Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx

CBA-09776; No of Pages 8

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Comparative Biochemistry and Physiology, Part A

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Blood oxygen affinity increases during digestion in the South Americanrattlesnake, Crotalus durissus terrificus

Rafael P. Bovo a,⁎, Adriana Fuga a, Mariana A. Micheli-Campbell a, José E. Carvalho b, Denis V. Andrade a

a Departamento de Zoologia, IB, Universidade Estadual Paulista (UNESP), Rio Claro, SP 13506900, Brazilb Instituto de Ciências Ambientais, Químicas e Farmacêuticas, Universidade Federal de São Paulo (UNIFESP), Diadema, SP 09972270, Brazil

⁎ Corresponding author at: Departamento de ZoolUniversidade Estadual Paulista (UNESP), Avenida 24-A13506900, Brazil. Tel.: +55 19 35264241; fax: +55 19 35

E-mail address: rpbovo@gmail.com (R.P. Bovo).

http://dx.doi.org/10.1016/j.cbpa.2014.10.0101095-6433/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Bovo, R.P., et al., Blterrificus, Comp. Biochem. Physiol., A (2014)

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 August 2014Received in revised form 20 October 2014Accepted 20 October 2014Available online xxxx

Keywords:Hb–O2 affinityBlood oxygen transportOxygen-binding propertiesPostprandialFeedingReptileViperidae

Digesting snakes experience massive increases in metabolism that can last for many days and are accompa-nied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding proper-ties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48 hafter the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general,oxygen–hemoglobin (Hb–O2) affinity was significantly increased 24 h post-feeding, and then returned to-ward fasting values within 48 h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemo-globin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandialincrease in Hb–O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderatealkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolitesat quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circu-lating lipids, however, was considerably increased, whichmay be related to theirmobilization, since lipids areknown to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that theexceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported bythe aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb–O2 load-ing is likely improved due to the increment in blood O2 affinity.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

A fundamental role of the blood is carrying oxygen from the gas-exchange organ to the metabolic active tissues (Willford et al., 1982;Schmidt-Nielsen, 2002). The amount of oxygen transported by theblood is a function of the content in the O2-binding proteins, such as he-moglobins, while the amount of oxygen loaded/unloaded under a givensituation is determined by the affinity between these carrier proteinsand the oxygen. In reptiles, as common to many other organisms, oxy-gen is transported by an iron-based protein, the hemoglobin (Hb),contained within nucleated red blood cells (Nikinmaa, 1990). Althoughblood oxygen affinity needs to be high enough to ensure adequate oxy-gen saturation at the lungs, it also needs to be low enough to ensure itsrelease to the tissues and, therefore, Hb–O2 affinity reflects such a com-promise (Brauner and Wang, 1997).

Reptiles, as usually true for ectothermic organisms, have low meta-bolic rates that, in general, are orders ofmagnitude lower than those ob-served in similar-sized endotherms (Brand et al., 1991). Accordingly,

ogia, Instituto de Biociências,, 1515, c.p. 199, Rio Claro, SP264300.

ood oxygen affinity increases, http://dx.doi.org/10.1016/j.c

compared to endotherm vertebrates, the cardiorespiratory system ofreptiles has a much lower capacity to transport oxygen along themulti-step cascade from the environment to the cells. However, the ordinarilylow metabolism of reptiles can be considerably elevated after meal in-gestion due to the specific dynamic action (SDA) (Kleiber, 1961;Andrade et al., 2005). Such response is particularly pronounced in spe-cies that feed infrequently on large prey items, as epitomized by mostambush hunting snake species (Andrade et al., 1997; Secor andDiamond, 1997). In these snakes, an increase in the postprandialmetab-olism up to 7–8 times above the resting (fasting) rate is a common oc-currence (Toledo et al., 2003; Wang et al., 2003) and even higherincrements have been reported (Secor and Diamond, 1997; Secor,2009). Thus, digesting snakes are faced with a severe challenge inmatching metabolic demand and oxygen supply and, therefore, signifi-cant cardiorespiratory adjustments come into play, including changes inblood parameters associated to the oxygen transport (Wang et al.,2005). These adjustments are particularly relevant since the postpran-dial metabolism for snakes remains elevated for days and seems to bealmost entirely aerobically supported (Benedict, 1932; Andrade et al.,1997, 2005; Wang et al., 2001a).

