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Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M)., (Copyright 2001 Elsevier Science.)

Embryo cryopreservation is a widely used and relatively well-established procedure. By contrast, ovarian tissue and unfertilized oocytes are only rarely cryopreserved, even though for germ line storage these often would be preferable to embryo cryopreservation. There are many reasons for this discrepancy. Unfertilized mature (MII) stage oocytes are more difficult to cryopreserve than cleavage stage embryos of the same species. Many factors contribute to this including the oocyte's surface to volume ratio, single membrane, temperature-sensitive metaphase spindle and zona, and its susceptibility to parthenogenetic activation and chill-injury. A completely different set of problems applies to primordial follicles. Oocytes in primordial follicles are very small and tolerate cryopreservation by slow cooling very well. The problem lies in the difficulty in producing mature oocytes from these primordial follicles. Better and/or more convenient cryopreservation procedures for both oocytes and ovarian tissue are being developed. This paper describes some of the advances in this area and outlines the relative merits and limitations of several currently available egg and ovarian tissue cryopreservation procedures.

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars: d-glucose, d(-)-fructose, d-sorbitol, sucrose, d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0. 1, 0.5, and 1 m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1 m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5 m) and 63 wt% (0.1 m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified., (Copyright 1999 Academic Press.)

Vitrification solutions which are used for cells or embryos generally contain cryoprotectant, physiological saline, and one or more macromolecular solutes. The macromolecules modify the vitrification tendencies of these solutions, but there is little detailed information on the vitrification properties of ethylene glycol solutions containing the additives PVP, Ficoll, and dextran. This study therefore added ethylene glycol to 0.9% NaCl in water (saline) and used differential scanning calorimetry to determine the lowest concentration at which the solution would remain vitreous when a warming rate of 10 degrees C/min was used. In the absence of other additives 59 wt% ethylene glycol (EG) in saline formed a stable glass. When ethylene glycol was replaced by the polymers Ficoll and/or dextran on a weight for weight basis, the resulting solution vitrified less readily than an EG-saline solution even though the total solute concentration was kept constant. The total solute concentration required to form a stable vitreous solution increased as the Ficoll 70,000 and 400,000 MW or dextran 78,000 MW content increased (5, 10, and 20 wt%). Ficoll and dextran had little or no effect on the glass transition and melting points of the solutions. In the presence of PVP vitrification occurred at a total solute concentration of 59 wt% (PVP 360,000 MW) or 60 wt% (PVP 40,000 MW) for all three tested PVP concentrations (5, 10, and 20 wt%). Although this indicates that PVP and EG have comparable vitrification properties, the melting and the glass transition temperature of the solutions rose as the PVP content increased. When 1 m sucrose was added to saline and 0, 5, 10, or 20 wt% PVP 40,000 MW vitrification was achieved with 31, 26, 23, and 15% EG, respectively, indicating that the total solute concentration required for vitrification could be estimated with reasonable accuracy from the sum of the individual components. We conclude that the tested polymers differ in how they interact with ethylene glycol-based vitrification solutions., (Copyright 1997 Academic Press.)

Three 1-yr-old swine and two 2.5-wk-old swine were fed a fat-free diet for 1 month and 5 months, respectively. The hepatic phosphatidylcholine and phosphatidylethanolamine were fractionated by silver ion thin-layer chromatography. A distinctive feature of the chromatographic procedure was the development of the chromatograms at low temperatures: -10 degrees C for phosphatidylcholine and 4 degrees C for phosphatidylethanolamine. The chromatographic fractions were hydrolyzed with phospholipase A(2), and the fatty acids were characterized. Significant concentrations of odd-chain saturated and unsaturated fatty acids were found in the swine deprived of fat for 5 months. The major molecular species of phosphatidylcholine in both groups contained monoenoic fatty acids: 16:0/18:1(n - 9), 18:0/18:1(n - 9), and 18:1(n - 9)/18:1(n - 9). Their concentrations changed only slightly with the diet. The molecular species of phosphatidylethanolamine were more sensitive to dietary changes. In the swine deprived of fat for 1 month, about 50% of the molecular species of phosphatidylethanolamine contained tetraenoic fatty acids: 16:0/20:4(n - 6), 18:0/20:4(n - 6), and 18:1(n - 9)/20:4(n - 6). The phosphatidylethanolamine of animals deprived of fat for 5 months contained only 3% molecular species with tetraenoic acids, 18:0/20:4(n - 6), but 36% molecular species with trienoic acids: 18:0/20:3(n - 9), 18:1(n - 9)/20:3(n - 9), 18:0/19:3(n - 8), 16:0/20:3(n - 9), and 17:0/20:3(n - 9). Doubly unsaturated species, such as 18:1(n - 9)/18:1(n - 9), 18:1(n - 9)/20:3(n - 9), and 18:1(n - 9)/20:4(n - 6), were found in both groups of swine, although their total concentrations were higher in the group deprived of fat for a longer period.

