Influence of euthanasia method on blood and gill variables in normoxic and hypoxic Gulf killifish Fundulus grandis.

In many experiments, euthanasia, or humane killing, of animals is necessary. Some methods of euthanasia cause death through cessation of respiratory or cardiovascular systems, causing oxygen levels of blood and tissues to drop. For experiments where the goal is to measure the effects of environmental low oxygen (hypoxia), the choice of euthanasia technique, therefore, may confound the results. This study examined the effects of four euthanasia methods commonly used in fish biology (overdose of MS-222, overdose of clove oil, rapid cooling and blunt trauma to the head) on variables known to be altered during hypoxia (haematocrit, plasma cortisol, blood lactate and blood glucose) or reflecting gill damage (trypan blue exclusion) and energetic status (ATP, ADP and ATP:ADP) in Gulf killifish Fundulus grandis after 24 h exposure to well-aerated conditions (normoxia, 7·93 mg O2 l-1 , c. 150 mm Hg or c. 20 kPa) or reduced oxygen levels (0·86 mg O2 l-1 , c. 17 mm Hg or c. 2·2 kPa). Regardless of oxygen treatment, fish euthanized by an overdose of MS-222 had higher haematocrit and lower gill ATP:ADP than fish euthanized by other methods. The effects of 24 h hypoxic exposure on these and other variables, however, were equivalent among methods of euthanasia (i.e. there were no significant interactions between euthanasia method and oxygen treatment). The choice of an appropriate euthanasia method, therefore, will depend upon the magnitude of the treatment effects (e.g. hypoxia) relative to potential artefacts caused by euthanasia on the variables of interest.

Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia.

Specific growth rate (G(S) ) and white skeletal muscle composition were measured in the mummichog Fundulus heteroclitus over a period of 28 days at four levels of dissolved oxygen (DO): severe hypoxia (c. 1.2 mg O(2) l(-1) ), moderate hypoxia (3.0 mg O(2) l(-1) ), normoxia (7.1 mg O(2) l(-1) ) and hyperoxia (10.6 mg O(2) l(-1) ). The G(S) was calculated over 0-8, 0-14, 0-28 and 14-28 days, and muscle protein, lactate dehydrogenase (LDH), DNA, RNA and water were measured at 0, 8, 14 and 28 days. Exposure of fish to severe hypoxia was associated with significantly reduced G(S) , lower muscle protein content and lower RNA:DNA compared with other DO treatments. When calculated over the first and second half of the 28 day exposure, however, G(S) of fish in severe hypoxia increased significantly during the second two-week interval, to the same rate as that of normoxic fish. Muscle LDH activity and water content were not significantly affected by DO level. Neither moderate hypoxia nor hyperoxia significantly affected G(S) or any biochemical variable. The results demonstrate that F. heteroclitus can tolerate wide variation in ambient oxygen concentration and, during prolonged exposure to severe hypoxia, shows significant compensation for the initial negative effects on growth. The capacity of F. heteroclitus to grow over a wide range of DO probably contributes to its ability to exploit habitats characterized by marked variation in oxygen availability.

Exercise- and hypoxia-induced anaerobic metabolism and recovery: a student laboratory exercise using teleost fish.

Anaerobic metabolism is recruited in vertebrates under conditions of intense exercise or lowered environmental oxygen availability (hypoxia), typically resulting in the accumulation of lactate in blood and tissues. Lactate will be cleared over time after the reoxygenation of tissues, eventually returning to control levels. Here, we present a laboratory exercise developed as part of an upper-level vertebrate physiology class that demonstrates the effects of exercise and hypoxia exposure on blood lactate in fish and the subsequent decrease in lactate during recovery. Typically, the results obtained by students demonstrate that both treatments cause significant increases in blood lactate concentrations (two to three times higher than control values) that decrease back to normal values within 3 h of recovery under normoxia. The procedures described are generally applicable to other fish species and provide an alternative to using humans or other mammalian species to investigate anaerobic metabolism.