Whereas duringmuscular exercise reptiles often incur in ametabolicacidosis that may cause a reduction in blood oxygen affinity via Bohr ef-fect (Nikinmaa, 1990), during digestion the pH of the blood undergoes

during digestion in the South American rattlesnake, Crotalus durissusbpa.2014.10.010

2 R.P. Bovo et al. / Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx

an alkalinization due to acid secretion into the lumen of the stomach(the so-called postprandial alkaline tide) (Rune, 1965; Coulson andHernadez, 1983). However, this response is attenuated in most reptilesby a respiratory acidosis caused by a relative hypoventilation (seeWanget al., 2001a). Thus, SDA in snakes is accompanied by increased rates ofmetabolism, changes in lung ventilation, and alterations in the acid-base equilibrium of the blood (Overgaard et al., 1999; Wang et al.,2001a, 2005). In this context, the properties of the blood associated tothe transport of oxygen may also be altered. Nonetheless, few and con-flicting data are available on the possible changes in blood oxygen affin-ity during digestion in reptiles. For example, while in Alligatormississippiensis the blood oxygen affinity remains unchanged during di-gestion (Bauer et al., 1981;Weber andWhite, 1986; Busk et al., 2000a),in Python molurus it is considerably increased (Overgaard and Wang,2002).

Herein, we examine the effects of feeding on blood gases, acid-basebalance, metabolites, and blood oxygen transport in the SouthAmerican rattlesnake, Crotalus durissus terrificus. This species is knownfor feeding infrequently on relatively large prey and for exhibiting amarked postprandial metabolic response (Andrade et al., 1997; Secorand Diamond, 1997).

List of abbreviations/acronymsctCO2 (CplCO2) plasma carbon dioxide contentpCO2 (PaCO2) arterial blood carbon dioxide partial pressureCaO2 arterial blood oxygen contentPaO2 arterial blood oxygen partial pressurepHa arterial blood pH[Hb] hemoglobin contentHct hematocritHb–O2 sat hemoglobin oxygen saturationODC oxygen dissociation curve[NTP] nucleoside triphosphates concentration (organic

phosphates)

2. Material and methods

2.1. Animals

A total of 10 South American rattlesnakes (mean 715 ± 108 g;Mass range 320–1065 g) were housed in individual wooden cages(30 × 29 × 27 cm), with sliding glass door front and side holes for ven-tilation, in a temperature controlled room (~28 ± 2 °C). Snakes werefed every other week with mice (Mus musculus) and were given freeaccess to water. Twenty days prior to experimentation, all animalswere fasted. Only non-ecdysial and healthy individuals were used inexperimental procedures. The research was performed under theBrazilian Institute for Environment and Natural Renewable Resourcesauthorization (IBAMA; license number 22028-1 and 35081-1) andwas also approved by the local University Ethical Committee for Ani-mal Experimentation (UNESP-IB-CEU: protocol 4361). All animalsoriginated from wild caught non-purposeful captures made by localsaround São Paulo state, Brazil, and delivered to our laboratory wherethey were housed for at least 3 months before experimentation began.

2.2. Surgical procedures

Snakes were anesthetized by CO2 inhalation until the loss of motorcontrol, which was verified by the abolition of the righting reflex (seeWang et al., 1993; Kohler et al., 1999; AVMA, 2013). Locally snakeswere treated with lidocaine hydrochloride (Pearson Lab) just before aventral–lateral incision (10.5 cm)wasmade above the heart, the under-lying tissues were removed and the vertebral artery was located and

Please cite this article as: Bovo, R.P., et al., Blood oxygen affinity increasesterrificus, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016/j.c

isolated. Then, this artery was cannulated occlusively, toward theheart, seeking to advance the tip of the cannula (PE 50–90) to theaorta. The cannula was externalized through the dorsal body wall andanchored in the underlying tissues. At the end of the surgery, the inci-sion was closed and the animals were treated with a broad-spectrumantibiotic (Baytril®). The cannulas were injected daily with saline solu-tion and filled with heparinized saline to prevent clogging thereof.

