Juvenile American Alligators Research Articles

Visual Obstruction has No Effect on Post Capture Corticosterone Levels of Juvenile American Alligators (Alligator mississippiensis)

Abstract Anecdotal observations suggest that visual obstruction after capture will calm crocodilians, leading many to hypothesize that eye covering reduces physiological stress. However, this has y...

The effects of embryonic hypoxic programming on cardiovascular function and autonomic regulation in the American alligator (Alligator mississippiensis) at rest and during swimming.

Reptilian embryos naturally experience fluctuating oxygen levels in ovo, and developmental hypoxia has been established to have long-term impacts on cardiovascular function in vertebrates. In the present study, we investigated the impact of developmental 21% (normoxia) and 10% O2 (hypoxia) on juvenile (4-year-old) American alligator cardiovascular function in animals at rest and during swimming. In both experimental groups, combined right aortic and right subclavian blood flow approximately doubled during swimming. Carotid blood flow increased during swimming in the hypoxia-programmed animals only, and both carotid and left aortic blood flow reached higher values in swimming hypoxic-programmed animals compared to the normoxic group. However, pulmonary blood flow, which increased two to threefold during swimming (in both groups), was higher in normoxic-programmed animals at both rest and swimming. The differences between programming groups were preserved after cholinergic blockade (atropine), but reduced by adrenergic receptor antagonists (propranolol and phentolamine). Propranolol and phentolamine also blunted the incremental increases in blood flows during swimming, which was especially clear in the hypoxia-programmed animals. Alteration in adrenergic control and relative cardiac size (which was increased in hypoxic-programmed alligators) may account for the differences between the experimental groups.

Effects of chronic exposure to 12‰ saltwater on the endocrine physiology of juvenile American alligator (Alligator mississippiensis)

American alligator (Alligator mississippiensis) habitats are prone to saltwater intrusion following major storms, hurricanes or droughts. Anthropogenic impacts affecting hydrology of freshwater systems may exacerbate saltwater intrusion into freshwater habitats. The endocrine system of alligators is susceptible to changes in the environment but it is currently not known how the crocodilian physiological system responds to environmental stressors such as salinity. Juvenile alligators were exposed to 12‰ saltwater for 5 weeks to determine the effects of chronic exposure to saline environments. Following 5 weeks, plasma levels of hormones [e.g. progesterone, testosterone, estradiol, corticosterone, aldosterone (ALDO), angiotensin II (ANG II)] were quantified using liquid chromatography and tandem mass spectrometry. Compared with freshwater-kept subjects, saltwater-exposed alligators had significantly elevated plasma levels of corticosterone, 11-deoxycortisol, 17α-hydroxyprogesterone, testosterone, 17β-estradiol, estrone and estriol whereas pregnenolone and ANG II were significantly depressed and ALDO levels were unchanged (slightly depressed). On the one hand, saltwater exposure did not affect gene expression of renal mineralocorticoid and glucorticoid and angiotensin type 1 (AT-1) receptors or morphology of lingual glands. On the other hand, saltwater exposure significantly reduced plasma glucose concentrations whereas parameters diagnostic of perturbed liver function (aspartate aminotransferase and alanine aminotransferase) and kidney function (creatinine and creatine kinase) were significantly elevated. Except for plasma potassium levels (K+), plasma ions Na+ and Cl- were significantly elevated in saltwater alligators. Overall, this study demonstrated significant endocrine and physiological effects in juvenile alligators chronically exposed to a saline environment. Results provide novel insights into the effects of a natural environmental stressor (salinity) on the renin-angiotensin-aldosterone system and steroidogenesis of alligators.

Salmonella Enterica Serovar Pomona Infection in Farmed Juvenile American Alligators ( Alligator Mississippiensis).

