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The discovery of glycerol as an effective cryoprotectant for spermatozoa led to research on cryopreservation of embryos. The first successful offspring from frozen-thawed embryos were reported in the mouse and later in other laboratory animals. Subsequently, these techniques were applied to domestic animals. Research in cryopreservation techniques have included studies concerning the type and concentration of cryoprotectant, cooling and freezing rates, seeding and plunging temperatures, thawing temperatures and rates, and methods of cryoprotectant removal. To date, successful results based on pregnancy rates have been obtained with cryopreserved cow, sheep, goat, and horse embryos but no success has been reported in swine. Post-thaw embryo survival has been shown to be dependent on the initial embryo quality, developmental stage, and species. The freezing techniques most frequently used in research and by commercial companies are identified as "equilibrium" cryopreservation. In this technique the embryos are placed in a concentrated glycerol solution (1.4 M in PBS supplemented with BSA) at room temperature and the glycerol is allowed to equilibrate for a 20-min period. During the cooling process the straws are seeded (-4 to -7 degrees C) and cooling is continued at a rate of 0.3 to 0.5 degree C/min to -30 degrees C when bovine embryos may be plunged into LN2. Sheep embryos are successfully frozen with ethylene glycol (1.5 M) or DMSO (1.5 M) rather than with glycerol. Horse embryos have been frozen in 0.5 rather than 0.25 cc straws but with cooling rates and seeding and plunging temperatures similar to those used with bovine embryos. Swine embryos have shown a high sensitivity to temperature and cryoprotectants probably due to their high lipid content and a temperature decrease to 15 or 10 degrees C causes a dramatic increase in the percentage of degenerated embryos. However, a recent study has shown that hatched pig blastocysts survived exposure below 15 degrees C. Recent research has shown that embryos may also be frozen by a "nonequilibrium" method. This rapid freezing by vitrification consists of dehydration of the embryo at room temperature by a very highly concentrated vitrification media (3.5 to 4.0 M) and a very rapid freeze that avoids the formation of ice allowing the solution to change from a liquid to a glassy state. Vitrification solutions consist of combinations of sucrose, glycerol, and propylene glycol. With this technique, 50% pregnancy rates have been reported with the bovine blastocyst.
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PMID:Status of cryopreservation of embryos from domestic animals. 160 26

The time requirements for permeation by glycerol and dehydration by sucrose before rapid freezing of Day-3 mouse embryos by direct transfer to -180 degrees C were studied. When the embryos were equilibrated in 2.0, 3.0, or 4.0 M-glycerol + 0.25 M-sucrose for 2.5 to 40 min, the post-thaw viability increased (P less than 0.001) with the length of equilibration period at 4 degrees C. At 20 degrees C the volume of embryos increased with the duration of equilibration up to 20 min (P less than 0.001), but the post-thaw viability was not affected. The effect of equilibration in glycerol-sucrose was determined at 20 degrees C for embryos which were previously permeated by glycerol, dehydrated by sucrose or left in PBS + 5% FCS. The survival of previously permeated embryos was not affected by equilibration for 1-16 min in glycerol-sucrose. The maximum survival rate was attained after shorter equilibration in glycerol-sucrose for embryos without pretreatment (4 min) than for those previously dehydrated (8 min). It is concluded that increases in the intracellular glycerol level are beneficial for the viability of rapidly frozen mouse embryos and previous or concomitant exposure to sucrose unfavourably affects glycerol permeation.
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PMID:Role of equilibration before rapid freezing of mouse embryos. 380 23

