.1016/j.cryobiol.2010.10.087
84. Absence of intracellular ice in rapidly cooled spermatozoa. G.J. Morris 1, F. Fonseca 2, 1 Asymptote Ltd., Cambridge, United Kingdom, 2 INRA, Thierval-Grignon, France We have demonstrated that rapid cooling of cryoprotected human and horse spermatozoa leads to loss of viability in the absence of intracellular ice. In this paper we examine the physical basis for the absence of intracellular ice in sperm cells cooled rapidly to 196 °C. In other cell types, for example oocytes and embryos, when
387
Intracellular ice is observed it forms at relatively high sub-zero temperatures, nucleated by a heterogeneous mechanism. It is interesting to speculate whether sperm cells lack heterogeneous nucleation sites. However the lack of heterogeneous nucleation sites within the cells would not necessarily prevent intracellular ice, homogenous ice nucleation would still occur at temperatures below 40 °C and the suppression of homogeneous nucleation requires further explanation. It has been demonstrated that water activity (Aw) is the determinant of homogeneous ice nucleation in aqueous solutions [Koop et al., Nature 406 (2000) 611–614]. This relationship is important as Aw is independent of the nature of the molecular species and can be determined for complex mixtures, such as may be predicted to exist within the intracellular environment of spermatozoa. Aw may be determined by direct measurement of the melting point Tm of a bulk solution and the homogeneous nucleation temperature established. It is generally assumed that the intracellular environment of the sperm cell has a low water content coupled with high protein levels. However, measured values on intracellular proteins levels are difficult to find in the literature, but protein levels could be greater than 20% w/v. We present a number of model cases in which the effect of protein concentration has been examined either in the absence or presence of glycerol. Using this simple approach values of intracellular Thom significantly below 60 °C may be estimated, the Tg of equivalent solutions may be measured to be approximately 30 °C. These data demonstrates that if heterogeneous nucleation sites are absent from sperm then intracellular vitrification would be expected. Conflict of interest: None. Source of funding: Asymptote Ltd.
doi:10.1016/j.cryobiol.2010.10.088
85. Embryo’s cryopreservation in cattle: Physical approach of foetal calf serum’s influence on cryopreservation solutions properties. Bruyere Pierre * 1, Baudot Anne 2, Guerin Pierre 1, Louis Gerard 2, Buff Samuel 1, 1 UPSP ‘Cryobio’ VetAgro Sup/Isara-Lyon, Université de Lyon, VetAgro Sup, Campus Vétérinaire, 1 avenue Bourgelat, 69280 Marcy l’Etoile, France, 2 INSERM U698, CHU X. Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France Slow-freezing methods are often used for cryopreservation of in-vitro produced bovine embryos. However, differences are observed between embryo’s survival rates, probably induced by the composition of the cryopreservation solutions used and the applied procedures [2]. Regarding the cryopreservation solutions, two aspects may explain this variability: differences in physical properties and/or differences in biological properties. In this experiment, a thermodynamic approach was engaged to characterise on a physical point of view the media designed for the cryopreservation of bovine embryos. This study was conducted with a differential scanning calorimeter (DSC Diamond, Perkin Elmer). Because bovine embryos are mostly cryopreserved by using a slow controlled freezing method, we measured two characteristic parameters: the heat of solidification Qmax (close to the maximal quantity of ice crystallized in percentage (w/w) of solution) [1] at slow cooling rate and the temperature Tm of the end of ice melting at slow warming rate. We investigated first the properties of four cryopreservation solutions used in bovine embryos cryopreservation and available from commercial sources: ViGROTM Ethylene Glycol Freeze Plus with 0.1 M sucrose (Bioniche Animal Health, Canada), an embryo freezing media with ethylene glycol (IMV Technologies, France), a patented cryopreservation solution from the UNCEIA (Union Nationale des Cooperatives d’Elevage et d’Insemination Animale, France) and SYNGROTM Holding (Bioniche Animal Health, Canada). This preliminary work was realised in order to determine if their thermodynamic properties differ enough to explain their unequal efficiency for the cryopreservation and the recovery of bovine embryos. Among the various components of cryopreservation solutions (about which information is often anecdotal), the choice of the macromolecular component differs from one to the others: Foetal Calf Serum (FCS), Bovine Serum Albumine, Polyvinyl alcohol. Because of its animal origin and the numerous industrial processes which are available in industry, the composition of FCS is very variable. Moreover, FCS is more and more discontinued due to issues relating to storage and biosecurity (risk of contaminating pathogens). Consequently, we decided to investigate secondly the effect of FCS on the thermodynamic properties of the solutions by testing three batches from different suppliers (Sigma, Fisher Scientific and VWR). For this study, a stock solution was defined without macromolecular component as follow: Dulbecco’s PBS, 1.5 M ethylene glycol, 0.1 M sucrose, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. This composition resulted from the synthesis of bibliographic investigations on bovine embryos cryopreservation. Cryopreservation solutions tested on the Differential Scanning Calorimeter were made by adding 20% (v/v) FCS to the stock solution. This work presents an evaluation of the characteristic physical parameters of bovine embryos cryopreservation solutions and a discussion about the effect of FCS on these parameters. Biological tests will soon complete this study in order to evaluate the biological properties and efficiency of the same cryopreservation solutions.
