Acute CO2 tolerance in fishes is associated with air breathing but not the Root effect, red cell ßNHE, or habitat.

High CO2 (hypercapnia) can impose significant physiological challenges associated with acid-base regulation in fishes, impairing whole animal performance and survival. Unlike other environmental conditions such as temperature and O2, the acute CO2 tolerance thresholds of fishes are not understood. While some fish species are highly tolerant, the extent of acute CO2 tolerance and the associated physiological and ecological traits remain largely unknown. To investigate this, we used a recently developed ramping assay, termed the Carbon Dioxide maximum (CDmax), that increases CO2 exposure until loss of equilibrium (LOE) is observed. We investigated if there was a relationship between CO2 tolerance and the Root effect, ß-adrenergic sodium proton exchanger (ßNHE), air-breathing, and fish habitat in 17 species. We hypothesized that CO2 tolerance would be higher in fishes that lack both a Root effect and ßNHE, breathe air, and reside in tropical habitats. Our results showed that CDmax ranged from 2.7 to 26.7 kPa, while LOE was never reached in four species at the maximum PCO2 we could measure (26.7 kPa); CO2 tolerance was only associated with air-breathing, but not the presence of a Root effect or a red blood cell (RBC) ßNHE, or fish habitat. This study demonstrates that the diverse group of fishes investigated here are incredibly tolerant of CO2 and that although this tolerance is associated with air-breathing, further investigations are required to understand the basis for CO2 tolerance.

Preferential intracellular pH regulation is a common trait amongst fishes exposed to high environmental CO2.

Acute (<96 h) exposure to elevated environmental CO2 (hypercarbia) induces a pH disturbance in fishes that is often compensated by concurrent recovery of intracellular and extracellular pH (pHi and pHe, respectively; coupled pH regulation). However, coupled pH regulation may be limited at CO2 partial pressure (PCO2 ) tensions far below levels that some fishes naturally encounter. Previously, four hypercarbia-tolerant fishes had been shown to completely and rapidly regulate heart, brain, liver and white muscle pHi during acute exposure to >4 kPa PCO2  (preferential pHi regulation) before pHe compensation was observed. Here, we test the hypothesis that preferential pHi regulation is a widespread strategy of acid-base regulation among fish by measuring pHi regulation in 10 different fish species that are broadly phylogenetically separated, spanning six orders, eight families and 10 genera. Contrary to previous views, we show that preferential pHi regulation is the most common strategy for acid-base regulation within these fishes during exposure to severe acute hypercarbia and that this strategy is associated with increased hypercarbia tolerance. This suggests that preferential pHi regulation may confer tolerance to the respiratory acidosis associated with hypercarbia, and we propose that it is an exaptation that facilitated key evolutionary transitions in vertebrate evolution, such as the evolution of air breathing.

The role of nitric oxide in the cardiovascular response to chronic and acute hypoxia in White Leghorn chicken (Gallus domesticus).

AIM: Prenatal hypoxia due to placental insufficiency results in deleterious phenotypes and compensatory mechanisms including increased sympathetic tone. Utilizing the embryonic chicken model, we investigated (i) changes in nitric oxide (NO)-mediated tone in response to chronic hypoxic development and (ii) the in vivo role of NO-mediated tone during acute hypoxic exposure, which has not been previously studied. We hypothesized that NO tone on the cardiovascular system would be unaffected by chronic hypoxic incubation in White Leghorn chicken (Gallus domesticus) embryos. METHODS: We measured arterial pressure, heart rate and femoral blood flow (via a Doppler flow probe) in response to acute hypoxia (10% O2 ) and pharmacological manipulations in normoxic- and hypoxic (15% O2 )-incubated embryos. This was performed at 70 and 90% of total incubation time (21 days). At 70% of incubation (day 15), blood volume and chorioallantoic membrane development are maximal; 90% of incubation (day 19) is 1 day prior to lung ventilation. RESULTS: Acute hypoxic exposure decreased femoral flow in both 90% groups, but increased femoral artery resistance in the hypoxic group. NO tone increased during development, but was not affected by hypoxic incubation. Inhibition of NO production by L-NAME (100 mg kg(-1) ) revealed that NO plays a significant role in the flow response to hypoxia. CONCLUSION: Chronic hypoxic incubation has no effect on cardiovascular NO tone during White Leghorn chicken development. In the intact animal, NO function during acute hypoxic stress is suppressed by hypoxic incubation, indicating that chronic hypoxic stress dampens the NO contribution.

Comparative cardiovascular physiology: future trends, opportunities and challenges.

