In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN₂) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN₂ has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.     

Effect of radiation on skin expansion and skin flap viability in pigs

The aim of the present study was to investigate the effect of radiation treatment both on skin tissue expansion with the chronic inflation of subcutaneous expanders and on skin flap viability in surgically delayed and expanded skin in the pig. One flank in each of six pigs (initially weighing 17 +/- 1.8 kg) was randomly assigned for radiation treatment, and the contralateral flank served as a nonirradiated control. Three mirror-image, 8 x 10 cm, rectangular templates were marked on each flank; these templates were randomly assigned to the construction of a delayed skin flap (group A), a skin flap raised on expanded skin (group B), or a skin flap raised on expanded skin with a capsulectomy before flap surgery (group C). Radiation treatment was performed using sequential radiation with three fractions per week (810 cGy/fraction) for 2 weeks, with a total dose of 4,860 cGy. Twelve weeks after radiation treatment, skin expanders (8 x 10 cm) were installed subcutaneously in the locations assigned for skin expansion. Skin expansion by the inflation of subcutaneous skin expanders with saline twice weekly was started 8 weeks later and lasted for 3 weeks. Two weeks after surgical delay and the last skin expansion, 8 x 20 cm skin flaps were raised on the locations assigned for delayed skin flaps, expanded skin flaps, and expanded skin flaps with a capsulectomy. Skin flap viability was assessed 24 hours later using a fluorescein dye-staining technique. Skin expansion by the inflation of subcutaneous expanders with saline was slower (p < 0.05) in the radiated skin (39 +/- 6 ml/filling) than in the nonirradiated control skin (51 +/- 6 ml/filling). Radiation reduced the overall area of expanded skin by 23 percent (p < 0.05) compared with the control. Radiation treatment also reduced skin viability by 36 percent (p < 0.05) in the delayed skin flaps, 27 percent (p = 0.10) in the expanded skin flaps, and 36 percent (p < 0.05) in the expanded skin flaps with a capsulectomy when compared with their contralateral, nonirradiated controls. There were no significant differences in skin viability among these three types of skin flaps within the radiated and nonirradiated groups. Taken together, these observations indicate that radiation treatment reduced the effectiveness of the surgical delay procedure, the amount of subcutaneous skin expansion (by an increase in skin area), and skin flap viability. However, a capsulectomy alone did not affect the viability of skin flaps raised on expanded skin.     

Effect of oxygen on viability and substrate utilization in Chromatium

Chromatium D can be exposed to oxygen for prolonged periods without any loss in motility or viability. Oxygen did not affect the rate of thiosulfate disappearance from the media, the oxidation of the inner sulfur atom of thiosulfate to sulfate, or the conversion of the outer sulfur atom of thiosulfate to intracellular sulfur, but it did inhibit the oxidation of intracellular sulfur to sulfate. Oxygen partially inhibited the uptake of pyruvate from the medium, but had little effect on the uptake of acetate. The distribution of label from pyruvate-2-¹⁴C into various cell fractions under aerobic conditions differed only slightly from that obtained under anaerobic conditions. Cells utilizing acetate-2-¹⁴C aerobically converted the majority of the metabolized acetate into a cell fraction with the solubility characteristics of poly-β-hydroxybutyric acid, whereas under anaerobic conditions the acetate was distributed throughout the other cell fractions. Oxygen completely prevented the synthesis of bacteriochlorophyll.     

Effect of anthracyclines and mitoxantrone on oxygen uptake and ATP intracellular concentration in rat heart slices

The clinical use of anthracycline antibiotics is severely limited by dose-dependent cardiotoxicity. This accounts for our interest in differences on respiratory control of cardiac cells induced by two new anticancer agents epirubicin and mitoxantrone. Cellular oxygen uptake was measured for increasing drugs concentrations with a Warburg manometric apparatus and intracellular ATP by high-pressure liquid chromatography in rat heart slices incubated for 60′ with anthracyclines or mitoxantrone. Epirubicin inhibits endogenous respiration by 18% versus control while doxorubicin and mitoxantrone reduce oxygen uptake by 34 and 46%, respectively. ATP intracellular concentration was significantly reduced by all anticancer agents but particularly by mitoxantrone 86%.These results may be related to the biochemical side effects produced by these drugs on bioenergetics and cellular respiratory control.     