The suitability of bromodeoxyuridine (BrdU) immunohistochemistry for the study of myosatellite cell proliferation in three subadult carp (Cyprinus carpio) stages (11, 15, 17 cm) was examined. They were injected intraperitoneally with BrdU and fixed one hour late. After fixation and dehydration, white myotomal muscle and small intestine samples were embedded in Histowax. Cross sections mounted on glass slides, were incubated with monoclonal antiBrdU antibody (1:100) after HCl denaturation. After washing twice in PBS, slides were incubated in goat antimouse IgG FITC secondary antibody (1:20). Single cell suspensions were obtained from gut samples. Cellular DNA partially denatured with 2 N HCl, were immunolabelled with monoclonal antibodies against BrdU. Bivariate distribution of BrdU/total cellular DNA content was measured by flow cytometry. Good visualization of BrdU labelled myosatellite cells (4-6%) and small intestine (8-9%) was obtained. Flow cytometric bivariate BrdU/DNA analysis gave evidence of the same proliferative rate in the gut samples. The applicability of these methods to fish tissues further extend the broad range of biological and biomedical investigation in which BrdU immunohistochemistry has been used.
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PMID:Use of 5'-bromodeoxyuridine immunohistochemistry to examine proliferative activity of fish tissues. 768 4

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, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) 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 (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.
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PMID:Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents. 1045 55

Preservation by vitrification of Day 7 and Day 8 bovine blastocysts derived from nuclear transfer with cumulus cells was compared with preservation of in vitro fertilized blastocysts. In Experiment 1, embryos were vitrified in PBS containing 60% ethylene glycol. In Experiment 2, they were vitrified in combination with partial dehydration using a solution of 39% ethylene glycol + 0.7 M sucrose and 8.6% Ficoll. In Experiment 1, survival and hatching rates were 44 and 95% for nuclear transferred embryos, and 78 and 55% for in vitro fertilized embryos, respectively. In Experiment 2, survival and hatching rates were 93 and 95% for nuclear transfer embryos, and 77 and 85% for in vitro fertilized embryos, respectively. It is concluded that Day 7 and Day 8 bovine blastocysts derived from cumulus cells could be cryopreserved without the loss of viability by a simple and efficient method using a combination of partial dehydration and vitrification.
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PMID:Efficient cryopreservation of bovine blastocysts derived from nuclear transfer with somatic cells using partial dehydration and vitrification. 1089 13

Mouse spermatozoa in 18% raffinose and 3.8% Oxyrase in 0.25 x PBS exhibit high motilities when frozen to -70 degrees C at 20-130 degrees C/min and then rapidly warmed. However, survival is <10% when they are frozen at 260 or 530 degrees C/min, presumably because, at those high rates, intracellular water cannot leave rapidly enough to prevent extensive supercooling and this supercooling leads to nucleation and freezing in situ (intracellular ice formation [IIF]). The probability of IIF as a function of cooling rate can be computed by coupled differential equations that describe the extent of the loss of cell water during freezing and from knowledge of the temperature at which the supercooled protoplasm of the cell can nucleate. Calculation of the kinetics of dehydration requires values for the hydraulic conductivity (Lp) of the cell and for its activation energy (Ea). Using literature values for these parameters in mouse sperm, we calculated curves of water volume versus temperature for four cooling rates between 250 and 2000 degrees C/min. The intracellular nucleation temperature was inferred to be -20 degrees C or above based on the greatly reduced motilities of sperm that underwent rapid cooling to a minimum temperature of between -20 and -70 degrees C. Combining that information regarding nucleation temperature with the computed dehydration curves leads to the conclusion that intracellular freezing should occur only in cells that are cooled at 2000 degrees C/min and not in cells that are cooled at 250-1000 degrees C/min. The calculated rate of 2000 degrees C/min for IIF is approximately eightfold higher than the experimentally inferred value of 260 degrees C/min. Possible reasons for the discrepancy are discussed.
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PMID:Is intracellular ice formation the cause of death of mouse sperm frozen at high cooling rates? 1196 14

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.
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PMID:Extra- and intracellular ice formation in mouse oocytes. 1597 68

