Temperature and photoperiod as environmental cues affect ... · Temperature and photoperiod as environmental cues affect body mass and thermoregulation in Chinese bulbuls, Pycnonotus

RESEARCH ARTICLE

Temperature and photoperiod as environmental cues affect bodymass and thermoregulation in Chinese bulbuls PycnonotussinensisShi-Nan Hu1 Ying-Yang Zhu1 Lin Lin1 Wei-Hong Zheng12 and Jin-Song Liu12

ABSTRACTSeasonal changes in temperature and photoperiod are importantenvironmental cues used by small birds to adjust their body mass(Mb) and thermogenesis However the relative importance of thesecues with respect to seasonal adjustments in Mb and thermogenesisis difficult to distinguish In particular the effects of temperature andphotoperiod on energy metabolism and thermoregulation are not wellknown in many passerines To address this problem we measuredthe effects of temperature and photoperiod on Mb energy intakeresting metabolic rate (RMR) organ mass and physiological andbiochemical markers of metabolic activity in the Chinese bulbul(Pycnonotus sinensis) Groups of Chinese bulbuls were acclimated ina laboratory to the following conditions (1) warm and longphotoperiod (2) warm and short photoperiod (3) cold and longphotoperiod and (4) cold and short photoperiod for 4 weeks Theresults indicate that Chinese bulbuls exhibit adaptive physiologicalregulation when exposed to different temperatures and photoperiodsMb RMR gross energy intake and digestible energy intake werehigher in cold-acclimated than in warm-acclimated bulbuls and in theshort photoperiod than in the long photoperiod The resultantflexibility in energy intake and RMR allows Chinese bulbulsexposed to different temperatures and photoperiods to adjust theirenergy balance and thermogenesis accordingly Cold-acclimatedbirds had heightened state-4 respiration and cytochrome c oxidaseactivity in their liver and muscle tissue compared with warm-acclimated birds indicating the cellular mechanisms underlying theiradaptive thermogenesis Temperature appears to be a primary cuefor adjusting energy budget and thermogenic ability in Chinesebulbuls photoperiod appears to intensify temperature-inducedchanges in energy metabolism and thermoregulation

KEY WORDS Acclimation Resting metabolic rate Cytochrome coxidase Mitochondria State-4 respiration

INTRODUCTIONOrganisms can generally adjust their morphology physiology andbehavior in response to changing environmental conditions(Swanson 1991a Zheng et al 2008ab 2014a) resulting inphenotypic changes that are reversible temporary and repeatable

(Piersma and Drent 2003 McKechnie et al 2006) In birdsphenotypic flexibility in metabolic power output is an importantcomponent of their thermoregulatory responses to the periodicallyelevated energy requirements they experience in seasonalenvironments (McKechnie et al 2007) The capacity forthermogenesis and energy intake is particularly important for thesurvival of small birds in winter (Swanson 2010) Temperature andphotoperiod are considered to be the most important environmentalfactors influencing an animalrsquos seasonal thermoregulation anddriving the evolution of a suite of morphological physiological andbehavioral adaptations (Zheng et al 2013a Swanson et al 2014Zhou et al 2016) Seasonal changes in temperature andphotoperiod are important environmental cues that small birds useto adjust morphological and physiological parameters such as bodymass (Mb) energy intake and thermogenic capacity (Swanson1990 Zheng et al 2008a Wu et al 2014a) Winter is anenergetically stressful period for birds living in temperate zonesbecause the cost of thermoregulation increases while food qualityand availability are reduced (Yuni and Rose 2005 Petit et al2014) To cope with this relatively large temperate resident birdslike ptarmigans (Lagopus spp) reduced their thermal conductancein winter partly through increasing their feather insulation and insome cases by increasing subcutaneous fat (Mortensen and Blix1986 Lees et al 2010) However the size of many bird specieslimits the effectiveness of such adjustments rather than reducethermal conductance small birds tend to cope with coldenvironments by increasing their capacity for thermogenesis(Swanson 1991ab Zheng et al 2008b 2014a)