The inaugural Kjell Johansen Lecture in the Zoophysiology Department of Aarhus University (Aarhus, Denmark) afforded the opportunity for a focused workshop comprising comparative cardiovascular physiologists to ponder some of the key unanswered questions in the field. Discussions were centred around three themes. The first considered function of the vertebrate heart in its various forms in extant vertebrates, with particular focus on the role of intracardiac shunts, the trabecular ('spongy') nature of the ventricle in many vertebrates, coronary blood supply and the building plan of the heart as revealed by molecular approaches. The second theme involved the key unanswered questions in the control of the cardiovascular system, emphasizing autonomic control, hypoxic vasoconstriction and developmental plasticity in cardiovascular control. The final theme involved poorly understood aspects of the interaction of the cardiovascular system with the lymphatic, renal and digestive systems. Having posed key questions around these three themes, it is increasingly clear that an abundance of new analytical tools and approaches will allow us to learn much about vertebrate cardiovascular systems in the coming years.

Effect of selection for commercially productive traits on the plasticity of cardiovascular regulation in chicken breeds during embryonic development.

Domesticated animal breeds have experienced profound anatomical and physiological changes as a result of human-driven genetic selection. In poultry, this selection process has resulted in many distinct phenotypes from the ancestral bird, the Red Junglefowl. Growth rate and egg-laying capacity are 2 traits that have been commercially prioritized, and this has resulted in a fast-growth breed, the broiler, and a prolific egg layer, the White Leghorn. In this study, we investigated basic cardiovascular physiology in these 3 breeds at 90% of incubation. We aimed to identify breed-specific features of arterial blood pressure and heart rate as well as the physiological mechanisms regulating them. Specifically, we investigated mechanisms mediated by the autonomic nervous system by means of cholinergic and adrenergic receptors. Our overriding hypothesis was that selection for rapid growth would require an acceleration of heart rate and arterial pressure development in broilers compared with White Leghorns and the ancestral breed. The embryonic broiler is characterized by resting relative hypertensive bradycardia, whereas the White Leghorn is hypotensive. All 3 breeds maintained resting arterial pressure and heart rate via a similar ß- and α-adrenergic receptor tone; however, cholinergic tone on heart rate was absent in the embryonic White Leghorn. Each breed responded differently to incubation in chronic hypoxic conditions (14% O(2)). White Leghorn relied on augmenting cholinergic heart rate tone, and broilers relied on reducing ß-adrenergic tone on pressure. We concluded that selection for rapid growth shifts cardiovascular regulatory plasticity to emphasize mechanisms that modulate pressure, and that selection for egg-laying capacity emphasizes mechanisms that modulate heart rate.

Physiological variability in yearling alligators: clutch differences at rest and during activity.

The adult phenotype of an organism is the result of its genotype, the environment, and the interaction between the two. Assessing the relative contribution of these factors to the final adult phenotype continues to occupy researchers. Studies have shown clutch effects early in development but few have investigated the persistence of clutch effects on a longer time scale. Five clutches of American alligators were reared for 1 year in a common environment then assessed for the presence of clutch effects as they related to morphological and physiological characteristics. After 1 year, significant clutch effects were evident in all size related variables despite open access to food. Additionally, lung and liver masses remained different between clutches after animal mass was taken into account. Although clutch had no effect on resting heart rate, it significantly contributed to mean arterial pressure. During swimming and exhaustive exercise, the resulting respiratory and metabolic acidoses were strongly dependent on clutch. Therefore, while the environment can have significant influences on the American alligator from hatching to death, the measureable contribution of genetics to the morphology and physiology of the organism remains evident, even after 1 year of common rearing conditions. It behooves researchers to acknowledge and control for clutch effects when designing experiments.

Chronic ingestion of Porphyromonas gingivalis induces systemic nitric oxide response in mice.

INTRODUCTION: Porphyromonas gingivalis induces nitric oxide (NO) production in various cells, systemic NO elevation being expected in chronic oral challenge. METHODS: Groups of BALB/c mice were inoculated orally with either live P. gingivalis ATCC 33277 or sterile broth on days 0, 2 and 4, with or without later administration of the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Plasma and tissues were harvested on day 42 for assays of tumor necrosis factor-alpha (TNF-alpha), nitrite and nitrate (NOx) and tissue NO, or histology and iNOS immunohistochemistry. RESULTS: No signs of gingivitis were observed, but plasma NOx was significantly elevated (P = 0.028) as was TNF-alpha (P = 0.079) in P. gingivalis-inoculated animals compared with controls, NOx being reduced when 1400W was used. NO production in organs showed a similar trend, with significant elevation in liver (P = 0.017) and kidneys (P = 0.027), whereas concomitant treatment of inoculated animals with 1400W caused significant reductions in NO in aorta (P = 0.008) and kidneys (P = 0.046). Sham-inoculated 1400W-treated animals had significantly increased plasma NOx (P = 0.004) and liver NO (P = 0.04). NOx in plasma correlated significantly with NO production in lungs (0.35, P = 0.032) and kidneys (0.47, P = 0.003). Immunohistochemistry demonstrated iNOS activity in many tissues in all groups. CONCLUSION: Repeated oral administration of P. gingivalis induced systemic NO and NOx production in mice, probably by activating iNOS as suggested by the response to 1400W.

