Temperature and adrenoceptors in the frog heart

1. Cardiac adrenergic receptors in a frog, Rana tigrina, were examined in winter and summer months using isolated atria preparation maintained at 24 degrees, 14 degrees and 6 degrees C. Treatments included an examination of the atrial responses to selective alpha and beta adrenergic agonists (phenylephrine and isoproterenol respectively) and antagonists (phentolamine and propranolol). 2. Basal atrial beating rates differed between summer and winter months and increased with temperature. 3. Phenylephrine produced dose-dependent increases in the atrial beating rate and tension in the winter frogs only at 6 degrees C. These increases were blunted by phentolamine. 4. Isoproterenol produced positive chronotropic effects of 14 degrees and 24 degrees C but not at 6 degrees C in both summer and winter frogs; these effects were abolished by propranolol. Further, at 6 degrees C, the contractile response of the atrial tissue to isoproterenol was very sensitive. 5. Data suggests that the alpha adrenoceptor might be physiologically important to the frog in the low temperature environment of the cold season, during which period the cardiac beta adrenergic activity would be minimal or even absent.     

The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures

Membrane fluidity in whole cells of Saccharomyces cerevisiae W303-1A was estimated from fluorescence polarization measurements using the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, over a wide range of temperatures (6-35 degrees C) and at seven levels of osmotic pressure between 1.38 MPa and 133.1 MPa. An increase in phase transition temperatures was observed with increasing osmotic pressure. At 1.38 MPa, a phase transition temperature of 12 +/- 2 degrees C was observed, which increased to 17 +/- 4 degrees C at 43.7 MPa, 21+/- 7 degrees C at 61.8 MPa, and 24 +/- 9 degrees C at an osmotic pressure of 133.1 MPa. From these results we infer that, with increases in osmotic pressure, the change in phospholipid conformation occurs over a larger temperature range. These results allow the representation of membrane fluidity as a function of temperature and osmotic pressure. Osmotic shocks were applied at two levels of osmotic pressure and at nine temperatures, in order to relate membrane conformation to cell viability.     

Activity and stability of a neutral protease from Vibrio sp. (vimelysin) in a pressure-temperature gradient.

The apparent second-order rate constant of hydrolysis of Fua-Gly-LeuNH2 by vimelysin, a neutral protease from Vibrio sp. T1800, was measured in a variable pressure-temperature gradient (0. 1-400 MPa and 5-40 degrees C). The apparent maximum rate was observed at approximately 15 degrees C and 150-200 MPa; the pressure-activation ratio (kcat/Km(max)/kcat/Km(0.1 MPa)) was reached about sevenfold. The pressure dependence of the kcat and Km parameters at constant temperature (25 degrees C) revealed that the pressure-activation below 200 MPa was mainly caused by a change in the kcat parameter. The change in the intrinsic fluorescence intensity of vimelysin was also measured in a pressure-temperature plane (0.1-400 MPa and -20 to +60 degrees C). The fluorescence intensity was found to decrease by increasing pressure and temperature, and the isointensity contours were more or less circular. The tangential lines to the contours at high temperatures and low to medium pressures seem to have slightly positive slopes, which was reflected by the higher residual activities left after incubations at higher temperatures and medium pressure (200 MPa and 50 degrees C) and by the almost intact secondary structure left after 1 h of incubation at 200 MPa and 40 degrees C, as studied by circular dichroism. These results were compared with the corresponding results for thermolysin, a moderately thermostable protease from Bacillus thermoproteolyticus. Apparent differences that might be related to the temperature adaptations of the respective source microbes are also discussed.     

