Breathing patterns and associated cardiovascular changes in intermittently breathing animals: (Partially) correcting a semantic quagmire.

Many animal species do not breathe in a continuous, rhythmic fashion, but rather display a variety of breathing patterns characterized by prolonged periods between breaths (inter-breath intervals), during which the heart continues to beat. Examples of intermittent breathing abound across the animal kingdom, from crustaceans to cetaceans. With respect to human physiology, intermittent breathing-also termed 'periodic' or 'episodic' breathing-is associated with a variety of pathologies. Cardiovascular phenomena associated with intermittent breathing in diving species have been termed 'diving bradycardia', 'submersion bradycardia', 'immersion bradycardia', 'ventilation tachycardia', 'respiratory sinus arrhythmia' and so forth. An examination across the literature of terminology applied to these physiological phenomena indicates, unfortunately, no attempt at standardization. This might be viewed as an esoteric semantic problem except for the fact that many of the terms variously used by different authors carry with them implicit or explicit suggestions of underlying physiological mechanisms and even human-associated pathologies. In this article, we review several phenomena associated with diving and intermittent breathing, indicate the semantic issues arising from the use of each term, and make recommendations for best practice when applying specific terms to particular cardiorespiratory patterns. Ultimately, we emphasize that the biology-not the semantics-is what is important, but also stress that confusion surrounding underlying mechanisms can be avoided by more careful attention to terms describing physiological changes during intermittent breathing and diving.

Short communication: Baroreflex function in embryonic emus (Dromiceius novaehollandiae).

The baroreflex involves cardiovascular homeostatic mechanisms that buffer the system against acute deviations in arterial blood pressure. It is comprised of the cardiac limb which involves adjustments in heart rate and the peripheral limb which involves adjustments in vascular resistance. This negative feedback loop mechanism has been investigated in numerous species of adult vertebrates, however our understanding of the maturation and functional importance of the reflex in developing animals remains poorly understood. In egglaying species, our knowledge of this mechanism is limited to the domestic chicken embryo and the embryonic alligator. While each of these species possess a cardiac baroreflex prior to hatching, they differ in the timing when it becomes functional, with the embryonic chicken possessing the reflex at 90% of incubation, while the alligator possesses the reflex at 70% of incubation. In an effort to determine if bird species might share similar patterns of active baroreflex function, we studied embryonic emus (Dromiceius novaehollandiae). However, we hypothesized that emus would possess a pattern of baroreflex function similar to that of the American alligator given the emu embryo possesses functional vagal tone at 70% of incubation, possibly indicating a more mature collection of cardiovascular control mechanism than those found in embryonic chickens. Our findings illustrate that emu embryos possess a hypotensive baroreflex at 90% of incubation. Therefore, our data fail to support our original hypothesis. While only two species of birds have been studied in this context, it could indicate that baroreflex function is not essential for cardiovascular homeostasis in birds for the majority of in ovo development.

Temperature and hypoxia trigger developmental phenotypic plasticity of cardiorespiratory physiology and growth in the parthenogenetic marbled crayfish, Procambarus virginalis Lyko, 2017.

Attempting to differentiate phenotypic variation caused by environmentally-induced alterations in gene expression from that caused by actual allelic differences can be experimentally difficult. Environmental variables must be carefully controlled and then interindividual genetic differences ruled out as sources of phenotypic variation. We investigated phenotypic variability of cardiorespiratory physiology as well as biometric traits in the parthenogenetically-reproducing marbled crayfish Procambarus virginalis Lyko, 2017, all offspring being genetically identical clones. Populations of P. virginalis were reared from eggs tank-bred at four different temperatures (16, 19, 22 and 25 °C) or two different oxygen levels (9.5 and 20 kPa). Then, at Stage 3 and 4 juvenile stages, physiological (heart rate, oxygen consumption) and morphological (carapace length, body mass) variables were measured. Heart rate and oxygen consumption measured at 23 °C showed only small effects of rearing temperature in Stage 3 juveniles, with larger effects evident in older, Stage 4 juveniles. Additionally, coefficients of variation were calculated to compare our data to previously published data on P. virginalis as well as sexually-reproducing crayfish. Comparison revealed that carapace length, body mass and heart rate (but not oxygen consumption) indeed showed lower, yet notable coefficients of variation in clonal crayfish. Yet, despite being genetically identical, significant variation in their morphology and physiology in response to different rearing conditions nonetheless occurred in marbled crayfish. This suggests that epigenetically induced phenotypic variation might play a significant role in asexual but also sexually reproducing species.

