Ramachandra V. Devireddy

Cryopreservation of Equine Sperm: Optimal Cooling Rates in the Presence and Absence of Cryoprotective Agents Determined Using Differential Scanning Calorimetry

Abstract Optimization of equine sperm cryopreservation protocols requires an understanding of the water permeability characteristics and volumetric shrinkage response during freezing. A cell-shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine sperm suspensions at cooling rates of 5°C/min and 20°C/min in the presence and absence of cryoprotective agents (CPAs), i.e., in the Kenney extender and in the lactose-EDTA extender, respectively. The equine sperm was modeled as a cylinder of length 36.5 μm and a radius of 0.66 μm with an osmotically inactive cell volume (Vb) of 0.6Vo, where Vo is the isotonic cell volume. Sperm samples were collected using water-insoluble Vaseline in the artificial vagina and slow cooled at ≤0.3°C/min in an Equitainer-I from 37°C to 4°C. By fitting a model of water transport to the experimentally obtained DSC volumetric shrinkage data, the best-fit membrane permeability parameters (Lpg and ELp) were determined. The combined best-fit parameters of water transport (at both 5°C/min and 20°C/min) in Kenney extender (absence of CPAs) are Lpg = 0.02 μm min−1 atm−1 and ELp = 32.7 kcal/mol with a goodness-of-fit parameter R2 = 0.96, and the best-fit parameters in the lactose-EDTA extender (the CPA medium) are Lpg[cpa] = 0.008 μm min−1 atm−1 and ELp[cpa] = 12.1 kcal/mol with R2 = 0.97. These parameters suggest that the optimal cooling rate for equine sperm is ∼29°C/min and is ∼60°C/min in the Kenney extender and in the lactose-EDTA extender. These rates are predicted assuming no intracellular ice formation occurs and that the ∼5% of initial osmotically active water volume trapped inside the cells at −30°C will form innocuous ice on further cooling. Numerical simulations also showed that in the lactose-EDTA extender, equine sperm trap ∼3.4% and ∼7.1% of the intracellular water when cooled at 20°C/min and 100°C/min, respectively. As an independent test of this prediction, the percentage of viable equine sperm was obtained after freezing at 6 different cooling rates (2°C/min, 20°C/min, 50°C/min, 70°C/min, 130°C/min, and 200°C/min) to −80°C in the CPA medium. Sperm viability was essentially constant between 20°C/min and 130°C/min.

Effect of Microscale Mass Transport and Phase Change on Numerical Prediction of Freezing in Biological Tissues

A numerical model incorporating the microscale heat and mass transport in biological tissue during freezing is developed. The heat transfer problem is formulated in a general one-dimensional coordinate system (cartesian, cylindrical or spherical), and a finite control volume discretization is used. The latent heat release for each control volume in the domain is determined by the cellular water transport and intracellular ice formation processes occurring there (a coupled thermal/biophysical approach). The coupled model is applied to two cryobiological freezing problems, with different geometry and boundary conditions. The temperature dependent thermal properties of water and the biophysical properties of two biological tissues, normal rat liver and Dunning AT-1 rat prostate tumor tissue are used to simulate both the micro and macroscale freezing processes. A major advantage of the coupled thermal/biophysical model is its unique ability to predict both the macroscale thermal response and the microscale biophysical response at various locations within the tissue domain during a freezing process, simultaneously

Cryopreservation of Collagen-Based Tissue Equivalents. II. Improved Freezing in the Presence of Cryoprotective Agents