CpG oligodeoxynucleotides (CpG ODN) are known to be a potent immunoadjuvant for a wide range of antigens. The aim of this study was to evaluate the role of CpG ODN co-encapsulated with rgp63 antigen in cationic liposomes (Lip-rgp63-CpG ODN) in immune response enhancement and protection in BALB/c mice against leishmaniasis. Lip-rgp63-CpG ODN prepared by using dehydration-rehydration vesicle (DRV) method significantly inhibited (P<0.001) Leishmania major infection in mice measured by footpad swelling compared to Lip-rgp63, rgp63 alone, rgp63 plus CpG ODN, PBS or control liposomes. The mice immunized with Lip-rgp63-CpG ODN also showed the lowest spleen parasite burden, highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 production compared to the other groups. The results indicate that co-encapsulation of CpG ODN in liposomes improves the immunogenicity of Leishmania antigen.
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PMID:The role of CpG ODN in enhancement of immune response and protection in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63) encapsulated in cationic liposome. 1762 72

The formation of ice crystals within cells (IIF) is lethal. The classical approach to avoiding it is to cool cells slowly enough so that nearly all their supercooled freezable water leaves the cell osmotically before they have cooled to a temperature that permits IIF. An alternative approach is to cool the cell rapidly to just above its ice nucleation temperature, and hold it there long enough to permit dehydration. Then, the cell is cooled rapidly to -70 degrees C or below. This approach, often called interrupted rapid cooling, is the subject of this paper. Mouse oocytes were suspended in 1.5M ethylene glycol (EG)/PBS, rapidly cooled (50 degrees C/min) to -25 degrees C and held for 5, 10, 20, 30, or 40 min before being rapidly cooled (50 degrees C/min) to -70 degrees C. In cells held for 5 min, IIF (flashing) occurred abruptly during the second rapid cool. As the holding period was increased to 10 and 20 min, fewer cells flashed during the cooling and more turned black during warming. Finally, when the oocytes were held 30 or 40 min, relatively few flashed during either cooling or warming. Immediately upon thawing, these oocytes were highly shrunken and crenated. However, upon warming to 20 degrees C, they regained most of their normal volume, shape, and appearance. These oocytes have intact cell membranes, and we refer to them as survivors. We conclude that 30 min at -25 degrees C removes nearly all intracellular freezable water, the consequence of which is that IIF occurs neither during the subsequent rapid cooling to -70 degrees C nor during warming.
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PMID:Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling. 1804 84

Liposomes as a lipid-based system have been shown to be an effective adjuvant formulation. In this study, the role of liposome charge in induction of a Th1 type of immune response and protection against leishmaniasis in BALB/c mice was studied. Liposomes containing rgp63 were prepared by Dehydration-Rehydration Vesicle (DRV) method. Neutral liposomes consisted of dipalmitoylphosphatidylcholine and cholesterol. Positively and negatively charged liposomes were prepared by adding dimethyldioctadecylammonium bromide (DDAB) or dicetyl phosphate (DCP) to the neutral liposome formulation, respectively. Female BALB/c mice were immunized subcutaneously with negatively, positively charged or neutral liposomes encapsulated with rgp63, rgp63 in soluble form or PBS, three times in 3week intervals. The extent of protection and type of immune response generated were studied in different groups of mice. The group of mice immunized with rgp63 encapsulated in neutral liposomes showed a significantly (P<0.01) smaller footpad swelling upon challenge with Leishmania major compared with positively or negatively charged liposomes. The mice immunized with neutral liposomes also showed a significantly (P<0.01) the lowest splenic parasite burden, the highest IgG2a/IgG1 ratio and IFN-gamma production and the lowest IL-4 level compared to the other groups. The results indicated that a Th1 type of immune response was induced in mice immunized with neutral liposomes more efficiently than positively charged liposomes and conversely negatively charged liposomes induced a Th2 type of immune response.
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PMID:The role of liposome charge on immune response generated in BALB/c mice immunized with recombinant major surface glycoprotein of Leishmania (rgp63). 1921 Oct 22


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