A fatal epizootic of salmonellosis occurred in farmed juvenile American alligators in Louisiana. Six animals were examined. Gross lesions included severe fibrinonecrotizing enterocolitis, necrotizing splenitis, coelomic effusion, and perivisceral and pulmonary edema. Microscopic examination revealed severe necrotizing enterocolitis and splenitis with intralesional bacteria and pneumocyte necrosis with fibrin thrombi. Salmonella enterica serovar Pomona was isolated from intestine and lung. Clinical salmonellosis is a rare finding in reptiles and salmonellosis caused by S. Pomona is not previously reported in American alligators. Since S. Pomona is a commonly isolated Salmonella serotype from patients with reptile-associated salmonellosis in the United States, and since alligator meat is consumed and the skin is exported to numerous countries, risk of human and animal infection should be considered.

Developmental plasticity of mitochondrial function in American alligators, Alligator mississippiensis.

The effect of hypoxia on cellular metabolism is well documented in adult vertebrates, but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20 to 90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 yr. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of Leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype.

Chronic Ingestion of Coal Fly-Ash Contaminated Prey and Its Effects on Health and Immune Parameters in Juvenile American Alligators (Alligator mississippiensis).

Coal-burning power plants supply approximately 37% of the electricity in the United States. However, incomplete combustion produces ash wastes enriched with toxic trace elements that have historically been disposed of in aquatic basins. Organisms inhabiting such habitats may accumulate these trace elements; however, studies investigating the effects on biota have been primarily restricted to shorter-lived, lower-trophic organisms. The American alligator (Alligator mississippiensis), a long-lived, top-trophic carnivore, has been observed inhabiting these basins, yet the health or immune effects of chronic exposure and possible accumulation remains unknown. In this study, we investigated how chronic dietary ingestion of prey contaminated with coal combustion wastes (CCWs) for 25months, and subsequent accumulation of trace elements present in CCWs, affected juvenile alligator immune function and health. Alligators were assigned to one of four dietary-treatment groups including controls and those fed prey contaminated with CCWs for one, two, or three times a week. However, no effect of Dietary Treatment (p>0.05) was observed on any immune parameter or hematological or plasma analyte we tested. Our results suggest that neither exposure to nor accumulation of low doses of CCWs had a negative effect on certain aspects of the immune and hematological system. However, future studies are required to elucidate this further.

Thermal acclimation in American alligators: Effects of temperature regime on growth rate, mitochondrial function, and membrane composition

We investigated the ability of juvenile American alligators (Alligator mississippiensis) to acclimate to temperature with respect to growth rate. We hypothesized that alligators would acclimate to cold temperature by increasing the metabolic capacity of skeletal muscles and the heart. Additionally, we hypothesized that lipid membranes in the thigh muscle and liver would respond to low temperature, either to maintain fluidity (via increased unsaturation) or to maintain enzyme reaction rates (via increased docosahexaenoic acid). Alligators were assigned to one of 3 temperature regimes beginning at 9 mo of age: constant warm (30°C), constant cold (20°C), and daily cycling for 12h at each temperature. Growth rate over the following 7 mo was highest in the cycling group, which we suggest occurred via high digestive function or feeding activity during warm periods and energy-saving during cold periods. The warm group also grew faster than the cold group. Heart and liver masses were proportional to body mass, while kidney was proportionately larger in the cold group compared to the warm animals. Whole-animal metabolic rate was higher in the warm and cycling groups compared to the cold group – even when controlling for body mass – when assayed at 30°C, but not at 20°C. Mitochondrial oxidative phosphorylation capacity in permeabilized fibers of thigh muscle and heart did not differ among treatments. Membrane fatty acid composition of the brain was largely unaffected by temperature treatment, but adjustments were made in the phospholipid headgroup composition that are consistent with homeoviscous adaptation. Thigh muscle cell membranes had elevated polyunsaturated fatty acids in the cold group relative to the cycling group, but this was not the case for thigh muscle mitochondrial membranes. Liver mitochondria from cold alligators had elevated docosahexaenoic acid, which might be important for maintenance of reaction rates of membrane-bound enzymes.

