Bone and shell contribution to lactic acid buffering of submerged turtles Chrysemys picta bellii at 3 degrees C

To evaluate shell and bone buffering of lactic acid during acidosis at 3 degrees C, turtles were submerged in anoxic or aerated water and tested at intervals for blood acid-base status and plasma ions and for bone and shell percent water, percent ash, and concentrations of lactate, Ca(2+), Mg(2+), P(i), Na(+), and K(+). After 125 days, plasma lactate concentration rose from 1.6 +/- 0.2 mM (mean +/- SE) to 155.2 +/- 10.8 mM in the anoxic group but only to 25.2 +/- 6.4 mM in the aerated group. The acid-base state of the normoxic animals was stable after 25 days of submergence. Plasma calcium concentration (?Ca(2+)) rose during anoxia from 3.2 +/- 0.2 to 46.0 +/- 0.6 mM and ?Mg(2+) from 2.7 +/- 0.2 to 12.2 +/- 0.6 mM. Both shell and bone accumulated lactate to concentrations of 135.6 +/- 35.2 and 163.6 +/- 5.1 mmol/kg wet wt, respectively, after 125 days anoxia. Shell and bone ?Na(+) both fell during anoxia but the fate of this Na(+) is uncertain because plasma ?Na(+) also fell. No other shell ions changed significantly in concentration, although the concentrations of both bone calcium and bone potassium changed significantly. Control shell water (27.8 +/- 0.6%) was less than bone water (33.6 +/- 1.1%), but neither changed during submergence. Shell ash (44.7 +/- 0.8%) remained unchanged, but bone ash (41.0 +/- 1.0%) fell significantly. We conclude that bone, as well as shell, accumulate lactate when plasma lactate is elevated, and that both export sodium carbonate, as well as calcium and magnesium carbonates, to supplement ECF buffering.     

Avenues of extrapulmonary oxygen uptake in western painted turtles (Chrysemys picta belli) at 10 degrees C

The major avenues of extrapulmonary oxygen uptake were determined on submerged western painted turtles (Chrysemys picta bellii) at 10 degrees C by selectively blocking one or more potential pathways for exchange. Previous work indicated that the skin, the cloaca, and the buccopharyngeal cavity can all contribute significantly in various species of turtles. O(2) uptake was calculated from the rate of fall in water P(O(2)) in a closed chamber. Two series of experiments were conducted: in Series 1, each of the potential avenues was mechanically blocked either singly or in combination; in Series 2, active cloacal and buccal pumping were prevented pharmacologically using the paralytic agent rocuronium. In addition in Series 2, N(2)-breathing preceded submergence in some animals and in one set of Series 2 experiments arterial blood was sampled and analyzed for pH, lactate, P(O(2)), and P(CO(2)). Results in both Series 1 and Series 2 revealed that prevention of cloacal and/or buccopharyngeal exchange did not significantly affect total O(2) uptake. Interfering with skin diffusion in Series 1, however, significantly reduced O(2) uptake by 50%. N(2)-breathing prior to submergence in Series 2 did not affect O(2) uptake in paralyzed turtles but significantly increased uptake in unparalyzed turtles without catheters. Blood analysis revealed that all submerged turtles developed lactic acidosis, but the rate of rise in lactate was significantly lower in paralyzed animals. We conclude that passive diffusion through the integument is the principal avenue of aquatic O(2) uptake in this species.     

Effects of experimental anemia on blood ion and acid-base status of turtles during submergence in aerated water at 3 degrees C

The importance of blood hemoglobin to aquatic oxygen uptake by turtles (Chrysemys picta bellii) submerged in aerated water at 3 degrees C was tested by comparing the responses of anemic turtles (hematocrit approximately 6%) to turtles with normal hematocrits (hematocrit approximately 33%). All turtles were submerged for 42 days and blood samples were collected at 0, 7, 21, 32 and 42 days. Blood was analyzed for pH, PCO(2), PO(2), hematocrit, hemoglobin concentration ([Hb]) and plasma was analyzed for concentrations of lactate, glucose, Na(+), K(+), Ca(2+) and Mg(2+). Plasma [HCO(3)(-)] was calculated. [Hb] correlated closely with hematocrit levels. [Lactate] reached higher final values in anemic turtles (34.5+/-5.3 mmol l(-1)) than in normal turtles (14.5+/-4.6 mmol l(-1)) indicating a greater reliance of the anemic animals on anaerobic metabolism. Both groups compensated for acidosis by reduced PCO(2) and anemic turtles also had increased [Ca(2+)] and [Mg(2+)]. Blood pH fell significantly in the anemic turtles but not in the controls. Although the data indicate that the anemic turtles relied more on anaerobic metabolism than the controls, the effect was much less than expected on the basis of the reduced blood O(2) carrying capacity. Possible compensatory mechanisms utilized by the anemic turtles to minimize anaerobic metabolism are discussed.     

The effect of freezing and storage at -60 degrees C on the viability of Mycobacterium leprae

Suspensions of M. leprae in 10% glycerol and 0.1% bovine serum albumin were quick-frozen and stored for various periods of time at −60 °C. Viability of the bacterial suspensions, measured by means of Shepard's mouse footpad technique, did not decrease progressively during prolonged storage, suggesting that the observed decreases in viability resulted from the freezing process rather than from storage. Considerable variability was noted in the loss of viability which resulted from freezing.     