Effect of potential-dependent potassium uptake on production of reactive oxygen species in rat brain mitochondria

The effect of potential-dependent potassium uptake on reactive oxygen species (ROS) generation in mitochondria of rat brain was studied. It was found that the effect of K⁺ uptake on ROS production in the brain mitochondria under steady-state conditions (state 4) was determined by potassium-dependent changes in the membrane potential of the mitochondria (ΔΨ_m). At K⁺ concentrations within the range of 0–120 mM, an increase in the initial rate of K⁺-uptake into the matrix resulted in a decrease in the steady-state rate of ROS generation due to the K⁺-induced depolarization of the mitochondrial membrane. The selective blockage of the ATP-dependent potassium channel (K _ATP ⁺ -channel) by glibenclamide and 5-hydroxydecanoate resulted in an increase in ROS production due to the membrane repolarization caused by partial inhibition of the potential-dependent K⁺ uptake. The ATP-dependent transport of K⁺ was shown to be ～40% of the potential-dependent K⁺ uptake in the brain mitochondria. Based on the findings of the experiments, the potential-dependent transport of K⁺ was concluded to be a physiologically important regulator of ROS generation in the brain mitochondria and that the functional activity of the native K _ATP ⁺ -channel in these organelles under physiological conditions can be an effective tool for preventing ROS overproduction in brain neurons.     

Effect of experimental ventilation of the skin on cutaneous oxygen uptake in the carp,Cyprinus carpio

• 1.1. Cutaneous O₂ uptake in the carp, Cyprinus carpio, was determined at various water flow rates across the skin (.V) ranging from 2.5 to 40 ml/min, using flow-through respirometers.
• 2.2. When thickness of water flow was 2mm, cutaneous O₂ uptake remained stable (about 3.8 nmol/cm²/min) at a .V of 20–40 ml/min and decreased with .V below 20 ml/min.
• 3.3. When thickness of water flow was 4 mm, cutaneous O₂ uptake decreased with .V below 40 ml/min.
• 4.4. Apparent water velocity (U') was calculated dividing .V by an area of a cross section of the water flow (0.5 and 1.0 cm² respectively). In both experiments, cutaneous O₂ uptake decreased with U' below 0.7 cm/sec.
• 5.5. This suggests that cutaneous O₂ uptake in the carp is limited at a low water velocity by a resistance of the hypoxic boundary layer.

     

On-line oxygen uptake rate and culture viability measurement of animal cell culture using microplates with integrated oxygen sensors

O2 uptake rates of animal cells (Chinese hamster ovary-CHO) were measured in 96-well microtiter plates by integrating with fluorescent sensors thereby measuring fluorescence intensity ratios of an O2-sensitive and an insensitive fluorophor. O2 consumption rate was estimated from measured dissolved O2 and from O2 mass transfer coefficient determined in advance. Specific uptake decreased with time from 3.2 x 10(-13) mol O2 cell(-1) h(-1) at 15 h cultivation to 1.8 x 10(-13) mol O2 cell(-1) h(-1) at 48 h. Specific O2 uptake was also determined by sampling from a spinner-flask culture giving identical values. A cell viability assay for cultures based on O2 measurements is described in which cells are incubated outside the fluorescence reader and then the dissolved O2 is measured only once at a fixed time after the start of incubation. This protocol can be directly applied for high-throughput measurements.     

Effect of age and sex on oxygen uptake by adult mosquitoes

Summary Newly emerged mosquitoes show a low respiratory rate which increases during the first two days of adult life. On the fourth day a temporary decrease occurs, after which there is no correlation between age and oxygen uptake. Males show a higher metabolic rate (calculated per mg body weight) than females at all temperatures studied.

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Résumé Le coefficient respiratoire des moustiques qui viennent d'émerger est apparemment assez bas; il augmente pendant les deux premières, journées de leur vie adulte. Dans la quatrième journée une diminution temporaire a lieu, après laquelle on ne décèle plus de corrélation entre l'âge et la consommation d'oxygène.Le coefficient métabolique des mâles (calculé en mg/poids de l'insecte) est plus élevé que celui des femelles à toutes les températures étudiées.

     

Effect of helium and argon on the oxygen uptake by lymphocytes

The effect of inert helium and argon gases on the tissue respiration has been studied on lymphocyte suspensions of white rats. It is shown that normoxic helium-oxygen mixture induces almost a two-fold increase of the O2 uptake by lymphocytes as compared with the control (air). No deviations in the value of the studied parameter are revealed in case of replacement of nitrogen from air by argon. Significance of the membrane structure in realization of effects of inert gases is under discussion.     