[14C]Captopril was given as a priming dose, followed by constant intravenous infusion for 4 or 6 hr, to three anesthetized dogs and three anesthetized monkeys. Blood, urine, and bile samples were collected during and after drug infusion. Pharmacokinetic evaluations were carried out exclusively on data obtained for unchanged captopril. The average total body clearance (ClT) and the renal clearance (ClR) of captopril, in milliliters per kilogram per hour, were 605 and 341 in the dog and 1135 and 944 in the monkey. The biliary clearance of captopril was negligible in both species. The greater difference between the ClR and ClT values in the dog compared to that in the monkey was the result of more extensive metabolism of captopril by the dog. Since almost all of the radioactive dose was recovered in urine in both species, captopril and its metabolites were almost exclusively eliminated by the kidneys. One primary reason that body and renal clearance values of captopril were much greater in the monkey than in the dog was that the net tubular secretion fo captopril was about three times greater in the monkey (82%) than in the dog (28%). The volume of distribution of captopril was higher in the monkey (3.6 liters/kg) than in the dog (2.6 liters/kg); the volume of the central compartment was about the same (0.5 liter/kg) for both species. The terminal half-life value was slightly higher in the dog (2.8 hr) than in the monkey (2.2 hr).

The rates of release of 14C-fluphenazine enanthate and 14C-fluphenazine decanoate were compared in two groups of five male dogs. Each dog was given a single dose (2 mg/kg im) of either the enanthate or decanoate ester in sesame oil. The times required to attain maximum concentrations of radioactivity in plasma were 3.8 +/- 0.5 days (+/-SE) for the enanthate ester and 10.6 +/- 1.1 days for the decanoate ester (p less than 0.001); maximum concentrations of radioactivity in the plasma at these times were 16.7 +/- 1.1 and 11.1 +/- 1.2 ng/ml, respectively (p less than 0.01). However, 35 days after dosing, the concentrations of radioactivity in plasma were greater for the decanoate ester than for the enanthate ester. The times required for 50% of the dose to be excreted in the urine and feces were 7.8 +/- 0.5 days for the enanthate ester and 22.6 +/- 4.4 days for the decanoate ester (p less than 0.05). The total amounts excreted in 35 days were 85.4 +/- 1.8 and 68.8 +/- 6.6% of the dose for the enanthate and decanoate esters, respectively; the average half-times for the rates of release of radioactivity from depot and body, as calculated from the data for total excretion, were 5.55 days for the enanthate ester and 15.4 days for the decanoate ester. Thirty-five days after dosing, the amount of the dose present in the injection site was 4.6 +/- 1.6% for the enanthate ester and 18.6 +/- 5.7% for the decanoate ester. Two groups of six dogs each were protected against the emetic effects of apomorphine more than twice as long by the decanoate ester than by the enanthate ester after the subcutaneous administration of single 8-mg/kg doses of either drug in sesame oil (p less than 0.05). Based on measurements of total radioactivity, it was concluded that the decanoate ester was released from the depot at less than one-half the rate of the enanthate ester.

14C-Fluphenazine base was administered intramuscularly in sesame oil to five male beagles (2 mg/kg). The concentration of radioactivity in plasma and the excretion of radioactivity in urine and feces were measured for 14 days. Maximum concentrations of radioactivity were found in plasma 2 hr after administration. These levels declined with elimination half-lives of 3.20 hr during the 2-12-hr interval after dosing and of 4.02 days during the 2-14-day interval. Most administered radioactivity was excreted during the first 2 days after dosing, predominantly in the feces. An average of 0.43% of the dose was present at the injection site 14 days after dosing; some residual radioactivity was found in the liver and in the ocular portion consisting of the combined retina, choroid, and sclera. 14C-Fluphenazine and its enanthate and decanoate esters were each administered intravenously to three different groups of intact dogs at doses of 1 mg/kg. Regardless of which compound was administered, concentrations of radioactivity in the plasma of these dogs were comparable. Thirty minutes after these dogs had been dosed with 14C-fluphenazine enanthate or 14C-fluphenazine decanoate, most radioactivity in the plasma was present as 14C-fluphenazine base and other unidentified metabolites; at this time, at least 79% of either of the two 14C-fluphenazine esters had been biotransformed. The excretion of radioactivity by these same three groups of dogs was very similar, regardless of which of the compounds was given. In 7 days, an average of only 3-4% of the dose was excreted in urine; the remainder was excreted in feces. 14C Fluphenazine and its enanthate and decanoate esters (1 mg/kg) were administered intravenously to dogs whose bile ducts had been cannulated. The amounts of radioactivity excreted in the urine and bile in 8 hr were very similar, as were the residual amounts of radioactivity present in selected tissues. Comparison of the data obtained from dogs given these three compounds intravenously (unformulated) or intramuscularly in sesame oil points to the following conclusions: (a) fluphenazine base per se does not provide slow-release characteristics unless it has been esterified, for example, with heptanoic or decanoic acid, and (b) intramuscular rather than intravenous administration of these two esters is responsible for producing their slow-release characteristics.