Evaluation of viability and apoptosis in horse embryos stored under different conditions at 5 degrees C

The aim of this study was to evaluate the viability (percentage of dead cells) and the incidence of DNA fragmentation of horse embryos after storage in three different media at 5 degrees C for 6 and 24 h. Forty embryos were stored in Emcare Holding Solution for 6 and 24 h, in Hams'F10 or Vigro Holding Plus for 24 h at 5 degrees C (n = 9-10 per group) and 10 embryos were evaluated immediately after collection. First, embryos were stained, immediately after collection or following storage, to detect dead cells (DAPI) and, subsequently, DAPI-stained embryos were fixed and stained to detect DNA fragmentation (TUNEL). Finally, all the fixed embryos were re-stained with DAPI to determine the total number of cells. The percentage of cells stained with both TUNEL and DAPI or TUNEL-only or DAPI-only were determined. The percent of dead cells (DAPI-labelled) per embryo increased with duration of storage, but no differences were detected between the storage media. The percentage of early apoptotic cells (TUNEL+/DAPI-) in fresh and stored embryo for 6 h or 24 h did not differ significantly (P > 0.05). There was a significant correlation between the percentage of cells labelled by TUNEL and DAPI (R = 0.87) (P < 0.001). These results suggest that cooled storage increases cell death but this does not appear to occur by induction of apoptosis and that DAPI staining proves to be a quick and reliable method for assessing embryo viability.     

Hemoprotozoa of freshwater turtles in Queensland

Blood smears from 27 turtles (15 Emydura signata, nine Elseya latisternum, and three Chelodina longicollis) from southeastern Queensland (Australia) were examined for infections by hemoprotozoan parasites between January and June 1999. Infections were found in 26 (96%) of the turtles. Twenty five (93%) were infected with the adeleorin coccidian Haemogregarina clelandi, eight (30%) with the hemosporidian Haemoproteus chelodinae, 11 (41%) with the kinetoplastid flagellate Trypanosoma chelodinae, and eight (30%) with a novel Trypanosoma sp. Despite the high prevalence and intensity of infections, there was no evidence of clinical disease in any of the turtles.     

Nucleotide binding by myosin in glycerol-extracted rabbit skeletal muscle fibres at temperatures between -35 degrees C and 10 degrees C

Glycerol-extracted rabbit psoas fibres were incubated at temperatures between -35 degrees C and +10 degrees C in a low-ionic-strength relaxing solution containing 50% ethyleneglycol, 100 microM [3H]MgATP, 1 mM [14C]mannitol and less than 0.01 microM Ca2+. The fibres were then rinsed in a solution containing 1 mM ATP and the bound nucleotide eluted in trichloroacetic acid; all these operations were carried out at the cold temperature. Residual bound nucleotide was eluted with trichloroacetic acid at room temperature. The fibres were found to bind approximately 180 microM nucleotide, which is consistent with binding to the enzymatic site of myosin. The eluate, obtained in the cold, was analysed on poly(ethyleneimine)-cellulose for its ATP and ADP content. At temperatures down to -22 degrees C most of the bound nucleotide was ADP and there was little variation of this fraction with temperature. As the temperature was lowered below -22 degrees C the ATP fraction rose sharply; by -35 degrees C it predominated. These results are similar in type to those found by Biosca et al. [(1984) Biochemistry 23, 1947-1953] on isolated subfragment 1, but are displaced to a much lower temperature range. Thus in a muscle fibre only a low thermal energy is needed for myosin to hold its nucleotide in a constant balance between ATP and ADP.     

The effect of abrupt changes in sodium chloride concentration upon the viability of gram-negative bacteria at 2 and 40 degrees C

“Osmotic” shock between 0.14 m and 4.5 m NaCl is highly temperature dependent for postlog populations of E. coli, A. aerogenes, and P. aeruginosa, suggesting that killing is not due solely to simple physical osmotic action. Thermal shock in postlog E. coli and A. aerogenes does not occur, although in P. aeruginosa populations when temperature fluctuations are repetitious appreciable loss of viability does occur. Thermal shock concomitant with osmotic shock, on the other hand, is lethal to postlog populations of all 3 species. Thus, during freezing-thawing concentrated solute is a prime candidate for causing death in postlog E. coli, A. aerogenes, and P. aeruginosa populations.     

Fluid storage of thrombocytes. In vitro viability of thrombocytes at 12 degrees C storage temperature

Platelet concentrates from ACD-blood were stored with and without agitation at +12 degrees C for 3 days. pH-values, hypotonic shock response, serotonin uptake and adenine nucleotides were investigated. In addition platelet shape was morphologically differentiated in discs with and without pseudopods, in spheres and irregular platelets. In accordance with platelet storage at +22 degrees C an optimum platelet number and gentle agitation were essential to maintain in vitro vitality at +12 degrees C for 3 days. The number of discoid platelets declined from 76% in fresh platelet concentrates to 25% after 3 days at +12 degrees C although pH-values did not fall below 6.8 in the agitated concentrates. A diminished post-transfusion survival could result from this, unless the shape change reverses in the circulation.