Resting metabolic rate (minimum maintenance metabolic rateRMR) in small birds is one of the fundamental physiologicalstandards for assessing the energy cost of thermoregulation(Clapham 2012) It has been demonstrated that a birdrsquos Mbenergy balance and RMR are all affected by temperature andphotoperiod (McKechnie 2008 McKechnie and Swanson 2010Wu et al 2014b) Low ambient temperature can increase the RMRof some birds (Tieleman et al 2003 McKechnie et al 2007)Short photoperiod either alone or in combination with cold canalso increase metabolic thermogenesis in birds (Saarela andHeldmaier 1987 Ni et al 2010 Swanson et al 2014) Thissuggests that temperature and photoperiod play an important role inmediating the metabolic rates of small birds (Swanson et al 2014)Under basal metabolic conditions the liver has been shown tocontribute 20ndash25 of total heat production in animals (Villarinet al 2003 Zheng et al 2008b) Skeletal muscle makes up nearly40 of the total Mb of birds and plays a key role in thermogenesis(Weber and Piersma 1996 Veacutezina et al 2006 2007) Adjustmentof cellular aerobic capacity by modulating the activities of keycatabolic enzymes in oxidative pathways may also thereforecontribute to the physiological phenotypes of small birds (LiknesReceived 14 July 2016 Accepted 13 December 2016

1School of Life and Environmental Sciences Wenzhou University Wenzhou325035 China 2Department of Biology Zhejiang Provincial Key Lab forSubtropical Water Environment and Marine Biological Resources ProtectionWenzhou 325035 China

Author for correspondence (ljswzueducn)

J-SL 0000-0002-1305-9955

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copy 2017 Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220 844-855 doi101242jeb143842

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and Swanson 2011 Swanson et al 2014 Zheng et al 2013b2014a) Variation in cellular metabolic intensity is often measuredby examining variation in state-4 respiration (reflecting oxidativephosphorylation capacity) or cytochrome c oxidase (COX) activity(a key regulatory enzyme of oxidative phosphorylation) (Zhenget al 2008b 2014ab)Chinese bulbuls Pycnonotus sinensis (Gmelin 1789) are small

passerine birds that inhabit vast areas of eastern and southern Asiaincluding central southern and eastern China (Zheng and Zhang2002) Elevated winter RMR in bulbuls is associated with elevatednutritional and exercise organ masses and heightened respiratoryenzyme activity in liver and muscle (Zheng et al 2008a 20102014a) We selected Chinese bulbuls as a study species becausethey are resident in Zhejiang Province where we are based becauseglobal warming appears to have allowed the species to colonizenortheastern and northwestern China and because previous studies(Zhang et al 2008 Zheng et al 2008a 2010 Ni et al 2010)provide critical background information required for our researchHowever the Chinese bulbul commonly encounters coldtemperatures and short photoperiods but the physiological andbiochemical mechanisms are unknown In this study we comparedselected morphological physiological and biochemical indices ofwild-caught Chinese bulbuls to different combinations oftemperature (warm and cold) and photoperiod (long day and shortday) We hypothesized that cold exposure and short day are keyfactors driving metabolic flexibility in Chinese bulbuls We alsohypothesized that there would be an interaction betweentemperature and photoperiod such that the greatest treatmenteffects would be observed in birds acclimated to both coldtemperatures and short days We predicted that Chinese bulbulsexposed to cold temperatures and short days would increase theirRMR and respiratory enzyme activity by and quantifying theseadjustments we aimed to improve understanding of themorphological physiological and biochemical responses of smallbird species to cold temperatures and short photoperiods

MATERIALS AND METHODSAnimalsThe Chinese bulbul is a common resident bird in Zhejiang Province(MacKinnon and Phillipps 2000) The Chinese bulbuls used in theexperiments were captured with mist nets in Wenzhou cityZhejiang Province China in June 2011 The climate in Wenzhouis warmndashtemperate with an average annual rainfall of 1700 mmacross all months with slightly more precipitation during winter andspring Mean daily maximum temperature ranges from 39degC in Julyto 8degC in January The mean temperature from June to August is32degC (Zheng et al 2008a) We determined bulbulMb to the nearest01 g immediately upon capture with a Sartorius balance (modelBT25S) We transported birds to the laboratory on the day of capture

and kept them in outdoor cages (50times30times20 cm) for 1 or 2 daysunder natural ambient temperature (28plusmn1degC) and photoperiodconditions before measurements began Food and water weresupplied ad libitum Birds were then kept in individual cages for atleast 2 weeks after which 28 birds were randomly assigned to one offour treatment groups (1) warm and long photoperiod (WL 30degC16 h light8 h dark) (2) warm and short photoperiod (WS 30degC 8 hlight16 h dark) (3) cold and long photoperiod (CL 10degC 16 hlight8 h dark) and (4) cold and short photoperiod (CS 10degC 8 hlight16 h dark) Each group of birds was acclimated to its respectivetreatment for 4 weeks (Ni et al 2010 Zheng et al 2013a) Allexperimental procedures were approved by the Animal Care andUse Committee of Wenzhou City Zhejiang Province China