Evaluating the effect of sample type on American alligator (Alligator mississippiensis) analyte values in a point-of-care blood analyser.

The assessment of wildlife health has been enhanced by the ability of point-of-care (POC) blood analysers to provide biochemical analyses of non-domesticated animals in the field. However, environmental limitations (e.g. temperature, atmospheric humidity and rain) and lack of reference values may inhibit researchers from using such a device with certain wildlife species. Evaluating the use of alternative sample types, such as plasma, in a POC device may afford researchers the opportunity to delay sample analysis and the ability to use banked samples. In this study, we examined fresh whole blood, fresh plasma and frozen plasma (sample type) pH, partial pressure of carbon dioxide (PCO2), bicarbonate (HCO3 (-)), total carbon dioxide (TCO2), base excess (BE), partial pressure of oxygen (PO2), oxygen saturation (sO2) and lactate concentrations in 23 juvenile American alligators (Alligator mississippiensis) using an i-STAT CG4+ cartridge. Our results indicate that sample type had no effect on lactate concentration values (F 2,65 = 0.37, P = 0.963), suggesting that the i-STAT analyser can be used reliably to quantify lactate concentrations in fresh and frozen plasma samples. In contrast, the other seven blood parameters measured by the CG4+ cartridge were significantly affected by sample type. Lastly, we were able to collect blood samples from all alligators within 2 min of capture to establish preliminary reference ranges for juvenile alligators based on values obtained using fresh whole blood.

Coronary blood flow in the anesthetized American alligator (Alligator mississippiensis)

Coronary circulation of the heart evolved early within ectothermic vertebrates and became of vital importance to cardiac performance in some teleost fish, mammals and birds. In contrast, the role and function of the coronary circulation in ectothermic reptiles remains largely unknown. Here, we investigated the systemic and coronary arterial responses of five anesthetized juvenile American alligators (Alligator mississippiensis) to hypoxia, acetylcholine, adenosine, sodium nitroprusside, isoproterenol, and phenylephrine. We recorded electrocardiograms, monitored systemic blood pressure, blood flows in both aortae, and blood flow in a major coronary artery supplying most of the right ventricle. Coronary arterial blood flow was generally forward, but there was a brief retrograde flow during a ventricular contraction. Blood pressure was significantly changed in all conditions. Acetylcholine decreased coronary forward flow, but this response was confounded by the concomitant lowered work of the ventricles due to decreased heart rate and blood pressure. Coronary forward flow was poorly correlated with heart rate and mean arterial pressure across treatments. Overall changes in coronary forward flow, significant and not significant, were generally in the same direction as mean arterial pressure and ventricular power, approximated as the product of systemic cardiac output and mean arterial pressure.

Quantification of intraskeletal histovariability in Alligator mississippiensis and implications for vertebrate osteohistology.

Bone microanalyses of extant vertebrates provide a necessary framework from which to form hypotheses regarding the growth and skeletochronology of extinct taxa. Here, we describe the bone microstructure and quantify the histovariability of appendicular elements and osteoderms from three juvenile American alligators (Alligator mississippiensis) to assess growth mark and tissue organization within and amongst individuals, with the intention of validating paleohistological interpretations. Results confirm previous observations that lamellar and parallel fibered tissue organization are typical of crocodylians, and also that crocodylians are capable of forming woven tissue for brief periods. Tissue organization and growth mark count varies across individual skeletal elements and reveal that the femur, tibia, and humerus had the highest annual apposition rates in each individual. Cyclical growth mark count also varies intraskeletally, but data suggest these inconsistencies are due to differing medullary cavity expansion rates. There was no appreciable difference in either diaphyseal circumference or cyclical growth mark circumferences between left and right element pairs from an individual if diaphyses were sampled from roughly the same location. The considerable intraskeletal data obtained here provide validation for long-held paleohistology assumptions, but because medullary expansion, cyclical growth mark formation, and variable intraskeletal growth rates are skeletal features found in tetrapod taxa living or extinct, the validations presented herein should be considered during any tetrapod bone microanalysis.