High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures

High hydrostatic pressure is a new technology in the food processing industry, and is used for cold pasteurization of food products. However, the pressure inactivation of food-borne microorganisms requires very high pressures (generally more than 400 MPa) and long pressure holding times (5 min or more). Carrying out pressure processing at low temperatures without freezing can reduce these parameters, which presently limit the application of this technology, in keeping the quality of fresh raw product. The yeast, Saccharomyces cerevisiae and the bacterium, Lactobacillus plantarum were pressurized for 10 min at temperatures between -20 and 25 degrees C and pressure between 100 and 350 MPa. Pressurization at subzero temperatures without freezing significantly enhanced the effect of pressure. For example, at a pressure of 150 MPa, the decrease in temperature from ambient to -20 degrees C allowed an increase in the pressure-induced inactivation from less than 1 log up to 7-8 log for each microorganism studied. However, for comparable inactivation levels, the kinetics of microorganism inactivation did not differ, which suggests identical inactivation mechanisms. Implications of water thermodynamical properties like compression, protein denaturation, as well as membrane phase transitions, are discussed.     

Tension responses to increased hydrostatic pressure in glycerinated rabbit psoas muscle fibres

A method developed to study the effect of increased hydrostatic pressure on the isometric tension of a single muscle fibre is described and experiments done at room temperature (18-22 degrees C) on glycerinated rabbit psoas muscle fibres are presented. Increase of pressure (range 1-10 MPa) caused little change in tension transducer response when a muscle fibre was relaxed. However, there was a reversible depression of isometric tension with an increase of pressure when a fibre was maximally calcium-activated or in rigor; the depression was around 15% for active tension and 30% for rigor tension, for an increase of pressure of 10 MPa (ca. 100 atm).     

Low temperature preservation and space medicine

Cold maintenance may be an option for compromised space-borne astronauts. Contemporary aneurysm surgery can involve cooling below 20 degrees C for nearly one hour. Dogs and baboons have survived blood-substituted hypothermia for 1-3 hours. Hamsters have recovered from partial-freezing below -1 degree C, and supercooling at -5 degrees C. Laboratory frogs have survived partial-freezing from -9 degrees C, while in nature, frogs may overwinter in these states. While some invertebrates can tolerate freezing to cryogenic temperatures, no vertebrate has survived complete freezing. The following studies (hypothermia and sub-zero experiments) were conducted to explore low temperature preservation of rodents, dogs and baboons.     

Coupling effects of osmotic pressure and temperature on the viability of Saccharomyces cerevisiae

The osmotic tolerance of cells of Saccharomyces cerevisiae as a function of glycerol concentration and temperature has been investigated. Results show that under isothermal conditions (25 degrees C) cells are resistant (94% viability) to hyperosmotic treatment at 49.2 MPa. A thigher osmotic pressure, cell viability decreases to 25% at 99 MPa. Yeast resistance to high osmotic stress (99 Mpa) is enhanced at low temperatures (5-11 degrees C). Therefore, the temperature at which hyperosmotic pressure is achieved greatly affects cell viability. These results suggest that temperature control is a suitable means of enhancing cell survival in response to osmotic dehydration.     

Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.     

Synergistic and antagonistic effects of combined subzero temperature and high pressure on inactivation of Escherichia coli

The combined effects of subzero temperature and high pressure on the inactivation of Escherichia coli K12TG1 were investigated. Cells of this bacterial strain were exposed to high pressure (50 to 450 MPa, 10-min holding time) at two temperatures (-20 degrees C without freezing and 25 degrees C) and three water activity levels (a(w)) (0.850, 0.992, and ca. 1.000) achieved with the addition of glycerol. There was a synergistic interaction between subzero temperature and high pressure in their effects on microbial inactivation. Indeed, to achieve the same inactivation rate, the pressures required at -20 degrees C (in the liquid state) were more than 100 MPa less than those required at 25 degrees C, at pressures in the range of 100 to 300 MPa with an a(w) of 0.992. However, at pressures greater than 300 MPa, this trend was reversed, and subzero temperature counteracted the inactivation effect of pressure. When the amount of water in the bacterial suspension was increased, the synergistic effect was enhanced. Conversely, when the a(w) was decreased by the addition of solute to the bacterial suspension, the baroprotective effect of subzero temperature increased sharply. These results support the argument that water compression is involved in the antimicrobial effect of high pressure. From a thermodynamic point of view, the mechanical energy transferred to the cell during the pressure treatment can be characterized by the change in volume of the system. The amount of mechanical energy transferred to the cell system is strongly related to cell compressibility, which depends on the water quantity in the cytoplasm.     