Single gavage with Porphyromonas gingivalis reduces acute systemic nitric oxide response in mice.

INTRODUCTION: Porphyromonas gingivalis, an important periodontal pathogen, can also induce host responses in distant tissues. P. gingivalis induces nitric oxide (NO) production in immune system cells and non-immune system cells, therefore NO might be involved in an acute systemic host response. METHODS: Eighteen female BALB/c mice were perorally inoculated with 10(8) colony-forming units live P. gingivalis ATCC 33277. Plasma nitrite and nitrate (NOx) and NO production in lungs, aorta, heart, liver, spleen, kidneys, and brain were measured at intervals after inoculation and compared with levels in 11 control animals. RESULTS: NOx levels were significantly (P = 0.017) lower at 7, 13, and 25 h after P. gingivalis inoculation. A similar trend in NO production occurred in most tested organs, but never reached statistical significance. The correlation between NOx in plasma and NO in liver was positive (Spearman correlation coefficient = 0.81, P = 0.0025) and marginal for kidney (0.58, P = 0.059). CONCLUSION: Single peroral inoculation of mice with P. gingivalis reduces the acute systemic NO response. As NO is important for host defense, the reduction of NO levels after exposure is likely to delay the host response, increasing the chances that infection with P. gingivalis will become established.

In vitro comparison of three materials as apical sealants of equine premolar and molar teeth.

REASONS FOR PERFORMING STUDY: Surgical endodontic therapy is a conservative dental technique used in horses with some degree of clinical success. Failure of this procedure can partially be explained by inadequate sealing of the root apices with resultant microleakage in the periapical area. OBJECTIVES: To assess and compare in vitro sealing ability of 3 different dental restorative materials used as apical sealants during equine surgical endodontics. METHODS: Thirty extracted equine cheek teeth were divided randomly into 3 groups and subjected to apicoectomy and apical sealing using 3 materials: reinforced zinc oxide-eugenol cement; intermediate restorative material (IRM); a resin-modified glass ionomer; and amalgam. After apical sealing, the teeth were submerged in a solution of Procion Brilliant Cresyl Blue stain for a period of 7 days. The teeth were then washed, embedded in resin, sectioned and assessed microscopically for dye leakage around the apical restorations. RESULTS: Although the materials proved effective as apical sealants, some dye leakage was encountered in all 3 groups with no statistical difference (P = 0.114). CONCLUSIONS AND POTENTIAL RELEVANCE: IRM, a resin-modified glass ionomer and amalgam all showed comparative features as apical sealants when used in vitro in equine teeth. IRM is currently regarded as the superior material in clinical situations due to its ease of handling and lesser sensitivity to environmental moisture during placement compared to the other 2 materials.

Skull size and cheek-tooth length in wild-caught and captive-bred chinchillas.

Chinchillas are herbivorous rodents with teeth that all grow continuously. In captivity, they are commonly affected by dental disease. As the range of dental disease occurring in wild chinchillas is unknown, the dentition of museum specimens originally obtained from the wild was assessed and compared with specimens prepared from captive-bred animals. Skulls from wild-caught chinchillas showed minimal evidence of dental disease and the teeth were all short, cheek-tooth lengths averaging 5.9 mm. Cheek-tooth lengths in zoo specimens (average 6.6 mm), clinically normal (average 7.4 mm) and captive-bred animals with dental disease (average 10 mm) were significantly elongated by comparison (P<0.0001). Captive-bred specimens showed a wide range of tooth-related lesions. These results suggest that some aspect of captivity is responsible for the development of dental disease in chinchillas. It is suggested that the diet (its physical form and composition) is the main aetiological factor, and that provision of a diet closely matching that of wild chinchillas should significantly reduce the incidence of dental disease in captive animals.

Dental disease in chinchillas in the UK.