Concentrations of trace elements in American alligators (Alligator mississippiensis) from Florida, USA

Concentrations of 28 trace elements (Li, Mg, Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, As, Se, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Tl, Hg, Pb, and Bi) in the livers of juvenile and adult American alligators inhabiting two central Florida lakes, Lake Apopka (LA), and Lake Woodruff National Wildlife Refuge (LW) and one lagoon population located in Merritt Island National Wildlife Refuge (MINWR; NASA), were determined. In juveniles from MINWR, concentrations of nine elements (Li, Fe, Ni, Sr, In, Sb, Hg, Pb and Bi) were significantly higher, whereas six elements (V, Fe, As, Sr, Hg and Bi) were elevated in adults (p<0.05) obtained from MINWR. Significant enrichment of some trace elements in adults, relative to juveniles, was observed at all three sampling areas. Specifically, Fe, Pb and Hg were significantly elevated in adults when compared to juveniles, suggesting age-dependent accumulation of these elements. Further, As, Se and Sn showed the same trend but only in animals collected from MINWR. Mean Fe concentrations in the livers of adults from LA, LW and MINWR were 1770μgg−1 DW, 3690μgg−1 DW and 5250μgg−1 DW, respectively. More than half of the adult specimens from LW and MINWR exhibited elevated hepatic Fe concentrations that exceed the threshold value for toxic effects in donkey, red deer and human. These results prompted us to express our concern on possible exposure and health effects in American alligators by some trace elements derived from NASA activities.

The Pulmonary Anatomy of Alligator mississippiensis and Its Similarity to the Avian Respiratory System

Using gross dissections and computed tomography we studied the lungs of juvenile American alligators (Alligator mississippiensis). Our findings indicate that both the external and internal morphology of the lungs is strikingly similar to the embryonic avian respiratory system (lungs + air sacs). We identified bronchi that we propose are homologous to the avian ventrobronchi (entobronchi), laterobronchi, dorsobronchi (ectobronchi), as well as regions of the lung hypothesized to be homologous to the cervical, interclavicular, anterior thoracic, posterior thoracic, and abdominal air sacs. Furthermore, we suggest that many of the features that alligators and birds share are homologous and that some of these features are important to the aerodynamic valve mechanism and are likely plesiomorphic for Archosauria.

Morphology and Histochemistry of Juvenile Male American Alligator (Alligator mississippiensis) Phallus

Phalli of male crocodilians transfer sperm to female cloaca during sexual intercourse, resulting in internal fertilization. For over a century there have been scientific descriptions of crocodilian phallus morphologies; however, little work has presented detailed cellular-level analyses of these structures. Here we present a histological investigation of the complex functional anatomy of the juvenile male American alligator phallus, including fibrous and vascular erectile structures, a variety of secretory epithelium morphologies, and observed immune cells. Using 3D reconstruction software, we show the shape and location of vascular erectile tissues within the phallus. Histochemical staining detected mucin-rich secretory cells in glandular epithelial cells of the phallic shaft and also of the semen-conducting ventral sulcus. Lymphoid aggregates, lymphocytes, and epithelial mucin coats suggest an active immune system in the phallus defending from both the external and intracloacal environments. These results better characterize the complexity of the alligator phallus and predict later reproductive functions during adulthood.