8-(Methylthio-14C or -35S)cyclic 3',5'-adenosine monophosphate (I) was given intravenously to rats (5 mg/kg) and orally and intravenously to dogs (0.25, 2.5, or 50 mg/kg). Oral doses were absorbed well but slowly. Plasma half-lives in dogs were about 3 hr after oral or intravenous doses of 0.25 or 2.5 mg/kg and ranged from 5 to 12 hr after oral or intravenous doseas of 50 mg/kg. Plasma glucose and insulin concentrations in dogs were increased by oral or intravenous doses of the compound. Regardless of the route, excretion of radioactivity by rats and dogs at all doses was chiefly in the urine (74-87% of the dose); the remainder was excreted in the feces or bile. Compound I was rapidly distributed to most tissues of dogs but entered the brain and certain portions of the eye slowly and to a limited extent. Urine and plasma of dogs and urine of rats contained I, 8-(methylthio)adenosine, and at least two other unidentified metabolites. Compound I and cyclic 3',5'-adenosine monophosphate were metabolized in vitro by the soluble fraction of dog liver to form 8-(methylthio)adenosine-5'-monophosphate and adenosine-5'-monophosphate, respectively. These compounds were further converted to 8-(methylthio)adenosine and adenosine, respectively. Compound I was metabolized in vitro more slowly than cyclic 3',5'-adenosine monophosphate.

The absorption of captopril (I), a new antihypertensive agent, was studied in mice and rats at doses (50 and 1350 mg/kg) administered in the diet in chronic toxicological studies. 3H- or 35S-Labeled I was administered by gavage and in the diet to male and female animals in a two-way crossover study. Animals received daily doses of nonradiolabeled I in the diet for 25 days, except on Days 15 and 22 when radiolabeled I was administered either by gavage or in the diet. Absorption of the total radioactivity in 2-month-old mice averaged 49 and 48%, respectively, of the 50- and 1350-mg/kg doses given in the diet and 57 and 65%, respectively, of the doses given by gavage. The bioavailability of I in 2-month-old mice averaged 48 and 39% (diet) and 44 and 59% (gavage) of the 50- and 1350-mg/kg doses, respectively. In 2-month-old rats, absorption of the total radioactivity averaged 41% of the 50-mg/kg dose given in the diet. In 2- and 15-month-old rats, minimum absorption of the 1350-mg/kg dose averaged 36 and 45% (diet) and 51 and 71% (gavage), respectively; the minimum bioavailability averaged 20 and 29% (diet) and 39 and 44% (gavage), respectively. These studies demonstrate adequate absorption and bioavailability of I over a wide range of doses from the drug-diet mixtures and by young and old animals and also illustrate a useful experimental design for the estimation of relative oral absorption of a drug administered continuously in the diet over several days.

Two-cell mouse embryos were frozen by direct plunging into liquid nitrogen after a 3-min exposure to solutions containing 0.25 M sucrose with 1.5, 3, or 4.5 M dimethyl sulfoxide (Me2SO), and 0, 4, 8, 16, or 32 mg/ml bovine serum albumin (BSA). In the absence of BSA, significantly more embryos were lost or damaged during freezing and thawing. Increasing the BSA concentration from 4 to 32 mg/ml had no significant effect on subsequent embryo viability in vivo or in vitro. Blastocyst formation in vitro was greater than 90% in embryos exposed to the cryoprotective solutions only. Although development to blastocysts was not significantly different to nonfrozen controls in most groups frozen in 3 and 4.5 M Me2SO (up to 92% blastocysts), it was significantly reduced when embryos were frozen in 1.5 M Me2SO (up to 65% blastocysts). The development to fetuses of embryos frozen in 3 M Me2SO (64 to 74% fetuses) was not significantly different from nonfrozen controls (68 to 79% fetuses) or embryos frozen by a conventional slow cooling method (70%). Frozen thawed two-cell embryos developed into normal adults which were able to reproduce normally. We conclude that this freezing method can efficiently cryopreserve early cleavage stage mouse embryos.