2.3. Experimental protocol

After surgery, snakes were kept in individual boxes (30 ×29× 27 cm) inside a climatic chamber BOD (Fanem, 347CD; andEletrolab, 122FC) at 30 °C. A 24 to 48 h period was then observed inorder to allow for the recovery of the animals from the surgical proce-dure (seeWang et al., 1993). After, an initial 3 ml sample of blood wastaken from each snake as representative for the fasting condition.Then, rattlesnakes were fed with a mouse equaling to 15% (±2%) ofthe snake body mass. Thereafter, we collected blood samples at 24and 48 h after feeding. For the control group, we followed the samesampling protocol but withholding feeding. All analyzes were per-formed with individual blood samples.

2.4. Hematological parameters and oxygen dissociation curves

Oxygen dissociation curves (ODC) were obtained by determiningthe oxygen content (ctO2) of individual samples of blood after theyhave been equilibrated with gas mixtures varying in their oxygen frac-tional concentration ~28 kPa; 9.5 kPa; 5.7 kPa; 3.7 kPa; 1.9 kPa;0.9 kPa. The Hb–O2/Hb ratio was calculated assuming fully Hb satura-tion at the PO2 of 28 kPa. For every blood sample, we obtained two ox-ygen equilibration curves at two different pH values, which weremanipulated by changing the PCO2 of the gas mixtures equilibratedwith the blood (≈0.953 and 3.812 kPa).

Blood samples (3ml)were placed in a tonometer coupled to a stirrerimmersed in a thermostatic bath maintained at 30 °C (Cameron Instru-ment Co., Model DEQ1). Gases mixtures were set by a Gas Mixing Unit(GF-3MP, Cameron Instruments) and a flow of 200 ml/min was sentto the tonometer for a period of 20 min at each level PO2. At the endof each exposure periods, approximately 40 μl of blood were collectedfor ctO2 determination, hematocrit, and pHmeasurement. All measure-ments were made in duplicate and the maximum level of variation ac-cepted between them was 5%.

Blood ctO2 was determined by injecting 20 μl of blood into a Tuckerchamber, previously filled with a solution of potassium ferricyanidedegassed at 40 °C. The change in O2 tension in the Tucker chamber(see Tucker, 1967),whichwasmonitored by a Clark electrode (Radiom-eter Copenhagen, BMS 3), was read directly from the digital display ofpH and blood gases monitor (Radiometer Copenhagen, PHM73). Theoxygen bound to the hemoglobin was determined by subtracting thecalculated dissolved oxygen in the blood (α) (see Christoforides andHedley-White, 1969), from the total ctO2. Thus, the saturation of hemo-globin was calculated as:

Hb–O2 sat 100 [(ctO2 − (α.PO2))/capO2]%, whereHb–O2 sat percentage saturation of hemoglobin;ctO2 total oxygen content in the blood;PO2 oxygen partial pressure;capO2 total oxygen capacity of hemoglobin;α solubility coefficient of oxygen in the plasma.

Blood pH in vitro was measured at the beginning and at the end oftheODCdeterminations. Arterial pH in vivowas determined for each in-dividual snake – the same ones used for ODC determination – at thesame protocol intervals (i.e., 24 h before and 24 h after feeding) underundisturbed conditions. All pH measurements were made with a

during digestion in the South American rattlesnake, Crotalus durissusbpa.2014.10.010

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microelectrode (PS-1204, Radiometer) mounted on a unit of blood gasanalysis (BMS Mk3, Radiometer) at 30 °C.

The Bohr effect was calculated as ΔlogP50/ΔpH. Hematocrit wasmeasured at the beginning and at the final of the experiments afterfour minutes of centrifugation at 14,890 RPM. The Hb content of theblood (Sigma Diagnostics, No. 525) and the level of organic phosphates(NTP concentrations: [NTP]) were measured spectrophotometrically.

