Application of silver iodide as an inducer of ice formation in cryopreservation of mammalian embryos

ABSTRACTS, 24th ANNUAL and reflection coefficient. o, to obtain an optimum correlation of the simulation with the experimental data. Experiments were performed in the temperature range from -7 to - lO”C, with an observed decrease in L, and a defined activation energy AE for cells which had been equilibrated in the Me,SO solution prior to examination. It was found that the movement of Me,SO across the plasma membrane was insufficient in magnitude to achieve the sensitivity required for evaluation of the parameters o and cr. (Sponsored by a grant from the Texas Advanced Technology Research Program.) SYMPOSIUM II-AQUEOUS SOLUTIONS AT LOW TEMPERATURES 13. Glasses in Aqueous Systems. TIMOTHY W. SCHENZ (Technical Center, General Foods Corp., 555 S. Broadway, Tarrytown, New York 10591). The characteristics and attributes of aqueous systems at low temperatures depend to a great extent on the physicochemical and thermomechanical properties of the unfrozen, amorphous matrix that surrounds the ice crystals. If this matrix is maintained as a kinetically metastable mechanical solid (a glass), then the changes that typically occur at higher temperatures can be prevented or greatly retarded. The optimum storage temperature or optimum formulation for a particular system is dictated by the characteristic glass transition temperature, T8’, of the aqueous system. The formation of aqueous glasses and their properties is influenced primarily by the nature and composition of the solutes in the unfrozen aqueous phase. The relationships between glass formation and the properties of the solutes, methodology for the measurement of glass transitions, and the functional uses of glasses in aqueous systems will be reviewed. 14. Antifreeze

Proteins

and Their Mode of Action ut

rhe Ice Surf&e. ROBERTFEENEY (Department of Food Science, University of California).

qf Ice during Systems. D. S. REID

15. Facrors Involved in the Propagation the Freezing ofBiological

(Department of Food Science, University of California, Davis). For ice to be present in a frozen system, a complex sequence of events has had to take place. Nucleation, the initiation of growth, is necessary. The nucleation process is affected by the composition of the system and by the presence or absence of a variety of catalytic moieties which can induce the heterogeneous nucleation process. Nucleation per se is a necessary but not sufficient prerequisite for the formation of ice in quantity in a frozen system. Ice has to propagate from the nuclei. The growth or propagation of ice is a process which is affected by system composition, by

MEETING

547

system temperature, and by the rate at which conditions are changing. The interrelationships between the kinetics of nucleus formation and the kinetics of crystal propagation are important in the light of the current understanding of the processes contributing to the observed phenomena. SESSION II-AQUEOUS SOLUTIONS AT LOW TEMPERATURES 16. Application of Silver Iodide as an Inducer of Ice Formation in Cryopreservation of Mammulian Embryos. T. KOJIMA, T. SOMA, AND N. OGURI

(Department of Animal Reproduction, National Institute of Animal Industry, Tsukuba Science City, Ibaraki, Japan). A method was devised for inducing ice crystal formation in extracellular solution using silver iodide. A latent heat occurred immediately before the temperature of the sample reached -7”C, when a 70-mm column of I .5 M dimethyl sulfoxide (Me,SO) was aspirated into a plastic straw followed by 3 mm of air and 10 mm of 1% suspension of silver iodide in distilled water (1% AgI). To examine the effect of silver iodide as an inducer of ice crystal formation in extracellular solution on in vitro development of frozen-thawed rabbit morulae, the straws were filled by successive aspiration of the following fractions: 175 pl of 1.5 M Me,SO containing the embryos, 7.5 pl of air, and 25 JLI of 1% AgI. The straws were cooled to -7°C at I”C/ min and held at -7°C for 10 min without initiating seeding. They were then cooled again to -30°C at l”C/min and plunged into liquid nitrogen. After rapid thawing, 100 of 109 (92%) embryos that were recovered developed into expanding blastocysts. To examine the viability of bovine Day 7 embryos frozen using the silver iodide seeding procedure after cervical transfer to appropriate heifers, the straws were loaded by successive aspiration of the following fractions: 175 pJ of 10% (v/v) glycerol containing the embryo, 7.5 p1 of air, and 25 pl of 1% AgI. The straws were cooled to - 7°C at 1”Cimin and held at - 7°C for 10 min without initiating seeding. They were then cooled to - 35°C at 0.3”Cimin and plunged into liquid nitrogen. After rapid thawing, the embryos recovered were immersed in 1.O M sucrose solution for 10 min and washed by modified PBS. The seven embryos were transferred to the total seven recipients simultaneously. Two pregnancies were produced, and then two normal male calves were born. The silver iodide method does not require special equipment or a manual procedure for seeding and avoids extreme temperature fluctuation of the samples that can accompany seeding procedures. Using the silver iodide method, it may be possible to seed simultaneously a large number of samples and to set samples horizontally in a cooling bath because of nonlimitation in setting of samples. This method may also make it possible to employ freezing vessels other than plastic straws. We need only to cool samples to a con-