"Bet hedging" against climate change in developing and adult animals: roles for stochastic gene expression, phenotypic plasticity, epigenetic inheritance and adaptation.

Animals from embryos to adults experiencing stress from climate change have numerous mechanisms available for enhancing their long-term survival. In this review we consider these options, and how viable they are in a world increasingly experiencing extreme weather associated with climate change. A deeply understood mechanism involves natural selection, leading to evolution of new adaptations that help cope with extreme and stochastic weather events associated with climate change. While potentially effective at staving off environmental challenges, such adaptations typically occur very slowly and incrementally over evolutionary time. Consequently, adaptation through natural selection is in most instances regarded as too slow to aid survival in rapidly changing environments, especially when considering the stochastic nature of extreme weather events associated with climate change. Alternative mechanisms operating in a much shorter time frame than adaptation involve the rapid creation of alternate phenotypes within a life cycle or a few generations. Stochastic gene expression creates multiple phenotypes from the same genotype even in the absence of environmental cues. In contrast, other mechanisms for phenotype change that are externally driven by environmental clues include well-understood developmental phenotypic plasticity (variation, flexibility), which can enable rapid, within-generation changes. Increasingly appreciated are epigenetic influences during development leading to rapid phenotypic changes that can also immediately be very widespread throughout a population, rather than confined to a few individuals as in the case of favorable gene mutations. Such epigenetically-induced phenotypic plasticity can arise rapidly in response to stressors within a generation or across a few generations and just as rapidly be "sunsetted" when the stressor dissipates, providing some capability to withstand environmental stressors emerging from climate change. Importantly, survival mechanisms resulting from adaptations and developmental phenotypic plasticity are not necessarily mutually exclusive, allowing for classic "bet hedging". Thus, the appearance of multiple phenotypes within a single population provides for a phenotype potentially optimal for some future environment. This enhances survival during stochastic extreme weather events associated with climate change. Finally, we end with recommendations for future physiological experiments, recommending in particular that experiments investigating phenotypic flexibility adopt more realistic protocols that reflect the stochastic nature of weather.

Dietary crude oil exposure during sex differentiation skewed adult sex ratio towards males in the zebrafish.

Dietary crude oil exposure has detrimental morpho-physiological effects in fishes, including endocrine disruption. However, little is known about how it influences sex differentiation and its potential for skewing sex ratios of populations. Appropriate sex ratio is important for maintaining effective population size and structure. Deviations of these ratios can compromise population growth and maintenance and may induce changes in a species' evolutionary trajectory. We assessed the potential of dietary exposure to crude oil (6.5, 11.4, and 17.5 mg/kg food) to alter sex differentiation in the zebrafish (Danio rerio) (20-35 days post fertilization (dpf)) and subsequently skew the adult (90 dpf) sex ratio. Multiple health- and fitness-related phenotypic traits (i.e., body mass and length, condition factor, heart rate, oxygen consumption, and their capacity to cope with hypoxia) were also assessed to better understand the effects of dietary crude oil exposure. We showed that dietary exposure to crude oil during the process of sex differentiation skewed sex ratio towards males (up to 0.34:1 female to male ratio in the highest oil concentration). Remarkably, this effect occurred independently of affecting physiological variables and female gonad characteristics, thus highlighting just how subtle the effects of dietary crude oil exposure can be. Our results suggest that, although fish were in an apparently healthy state during experimentation, sex ratio was still impacted, potentially compromising the resilience of the population. Therefore, considering how complex chemical mixtures affect organisms at several levels (molecular-individual) in experimental designs is warranted to better understand the implications of the exposures and the hazards that populations face in the wild.

Zebrafish (Danio rerio) shoaling in light and dark conditions involves a complex interplay between vision and lateral line.