In vitro reactivity of ventral aorta to acetylcholine and noradrenaline in yellow freshwater eel (Anguilla anguilla L.) acclimatized to 10.1 MPa hydrostatic pressure

We examined in vitro vascular reactivity of eels previously acclimatized to 10.1 MPa hydrostatic pressure (HP) for 21 days. The isometric tension developed by ventral aortic rings was measured at atmospheric pressure. Dose-response curves for either acetylcholine (ACh) or noradrenaline (NA), as well as contractions evoked by 80 mM K+, were compared with time-matched experiments conducted on rings obtained from control eels. Results showed that neither the optimal tension nor the maximal force of the K+-evoked contraction were significantly modified, suggesting that acclimatization to high HP did not change the vascular smooth muscle contractile machinery. The dose-response curve to ACh was not significantly changed. Conversely, although NA always relaxed aortic rings, the response of acclimatized eels was significantly reduced over the entire range of the agonist concentration tested (10(-8) to 10(-3) M), except for the lowest one (10(-9) M). The maximal amplitude of the NA-induced relaxation was significantly reduced in aortic rings from acclimatized eels as compared with non-acclimatized samples (339.3 +/- 86.5 vs. 744.3 +/- 72.1 mg x mg(-1) dry weight, P < 0.005). Our results suggest that acclimatization to high HP could selectively alter the control of vascular tone by catecholamines.     

Metabolic depression induced by urea in organs of the wood frog, Rana sylvatica: effects of season and temperature

It has long been suspected that urea accumulation plays a key role in the induction or maintenance of metabolic suppression during extended dormancy in animals from diverse taxa. However, little evidence supporting that hypothesis in living systems exists. We measured aerobic metabolism of isolated organs from the wood frog (Rana sylvatica) in the presence or absence of elevated urea at various temperatures using frogs acclimatized to different seasons. The depressive effect of urea on metabolism was not consistent across organs, seasons, or temperatures. None of the organs from summer frogs, which were tested at 20 degrees C, or from winter frogs tested at 4 degrees C were affected by urea treatment. However, liver, stomach, and heart from spring frogs tested at 4 degrees C had significantly lower metabolic rates when treated with urea as compared with control samples. Additionally, when organs from winter frogs were tested at 10 degrees C, metabolism was significantly decreased in urea-treated liver and stomach by approximately 15% and in urea-treated skeletal muscle by approximately 50%. Our results suggest that the presence of urea depresses the metabolism of living organs, and thereby reduces energy expenditure, but its effect varies with temperature and seasonal acclimatization. The impact of our findings may be wide ranging owing to the number of diverse organisms that accumulate urea during dormancy. J. Exp. Zool. 309A:111-116, 2008. (c) 2008 Wiley-Liss, Inc.     

Thermal stress and Ca-independent contractile activation in mammalian skeletal muscle fibers at high temperatures.

Temperature dependence of the isometric tension was examined in chemically skinned, glycerinated, rabbit Psoas, muscle fibers immersed in relaxing solution (pH approximately 7.1 at 20 degrees C, pCa approximately 8, ionic strength 200 mM); the average rate of heating/cooling was 0.5-1 degree C/s. The resting tension increased reversibly with temperature (5-42 degrees C); the tension increase was slight in warming to approximately 25 degrees C (a linear thermal contraction, -alpha, of approximately 0.1%/degree C) but became more pronounced above approximately 30 degrees C (similar behavior was seen in intact rat muscle fibers). The extra tension rise at the high temperatures was depressed in acidic pH and in the presence of 10 mM inorganic phosphate; it was absent in rigor fibers in which the tension decreased with heating (a linear thermal expansion, alpha, of approximately 4 x 10(-5)/degree C). Below approximately 20 degrees C, the tension response after a approximately 1% length increase (complete < 0.5 ms) consisted of a fast decay (approximately 150.s-1 at 20 degrees C) and a slow decay (approximately 10.s-1) of tension. The rate of fast decay increased with temperature (Q10 approximately 2.4); at 35-40 degrees C, it was approximately 800.s-1, and it was followed by a delayed tension rise (stretch-activation) at 30-40.s-1. The linear rise of passive tension in warming to approximately 25 degrees C may be due to increase of thermal stress in titin (connectin)-myosin composite filament, whereas the extra tension above approximately 30 degrees C may arise from cycling cross-bridges; based on previous findings from regulated actomyosin in solution (Fuchs, 1975), it is suggested that heating reversibly inactivates the troponin-tropomyosin control mechanism and leads to Ca-independent thin filament activation at high temperatures. Additionally, we propose that the heating-induced increase of endo-sarcomeric stress within titin-myosin composite filament makes the cross-bridge mechanism stretch-sensitive at high temperatures.     