548

ABSTRACTS, 24th ANNUAL

stant temperature only slightly below that of the freezing point of the freezing solution to induce ice crystal formation and to hold the samples at that temperature for 10 min to restore the thermal equilibrium and establish osmotic equilibrium. 17. Extended Phase Diagrams for the Ternary Solutions H,O-NACI-Glycerol and H,O-NACIHydroxyethylstarch (HES) Determined by DSC. M. JOCHEM AND CH. KGRBER (Helm-

holtz-Institut fur Biomedizinische Technik an der RWTH Aachen, D-5100 Aachen, West Germany). Phase transformations occurring in the solutions H,O-NaCl-glycerol and H,O-NaCI-HES on nonequilibrium cooling and heating conditions were examined by means of differential scanning calorimetry (DSC). Glass transition, devitrification, eutectic melting and primary ice melting were evaluated to obtain data regarding the stability of the amorphous phases as well as for the composition of the residual solution remaining after crystallization. It was found that for many initial compositions and cooling/heating conditions used in cryobiology only primary ice crystallizations occurs. Therefore the description of the ternary phase-change behavior may become less complicated for nonequilibrium conditions than in the case of assumed equilibrium. The results were used to modify and extend the equilibrium phase-diagrams to make them applicable to problems with boundary conditions which exclude thermodynamic equilibrium and result in the formation of metastable phases. The application of the phase diagrams is important for obtaining information about the fraction of ice formed and the changes in composition and concentration during the cooling/heating process. 18. The Influence

of Holding Temperature Phenomenon of “Time Dependency” drated Lysozyme Glasses as Measured ferential Scanning Calorimetry. JAN LANCZYKANDJOHNG. BAUST (Luyet

on the in Hyby Dif

P. WOCenter

for Cryobiological Research, State University of New York, Binghamton, New York 13901). The phenomenon of “time dependency” was investigated in hydrated hen egg white lysozyme glasses using differential scanning calorimetry. Both the subambiant holding temperature and holding time were varied in an effort to vary the amount of freezing water in the system. The amount of nonfreezing water measured in the sample decreased with increasing holding time (time dependency), a similar decrease in the amount of nonfreezing water in the sample was observed by lowering the holding temperature. 19. PNMR

and DSC Analyses

of Cryoprotectant

MEETING Mixtures. JOHN M. WASYL~K, RAYMOND L. SZYMANSKI,.~AN WOLANCZYK, ANDJOHNG. BAUST (Center for Cryobiological Research,

State University of New York at Binghamton, Binghamton, New York 13903). In an attempt to measure the time course of ice accumulation in freeze tolerant insects along with the relative contribution of soluble and nonsoluble components to the cold hardening process, it was observed that simple and complex mixtures of cryoprotective solutions demonstrated nonidealistic freezing behavior. Characteristics of the nonidealistic behavior have been studied by various techniques including ‘H nuclear magnetic resonance (NMR) and differential scanning calorimetry (DSC). NMR studies have suggested that an increase in proton mobility occurs after freezing (- 30 to - 50°C). DSC experiments have been correlated to this temperature range in an effort to detect changes in associated proton energy. 20. Differential Scanning Calorimetric Analysis of Antifreeze Protein Activity in the Common Mealworm, Tenebrio molitor. THOMAS N. HANSEN, CHRISTOPHER J. PIO, AND JOHN G. BAUST. (Center for Cryobiological Research,

State University of New York, Binghamton, New York 13901). Hemolymph samples from the common mealworm adult, Tenebrio moliror, were analyzed for antifreeze protein (AFP) activity using differential scanning calorimetry. Effects of temperature and photoperiod on AFP activity were tested under various dual conditions (25 vs 5°C 16:8 vs. 8:16 L:D photoperiod). Individual samples (l-4 pJ) were sequentially frozen and warmed to a series of partial-melt temperatures (- 0.1 to -2.O”C) to mimic changes in ice crystal size. The difference between the partial-melt temperature and the onset of the freezing exotherm during subsequent cooling was taken as a quantitative measure of AFP activity. Cold and warm acclimated animals showed similar amounts of antifreeze activity. Long-day acclimated animals showed higher amounts of antifreeze protein activity than short-day acclimated animals. The results suggest that AFP production is not triggered by overwintering conditions in adult T. molitor. 21. Differential

Scanning Calorimetric and Mass Spectrometric Analysis of Mammalian Tissue Samples. J. G. BALJST, S. R. MAY,* J. P. WOLANCZYK, S. A. LIVESEY,* AND J. G. LINNER* (Center for Cryobiological Research,

University Center at Binghamton, State University of New York, Binghamton, New York, 13901; *LifeCell Corporation, The Woodlands, Texas; and Cryobiological Research Center,