Abstract:Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique… Show more
“…experimental data can be reconciled (Devireddy et al 1999, Curry 2000. The best-fit parameters obtained in this study with the DSC water transport data (shown in Table 1) during freezing of bovine spermatozoa confirm that this is indeed the case; L pg ¼ 0.02-0.036 mm/min-atm and E Lp ¼ 26.4-42.1 kcal/mol.…”
Section: Discussionsupporting
confidence: 72%
“…A dissimilarity between the suprazero and subzero water transport parameters was also found for mouse (Devireddy et al 1999), human (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003) and boar sperm cells. This discrepancy between the membrane permeabilities may be associated with possible changes in the sperm cell plasma membrane during suprazero cooling, including a lipid-phase transition of 0 -4 8C (Noiles et al 1995) and/or cold-shock damage or 'chilling' injury during cooling (Blok et al 1976, Watson 1981, Steponkus 1984.…”
Section: Effect Of Extracellular Ice On Bovine Sperm Membrane Transpomentioning
confidence: 81%
“…We chose to study membrane transport in the presence of glycerol, since bovine sperm is routinely cryopreserved in the presence of 4 -8% v/v glycerol (Rodriguez et al 1975, Robbins et al 1976; for a review of various bovine sperm-preservation media, see Vishwanath & Shannon 2000). Stepwise addition of CPAs was performed at 25 8C to minimize the osmotic injury and to lessen the volumetric excursions of bovine spermatozoa during the CPA-loading process (Liu & Foote 1998, Devireddy et al 1999. At room temperature (25 8C), a stock of 1.4 M CPA was added to the sperm cells in five equal volume steps at 5-min intervals such that the final concentration of the CPA was 0.7 M. The equilibration time and the number of steps were chosen on the basis of equations developed by Kedem and Katchalsky (1958), and utilizing the suprazero (obtained in the absence of extracellular ice) membrane permeability parameter values (L p , 0.7 m m/min-atm and E a , 10 kcal/mol) reported in the literature for bovine sperm , Chaveiro et al 2004.…”
Section: Loading Of Glycerol and Clcmentioning
confidence: 99%
“…At cooling rates over 60 8C/min, the amount of water trapped inside the tissue cells increases rapidly with increasing cooling rate. A more detailed analysis of the water transport simulations was also performed, as described in earlier studies (Devireddy et al 1999, Thirumala et al 2003, to determine the optimal rate of freezing bovine sperm in the three freezing media investigated. Briefly, we found that the predicted optimal rates of freezing obtained by analyzing the water transport simulations are 43 8C/min, 57 8C/min and 62 8C/min for bovine sperm frozen in the absence of CPAs, in the presence of glycerol and in the presence of CLC respectively.…”
Section: Optimal Rates Of Cooling Bovine Spermatozoamentioning
confidence: 99%
“…However, a novel method using a differential scanning calorimeter (DSC) has enabled the measurement of the water transport response during freezing of spermatozoa of several species, including mouse (Devireddy et al 1999), man (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003), Pacific oyster (He et al 2004), boar , green swordtail and southern platyfish (Pinisetty et al 2005). This DSC technique was used in this study to measure the membrane permeability parameters of bovine spermatozoa at a cooling rate of 20 8C/min in three different media: 1. in the absence of any cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol.…”
Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L pg ) and activation energy (E Lp )). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 8C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 mm and radius 0.4 mm, with osmotically inactive cell volume (V b ) of 0.61 V o , where V o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L pg and E Lp ) were determined. The predicted best-fit permeability parameters ranged from L pg 5 0.02 to 0.036 mm/min-atm and E Lp 5 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 8C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.