Measurement of metabolic rateMetabolic rate of the birds was estimated by measuring their oxygenconsumption in an open-circuit respirometry system (S-3AI AEITechnologies Pittsburgh PA USA) Metabolic chambers were15 l in volume and made of plastic A perch was provided for thebird to stand on (Smit and McKechnie 2010 Zheng et al 2014a)Chamber temperature was regulated by a temperature-controlledcabinet (BIC-300 artificial climate incubator Shanghai BoxunMedical Biological Instrument Corp China) capable of regulatingtemperature to plusmn05degC Water vapor and CO2 were scrubbed fromthe air passing through the chamber in a silica gelsoda limesilicacolumn before passing through the oxygen analyzer We measuredthe oxygen content of excurrent gas from metabolic chambers withan oxygen sensor (N-22M AEI Technologies) We used a flowcontrol system (R-1 AEI Technologies) to set the flow of excurrentgas to 300 ml minminus1 during metabolic rate measurements Thismaintained a fractional concentration of O2 in the respirometrychamber of about 20 calibrated to plusmn1 accuracy with a generalpurpose thermal mass flow-meter (TSI Series 4100 TSI IncShoreview MN USA) (McNab 2006) Oxygen consumption rateswere measured at 30plusmn05degC which is within the thermal neutralzone of Chinese bulbuls (Zheng et al 2008a) Baseline O2

concentrations were obtained before and after each test (Li et al2010) All measurements of gas exchange were obtained during theresting phase of the birdsrsquo circadian cycles (between 2000 h and2400 h) in darkened chambers when individual birds couldreasonably be expected to be postabsorptive Resting metabolic rate(RMR) is the energy required to perform vital body functions whilethe body is at rest Because it is doubtful that true RMRs can beachieved in the laboratory the term RMR is often used to refer tosuch measurements even when the standard conditions for RMRhave been met (Swanson 2010) Food was removed 4 h before eachmeasurement to minimize the heat increment associated withfeeding We first ensured that birds were perching calmly in thechamber and started recording oxygen consumption at least 1 hlater Each animal was generally in the metabolic chamber for atleast 2 h The data obtained were used to calculate 5 min runningmeans of instantaneous oxygen consumption over the entire testperiod using eqn 2 of Hill (1972) The lowest 5 min mean recordedover the test period was considered the RMR (Smit andMcKechnie2010) All values for oxygen consumption were expressed asml O2 hminus1 and corrected to STPD conditions (Schmidt-Nielsen1997) Body temperature was measured during metabolicmeasurements using a lubricated thermocouple inserted into thecloaca to a depth of 1ndash2 cm a depth at which a slight withdrawal ofthe thermocouple did not cause a change in the readingThermocouple outputs were digitized using a thermocouple meter(Beijing Normal University Instruments Co) Mb was measured to

List of symbols and abbreviationsCL cold and long photoperiodCOX cytochrome c oxidaseCS cold and short photoperiodDEI digestible energy intakeFE feces energyGEI gross energy intakeMb body massRMR resting metabolic rateWL warm and long photoperiodWS warm and short photoperiod

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the nearest 01 g before and after experiments and mean Mb wasused in calculations All measurements were made daily between2000 h and 2400 h

Energy budgetWe regarded digestible energy intake as an index of total dailyenergy expenditure Food and water were available ad libitumthroughout the experiment and replenished daily We collected foodresidues and feces once for 3 days prior to temperature andphotoperiod acclimation (week 0) and thereafter weekly (every7 days) throughout the 4 week experimental period We separatedthe residues manually and oven-dried then at 60degC to constant massWe then determined their caloric content with a C200 oxygen bombcalorimeter (IKA Staufen im Breisgau Germany) We calculatedgross energy intake (GEI) feces energy (FE) digestible energyintake (DEI) and energy digestibility according to the methodsdescribed in Grodzinski and Wunder (1975) and Ni et al (2010)

GEI = dry food intake caloric value of dry food eth1THORNwhere GEI is in kJ dayminus1 dry food intake is in g dayminus1 and thecaloric value of dry food is in kJ gminus1

FE = dry mass of feces caloric value of dry feces eth2THORNwhere FE is in kJ dayminus1 dry mass of feces is in g dayminus1 and thecaloric value of dry feces is in kJ gminus1