Vertebrate osmoregulation: a student laboratory exercise using teleost fish.

Here, we describe a laboratory experiment as part of an upper-level vertebrate physiology course for biology majors to investigate the physiological response of vertebrates to osmoregulatory challenges. The experiment involves measuring plasma osmolality and Na+-K+-ATPase activity in gill tissue of teleost fish acclimated to water of differing salinity. We describe results obtained using the widely available goldfish (Carassius auratus) and a common baitfish, the Gulf killifish (Fundulus grandis). The procedures described are generally applicable to other fish species, and they provide an alternative to the experimental use of humans or other mammalian species to investigate osmoregulation mechanisms. In addition to reenforcing the conceptual material covered in lecture, this laboratory exercise trains students in a wide range of laboratory and analytical skills, such as calculating and performing dilutions, pipetting, tissue sampling and homogenizing, preparing standard curves, conducting enzymatic assays, and analyzing and interpreting results. Typical student results are presented and discussed, as are common experimental and conceptual mistakes made by students.

Structure and sequence conservation of a putative hypoxia response element in the lactate dehydrogenase-B gene of Fundulus.

Many aquatic habitats are characterized by periodic or sustained episodes of low oxygen concentration, or hypoxia, and organisms that survive in these habitats do so by utilizing a suite of behavioral, physiological and biochemical adjustments to low oxygen (1-3). In the killifish Fundulus heteroclitus, one response to prolonged exposure to hypoxia is an increase in the activity of lactate dehydrogenase-B (LDH-B), the terminal enzyme of anaerobic glycolysis, in liver tissue (4). An increase in glycolytic enzyme activity also occurs in mammalian cells during hypoxia, a process due, in part, to increased rates of gene transcription mediated by the hypoxia-inducible transcription factor, HIF-1 (5). Given that a homolog of HIF-1 has been identified in fish (6), we hypothesized that HIF might be involved in the observed up-regulation of LDH-B in F. heteroclitus. Herein, we describe the presence of DNA elements in intron 2 of the Ldh-B gene from F. heteroclitus that resemble hypoxia response elements (HRE) describedfor mammalian genes (7-10). Specifically, over a region of approximately 50 base pairs we identified two consensus HIF-1 binding sites, as well as DNA elements that may bind other transcription factors (e.g., cyclic AMP response elements; CRE). We found that these sites were perfectly conserved among geographically diverse populations of F. heteroclitus, as well as being highly conserved among multiple species in the genus Fundulus. The spacing, orientation, and sequence conservation of these putative regulatory elements suggest that they may be functionally involved in the hypoxic regulation of Ldh-B in these fish.

Acclimation to hypoxia increases survival time of zebrafish, Danio rerio, during lethal hypoxia.

Survivorship of zebrafish, Danio rerio, was measured during lethal hypoxic stress after pretreatment in water at either ambient oxygen or at a lowered, but nonlethal, level of oxygen. Acclimation to nonlethal hypoxia (pO(2) congruent with 15 Torr; ca. 10% air-saturation) for 48 hr significantly extended survival time during more severe hypoxia (pO(2) congruent with 8 Torr; ca. 5% air-saturation) compared to survival of individuals with no prior hypoxic exposure. The magnitude of the acclimation effect depended upon the sex of the fish: hypoxia pretreatment increased the survival times of males by a factor of approximately 9 and that of females by a factor of 3 relative to controls. In addition, survival time of control and hypoxia acclimated fish depended upon when in the year experiments were conducted. Survival times were 2-3 times longer when measured in the late fall or winter compared to survival times measured during the spring or summer. These results demonstrate a direct survival benefit of short-term acclimation to hypoxia in this genetically tractable fish. The fact that the acclimation effect depended upon the sex of the fish and the season during which experiments were conducted demonstrates that other genetic and/or environmental factors affect hypoxia tolerance in this species. J. Exp. Zool. 289:266-272, 2001.

Oxygen consumption, blood lactate and inter-individual variation in the gulf killifish, Fundulus grandis, during hypoxia and recovery.