Seasonal variation in plasma thyroid hormone concentrations in coastal versus inland populations of juvenile American alligators ( Alligator mississippiensis): Influence of plasma iodide concentrations

Thyroid hormones, essential for normal growth and health, are associated with changes in temperature, photoperiod, and reproduction. Iodide, a necessary element for thyroid hormone production, varies in diet, and is more abundant in estuarine environments, which could alter thyroid hormone variation. However, associations between thyroid hormone concentrations in animals from marine versus freshwater environments, which could become more pertinent with rising sea levels associated with global climate change, are not well studied. To determine the importance of dietary iodide in seasonal variation of plasma thyroid hormone concentrations, we analyzed seasonal variation of plasma thyroxine ( T 4) and triiodothyronine ( T 3) concentrations in juvenile alligators from an estuarine habitat (Merritt Island National Wildlife Refuge; MI) and a freshwater habitat (Lake Woodruff National Wildlife Refuge; LW) and compared these results to plasma inorganic iodide (PII) concentrations. Alligators from MI did not display seasonal variation in plasma T 4, but exhibited a seasonal pattern in plasma T 3 concentrations similar to alligators from LW. Plasma thyroid hormone concentrations were consistently higher at MI than at LW. PII concentrations were correlated with plasma T 4 and T 3 concentrations in juvenile alligators from LW but not MI. The data on plasma T 4 and T 3 concentrations suggest altered iodide metabolism in estuarine alligators. Differences in thyroid hormone concentrations between the populations could be due to differences in dietary iodide, which need to be further evaluated.

Mortality of American Alligators Attributed to Cannibalism

Abstract Mortality of juvenile (<122 cm total length; TL) American Alligators (Alligator mississippiensis) attributed to cannibalism on Orange Lake, Florida was examined. Alligator web tags used in mark–recapture studies were found in 12% of 267 stomachs sampled from alligators ≥168 cm TL. Captive alligators retained 76% of force-fed tags during a 588-d tag-retention trial. Models relating the probability of tag recovery to the annual probabilities of juvenile survival, cannibalism, tag retention, adult survival, and adult harvest suggested that cannibalism may on average remove 6–7% of the juvenile alligator population annually. Vulnerability continued to 140 cm TL (age 6–8 yr). Cannibalism of juveniles may serve to regulate the alligator population on Orange Lake. Alligator cannibalism may vary widely among populations, depending on demography and environmental conditions. The role and importance of cannibalism in alligator population dynamics should be more fully assessed and environmental and populati...

Influences of Sex, Incubation Temperature, and Environmental Quality on Gonadal Estrogen and Androgen Receptor Messenger RNA Expression in Juvenile American Alligators (Alligator mississippiensis)1

Gonadal steroid hormone receptors play a vital role in transforming ligand signals into gene expression. We have shown previously that gonads from wild-caught juvenile alligators express greater levels of estrogen receptor 1 (ESR1) than estrogen receptor 2 (ESR2). Furthermore, sexually dimorphic ESR2 mRNA expression (female > male) observed in animals from the reference site (Lake Woodruff, FL, USA) was lost in alligators from the contaminated Lake Apopka (FL, USA). We postulated that environmental contaminant exposure could influence gonadal steroid hormone receptor expression. Here, we address questions regarding gonadal estrogen and androgen receptor (AR) mRNA expression in 1-yr-old, laboratory-raised alligators. What are relative expression levels within gonads? Do these levels vary between sexes or incubation temperatures? Can contaminant exposure change these levels? We observed a similar pattern of expression (ESR1 > AR > ESR2) in ovary and testis. However, both incubation temperature and environment modulated expression. Males incubated at 33.5 degrees C expressed greater AR levels than females incubated at 30 degrees C; dimorphic expression was not observed in animals incubated at 32 degrees C. Compared to Lake Woodruff alligators, Lake Apopka animals of both sexes showed lesser ESR2 mRNA expression levels. Employing cluster analyses, we integrated these receptor expression patterns with those of steroidogenic factors. Elevated ESR2 and CYP19A1 expressions were diagnostic of alligator ovary, whereas elevated HSD3B1, CYP11A1, and CYP17A1 expressions were indicative of testis. In contrast, AR, ESR1, and NR5A1 showed variable expressions that were not entirely associated with sex. These findings demonstrate that the mRNA expression of receptors required for steroid hormone signaling are modified by exposure to environmental factors, including temperature and contaminants.