DEI frac14 GEI FE eth3THORN

Digestibility frac14 DEI=GEI 100 eth4THORN

Measurement of organ massesBirds were killed by cervical dislocation at the end of the 4 weekexperimental period and their brain heart lungs liver kidneysstomach small intestine rectum and pectoral muscle extracted andweighed to the nearest 01 mg Sub-samples of liver and musclewere used for the preparation of mitochondria (Zheng et al 2014a)We dried internal organs including the remaining part of the liverand muscle to a constant mass over 2 days at 75degC after whichthese were reweighed to the nearest 01 mg (Williams and Tieleman2000 Liu and Li 2006)

Preparation of mitochondriaLiver and pectoral muscle sub-samples were cleaned of anyadhering tissue blotted dry and weighed before being placed inice-cold sucrose-buffered medium Both liver and pectoral musclesamples were then coarsely chopped with scissors after which liversamples were rinsed and resuspended in 5 volumes of ice-coldmedium (250 mmol lminus1 sucrose 5 mmol lminus1 TrisHCl 1 mmol lminus1

MgCl2 and 05 mmol lminus1 EDTA pH 74 4degC) (Rasmussen et al2004) Pectoral muscle samples were treated with proteinase for5ndash10 min then resuspended in 10 volumes of ice-cold medium(100 mmol lminus1 KCl 50 mmol lminus1 TrisHCl 5 mmol lminus1 MgSO4 and1 mmol lminus1 EDTA pH 74 4degC) Liver and muscle preparationswere then homogenized in a Teflonglass homogenizerHomogenates were centrifuged at 600 g for 10 min at 4degC in anEppendorf centrifuge and resultant pellets of nuclei and cell debrisdiscarded The supernatants were then centrifuged at 12000 g for10 min at 4degC The resultant pellets were suspended respun at12000 g resuspended and the final pellets obtained were placed inice-cold medium (21 wv for liver and 41 wv for muscle) (Zheng

et al 2008b 2013b) We determined the protein content ofmitochondria by the Folin phenol method with bovine serumalbumin as standard (Lowry et al 1951)

Mitochondrial respiration and enzyme activityState-4 respiration in liver and muscle mitochondria was measuredat 30degC in 196 ml of respiration medium (225 mmol lminus1 sucrose50 mmol lminus1 TrisHCl 5 mmol lminus1 MgCl2 1 mmol lminus1 EDTA and5 mmol lminus1 KH2PO4 pH 72) with a Clark electrode (DW-1Hansatech Instruments Ltd Kingrsquos Lynn UK) essentially asdescribed by Estabrook (1967) State-4 respiration was measuredover a 1 h period under substrate-dependent conditions withsuccinate as the substrate (Zheng et al 2013b 2014a) Theactivity of COX in liver and muscle was measured polarographicallyat 30degC using a Clark electrode according to Sundin et al (1987)We express state-4 respiration and COX activity measurements asmean mass-specific values (micromol O2 minminus1 gminus1 tissue) (Wiesingeret al 1989 Zheng et al 2013b 2014a)

StatisticsStatistical analyses were performed using the SPSS package(version 120) All variables were tested for normality using theKolmogorovndashSmirnov test Non-normally distributed data werenormalized by transforming them to their natural logarithm Two-way repeated-measures (RM)-ANOVA was used to determine thesignificance of changes in Mb GEI FE DEI and digestibility overtime Tukeyrsquos post hoc tests were used to determine the significanceof differences among different days of acclimation The significanceofMb and digestibility on the same day among different groups wasevaluated with a two-way ANOVA Direct comparisons of GEI FEand DEI on the same day among different groups were made with atwo-way ANCOVAwithMb as the covariate To test whether RMRdiffered between temperature-acclimated birds and also betweenphotoperiod-acclimated birds we performed an ANCOVA usingthe Tukeyrsquos post hoc test for multiple comparisons among groupsThis design used the treatment (cold warm long photoperiod andshort photoperiod) as the independent variable and log RMR as thedependent variable (Maldonado et al 2009) Because total RMRwas correlated with Mb the effect of Mb was removed using Mb asthe covariate For the analysis of organ masses we used Mb minuswet organ mass for the organ in question to avoid statisticalproblems with part-whole correlations (Christians 1999) Apreliminary model was run to test for homogeneity of slopes ofthe dependent variable versus the covariate among treatments Theeffects of temperature and photoperiod on mitochondrial proteinmitochondrial state-4 respiration and COX activity in liver andmuscle were also analyzed with a Tukeyrsquos post hoc test for multiplecomparisons among groups Allometric and residual correlationswere used to evaluate the relationship between RMR and dry organmass (controlled for Mb minus wet mass of the organ) and least-squares linear regression to evaluate the relationship between logMb

and log RMR between log Mb log GEI and log DEI between logRMR log GEI and log DEI and between log RMR log state-4respiration and log COX activity Data are reported as meansplusmnsem