2.5. Blood gases and acid–base parameters

Arterial PO2 (PaO2) and pH (pHa)weremeasured using Radiometerelectrodes (E5046-0, PS-1 204, respectively) mounted in a BMS Mk3unit. Electrodes were kept at the temperature of the experimental an-imals by a custom made adaptation of the BMS Mk3 unit and elec-trodes were calibrated immediately before each sample analysis.Outputs from the electrodes were displayed on a Radiometer PHM73. Arterial O2 content (CaO2) and Hb–O2 saturation were measuredand calculated as described above (see Section 2.4). Plasma CO2 con-tent (CplCO2) was measured according to Cameron (1971) at 40 °C.Arterial PCO2 (PaCO2) and plasma HCO3

− content ([HCO3−]pl) were cal-

culated using the Henderson–Hasselbalch equation adopting pK′ andCO2 solubility values calculated from Heisler (1989). Plasma osmolal-ity was measured in 10 μl plasma samples using a vapor pressure Os-mometer (5500; Wescor, Logan, UT, USA).

2.6. Metabolites

Immediately after collection, blood samples (0.5 ml) were split into2 subsamples (0.25ml each) used for plasma extraction (after centrifu-gation at 7000 RPM, 5min, 4 °C), or deproteinized plasma extraction. Inthe last case, the blood samplewas added to a small centrifuge tube con-taining 2 volumes of 0.6 M perchloric acid (PCA) with 2 mmol·l− 1 ofEDTA before separation by centrifugation. Deproteinized plasma sampleswere used tomeasure plasma glucose and lactate concentration,while in-tegral plasma was used for measuring osmolality, and protein and totallipids concentration. All sampleswere frozen in liquid nitrogen at themo-ment of extraction and then stored in a freezer at−85 °C until analysis.

Plasma soluble proteinwas assayed according to Lowry et al. (1951),using Folin–Ciocalteau reaction and bovine serum albumin as a stan-dard. For L-lactate and D-glucose determinations we used enzymaticmethods based on absorbance changes of nicotinamide adenine dinu-cleotide (NAD+) or nicotinamide adenine dinucleotide phosphate(NADP), at 340 nm and 25 °C, according to Keppler and Decker(1984), Bergmeyer (1984, 1985), and Passonneau and Lowry (1993),in previously neutralized plasma samples. All procedureswere conduct-ed in a plate spectrophotometer (SpectraMax 250, Molecular Device,

Table 1In vitro parameters associated to oxygen transport by the blood of fed and control South Ameri(control group). * Indicates the occurrence of significant differences compared to fasting (−24

Time pH (in vitro) P50 (kPa) nHa Bohr effect (ΔlogP50/ΔpH)

Fed−24 h 7.36 ± 0.02 8.64 ± 0.63 2.86 ± 0.15 −0.38 ± 0.02

7.76 ± 0.03 6.07 ± 0.39 2.81 ± 0.224 h 7.48 ± 0.03 6.88 ± 0.29* 2.76 ± 0.14 −0.39 ± 0.03

7.86 ± 0.04 4.86 ± 0.28* 2.76 ± 0.1148 h 7.34 ± 0.01 8.37 ± 0.55* 2.72 ± 0.11 −0.44 ± 0.04

7.76 ± 0.01 5.4 ± 0.16 2.64 ± 0.13

Control−24 h 7.38 ± 0.01 7.26 ± 0.28 2.65 ± 0.24 −0.53 ± 0.14

7.71 ± 0.06 5.26 ± 0.17 3 ± 0.4724 h 7.25 ± 0.03 8.1 ± 0.27* 2.71 ± 0.23 −0.62 ± 0.08

7.56 ± 0.07 5.29 ± 0.07 3.25 ± 0.3248 h 7.32 ± 0.05 8.23 ± 0.15* 3.05 ± 0.21 −0.61 ± 0.06

7.69 ± 0.04 4.97 ± 0.14 3.8 ± 0.56

Please cite this article as: Bovo, R.P., et al., Blood oxygen affinity increasesterrificus, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016/j.c

Sunnyvale, CA, USA), at least in duplicate, and the results wereexpressed in mg or μmol per ml of plasma. We confirmed the efficacyof all protocols by producing standard curves based on high purity re-agents (Sigma-Aldrich Corp., St. Louis, MO, USA).