Effects of caffeine at different temperatures on contractile properties of slow-twitch and fast-twitch rat muscles

The slow-twitch soleus muscle (SOL) exhibits decreased twitch tension (cold depression) in response to a decreased temperature, whereas the fast-twitch extensor digitorum longus (EDL) muscle shows enhanced twitch tension (cold potentiation). On the other hand, the slow-twitch SOL muscle is more sensitive to twitch potentiation and contractures evoked by caffeine than the fast-twitch EDL muscle. In order to reveal the effects of these counteracting conditions (temperature and caffeine), we have studied the combined effects of temperature changes on the potentiation effects of caffeine in modulating muscle contractions and contractures in both muscles. Isolated muscles, bathed in a Tyrode solution containing 0.1-60 mM caffeine, were stimulated directly and isometric single twitches, fused tetanic contractions and contractures were recorded at 35 degrees C and 20 degrees C. Our results showed that twitches and tetani of both SOL and EDL were potentiated and prolonged in the presence of 0.3-10 mM caffeine. Despite the cold depression, the extent of potentiation of the twitch tension by caffeine in the SOL muscle at 20 degrees C was by 10-15 % higher than that at 35 degrees C, while no significant difference was noted in the EDL muscle between both temperatures. Since the increase of twitch tension was significantly higher than potentiation of tetani in both muscles, the twitch-tetanus ratio was enhanced. Higher concentrations of caffeine induced contractures in both muscles; the contracture threshold was, however, lower in the SOL than in the EDL muscle at both temperatures. Furthermore, the maximal tension was achieved at lower caffeine concentrations in the SOL muscle at both 35 degrees C and 20 degrees C compared to the EDL muscle. These effects of caffeine were rapidly and completely reversed in both muscles when the test solution was replaced by the Tyrode solution. The results have indicated that the potentiation effect of caffeine is both time- and temperature-dependent process that is more pronounced in the slow-twitch SOL than in the fast-twitch EDL muscles.     

Role of temperature in regulation of ovarian cycle in bull frog Rana tigerina

Effect of temperature on the ovarian cycle was studied in R. tigerina by exposing them to (1) constant low (22 degrees C) temperature during preparatory (active vitellogenic growth) phase (March-May) when the mean ambient temperature ranged from 26 degrees-28 degrees C and (2) to constant high (30 degrees +/- 1 degrees C) temperature during postbreeding regression phase (August-November) when the mean ambient temperature ranged from 22 degrees-26 degrees C. The ovaries of initial controls (biopsy samples taken prior to the commencement of the experiment) in March contained only first growth phase (FGP) oocytes with a maximum size range of 361-480 microns in diameter. In the frogs exposed to constant low temperature for 2 months, only 7% of FGP oocytes were recruited to second growth phase (SGP) with a mean largest diameter of 631 microns compared to 31% large SGP oocytes with a mean diameter of 1114 microns in the frogs collected from natural fields. The number of atretic follicles (AF) was lower and fat body weights were significantly higher in low temperature exposed frogs. The exposure of the frogs to constant high temperature during postbreeding months caused an increase in the mean diameter and number of large FGP oocytes, numerical increase in AF and decrease in fat body weights over corresponding controls maintained at room temperature. The pituitary gonadotrophs of these frogs showed stimulatory changes such as increase in cell size and appearance of secretory granules in the cytoplasm. The results suggest that in R. tigerina high temperature stimulates oocyte growth while low temperature retards it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of intestinal flora of laboratory-reared leopard frogs (Rana pipiens) to cold and fasting.