“…experimental data can be reconciled (Devireddy et al 1999, Curry 2000. The best-fit parameters obtained in this study with the DSC water transport data (shown in Table 1) during freezing of bovine spermatozoa confirm that this is indeed the case; L pg ¼ 0.02-0.036 mm/min-atm and E Lp ¼ 26.4-42.1 kcal/mol.…”
Section: Discussionsupporting
confidence: 72%
“…A dissimilarity between the suprazero and subzero water transport parameters was also found for mouse (Devireddy et al 1999), human (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003) and boar sperm cells. This discrepancy between the membrane permeabilities may be associated with possible changes in the sperm cell plasma membrane during suprazero cooling, including a lipid-phase transition of 0 -4 8C (Noiles et al 1995) and/or cold-shock damage or 'chilling' injury during cooling (Blok et al 1976, Watson 1981, Steponkus 1984.…”
Section: Effect Of Extracellular Ice On Bovine Sperm Membrane Transpomentioning
confidence: 81%
“…We chose to study membrane transport in the presence of glycerol, since bovine sperm is routinely cryopreserved in the presence of 4 -8% v/v glycerol (Rodriguez et al 1975, Robbins et al 1976; for a review of various bovine sperm-preservation media, see Vishwanath & Shannon 2000). Stepwise addition of CPAs was performed at 25 8C to minimize the osmotic injury and to lessen the volumetric excursions of bovine spermatozoa during the CPA-loading process (Liu & Foote 1998, Devireddy et al 1999. At room temperature (25 8C), a stock of 1.4 M CPA was added to the sperm cells in five equal volume steps at 5-min intervals such that the final concentration of the CPA was 0.7 M. The equilibration time and the number of steps were chosen on the basis of equations developed by Kedem and Katchalsky (1958), and utilizing the suprazero (obtained in the absence of extracellular ice) membrane permeability parameter values (L p , 0.7 m m/min-atm and E a , 10 kcal/mol) reported in the literature for bovine sperm , Chaveiro et al 2004.…”
Section: Loading Of Glycerol and Clcmentioning
confidence: 99%
“…At cooling rates over 60 8C/min, the amount of water trapped inside the tissue cells increases rapidly with increasing cooling rate. A more detailed analysis of the water transport simulations was also performed, as described in earlier studies (Devireddy et al 1999, Thirumala et al 2003, to determine the optimal rate of freezing bovine sperm in the three freezing media investigated. Briefly, we found that the predicted optimal rates of freezing obtained by analyzing the water transport simulations are 43 8C/min, 57 8C/min and 62 8C/min for bovine sperm frozen in the absence of CPAs, in the presence of glycerol and in the presence of CLC respectively.…”
Section: Optimal Rates Of Cooling Bovine Spermatozoamentioning
confidence: 99%
“…However, a novel method using a differential scanning calorimeter (DSC) has enabled the measurement of the water transport response during freezing of spermatozoa of several species, including mouse (Devireddy et al 1999), man (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003), Pacific oyster (He et al 2004), boar , green swordtail and southern platyfish (Pinisetty et al 2005). This DSC technique was used in this study to measure the membrane permeability parameters of bovine spermatozoa at a cooling rate of 20 8C/min in three different media: 1. in the absence of any cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol.…”
Recent experimental data show that incubating bovine sperm with cholesterol-loaded cyclodextrin (CLC) before cryopreservation increases the percentages of motile and viable cells recovered after freezing and thawing, compared with control sperm. In the present study, we report the effect of incubating bovine sperm with CLC on the subzero water transport response and the membrane permeability parameters (reference membrane permeability (L pg ) and activation energy (E Lp )). Water transport data during freezing of bovine sperm cell suspensions were obtained at a cooling rate of 20 8C/min under three different conditions: 1. in the absence of cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; and 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol. With previously published values, the bovine sperm cell was modeled as a cylinder of length 39.8 mm and radius 0.4 mm, with osmotically inactive cell volume (V b ) of 0.61 V o , where V o is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained data, the best-fit water transport parameters (L pg and E Lp ) were determined. The predicted best-fit permeability parameters ranged from L pg 5 0.02 to 0.036 mm/min-atm and E Lp 5 26.4 to 42.1 kcal/mol. These subzero water transport parameters are significantly different from the suprazero membrane permeability values (obtained in the absence of extracellular ice) reported in the literature. Calculations made of the theoretical response of bovine spermatozoa at subzero temperatures suggest that the optimal cooling rate to cryopreserve bovine spermatozoa is 45-60 8C/min, agreeing quite closely with experimentally determined rates of freezing bovine spermatozoa.
In the present study a well-established differential scanning calorimeter (DSC) technique is used to measure the water transport phenomena during freezing of stromal vascular fraction (SVF) and adipose tissue derived adult stem (ADAS) cells at different passages (Passages 0 and 2). Volumetric shrinkage during freezing of adipose derived cells was obtained at a cooling rate of 20 degrees C/min in the presence of extracellular ice and two different, commonly used, cryoprotective agents, CPAs (10% DMSO and 10% Glycerol). The adipose derived cells were modeled as spheres of 50 microm diameter with an osmotically inactive volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the "best-fit" membrane permeability parameters (reference membrane permeability to water, Lpg or Lpg[cpa] and the activation energy, ELp or ELp[cpa]) were determined. The "best-fit" membrane permeability parameters for adipose derived cells in the absence and presence of CPAs ranged from: Lpg=23.1-111.5x10(-15) m3/Ns (0.135-0.652 microm/min-atm) and ELp=43.1-168.8 kJ/mol (9.7-40.4 kcal/mol). Numerical simulations of water transport were then performed under a variety of cooling rates (5-100 degrees C/min) using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing adipose derived cells in the presence and absence of CPAs.
Now that transgenic strains of Xenopus laevis and X. tropicalis can be generated efficiently and with genomic sequence resources available for X. tropicalis, early amphibian development can be studied using integrated biochemical and genetic approaches. However, housing large numbers of animals generated during genetic screens or produced as novel transgenic lines presents a considerable challenge. We describe a method for cryopreserving Xenopus sperm that should facilitate low maintenance, long-term storage of male gametes. By optimising the cryoprotectant, the rates of cooling and thawing, and conditions for fertilisation, sperm from the equivalent of one-eighth of a X. laevis testis or of two X. tropicalis testes have been cryopreserved and used to fertilise eggs of both species after thawing. Sperm undergo a substantial loss of viability during a freeze-thaw cycle, but sufficient survive to fertilise eggs. Gametes of mutagenised frogs are being stored in connection with a screen for developmental mutations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.