83. Non-equilibrium ice formation affects the survival of bull sperm at rapid cooling rates. Jon E. Green 1, Idoia Celorrio Fernandez 2, Brian W.W. Grout * 3, Fernanda Fonseca 4, 1 Sustainability Research Institute, University of East London, 4-6 University Way, London E16 2RD, UK, 2 ABEREKIN, S.A., Centro de Inseminación, Parque Tecnológico, Edificio n ° 600, 48160, Derio, Bizkaia, Spain, 3 Faculty of Life Sciences, University of Copenhagen, Hoejbakkegaard Alle 21, 2630 Taastrup, Denmark, 4 Chargée de Recherche, UMR 782 Génie et Microbiologie des Procédés Alimentaires INRA, AgroParisTech, F- 78850 Thiverval-Grignon, France A pooled semen population was prepared from three bulls of different breeds, each recorded as routinely producing semen samples with high viability following cryopreservation. The semen was prepared for cryopreservation using a glycerol-based extender (BioxcellÒ, IMV Technologies) and samples were frozen in 0.25 ml straws at rates of 2, 5, 10, 20, 40, 60, 80, 300 and 500 °C min 1 and by direct immersion in liquid nitrogen. Samples were thawed by plunging into a water bath at 36 °C and recovery estimated by recording progressive motility and viability. Progressive motility and viability were highest following cooling at 20 °C min 1 and decreased markedly and progressively between 80 °C min 1 and the faster cooling rates. This reduction in performance at faster cooling rates coincided with nonequilibrium development of the ice fraction in the extender, visualised by Differential Scanning Calorimetry using comparable cooling rates. This pattern of sperm cell survival and ice fraction formation mirrors the previous study with equine material [1], and comparable ice fraction development has also been recorded in glycerol/NaCl solutions [2]. The data adds support to the argument that, at rapid and ultra-rapid cooling rates, ice formation in glycerol-based solutions is significantly influenced by increased viscosity, leading to variable ice formation and deviation of the concentration of the extracellular matrix from equilibrium values. Thawing from conditions where the magnitude of deviation is high i.e. very rapid rates and ultra-low temperatures, will impose severe osmotic shock on the spermatozoa, leading to the loss of viability. Conflict of interest: None declared. Source of funding: Funding for the study was provided proportionately by the University of Copenhagen, INRA and ABEREKIN, S.A.
References [1] Morris GJ, Goodrich M, Acton E, Fonseca F. The high viscosity encountered during freezing in glycerol solutions: effects on cryopreservation. Cryobiology 2007;52:323–34. [2] Morris GJ, Faszer K, Green JE, Draper D, Grout BWW, Fonseca F. Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology 2006;68:804–12. doi:10.1016/j.cryobiol.2010.10.087
84. Absence of intracellular ice in rapidly cooled spermatozoa. G.J. Morris 1, F. Fonseca 2, 1 Asymptote Ltd., Cambridge, United Kingdom, 2 INRA, Thierval-Grignon, France We have demonstrated that rapid cooling of cryoprotected human and horse spermatozoa leads to loss of viability in the absence of intracellular ice. In this paper we examine the physical basis for the absence of intracellular ice in sperm cells cooled rapidly to 196 °C. In other cell types, for example oocytes and embryos, when
387
Intracellular ice is observed it forms at relatively high sub-zero temperatures, nucleated by a heterogeneous mechanism. It is interesting to speculate whether sperm cells lack heterogeneous nucleation sites. However the lack of heterogeneous nucleation sites within the cells would not necessarily prevent intracellular ice, homogenous ice nucleation would still occur at temperatures below 40 °C and the suppression of homogeneous nucleation requires further explanation. It has been demonstrated that water activity (Aw) is the determinant of homogeneous ice nucleation in aqueous solutions [Koop et al., Nature 406 (2000) 611–614]. This relationship is important as Aw is independent of the nature of the molecular species and can be determined for complex mixtures, such as may be predicted to exist within the intracellular environment of spermatozoa. Aw may be determined by direct measurement of the melting point Tm of a bulk solution and the homogeneous nucleation temperature established. It is generally assumed that the intracellular environment of the sperm cell has a low water content coupled with high protein levels. However, measured values on intracellular proteins levels are difficult to find in the literature, but protein levels could be greater than 20% w/v. We present a number of model cases in which the effect of protein concentration has been examined either in the absence or presence of glycerol. Using this simple approach values of intracellular Thom significantly below 60 °C may be estimated, the Tg of equivalent solutions may be measured to be approximately 30 °C. These data demonstrates that if heterogeneous nucleation sites are absent from sperm then intracellular vitrification would be expected. Conflict of interest: None. Source of funding: Asymptote Ltd.