Dependence of oxygen uptake on ambient PO 2 in isolated perfused frog skin

Summary Rates of O₂ uptake across isolated perfused skin of bullfrogs (Rana catesbeiana) were measured in relation to blood flow at three levels of ambient O₂ tension: normoxia (O₂ tension=152 torr), hypoxia (12% O₂, 87 torr) and hyperoxia (42% O₂, 306 torr). At bulk perfusion rates ranging from 3.4 to 10.1 l·cm^-2·min^-1, O₂ uptake was positively correlated with hemoglobin delivery rate in both normoxia and hyperoxia, but was independent of delivery rate in hypoxia. Mean O₂ uptake in normoxia was 3.8 nmol O₂·cm^-2·min^-1 at a delivery rate of 9.8 nmol·cm^-2·min^-1 and 6.5 nmol O₂·cm^-2·min^-1 at a delivery rate of 28.3 nmol·cm^-2·min^-1. At any given bulk perfusion rate, oxygen uptake averaged about 49% lower in hypoxia than in normoxia, decreasing in proportion to the reduction of O₂ tension difference between medium and blood. In hyperoxia, O₂ uptake did not increase proportionally with the difference in O₂ tension between blood and medium, averaging only 50% higher at a 2.4-fold greater O₂ tension difference. Cutaneous diffusing capacity for O₂ averaged 0.041 nmol O₂·cm^-2·torr^-1·min^-1 during the first hour of perfusion in normoxia, and was not affected by reduction of ambient O₂ tension. The results indicate that cutaneous O₂ uptake in hypoxia is highly diffusion limited, and consequently, increases in cutaneous perfusion can not effectively compensate for reduction of ambient O₂ tension. In hyperoxia, O₂ uptake may be substantially perfusion limited because of reduced blood O₂ capacitance at high O₂ saturations.Abbreviations O₂ capacitance - C _Hb hemoglobin concentration - D diffusing capacity - PO₂ medium-blood PO₂ difference - Hb flow, hemoglobin delivery rate - Hepes N-[2-Hydroxyethyl]piperacine-N-[2 ethanesulfonic acid] - L _diff extent of diffusion limitation - MO₂ oxygen uptake rate - PO₂ oxygen tension - S O₂ saturation     

Effect of capsulectomy on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin in the pig

Skin flaps constructed on expanded skin usually include the underlying capsular tissue. It has been hypothesized that capsulectomy may jeopardize the viability of the expanded skin flap. The experiments reported herein were designed to test this hypothesis. Specifically, we studied the hemodynamics and viability of random-pattern skin flaps (8 X 20 cm) raised on delayed bipedicle flaps (group A) and on expanded skin pockets with capsulectomy at the time of flap elevation (group B) or with intact underlying capsular tissue (group C). Each group was randomly assigned to each flank in 16 pigs. Skin pockets were expanded by inflation of subcutaneous silicone tissue expanders with sterile saline (299 +/- 7 ml; X +/- SEM) over a period of 3 weeks. At the end of this period, the bipedicle flaps were constructed. Eight days later, random-pattern skin flaps were raised on bipedicle flaps and skin pockets. The length and area of skin flap viability, judged by the fluorescein dye test performed 1 day postoperatively, were not significantly different (p greater than 0.05) among groups A, B, and C (n = 31 to 32). There also were no significant differences (p greater than 0.05) in total skin capillary blood flow measured 1 day postoperatively (A = 2.6 +/- 0.4, B = 2.4 +/- 0.4, and C = 2.7 +/- 0.6 ml/min per flap; n = 15 to 16) and in skin viability assessed 7 days postoperatively (A = 74 +/- 2, B = 75 +/- 2, and C = 76 +/- 2 percent; n = 16) among delayed skin flaps and skin flaps raised on expanded skin pockets with or without capsulectomy. The results of this flap viability study were confirmed in 5 minipigs in a separate experiment. We conclude that capsulectomy did not have a detrimental effect on the hemodynamics and viability of random-pattern skin flaps raised on expanded skin. Furthermore, we hypothesize that skin flaps raised on expanded skin are similar to delayed skin flaps in that the skin blood flow is optimally augmented; therefore, the capsular tissue does not add significant blood supply to the overlying skin.