For the quantification of total lipids we used standard proceduresmodified from Frings and Dunn (1970), Frings et al. (1972), andKnight et al. (1972), based on the phosphovanilin reaction.We calculat-ed total lipids from standard curves made with known volumes of codliver oil (Sigma-Aldrich Corp., St. Louis, MO, USA) processed as sample.All measurements were made in duplicate and expressed in milligramsper ml of plasma.

2.7. Statistics

ANOVA One Way RM with Holm–Sidak post hoc test was used tocompare measurements taken at fasting, and at 24 and 48 h after feed-ing (or supposed feeding in the case of control group) within the samegroup (fed or control), once the assumptions of normality and homo-scedasticity of variancewere verified. Differences between fed and con-trol, at any givenmoment, were verifiedwith t-test. All testswere basedon Sokal and Rohlf (1995) and the level of significance was 0.05. Allvalues are presented as mean ± standard error.

3. Results

3.1. Blood oxygen transport

Blood oxygen affinity in vitro was significantly increased 24 h afterfeeding, with a decrease (left-shift) in the P50 values for both pH, acid(F2,5 = 11,59; p ≤ 0.001; post-hoc test: p ≤ 0.001) and alkaline(F2,5 = 12.52; p ≤ 0,05; post-hoc test: p ≤ 0.001) (see Table 1). Forboth pH, the blood oxygen affinity decreased again 48 h after feeding,with P50 values increasing, i.e., shifting to the right side, and then show-ing a tendency to return to fasting values (although still exhibiting sig-nificant differences between fasting and 48 h after feeding for alkalinepH; post-hoc test: p≤ 0.02; Table 1). In the control group, blood oxygenaffinity remained unchanged following 24 and 48 h after the supposedfeeding in alkaline pH (F2,3 = 1.22; p = 0.35), but decreased signifi-cantly in acid pH (post hoc test: p b 0.05 for both comparisons,i.e., fasting vs 24 and fasting vs 48 h; Table 1).

Considering the significant increase in the blood oxygen affinity 24 hpost-feeding, we estimated (from values of the Bohr effect, see Table 1)oxygen dissociation curves (ODC's) for values of arterial pH in vivo forfasting rattlesnakes and 24 h postprandial (Fig. 1). For the fed group,the P50 in vivo was significantly higher 24 h after feeding (t = 8.05;d.f. = 5; p b 0.001) and did not differ in the control group (t = 2.73;

can rattlesnakes, Crotalus durissus terrificus. Mean ± S.E.M.; n = 6 (fed group) and n = 4h). pH was manipulated by equilibrating the blood with 2 and 4% CO2 (see Section 2).

[NTP] (mmol l−1) [NTP]/[Hb] Hct (%) Osmolality (mmol·kg−1)

2.56 ± 0.21 0.81 ± 0.12 20 ± 0.7 301.7±13.4

2.2 ± 0.29 0.88 ± 0.15 17.8 ± 1* 314.7±27.1

2.12 ± 0.39 0.88 ± 0.12 17.8 ± 0.6* 308.64±30.6

1.83 ± 0.2 0.47 ± 0.03 20.4 ± 1.1 314.7±21.7

1.6 ± 0.05 0.45 ± 0.04 20.1 ± 1 305.5±14.1

1.4 ± 0.12 0.38 ± 0.05 16.3 ± 1.2* 315.7±20.8

during digestion in the South American rattlesnake, Crotalus durissusbpa.2014.10.010

Fig. 1. Oxygen dissociation curves of the blood of the South American rattlesnake Crotalusdurissus terrificus estimated for arterial pH values in vivo, fasting (gray line) and 24 h(black line) after feeding. Circles mean PO2 values for which hemoglobin saturation wasestimated.

4 R.P. Bovo et al. / Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx

d.f. = 3; p = 0.07) considering the same period of time (Fig. 1). TheBohr effect remained unchanged for both fed (F2,5 = 0.51, p = 0.61)and control (F2,3 = 0.99, p = 0.42) rattlesnakes (Table 1).