The importance of the M. diaphragmaticus to the duration of dives in the American alligator ( Alligator mississippiensis)

We tested the hypothesis that the crocodilian M. diaphragmaticus extends the duration of dives by disabling this muscle in a group of juvenile American alligators and comparing the duration of their dives to the duration of the dives of animals in which the muscle remained intact. We studied the groups while they were fasting, 1 h after they had eaten a meal with a density that was either greater or less than water, and at 22 and 28 °C. We found that the duration of dives was longer for the control group compared to animals without a functional M. diaphragmaticus, both when fasting and after having consumed the denser meal. The warmer temperature significantly decreased the duration of the dives for both groups, as did eating in general. The preponderance of these data indicates that transection of the diaphragmaticus reduced time spent underwater, but the mechanism for this reduction is unknown. Lack of a functional diaphragmaticus could impair the animals’ ability to inspire sufficient air to support the dive, but we think this explanation is unlikely because both groups were able to float at the surface and thus needed to reduce lung volume to dive. An alternative explanation is that the effect on duration is a consequence of an impairment of a locomotor rather than ventilatory function of the muscle.

Recruitment of the diaphragmaticus, ischiopubis and other respiratory muscles to control pitch and roll in the American alligator (Alligator mississippiensis)

We used electromyography on juvenile American alligators to test the hypothesis that the following muscles, which are known to play a role in respiration, are recruited for aquatic locomotion: M. diaphragmaticus, M. ischiopubis, M. rectus abdominis, M. intercostalis internus, and the M. transversus abdominis. We found no activity with locomotion in the transversus. The diaphragmaticus, ischiopubis, rectus abdominis and internal intercostals were active when the animals executed a head-down dive from a horizontal posture. Weights attached to the base of the tail resulted in greater electrical activity of diaphragmaticus, ischiopubis and rectus muscles than when weights were attached to the head, supporting a role of this musculature in locomotion. The diaphragmaticus and rectus abdominis were active unilaterally with rolling maneuvers. Although the function of these muscles in locomotion has previously been unrecognized, these data raise the possibility that the locomotor function arose when Crocodylomorpha assumed a semi-aquatic existence and that the musculoskeletal complex was secondarily recruited to supplement ventilation.

Gene expression patterns in juvenile American alligators ( Alligator mississippiensis) exposed to environmental contaminants

Reproductive and developmental abnormalities have been reported in the American alligator ( Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17β (E 2) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor β) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations of T, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E 2 concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator.

The Right‐to‐Left Shunt of Crocodilians Serves Digestion

Abstract All amniotes except birds and mammals have the ability to shunt blood past the lungs, but the physiological function of this ability is poorly understood. We studied the role of the shunt in digestion in juvenile American alligators in the following ways. First, we characterized the shunt in fasting and postprandial animals and found that blood was shunted past the lungs during digestion. Second, we disabled the shunt by surgically sealing the left aortic orifice in one group of animals, and we performed a sham surgery in another. We then compared postprandial rates of gastric acid secretion at body temperatures of 19 degrees and 27 degrees C and rates of digestion of bone at 27 degrees C. Twelve hours after eating, maximal rates of gastric acid secretion when measured at 19 degrees and 27 degrees C were significantly less in the disabled group than in sham-operated animals. Twenty-four hours postprandial, a significant decrease was found at 27 degrees C but not at 19 degrees C. For the first half of digestion, dissolution of cortical bone was significantly slower in the disabled animals. These data suggest the right-to-left shunt serves to retain carbon dioxide in the body so that it can be used by the gastrointestinal system. We hypothesize that the foramen of Panizza functions to enrich with oxygen blood that is destined for the gastrointestinal system to power proton pumps and other energy-demanding processes of digestion and that the right-to-left shunt serves to provide carbon dioxide to gastrointestinal organs besides the stomach, such as the pancreas, spleen, upper small intestine, and liver.