We know little about how - or even if in some species - fish shoal in darkness. We hypothesized that 'dark shoaling' occurs in zebrafish and therefore must depend upon lateral line sensory input. Shoaling in groups of five adult zebrafish was analyzed with motion tracking software. We measured average inter-individual distance, time near the arena wall (thigmotaxis zone) and total distance traveled under normal room light, and in near-complete darkness (infrared light at 850 nm). These observations were repeated in fish treated with cobalt chloride (CoCl2), which ablates lateral line function. In untreated controls, dark shoaling was reduced compared to in light, but nonetheless still present. Elimination of lateral line sensory input by CoCl2 treatment similarly reduced, but did not eliminate, shoaling under both light and dark. Our findings indicate that normal zebrafish shoaling in light or dark requires both visual and lateral line inputs, with neither alone sufficient for normal shoaling.

Acid-base and hematological regulation in chicken embryos during internal progressive hypercapnic hypoxia.

Development of the capacity to mitigate potential disturbances to blood physiology in bird embryos is incompletely understood. We investigated regulation of acid-base and hematology in day 15 chicken embryos exposed to graded intrinsic hypercapnic hypoxia created by varying degrees of water submersion. Metabolic acidosis with additional respiratory or metabolic acidosis occurred at 2 h according to magnitude of submersion. Acid-base disturbance was partially compensated by metabolic alkalosis at 6 h, but compensatory metabolic alkalosis was absent at 24 h. Following submersion with only air cell exposed to air, both hypercapnic respiratory acidosis and metabolic acidosis occurred within 10 min. Subsequently, both forms of acidosis created lethal levels of [HCO3-] at ∼120 min. Blood hematology showed small but significant effects associated with induced acid-base disturbance. Increased Hct occurring during partial egg submersion lasting 24 h was attributed to an increase in MCV. By day 15 of development chicken embryos are able to partially compensate for and withstand all but severe induced internal hypoxic hypercapnia.

Insufficient reporting of experimental variables as a cause for nonreproducibility in animal physiology? A case study.

Nonreproducibility in scientific investigations has been explained by inadequately reporting methodology, honest error, and even misconduct. We hypothesized that, within the field of animal physiology, the most parsimonious explanation for nonreproducibility is inadequate reporting of key methodological details. We further hypothesized that implementation of relatively recently released reporting guidelines has positively impacted journal article quality, as measured by completeness of the methodology descriptions. We analyzed 84 research articles published in five primarily organismal animal physiology journals in 2008-2010 (generally before current guidelines) and 2018-2020. Compliance for reporting 34 variables referring to biology, experiments, and data collection was assessed. Reporting compliance was just ∼61% in 2008-2010, rising only slightly to 67.5% for 2018-2020. Only 21% of the reported variables showed significant differences across the period from 2008-2020. We conclude that, despite attempts by societies and journals to promote greater reporting compliance, such efforts have so far been relatively unsuccessful in the field of animal physiology.

Physiological Regulation of Growth, Hematology and Blood Gases in Chicken Embryos in Response to Low and High Incubation Humidity.

Variations from a relative humidity (RH) of ∼50-60% can unfavorably alter chicken embryo development, but little is known of whether the embryo can mitigate these effects through physiological regulation. We examined effects of Low RH (25-35%), and High RH (85-93%) compared to Control RH (50-60%) on hatchability, embryonic growth, hematology and blood gases and pH. Mean hatchability was not affected by RH. Yet, Low RH decreased wet body mass of advanced embryos (days 17-19; d17-19), with lowered body water content compared with embryos of Control and High RH. However, dry body mass of developing (d11-19) embryos was not different between the three RH groups. Mean blood osmolality across development was higher in Low RH embryos and lower in High RH embryos compared with Control embryos. Mean blood lactate was higher in both Low and High RH embryos compared to Control embryos. Unexpectedly, hematological respiratory variables (Hct, [RBC], MCV, [Hb]) and blood gas variables (Po2, Pco2, pH, [HCO3 -]) across development were not affected by RH. Mean wet body mass at hatch (d20-22) was larger in High RH embryos compared with Low RH embryos, but mean wet and dry body mass upon euthanasia on d22 was unaffected. The ability of the three populations to physiologically regulate blood respiratory variables and blood acid-base balance was then examined by observing their responses to intrinsic hypoxemia and hypercapnia created by controlled partial egg submersion in water. Hct and [RBC] responses were less disturbed by submersion in High RH embryos compared with both Control and Low RH embryos, which showed major disturbance. Acid-base regulatory responses did not differ between RH groups. We conclude that, while different incubation RHs cause large differences in tissue water content and body mass, most hematological and acid-base regulatory capabilities are regulated near Control values.