Rates of oxygen consumption (M(O(2))) for Fundulus grandis, the gulf killifish, were measured in air-saturated water, at four progressively lower levels of oxygen and upon normoxic recovery. The pattern of M(O(2)) versus oxygen partial pressure (P(w)O(2)) was that of an oxygen regulator, with a critical oxygen pressure (P(c)) of 34 torr (1 torr=133.3 Pa). Below this value, M(O(2)) decreased and the concentration of blood lactate increased, indicating anaerobic metabolism during hypoxia. Recovery was characterized by elevated M(O(2)) compared to the initial normoxic exposure, coupled with the rapid clearance of blood lactate. Variation in M(O(2)) among the individual fish was appreciable and, in general, it was greater at higher levels of P(w)O(2). This inter-individual variation was significantly larger than the variation between replicate measures of M(O(2)) for a given individual, i.e. it cannot be attributed solely to random error. Furthermore, values for M(O(2)) during normoxia were found to be repeatable when the same fish were used in multiple experimental trials. The observation of significant, repeatable inter-individual variation in M(O(2)) suggests that such variation is a real and potentially important feature of fish metabolism.

Protein synthesis increases after fertilization of sea urchin eggs in the absence of an increase in intracellular pH.

We have reevaluated the presumed requirement for an elevated intracellular pH (pHi) in the acceleration of protein synthesis which follows fertilization of eggs of the sea urchin Lytechinus pictus. Zygotes were transferred to sea water at a low pH (6.8) containing a permeant weak acid at times ranging from 5 min to as early as 30 sec postinsemination, to reverse or prevent the rise in pHi that normally ensues upon fertilization. Using the fluorescent pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we show that transfer of zygotes at 1 min or earlier after fertilization essentially blocks the rise in pHi. Under these conditions, both the incorporation of radiolabeled leucine into protein and the assembly of ribosomes into polysomes increase substantially (> 50% of control values). We also assessed leucine incorporation during incubation of eggs and zygotes in sodium-free sea water or sea water containing amiloride, two additional treatments that block the pHi rise. In the presence of amiloride, leucine incorporation increased upon fertilization, whereas little or no increase was observed in sodium-free sea water. We provide evidence that the low rates of leucine incorporation in sodium-free sea water result from the tendency for this experimental condition to lower the pHi to values significantly lower than the pHi in unfertilized eggs. These findings call into doubt the belief that the pHi rise at fertilization is a necessary prerequisite for the acceleration of bulk protein synthesis. These observations support the view that pHi is only one of several signals involved in the turn on of protein synthesis at the time of fertilization of sea urchin eggs.

Biochemical Correlates of Estivation Tolerance in the Mountainsnail Oreohelix (Pulmonata: Oreohelicidae).

Biochemical changes occurring over 7 months of estivation were studied in two species of land snail, Oreohelix strigosa (Gould) and O. subrudis (Reeve), to determine whether differential mortality during estivation is related to different energetic strategies. Laboratory-maintained snails, which were fed ad libitum prior to estivation, were compared with snails collected from the field and induced to estivate without augmenting their energy reserves. In all groups, polysaccharide was catabolized early in estivation, and protein was the primary metabolic substrate after polysaccharide reserves were depleted. Lipid was catabolized at a low rate throughout estivation. Rates of catabolism were largely statistically equivalent between species. Urea and purine bases accumulated during estivation as a result of protein catabolism, with the former being quantitatively more important. In both laboratory-maintained and field-collected snails, the rate of urea accumulation was greater in O. subrudis, resulting in higher tissue urea contents in this species at the end of the 7-month experiment. The tissue concentrations of urea at 7 months ranged from about 150 to 300 mM and were positively correlated (r = 0.99, P = 0.006) with mortality in these snails. Methylamine compounds, a class of compounds that can offset disruptive effects of elevated urea, were measured in one group of O. strigosa at 7 months of estivation and found to be low relative to urea levels. We suggest, therefore, that in the absence of elevated levels of counteracting compounds, urea may reach toxic levels and may be one factor limiting the duration of estivation that is survived by these land snails.