In Part I of this study we determined an optimal cooling rate for cryopreservation of collagen-based tissue equivalents (TEs) that preserves both the postthaw cell viability and mechanical properties, but results in tissue contraction and an overall loss of opacity. The empirically determined optimal cooling rate (5 degrees C/min) was obtained in a freezing medium consisting solely of phosphate-buffered saline (PBS) at physiological concentration (1x). In the present study we report the effect of freezing on TEs in the presence of PBS and two cryoprotective agents (CPAs) (glycerol and dimethyl sulfoxide [Me(2)SO]), at two different concentrations (0.5 and 1.0 M), to two different end temperatures (-80 and -160 degrees C), at a cooling rate of 5 degrees C/min. The controlled rate freezing experiments, postthaw cell viability, and mechanical property measurements were performed as described in Part I of this study. In addition to studying the effect of CPAs on the postthaw properties of TEs, we also investigated (1). the effect of freezing TEs attached to the substrate (as opposed to detached and floating in medium) to determine differences when freezing TEs subject to static mechanical stress via a mechanical constraint to contraction; (2). the effect of freezing glutaraldehyde-fixed TEs to determine differences in freezing-mediated damage to the microstructure; and (3). the effect of freezing more mature TEs that were incubated for 4 weeks in growth factor-supplemented medium as opposed to 2 weeks in basal medium. All TEs frozen at 5 degrees C/min to -80 degrees C in the presence of 0.5 M glycerol or Me(2)SO in PBS were found to be optimally cryopreserved in terms of maintaining opacity and structure as well as cell viability and mechanical properties as compared with unfrozen TEs. The postthaw mechanical properties were adversely affected by freezing to the lower end temperature of -160 degrees C in the presence of CPAs, with the samples frozen in the 1.0 M concentration of CPAs exhibiting a total loss of structural integrity on thawing. Furthermore, TEs frozen attached to the substrate showed decreased opacity and significant contraction as compared with TEs frozen detached from the substrate, as did cross-linked samples frozen without CPA.

Cellular Biophysics During Freezing of Rat and Mouse Sperm Predicts Post-thaw Motility

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5°C/min and 20°C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters Lpg and ELp. A “combined fit” (to 5°C/min and 20°C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded Lpg = 0.007 μm min−1 atm−1 and ELp = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded Lpg = 0.005 μm min−1 atm−1 and ELp = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%–15% normalized cell water at −30°C during freezing in cryopreservation media. These predicted rates range from 53°C/min to 70°C/min and from 28°C/min to 36°C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50°C/min and 80°C/min for rat and at 20°C/min with a sharp drop at 50°C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

Comparison of the permeability properties and post-thaw motility of ejaculated and epididymal bovine spermatozoa ☆

There are very few experimental reports on the comparative water transport (membrane permeability) characteristics of ejaculated and epididymal mammalian spermatozoa during freezing. In the present study, we report the effects of cooling ejaculated and epididymal bovine sperm from the same males with and without the presence of a cryoprotective agent, glycerol. Water transport data during freezing of ejaculated and epididymal bovine sperm suspensions were obtained at a cooling rate of 20 degrees C/min under two different conditions: (1) in the absence of any cryoprotective agents, CPAs and, (2) in the presence of 0.7 M glycerol. Using values published in the literature, we modeled the spermatozoa as a cylinder of length 39.8 microm and a radius of 0.4 microm with an osmotically inactive cell volume, V(b), of 0.61 V(o), where V(o) is the isotonic cell volume. The subzero water transport response is analyzed to determine the variables governing the rate of water loss during cooling of bovine spermatozoa, i.e. the membrane permeability parameters (reference membrane permeability, L(pg) and activation energy, E(Lp)). The predicted best-fit permeability parameters ranged from, L(pg)=0.021-0.038 microm/min-atm and E(Lp)=27.8-41.1 kcal/mol. The subzero water transport response and consequently the subzero water transport parameters are not significantly different between the ejaculated and epididymal bovine spermatozoa under corresponding cooling conditions. If this observation is found to be more generally valid for other mammalian species as well, then in the future the sperm extracted from the testes of a postmortem male could be optimally cryopreserved using procedures similar to those derived for ejaculated sperm.

Phase change behavior of biomedically relevant solutions

Understanding the phase change behavior of biomedically relevant solutions is critical to the development of cryopreservation and cryosurgery protocols. In the present study, the phase change characteristics, particularly thermodynamically non-equilibrium phenomena including supercooled ice nucleation and eutectic formation, of various compositions and concentrations of biological solutions with/without glycerol as a cryoprotective agent (CPA) were investigated using a differential scanning calorimeter (DSC) and cryomicroscopy. The eutectic transitions (crystallization and melting) were observed in not only water-NaCl binary solutions, but also phosphate buffered saline (PBS) solutions. The eutectic transitions in both water-NaCl and PBS solutions were depressed during freezing far below their thermodynamic equilibrium eutectic temperatures. Contrary to freezing, eutectic melting starts very close to the thermodynamic equilibrium eutectic temperature during thawing. However, the eutectic transitions disappeared when a small amount of glycerol (∼0.1M) was added.Copyright