Cryopreservation of boar semen: equilibrium freezing in the cryomicroscope and in straws

Supercooling causes very abrupt temperature and osmotic changes and can thus lead to freezing damage. Supercooling can be prevented by seeding, using a sample volume and geometry that allows rapid spreading of the ice throughout the sample. In a split-sample comparison of such samples on the cooling stage of a cryomicroscope and seeded at -5 and -15 degrees C, respectively, the percentages of membrane-intact sperm and sperm with acrosomes with a 'normal apical ridge' (NAR) were 72.5+/-3.8 and 75.8+/-2.0 versus 46.3+/-4.8 and 36.0+/-3.7 (means+/-S.E.M., n=4). In ejaculates of 15 unselected AI boars, after seeding at -5 degrees C, the post-thaw % live and % NAR were 66.3+/-10.4 and 74.8+/-7.5, respectively. Our present research is aimed at translating these findings to freezing in straws and at a high sperm concentration. We have designed a novel type of freezing apparatus for controlled-rate freezing of straws, in which supercooling can be effectively prevented in the entire straw. In a split-sample comparison of semen frozen in straws at a sperm concentration of 1.5 x 10(9) cells/ml with nine ejaculates from eight unselected AI boars, we found 54.8+/-1.9% versus 40.7+/-1.7% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and a conventional freezing apparatus, respectively. With bull semen (eight ejaculates from six bulls), we obtained 67.3+/-3.0% versus 59.3+/-2.9% (means+/-S.E.M.) membrane-intact sperm for the new apparatus and conventional freezing, respectively. Additionally, the temperature curve after ice nucleation is of great importance. We have developed a model that allows us to predict that optimal cryopreservation requires a non-linear cooling curve in which the cooling rate varies as a function of subzero temperature.     

Theoretical analysis of specimen cooling rate during impact freezing and liquid-jet freezing of freeze-etch specimens.

We have carried out a theoretical analysis of specimen cooling rate under ideal conditions during impact freezing and liquid-jet freezing. The analysis shows that use of liquid helium instead of liquid nitrogen as cooling medium during impact freezing results in an increase in a specimen cooling rate of no more than 30-40%. We have further shown that when both impact freezing and liquid-jet freezing are conducted at liquid nitrogen temperature, the two methods give approximately the same specimen cooling rate under ideal conditions except for a thin outer layer of the specimen. In this region impact freezing yields the highest cooling rate.     

Intracellular solute gradients during osmotic water flow: An electron-microprobe analysis

Summary In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freezedried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.     

A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.     

A histological analysis of liver injury in freezing storage

As part of a more extensive study on the use of high subzero freezing for cryopreservation of mammalian livers we have tried to single out the effects of freezing and thawing on tissue damage. We compared the morphology of livers after freezing and thawing with what we considered an optimal high subzero cryopreservation protocol with the morphology of livers preserved under the same thermal conditions and in the same solution in a supercooled state, without freezing. The results show that while hepatocytes survive high subzero cryopreservation, detachment of endothelial cells occurs in every freezing experiment. On the other hand, the endothelial cells in livers that are not frozen are intact. This suggests that endothelial cell damage is caused by freezing and may be an important factor in high subzero freezing cryopreservation of the liver.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.     

A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dimethylsulphoxide (DMSO) as cryoprotectant were in the range -29 degrees C to -31 degrees C and 17.5% to 20% respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

A method for freezing bovine lymphocytes

A simple two-stage technique for preserving bovine lymphocytes is described. Lymphocytes from animals chosen at random were used. The experiments indicate that the optimum temperature for freezing and the optimum concentration of dime-thylsulphoxide (DMSO) as cryoprotectant were in the range -29 °C to -31 °C and 17.5 % to 20 % respectively. These concentrations of DMSO are much greater than those reported in most other studies.     

A mathematical analysis of water and solute transport across the root of Ricinus communis

Summary A mathematical analysis of the relationship between the flux of water, f_H2_O, and the flux of potassium f_K, to the xylem of exuding root systems of Ricinus communis, is presented. Previous analyses (Baker and Weatherley, 1969; Minchin and Baker, 1969) have indicated the presence of a water dependent and a water independent f_K both of which vary with the external concentration of potassium, Cm, supplied as potassium nitrate.The present analysis reveals that whereas at Cm values<1 mM both components of f_K contribute ions to the osmotically active solutions within the osmotic barrier, at Cm values>1 mM only the water dependent f_K is responsible for the osmotic work. This suggests that the ions are released within different regions of the stele. It is proposed that at cm values<1 mM both components are released from the inner stelar tissues whilst at higher Cm values the water dependent f_K is released from the outer stelar tissues. This requires that the solute permeability of the plasmalemma of the outer stelar tissues increases markedly at or about Cm values of 1 mM.It is postulated that the required separation of the two f_K components within the stelar symplasm at Cm values>1 mM is due to the water independent f_K being in a bound state, possibly being transported along a chain of binding sites whilst the water dependent f_K is in a free state within the aqueous phase of the cytoplasm.     

Formation of muscle proteins during freezing

The effect of different conditions on the formation and properties of cryogels prepared by the freezing-thawing procedure from suspensions and solutions of the carp (Cyprinus carpio) myofibrillar proteins was studied. The freezing of water solutions and suspensions of the native myofibrillar proteins resulted in the formation of the structures mainly stabilized by non-covalent bonds. When muscle proteins were denatured prior to the freezing they formed the structures stabilized by both non-covalent and covalent disulfide bonds.     

The role of the cytoskeleton during neuronal polarization

The formation of an axon and dendrites, neuronal polarization, is a prerequisite for neurons to integrate and propagate information within the brain. During the past years progress has been made toward understanding the initial stage of neuronal polarization, axon formation. First, the physiological role of some candidate regulators of neuronal polarity has been affirmed, including Sad kinases, the Rho-GTPase Cdc42, and the actin regulators Ena/VASP proteins. Second, recent studies have revealed microtubule stabilization as a mechanism complementary to actin dynamics underlying neuronal polarization. Moreover, stable microtubules in the axon may form a landmark to confer identity to the axon. This review highlights the recent advances in understanding the intracellular mechanisms underlying neuronal polarization and discusses them in the context of putative cytoskeletal effectors.     

A simple instrument for studying the polarization of fluorescence

The construction, theory, and operation of a simple, easily constructed photoelectric polariscope are described. An analysis of the various sources of random and systematic error shows that the apparatus is capable of measuring the degree of polarization of fluorescent radiation to an accuracy of 2–3% or better.Results of the measurement of the degree of polarization of the fluorescence of conjugates of bovine serum albumin with 1,5-dimethylaminonaphthalene sulfonyl chloride and of legumin with β-anthracene isocyanate are given. These results are used to compute rotational relaxation times of these protein conjugates by the method of Weber, and are shown to be in good agreement with values previously reported in the literature.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.