Metabolic responses to crude oil during early life stages reveal critical developmental windows in the zebrafish (Danio rerio).

Morphological effects of crude oil exposure on early development in fishes have been well documented, but crude oil's metabolic effects and when in early development these effects might be most prominent remains unclear. We hypothesized that zebrafish (Danio rerio) exposed to crude oil as a high energy water accommodated fraction (HEWAF) would show increased routine oxygen consumption (MO2) and critical oxygen tension (PCrit) and this effect would be dependent upon day of HEWAF exposure, revealing critical windows of development for exposure effects. Zebrafish were exposed to 0%, 10%, 25%, 50% or 100% HEWAF for 24 h during one of the first six days post-fertilization (dpf). Survival rate, body mass, routine MO2, and PCrit were then measured at 7 dpf. Survival rate and especially body mass were both decreased based on both exposure concentration and day of crude oil exposure, with the largest decrease when HEWAF exposure occurred at 3 dpf. HEWAF effects on routine MO2 also differed depending upon exposure day. The largest effect occurred at 3 dpf, when MO2 increased significantly by ~60% from 10.1 ± 0.8 µmol O2/g/h compared to control group value of 6.3 ± 0.4 µmol O2/g/h. No significant effects of HEWAF exposure on any day were evident for PCrit (85 ± 4 mmHg in the control population). Overall, the main effects on body mass and MO2 measured at 7 dpf occurred when HEWAF exposures occurred at ~3 dpf. This critical window for metabolism in zebrafish larvae coincides with time of hatching, which may represent an especially vulnerable period in development.

Metabolic cost of development, regeneration, and reproduction in the planarian Schmidtea mediterranea.

Planaria are known for their ability to completely regenerate upon fissioning or experimental amputation. Yet, metabolic costs of regeneration have not been directly measured in planaria. Our goal was to establish the relationships between oxygen consumption (V̇O2), regeneration, and reproductive mode for asexual and sexual strains of Schmidtea mediterranea. We hypothesized that V̇O2 would vary by regeneration day for both sexual and asexual S. mediterranea, reflecting different costs of tissue reconstruction, but with an additional cost for regenerating sexual organs. Testes regeneration and body mass, as indicators of regeneration progress, and routine mass-specific V̇O2 as a function of maturity, regeneration, and reproductive mode, were measured over a 22-day regeneration period. Testes growth was highest in sexually mature adults, ~1/2 that in 14-day post-amputation sexual adults, and not detectable in juveniles and hatchlings. Mass-specific routine V̇O2 in sexuals was highest in mature controls at ~23 µl O2/g/h, but only half that in juveniles, hatchlings, and 14 day post-amputation adults. Both intact and 14-day post-amputation asexuals had a mass-specific routine V̇O2 of ~10-12 µl O2/g/h. The sum of V̇O2 of all amputated sections was ~100% higher than pre-amputation levels in the first 6 days of regeneration in asexuals, but not sexuals. There was no significant difference in V̇O2 of head, middle, and tail sections during regeneration. Overall, the highest metabolic costs associated with regeneration occurred during the initial 1-6 days of regeneration in both strains, but regeneration costs for sexual structures were not reflected in major V̇O2 differences between sexual and asexual strains.

Hypoxia- and hyperoxia-related gene expression dynamics during developmental critical windows of the tropical gar Atractosteus tropicus.