Kinetic properties of hexokinase under near-physiological conditions. Relation to metabolic arrest in Artemia embryos during anoxia.

Previous analyses of glycolytic metabolites in Artemia embryos indicate that an acute inhibition of glucose phosphorylation occurs during pHi-mediated metabolic arrest under anoxia. We describe here kinetic features of hexokinase purified from brine shrimp embryos in an attempt to explain the molecular basis for this inhibition. At saturating concentrations of cosubstrate, ADP is an uncompetitive inhibitor toward glucose and a partial noncompetitive inhibitor toward ATP (Kis = 0.86 mM, Kii = 1.0 mM, Kid = 1.9 mM). With cosubstrates at subsaturating concentrations, the uncompetitive inhibition versus glucose becomes noncompetitive, while inhibition versus ATP remains partial noncompetitive. The partial noncompetitive inhibition of ADP versus ATP is characterized by a hyperbolic intercept replot. These product inhibition patterns are consistent with a random mechanism of enzyme action that follows the preferred order of glucose binding first and glucose-6-P dissociating last. We propose that inhibition by glucose-6-P (Kis = 65 microM) occurs primarily by competing with ATP at the active site, resulting in the formation of the dead-end complex, enzyme-glucose-glucose-6-P. Versus glucose, inhibition by glucose-6-P is uncompetitive at pH 8.0 and noncompetitive at pH 6.8. Over a physiologically relevant pH range of 8.0 to 6.8 alterations in Km and Ki values do not account for the reduction in glucose phosphorylation, and no evidence suggests that Artemia hexokinase activity is modulated by reversible binding to intracellular structures. Total aluminum in the embryos is 4.01 +/- 0.36 micrograms/g dry weight, or, based upon tissue hydration, 72 microM. This concentration of aluminum dramatically reduces enzyme activity at pH values less than 7.2, even in the presence of physiological metal ion chelators (citrate, phosphate). When pH, aluminum, citrate, phosphate, substrates, and products were maintained at cellular levels measured under anoxia, we can account for a 90% inhibition of hexokinase relative to activity under control (aerobic) conditions.

Thermal dependence of maximum Ca2+-activated force in skinned muscle fibres of the toad Bufo marinus acclimated at different temperatures.

Mechanically skinned muscle fibres from the twitch region of the iliofibularis muscle of cool- (16 +/- 1 degree C) and warm- (32 +/- 1 degree C) acclimated cane toads (Bufo marinus) were activated maximally by Ca2+ in solutions of different pH and at different temperatures (approx. 1-35 degrees C). Acclimation of up to 12 weeks at 16 degrees C and up to 8 weeks at 32 degrees C did not modify the marked thermal dependence of isometric force in the skeletal muscle fibres of the cane toad. The prominent decline of maximum Ca2+-activated force at lower temperatures, a property which is not characteristic of muscles from other anurans, was associated with an obvious decline in fibre stiffness at temperatures below about 20 degrees C, regardless of the temperatures at which the toads were kept prior to experimentation. The results suggest that the decline of isometric force at lower temperatures is due both to a reduction in the number of cross-bridges and to a decrease in the force output per cross-bridge. The maximum Ca2+-activated force response increased when fibres were activated in solutions of increasing pH at all temperatures investigated. This trend is expected to have a compensatory effect on the thermal dependence of the maximum Ca2+-activated force under physiological conditions, because of the elevation of intracellular pH as temperature declines. The isometric force did not depend on the concentration of the zwitterionic species of the pH buffer in solutions. The skinned fibre preparation developed a Ca2+-insensitive residual force following maximal activation. The increment in residual force followed a linear relationship with the duration of activation at a given temperature and a power relationship of activation temperature for a given duration of activation. Fibres from warm-acclimated animals developed less residual force following activations at 15 degrees C than did fibres from cool-acclimated animals, suggesting that thermal acclimation may substantially reduce the magnitude of this phenomenon at temperatures below 20 degrees C.