The bacterial flora of the large intestine was examined in 35 laboratory-reared leopard frogs (Rana pipiens) subjected to one of the following four treatments: (i) normal feeding at 21 degrees C (10 frogs); (ii) fasting for 2 weeks at 21 degrees C (8 frogs); (iii) chilling for 1 week at 4 degrees C (9 frogs); and (iv) simulated hibernation for 3 weeks at 4 degrees C (8 frogs). Bacteria from the intestinal contents and mucosa were counted microscopically and by colony counting after strictly anaerobic culturing. The predominant bacteria were isolated and partially characterized. Fasting for 2 weeks produced no significant changes in total counts or in the types of bacteria cultured. Chilling, whether rapid or in the course of simulated hibernation, was associated with a decrease in the numbers and variety of bacteria. Thus it appears that the lowering of temperature rather than the absence of food is the important factor in the reduction of bacterial flora seen in hibernating frogs. However, the bacteria showed some adaptation to the low temperature, as the longer the host had been at 4 degrees C, the higher the proportion of bacteria which could grow when cultured at that temperature.     

Temperature and pH/CO(2) modulate respiratory activity in the isolated brainstem of the bullfrog (Rana catesbeiana)

The effects of temperature and pH/CO(2) were examined in isolated brainstem preparations from adult North American bullfrogs (Rana catesbeiana). These experiments were undertaken to determine the effects of temperature on fictive breathing, central pH/CO(2) chemoreception, and to examine potential alphastat regulation of respiration in vitro. Adult bullfrog brainstem preparations were isolated, superfused with an artificial cerebrospinal fluid (aCSF) and respiratory-related neural activity was recorded from cranial nerves V, X and XII. In Series I experiments (N=8), brainstem preparations were superfused with aCSF equilibrated with 2% CO(2) at temperatures ranging from 10 to 30 degrees C. Neural activity was present in all preparations in the temperature range of 15-25 degrees C, but was absent in most preparations when aCSF was at 10 or 30 degrees C. The absence of fictive breathing at high (30 degrees C) temperatures was transient since fictive breathing could be restored upon returning the preparation to 20 degrees C. In Series II experiments (N=10), preparations were superfused with aCSF equilibrated with 0%, 2% and 5% CO(2) at temperatures of 15, 20 and 25 degrees C. Fictive breathing frequency (f(R)) was significantly dependent upon aCSF pH at all three temperatures, with slopes ranging from -0.82 min(-1) pH unit(-1) (15 degrees C) to -3.3 min(-1) pH unit(-1) (20 degrees C). There was a significant difference in these slopes (P<0.02), indicating that central chemoreceptor sensitivity increased over this temperature range. Fictive breathing frequency was significantly dependent upon the calculated alpha-imidazole (alpha(Im)) ionization (P<0.05), consistent with the alphastat hypothesis for the effects of temperature on the regulation of ventilation. However, most of the variation in f(R) was not explained by alpha(Im) (R(2)=0.05), suggesting that other factors account for the regulation of fictive breathing in this preparation. The results indicate that the in vitro brainstem preparation of adult bullfrogs has a limited temperature range (15-25 degrees C) over which fictive breathing is consistently active. Although there is a close correspondence of ventilation in vitro and in vivo at low temperatures, these data suggest that, as temperature increases, changes in ventilation in the intact animal are likely to be more dependent upon peripheral feedback which assumes a greater integrative role with respect to chemoreceptor drive, respiratory frequency and tidal volume.     

Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs

The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in amphibian declines worldwide. In vitro laboratory studies and those done on wild populations indicate that Bd grows best at cool temperatures between 17 and 25 degrees C. In the present study, we tested whether moderately elevating the ambient temperature to 30 degrees C could be an effective treatment for frogs infected with Bd. We acquired 35 bullfrogs Rana catesbeiana from breeding facilities and 36 northern cricket frogs Acris crepitans from the wild and acclimated them to either 23 or 26 degrees C for 1 mo. Following the acclimation period, frogs were tested for the presence of Bd using qPCR TaqMan assays. The 12 R. catesbeiana and 16 A. crepitans that tested positive for Bd were subjected to 30 degrees C for 10 consecutive days before returning frogs to their starting temperatures. Post-treatment testing revealed that 27 of the 28 frogs that had tested positive were no longer infected with Bd; only a single A. crepitans remained infected following treatment. This result indicates that elevating ambient temperature to a moderate 30 degrees C can be effective as a treatment for Bd infection in captive amphibians, and suggests that heat may be a superior alternative to antifungal drugs.     

High pressure nmr study of dihydrofolate reductase from a deep-sea bacterium Moritella profunda.

We have investigated the effect of pressure and temperature on the structural and thermodynamic stability of a protein dihydrofolate reductase from a deep-sea bacterium Moritella profunda in its folate-bound form in the pressure range between 3 and 375 MPa and the temperature range between -5 and 30 degrees C. The on-line cell variable pressure 1H NMR spectroscopy has been used to analyze the chemical shift and signal intensity in one-dimensional 1H NMR spectra. Thermodynamic analysis based on signal intensities from protons in the core part indicates that the thermodynamic stability of Moritella profunda DHFR is relatively low over the temperature range between -5 and 30 degrees C (deltaG0=15.8 +/- 4.1 kJ/mol at 15 degrees C), but is well adapted to the living environment of the bacterium (2 degrees C and 28 MPa), with the maximum stability around 5 degrees C (at 0.1 MPa) and a relatively small volume change upon unfolding (deltaV= 66 +/- 19 ml/mol). Despite the relatively low overall stability, the conformation in the core part of the folded protein remains intact up to approximately 200 MPa, showing marked stability of the core of this protein.     

Temperature acclimation and oxygen binding properties of blood of the European eel, Anguilla anguilla

Temperature acclimation of the European eel, Anguilla anguilla, resulted in red cell GTP/Hb molar ratios of 1.20, 1.77 and 0.80 at 2, 17 and 29 degrees C, respectively. A small increase in blood oxygen capacity was present in 29 degrees C acclimated eels. The CO2 Bohr effect and the shape of the oxygen binding curve (n-Hill) were invariant with both temperature and GTP/Hb. The significant differences in the GTP/Hb ratio corresponded with a strong enhancement of the temperature effect on blood oxygen affinity between 2 and 17 degrees C and a similarly strong compensation between 17 and 29 degrees C. Predicted in vivo P50 values were 3.0, 13.8 and 17.6 mmHg at 2 degrees C, 17 and 29 degrees C, respectively. The adaptational value of these findings are discussed in relation to standard metabolic rates at the various temperatures. A tentative hypothesis is proposed that the present study confirms and expands earlier work and supports the contention that adjustments in blood oxygen affinity of thermally acclimated teleosts serve to provide them with an unloading O2 tension for diffusion closely matching the standard oxygen requirements at the various temperatures.     

The effect of short term thermal stress on the heart and respiration rates of conscious and anaesthetized chickens

Heart and respiration rates were measured in eight 6-week-old, White Rock chicks at different ambient temperatures: 24--26 degrees C (neutral), 6.5--8.5 degrees C (low) and 3,95--43.5 degrees C (high). The animals were exposed to these temperatures for 10 min. In both groups the low ambient temperature did not influence the respiration rate, whereas the high temperature caused a significant increase of the respiration rates both in the conscious and anaesthetized birds. In both groups no significant changes in the heart rate at different temperatures were found. Statistically significant differences in the heart and respiration rates between the conscious and anaesthetized chickens were noted only at the low environmental temperature.