3.2. Blood gases and acid–base parameters

Whereas arterial oxygen pressure (PaO2) did not change withtime in both fed (F2,5 = 2.14; p = 0.16) and control (F2,3 = 1.12;

Fig. 2. Effects of feeding on parameters related to blood oxygen transport in the South Americsignificant difference from the value measured at−24 h (i. e., before feeding). See list of abbre

Please cite this article as: Bovo, R.P., et al., Blood oxygen affinity increasesterrificus, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016/j.c

p = 0.38) groups, the arterial oxygen content (CaO2) was signifi-cantly lower after 48 h in both groups (post-hoc test: p ≤ 0.05 forall comparisons) (Fig. 2). Hb–O2 saturation was significantly higher24 h after feeding (post-hoc test: p ≤ 0.05; Fig. 2).

Hematocrit (Hct) showed a general pattern of decreases along theexperiments for both fed and control groups (p ≤ 0.05 in all cases ofsignificant differences; Table 1). Hemoglobin concentration ([Hb])was reduced after feeding (post-hoc test: p ≤ 0.05) but remained un-changed for the control group (F2,3 = 2.24; p = 0.18) (Fig. 2). NTPconcentrations did not change with time for both fed snakes(F2,5 = 3.03, p = 0.1) and control ones (F2,3 = 2.03, p = 0.21)(Table 1). The [NTP]/[Hb] ratio (F2,5 = 0.53, p = 0.6 for fed groupand F2,6 = 0.93, p = 0.46 for control) and the Hill index ofcooperativity (nHa) (for acid pH: F2,5 = 0.53, p = 0.6 for fed groupand F2,3 = 3.94, p = 0.08 for control group; for alkaline pH: F2,5 =0.27, p = 0.76 for fed group and F2,3 = 2.81, p = 0.13 for controlgroup) remained unchanged (Table 1).

Plasma CO2 content (CplCO2) and plasma bicarbonate concentra-tion ([HCO3

−]pl) were both increased 24 h after feeding (post-hoctest: p ≤ 0.05 for all cases of significant differences; Fig. 3). ArterialCO2 pressure (PaCO2) did not change with time for both fed(F2,5 = 0.79; p = 0.47) and control group (F2,3 = 1.49; p = 0.29)(Fig. 3). Arterial pH remained unchanged for both fed snakes(F2,5 = 1.98; p = 0.19) and unfed ones (F2,3 = 0.12; p = 0.88)(Fig. 3).

3.3. Metabolites

The concentration of plasma lipids for fed snakes increased signifi-cantly at 48 h post-feeding in comparison to fasting (p b 0.05), andremained unchanged for the control group (p = 0.13; Fig. 4). The

an rattlesnake, Crotalus durissus terrificus. Data is presented as mean ± SE. An * indicatesviations for the meaning of each variable.

during digestion in the South American rattlesnake, Crotalus durissusbpa.2014.10.010

Fig. 3. Effects of feeding on acid–base parameters in the blood of the South American rattlesnake, Crotalus durissus terrificus. Data is presented asmean± SE. An * indicates significant dif-ference from the value measured at−24 h (i. e., before feeding). See list of abbreviations for the meaning of each variable.

5R.P. Bovo et al. / Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx

concentrations of protein, lactate, glucose (Fig. 4), and osmolality(Table 1) in the plasma were not significantly altered for fed or controlsnakes (p N 0.05, in all cases).

4. Discussion

Digestion in snakes, particularly in those that feed infrequently onrelatively large prey, is a challenging enterprise in the sense that it im-poses a long period of quite elevated rates of aerobic metabolism to an-imals that typically function at low metabolic levels. The immediatesurrogates of this response are the accompanying adjustments in themultistep chain of the gas transport cascade. However, digestion isalso accompanied by other responses that bear the potential to interferewith gas transport, such as the case of the alkaline tide brought about bythe acid secretion to the stomach. As a consequence, meal digestion en-compasses concurrent changes in metabolic activity, cardiorespiratoryfunction, blood pH, gases, and metabolites, which can affect/be affectedby each other. In the following discussion, we tried to explore such as-pects for C. d. terrificus.