Aquatic hypoxia is both a naturally-occurring and anthropogenically-generated event. Fish species have evolved different adaptations to cope with hypoxic environments, including gill modifications and air breathing. However, little is known about the molecular mechanisms involved in the respiration of embryonic and larval fishes during critical windows of development. We assessed expression of the genes hif-1α, fih-1, nhe1, epo, gr and il8 using the developing tropical gar as a piscine model during three developmental periods (fertilization to hatch, 1 to 6 days post hatch (dph) and 7 to 12 dph) when exposed to normoxia (~7.43 mg/L DO), hypoxia (~2.5 mg/L DO) or hyperoxia (~9.15 mg/L DO). All genes had higher expression when fish were exposed to either hypoxia or hyperoxia during the first two developmental periods. However, fish continuously exposed to hypoxia had increased expression of the six genes by hatching and 6 dph, and by 12 dph only hif-1α still had increased expression. The middle developmental period was the most hypoxia-sensitive, coinciding with several changes in physiology and morphology. The oldest larvae were the most resilient to gene expression change, with little variation in expression of the six genes compared. This study is the first to relate the molecular response of an air-breathing fish to oxygen availability to developmental critical windows and contributes to our understanding of some molecular responses of developing fish to changes in oxygen availability.

Morphological and cardiac alterations after crude oil exposure in the early-life stages of the tropical gar (Atractosteus tropicus).

Fish development can be affected by environmental pollutants such as crude oil (anthropogenic or natural sources), causing alterations especially in cardiac function and morphology. Most such studies have focused on saltwater species, whereas studies in freshwater fishes are scant. The objective of the current study was to evaluate the effects of crude oil exposure (as 0, 5, 10, 15, or 20% high-energy water accommodated fractions, HEWAF) on cardiac function and edema formation during two early periods of development (embryo and eleuteroembryo, 48 h each) individually using the tropical gar Atractosteus tropicus as a model. Embryos did not exhibit alterations in body mass, total length, condition factor, and cardiac function as a function of oil. In contrast, eleuteroembryos proved to be more sensitive and exhibited increased body mass, total length, and condition factor, decreased heart rate and phenotypic alterations such as cardiac dysmorphia (tubular hearts) and spine curvature at high concentrations of HEWAF. Moreover, edema formation was observed in both stages This study shows different functional responses of A. tropicus after crude oil exposure and provides useful information of the developmental impacts of these compounds on the early life stages of freshwater tropical fishes.

Beyond the Chicken: Alternative Avian Models for Developmental Physiological Research.

Biomedical research focusing on physiological, morphological, behavioral, and other aspects of development has long depended upon the chicken (Gallus gallus domesticus) as a key animal model that is presumed to be typical of birds and generally applicable to mammals. Yet, the modern chicken in its many forms is the result of artificial selection more intense than almost any other domesticated animal. A consequence of great variation in genotype and phenotype is that some breeds have inherent aberrant physiological and morphological traits that may show up relatively early in development (e.g., hypertension, hyperglycemia, and limb defects in the broiler chickens). While such traits can be useful as models of specific diseases, this high degree of specialization can color general experimental results and affect their translational value. Against this background, in this review we first consider the characteristics that make an animal model attractive for developmental research (e.g., accessibility, ease of rearing, size, fecundity, development rates, genetic variation, etc.). We then explore opportunities presented by the embryo to adult continuum of alternative bird models, including quail, ratites, songbirds, birds of prey, and corvids. We conclude by indicating that expanding developmental studies beyond the chicken model to include additional avian groups will both validate the chicken model as well as potentially identify even more suitable avian models for answering questions applicable to both basic biology and the human condition.

Form and Function of the Vertebrate and Invertebrate Blood-Brain Barriers.

The need to protect neural tissue from toxins or other substances is as old as neural tissue itself. Early recognition of this need has led to more than a century of investigation of the blood-brain barrier (BBB). Many aspects of this important neuroprotective barrier have now been well established, including its cellular architecture and barrier and transport functions. Unsurprisingly, most research has had a human orientation, using mammalian and other animal models to develop translational research findings. However, cell layers forming a barrier between vascular spaces and neural tissues are found broadly throughout the invertebrates as well as in all vertebrates. Unfortunately, previous scenarios for the evolution of the BBB typically adopt a classic, now discredited 'scala naturae' approach, which inaccurately describes a putative evolutionary progression of the mammalian BBB from simple invertebrates to mammals. In fact, BBB-like structures have evolved independently numerous times, complicating simplistic views of the evolution of the BBB as a linear process. Here, we review BBBs in their various forms in both invertebrates and vertebrates, with an emphasis on the function, evolution, and conditional relevance of popular animal models such as the fruit fly and the zebrafish to mammalian BBB research.