doi:10.1016/j.cryobiol.2010.10.088
85. Embryo’s cryopreservation in cattle: Physical approach of foetal calf serum’s influence on cryopreservation solutions properties. Bruyere Pierre * 1, Baudot Anne 2, Guerin Pierre 1, Louis Gerard 2, Buff Samuel 1, 1 UPSP ‘Cryobio’ VetAgro Sup/Isara-Lyon, Université de Lyon, VetAgro Sup, Campus Vétérinaire, 1 avenue Bourgelat, 69280 Marcy l’Etoile, France, 2 INSERM U698, CHU X. Bichat, 46 rue Henri Huchard, 75877 Paris Cedex 18, France Slow-freezing methods are often used for cryopreservation of in-vitro produced bovine embryos. However, differences are observed between embryo’s survival rates, probably induced by the composition of the cryopreservation solutions used and the applied procedures [2]. Regarding the cryopreservation solutions, two aspects may explain this variability: differences in physical properties and/or differences in biological properties. In this experiment, a thermodynamic approach was engaged to characterise on a physical point of view the media designed for the cryopreservation of bovine embryos. This study was conducted with a differential scanning calorimeter (DSC Diamond, Perkin Elmer). Because bovine embryos are mostly cryopreserved by using a slow controlled freezing method, we measured two characteristic parameters: the heat of solidification Qmax (close to the maximal quantity of ice crystallized in percentage (w/w) of solution) [1] at slow cooling rate and the temperature Tm of the end of ice melting at slow warming rate. We investigated first the properties of four cryopreservation solutions used in bovine embryos cryopreservation and available from commercial sources: ViGROTM Ethylene Glycol Freeze Plus with 0.1 M sucrose (Bioniche Animal Health, Canada), an embryo freezing media with ethylene glycol (IMV Technologies, France), a patented cryopreservation solution from the UNCEIA (Union Nationale des Cooperatives d’Elevage et d’Insemination Animale, France) and SYNGROTM Holding (Bioniche Animal Health, Canada). This preliminary work was realised in order to determine if their thermodynamic properties differ enough to explain their unequal efficiency for the cryopreservation and the recovery of bovine embryos. Among the various components of cryopreservation solutions (about which information is often anecdotal), the choice of the macromolecular component differs from one to the others: Foetal Calf Serum (FCS), Bovine Serum Albumine, Polyvinyl alcohol. Because of its animal origin and the numerous industrial processes which are available in industry, the composition of FCS is very variable. Moreover, FCS is more and more discontinued due to issues relating to storage and biosecurity (risk of contaminating pathogens). Consequently, we decided to investigate secondly the effect of FCS on the thermodynamic properties of the solutions by testing three batches from different suppliers (Sigma, Fisher Scientific and VWR). For this study, a stock solution was defined without macromolecular component as follow: Dulbecco’s PBS, 1.5 M ethylene glycol, 0.1 M sucrose, 100 IU/mL penicillin and 0.1 mg/mL streptomycin. This composition resulted from the synthesis of bibliographic investigations on bovine embryos cryopreservation. Cryopreservation solutions tested on the Differential Scanning Calorimeter were made by adding 20% (v/v) FCS to the stock solution. This work presents an evaluation of the characteristic physical parameters of bovine embryos cryopreservation solutions and a discussion about the effect of FCS on these parameters. Biological tests will soon complete this study in order to evaluate the biological properties and efficiency of the same cryopreservation solutions.