4.1. Hematological parameters

Hematocrit (Hct) and [Hb] were reduced during the rattlesnake di-gestion for both control and fed groups. Such decreases were previouslynoted in similar studies (Overgaard et al., 1999; Secor et al., 2001) andhad been generally accepted as a consequence of the serial blood sam-pling. However, as already pointed out by Overgaard and Wang(2002), sampling alone could not cause this level of Hct and [Hb]

Please cite this article as: Bovo, R.P., et al., Blood oxygen affinity increasesterrificus, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016/j.c

change. As also noted by those authors, large fluid shifts occurringduring meal digestion (see Starck and Beese, 2001), may alter watercontents on the intra and extracellular compartments and, at least par-tially, be involved in the observed changes in Hct and [Hb]. Intuitively,an increase in the O2 carrying capacity of the blood would be beneficialduring states of metabolic increase, as documented in fed dogs and am-phibians (Kurata et al., 1993;Wang et al., 1995; Busk et al., 2000a), or atleast that they remained unchanged, as in alligators (Busk et al., 2000b).The reasons forwhy this is not observed in fed snakes are still uncertain.The [NTP] decreased through the experiments but the [NTP]/[Hb] ratiodid not. Thus, the influence of changes in the allosteric interaction be-tween [NTP] and [Hb] on the blood oxygen affinity during the digestionof the rattlesnakes can be ruled out. This result is dissimilar from whatoccurs in P. molurus, in which the increase in Hb–O2 affinity can bemainly ascribed to a reduced [NTP] level (Overgaard and Wang, 2002).Therefore, the change noticed in the Hb–O2 affinity in rattlesnakes fol-lowing feeding is more dependent of changes in arterial pH, caused byan alkaline tide via Bohr effect, than previously noticed in P. molurus(see discussion below). Indeed, the sensitivity of blood oxygen affinityto changes in pH in C. d. terrificus (see Table 1) is considerably higherthan observed in P. molurus (Overgaard and Wang, 2002).

4.2. Oxygen transport

Blood oxygen affinity, calculated from pH values in vivo for fastingrattlesnakes, was lower (P50 = 59.69 mm Hg) than values compiledby Pough (1977) for 42 snake species. On the other hand, the Bohr Effect

during digestion in the South American rattlesnake, Crotalus durissusbpa.2014.10.010

Fig. 4. Plasma concentration of lactate, glucose, protein, and total lipids of fed and control South American rattlesnakes, Crotalus durissus terrificus. Mean ± S.E.M.; An * indicates the oc-currence of significant difference from the value measured at −24 h (i. e., before feeding).

6 R.P. Bovo et al. / Comparative Biochemistry and Physiology, Part A xxx (2014) xxx–xxx

for fasting and fed rattlesnakes did not differ substantially from thevalues also compiled by Pough (1980) for nine snake species.

The P50 in vivo for the rattlesnakes decreased from 7.93 kPa, duringfasting, to 6.33 kPa 24 h after feeding. This drop of 1.60 kPa was higherthan that found by Overgaard and Wang (2002) in P. molurus(=1.07 kPa) and it may be due to the occurrence of amore pronouncedalkaline tide in C. d. terrificus. In fact, while arterial pH of the rattlesnakesincreased from 7.55, during fasting, to 7.62 24 h after feeding, this vari-ation in P.moluruswas virtually absent (from7.60 for fasting individualsto 7.62 for 48 h after feeding; Overgaard and Wang, 2002). Therefore, amore pronounced alkaline tide associated to a higher sensitivity of theblood to changes in pH in the rattlesnakes may be responsible for thegreater changes in the post-prandial Hb–O2 affinity observed for thisspecies (present study) compared to P. molurus (Overgaard and Wang,2002).

Although the left shift of the oxygen dissociation curve during di-gestion of the rattlesnakes facilitates oxygen uptake in the lungs, itcould represent a possible problem during its delivery to the metabo-lizing tissues. This might indicate that during the postprandial meta-bolic increase, the oxygen uptake, rather than its delivery, is themost relevant factor to be adjusted by rattlesnakes. In this context, apossible explanation for this to occur can be a decrease in pulmonarytime transit for the blood during digestion due to increased heart rateand arterial pressure (see Wang et al., 2001b), which would impairadequate hemoglobin saturation because diffusion-limited pulmo-nary gas exchange. This effect may be specifically important consider-ing that the lungs of reptiles have low diffusion capacity (Glass, 1991;Hopkins et al., 1995) and, particularly, in the case of digesting snakes,

Please cite this article as: Bovo, R.P., et al., Blood oxygen affinity increasesterrificus, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016/j.c

a large and bulky prey item in the stomach may disturb lungventilation.