Dietary Exposure to Low Levels of Crude Oil Affects Physiological and Morphological Phenotype in Adults and Their Eggs and Hatchlings of the King Quail (Coturnix chinensis).

Despite the current knowledge of the devastating effects of external exposure to crude oil on animal mortality, the study of developmental, transgenerational effects of such exposure has received little attention. We used the king quail as an animal model to determine if chronic dietary exposure to crude oil in a parental population would affect morpho-physiological phenotypic variables in their immediate offspring generation. Adult quail were separated into three groups: (1) Control, and two experimental groups dietarily exposed for at least 3 weeks to (2) Low (800 PAH ng/g food), or (3) High (2,400 PAH ng/g food) levels of crude oil. To determine the parental influence on their offspring, we measured metabolic and respiratory physiology in exposed parents and in their non-exposed eggs and hatchlings. Body mass and numerous metabolic (e.g., O2 consumption, CO2 production) and respiratory (e.g., ventilation frequency and volume) variables did not vary between control and oil exposed parental groups. In contrast, blood PO2, PCO2, and SO2 varied among parental groups. Notably, water loss though the eggshell was increased in eggs from High oil level exposed parents. Respiratory variables of hatchlings did not vary between populations, but hatchlings obtained from High oil-exposed parents exhibited lower capacities to maintain body temperature while exposed to a cooling protocol in comparison to hatchlings from Low- and Control-derived parents. The present study demonstrates that parental exposure to crude oil via diet impacts some aspects of physiological performance of the subsequent first (F 1 ) generation.

Analysis of the potential behavioral impact of methanol when used as a solvent: Dataset from zebrafish (Danio rerio) behavioral research.

Toxicants are commonly administered to experimental organisms using solvents as vehicles. One common vehicle for dissolving toxicants is methanol (CH3OH), a solvent which on its own is capable of altering physiology and behavior high concentrations. This dataset describes behavioral results in zebrafish (Danio rerio) individually exposed to methanol (0.25%, 2.5% vol/vol), or control water, for 30 min prior to behavioral testing. Zebrafish were placed into an open field arena to examine locomotion and zone preference, which was recorded and quantified with motion-tracking software (EthoVision XT). Time spent in the outer ("thigmotaxis") zone of the arena is a proxy for increased anxiety-like behavior in zebrafish. Additionally, a novel object was placed into the center of the arena to quantify relative increases in boldness/exploration between the methanol and control groups. There were no differences in time spent in any zone of the arena or distance travelled between either group, in either test.

Embryotoxicity and Physiological Compensation in Chicken Embryos Exposed to Crude Oil.

Terrestrial, marine, or aquatic oil spills can directly or indirectly contaminate bird eggs. We hypothesized that chicken embryos exposed to crude oil can physiologically compensate to mitigate the potentially toxic effect of lower doses of oil. Embryos exposed to 0, 1, 3, or 5 µL of oil on embryonic days 4 and 10 were initially analyzed for mortality. All oil doses decreased day 4 embryo survival, but only the 2 highest oil doses lowered survival when applied on day 10. Thus, day 15 embryos treated with 1, 3, and 5 µL of source oil on day 10 had arterialized blood analyzed. The hematological variables hematocrit, red blood cell concentration ([RBC]), and hemoglobin concentration increased in response to 1 µL, were unchanged by 3 µL, and decreased by 5 µL of oil treatment. No changes occurred in arterialized blood gas variables (partial pressure of O2 [PO2 ], pH, bicarbonate concentration) for 1 and 3 µL embryos, but 5 µL of oil decreased PO2 and caused metabolic acidosis. Increased blood lactate in embryos treated with 3 and 5 µL of oil was correlated with decreased hematocrit and [RBC] and increased body mass, the latter likely reflecting edema. We conclude that embryos in middle development physiologically compensated for negative effects of lower doses of crude oil but that higher doses of oil were harmful to the embryos at all developmental stages. Environ Toxicol Chem 2021;40:2347-2358. © 2021 SETAC.