Dental abnormalities are common in chinchillas, although knowledge of the lesions responsible for the clinical signs is incomplete. Animals bred in the UK were examined to gain further knowledge of dental disease in this chinchilla population. Dental abnormalities, particularly those related to tooth elongation, were detected on careful external examination of 35 per cent of apparently healthy chinchillas. Incisor abnormalities were seen on clinical examination in 55 per cent of chinchillas presented because of clinical illness. In all but one case, this occurred secondarily to crown elongation of the cheek teeth or to the absence of opposing teeth, rather than being a primary skeletal problem. Clinical signs commonly attributed to malocclusion, such as ventral mandibular swelling, weight loss, dysphagia, altered chewing pattern and changed food preferences, were not specific to malocclusion, being seen associated with coronal spike formation, root elongation and advanced periodontal lesions. Caries and resorptive lesions rarely caused clinical signs in this population, but were identified during 37 per cent of postmortem examinations. Congenital absence of teeth, skeletal malocclusion and pathological loss of teeth all resulted in significant clinical signs, but were rare. It is concluded that provision of a diet with physical properties more closely matching that of wild chinchillas should improve the dental health of captive animals.

Physiological variability in neonatal armadillo quadruplets: within- and between-litter differences.

The role of genetics on neonatal physiological variability was examined in the nine-banded armadillo (Dasypus novemcinctus). Since armadillos give birth to only monozygous quadruplets, the genetic variation within litters is essentially zero. Quadruplets born in captivity were isolated and weighed within 8 h of birth. Oxygen consumption (V.(O2)) was measured in resting neonates by flow-through respirometry, heart rate obtained from an electrocardiogram and ventilation was measured by impedance techniques. Following the measurements, neonates were returned to the mother. Measurements were repeated at 4 and 8 days after birth. Mean heart rate significantly increased from 132 beats min(-1) on the day of birth to 169 beats min(-1) on day 8. Mean ventilation rate significantly decreased from 81 breaths min(-1) on the day of birth to 54 breaths min(-1) on day 8. During this same developmental period, mean mass significantly increased from 100 g to 129 g, and mean mass-specific oxygen consumption significantly decreased from 32.2 ml O(2)kg(-1)min(-1) to 28.6 ml O(2)kg(-1) min(-1). For all variables measured, within-litter variability was always significantly less than between-litter variability, confirming a 'sibling effect' that we attribute to the genetic components determining physiological characters.

Role of nitric oxide in the systemic and pulmonary circulation of anesthetized turtles (Trachemys scripta).

In reptiles the influence of local vascular factors on blood flow regulation is vaguely understood. The aim of this study was to investigate the role of nitric oxide (NO) on vascular function in anesthetized Trachemys scripta. The experimental protocol consisted of serial injections of sodium nitroprusside (SNP; 25 microg. kg(-1)), L-arginine (185 mg. kg(-1)) and L-NAME (50 mg. kg(-1)). SNP induced a systemic vasodilation (0.05 to 0.02 kPa. min. kg. mL(-1), P = 0.015), with no change in pulmonary vascular resistance (0.07 versus 0.08 kPa. min. kg. mL(-1), P > 0.05). L-Arg had no effect on resistances but increased cardiac output by 17%. L-NAME increased systemic resistance (33% increase; P = 0.01) while pulmonary resistance was unchanged. These effects are consistent with in vivo and in vitro studies on the systemic vasculature of different reptilian species, suggesting that NO has an important role in maintaining systemic vascular tone. The pulmonary vasculature did not respond to NO due to either a lack of an endogenous NO tone or a relaxed state of the pulmonary vasculature. The importance of NO-based mechanisms versus other neuro-humoral modulators in the reptilian circulation remains uncertain. However, as established in prior studies, cholinergic control of the proximal pulmonary artery is the main regulator of pulmonary resistance while systemic resistance depends on a more complex suite of neural, humoral and local effectors that include NO.

Control of blood pressure mediated by baroreflex changes of heart rate in the chicken embryo (Gallus gallus).

Pharmacological manipulation of peripheral resistance via sodium nitroprusside and phenylephrine was used to study baroreflex function over the latter two-thirds of incubation in embryonic chickens. From day 9 to day 19 of incubation, there is a positive linear relation between heart rate and blood pressure, indicating the feedforward action of arterial pressure on heart rate. A reciprocal relationship between blood pressure and heart rate became pronounced during the last 3 days of incubation. For the purpose of the study, gain of the baroreflex was calculated as maximal gain (only those embryos that demonstrated the response) or average gain (all embryos). Maximal gain increased progressively from 13 +/- 7 beats. min(-1). kPa(-1) at 18 days to 105 +/- 83 beats. min(-1). kPa(-1) in 2-day-old hatchlings. The percentage of embryos older than 18 days with baroreflex responses increased from 33% on day 19 to 56% on day 21, indicating that baroreflex regulation begins late in incubation ( approximately 90% incubation time), and the gain of this reflex exhibits a maturation over the final 3 days of incubation.