4.3. Arterial blood gases and acid base status

Despite the reduction in Hct and [Hb], CaO2 for fed snakes were keptalmost unaltered at 24 h post-feeding, which indicates a greater degreeof Hb–O2 saturation (see Fig. 2) and most likely is related to the incre-ment in Hb–O2 affinity discussed above. PaO2 did not change signifi-cantly on the course of 48 h post-feeding, which is in agreement withprevious studies (Wang et al., 1995; Busk et al., 2000b; Overgaard andWang, 2002). Digestion effects on the acid–base status of C. d. terrificusdid conform to the nowwell established pattern of ametabolic alkalosispartially compensated by a respiratory alkalosis (seeWang et al., 2001a,2005, and references therein). Thus, digesting snakes presented an in-crement in plasma [HCO3

−], whichwas counteracted by a concurrent in-crease in PaCO2 and, as a result, the increment in blood pH wasminimized (Fig. 3). Where the extra base load was originated from theimbalance associated to the acid gastric secretion and the incrementin PaCO2 was brought into play by a relative hypoventilation (Wanget al., 2005; Skovgaard et al., 2010).

4.4. Metabolites

Meal digestion did not cause changes in the plasma osmolality ofC. d. terrificus, which agrees with previous results in Python, Alligator,and anurans (Secor and Diamond, 1995; Busk et al., 2000a; Andersenet al., 2003). With the increase in acid secretion during digestion, the

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concomitant secretion of osmotic active solutes (as chloride) could leadto a reduction in extra-cellular fluids (Busk et al., 2000a,b). However,our results suggest thatwater balance is well regulated during digestionand probably depends on the interaction with other compartments inthe extra-cellular medium (see also Busk et al., 2000a).

Fed rattlesnakes up to 48 h post-feeding did not exhibit any signifi-cant change in the plasma content for protein and glucose. The meanvalues for both metabolites are within the range reported for othersnakes (Secor and Diamond, 1995), alligators (Busk et al., 2000b), andanurans (Busk et al., 2000a) during the transition from short fasting topostprandial conditions. This response is intriguing since the increasein carbohydrates and amino-acids uptake capacity, driven by the up-regulation on intestinal membrane transporters (see Secor andDiamond, 1995; Ott and Secor, 2007), could be expected to alter the cir-culating levels of glucose and protein. Perhaps, due to the proteinaceousnature of the rattlesnake meals, changes in glucose would only happenon later course coupled with gluconeogenesis. Finally, as we were ableto follow the snakes only up to 48 h post-feeding, it is possible that in-testinal absorptionwas too incipient at these very initial stages of diges-tion. Indeed, a rattlesnake digesting a meal under the conditions of thisstudy will take approximately at least 73 h to fully complete the SDA(Andrade et al., 1997).

Plasma lactate remained unchanged in fed rattlesnakes and this is ingood agreementwith previous results reported for other reptiles (Wanget al., 2005; McCue, 2006) corroborating the idea that SDA, even in ex-ceptional cases as infrequently feeding snakes, is almost entirely sup-ported by the aerobic pathway. Circulating levels of lipids weresignificantly increased during the digestion of C. d. terrificus. Such resultis most likely linked to the mobilization of lipids during the intestinalup-regulation that follows feeding in infrequently feeding animals(McCue, 2010). As Starck et al. (2004) described for Python regius, theincorporation of lipid droplets into the enterocytes are an importantchange observed on the gastrointestinal tract after fasting snakesbeing fed.

Acknowledgments

Financial support was provided by Sao Paulo Research Founda-tion (FAPESP) (grants 2004/03760-7 to RPB, 2004/05469-8 to JEC,and 2000/08296-6 to DVA), National Council for Scientific and Tech-nological Development (CNPq) (grant 302045/2012-0 to DVA), andFundação para o Desenvolvimento da Unesp (Fundunesp) (grant00077/03-DFP to DVA). We are grateful to Augusto S. Abe forgranting access to animals and equipment, and also to MarshallMcCue and one anonymous reviewer who contributed appreciablyto improve the quality of our manuscript.

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