RESULTSMb and RMRPrior to acclimation no significant difference in Mb was foundamong the four treatment groups (ANOVA F324=0399 P=0755Fig 1A) However Mb was significantly affected by temperature(RM-ANOVA F496=4069 P=0015) and photoperiod (RM-ANOVA F496=4789 P=0007) but not the interaction between

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temperature and photoperiod (RM-ANOVA F496=1478 P=0233)during acclimation (Fig 1A) Mb was significantly higher in coldgroups than in warm groups (P=0021) and in short photoperiodthan in long photoperiod (P=0048) Comparisons of RMRmeasured at 30degC revealed that means were statistically differentamong groups (ANOVA mass-independent F324=3056 P=0048ANCOVA total F323=4404 P=0014 Fig 1B) RMR wassignificantly higher in the cold group than in the warm group(P=0001) and averaged 18 higher than that of warm-acclimatedbirds Homogeneity of slopes test showed that slopes weresignificantly different for temperature acclimation An analysis ofthe temperature acclimation data for the long photoperiod groupsindicated that log RMR showed a poor and non-significantassociation with log Mb (r2=0102 P=0266 Fig 1C) When alltemperature acclimation data were pooled for the short photoperiodlog RMR showed a positive and significant correlation with logMb

(r2=0503 P=0045 Fig 1D)

Energy intake and digestibilityGEI was significantly affected by temperature (RM-ANOVAF496=23251 Plt0001) photoperiod (RM-ANOVA F496=2979P=0023) and the interaction between temperature and photoperiod(RM-ANOVA F496=5732 Plt0001 Fig 2A) No groupdifferences in GEI were found prior to cold acclimationHowever a significant decrease in GEI (Plt0001) was apparent inthe warm groups after day 7 of acclimation to 30degC and thesedecreases were sustained for the 28 day duration of the experiment(Fig 2A) When all temperature acclimation data were pooled forlong photoperiod there was no significant relationship between logGEI and log Mb (r2=0145 P=0179 Fig 3A) there was asignificant positive relationship between log GEI and log RMR(r2=0448 P=0009 Fig 3C) An analysis of the temperatureacclimation data for the short photoperiod indicated that log GEIshowed a positive and significant correlation with logMb (r

2=0445P=0009 Fig 3B) and log RMR (r2=0402 P=0014 Fig 3D)FE was also significantly affected by temperature (RM-ANOVA

F496=15495 Plt0001) and there was a significant interaction

between temperature and photoperiod (RM-ANOVA F496=4037P=0009 Fig 2B) Bulbuls acclimated to 30degC had a significantlysmaller FE than those acclimated to 10degC (ANCOVA day 7 14 21and 28 all Plt0001 Fig 2B)

Temperature (RM-ANOVA F496=21267 Plt0001) andphotoperiod (RM-ANOVA F496=4208 P=0004) alsosignificantly affected DEI and there was a significant interactionbetween temperature and photoperiod (RM-ANOVA F496=5769Plt0001 Fig 2C) A significant difference in DEI (Plt0001) wasalso apparent in the warm groups after day 7 of acclimation to 30degCand these differences were sustained for the 28 day duration of theexperiment (Fig 2C) An analysis of the temperature acclimation datafor the long photoperiod indicated that log DEI had a non-significantassociation with log Mb (r2=0058 P=0408 Fig 4A) but asignificant association with log RMR (r2=0325 P=0033 Fig 4C)There was a significant positive linear relationship between log DEIand logMb (r2=0426 P=0011 Fig 4B) and between log DEI andlog RMR (r2=0423 P=0012 Fig 4D) for the short photoperiod

Digestibility was significantly affected by temperature (RM-ANOVA F496=2208 Plt0001) photoperiod (RM-ANOVAF496=4478 Plt0001) and the interaction between temperatureand photoperiod (RM-ANOVA F496=2648 P=0038 Fig 2D)Bulbuls acclimated to the warm showed a greater energydigestibility than those under cold conditions after day 14(Plt0001) and these increases were sustained (Plt0001) for the28 day duration of the experiment (Fig 2D)

Organ and muscle massThe ANCOVA revealed that organ masses were affecteddifferentially by the experimental treatment liver mass wassignificantly affected (wet mass F323=4341 P=0015 dry massF323=6061 P=0003) and the post hoc analysis revealed thatbirds in the CS treatment had the heaviest livers among thegroups (Table 1) Stomach mass was also affected by theexperimental treatment (wet mass F323=7988 P=0001 drymass F323=5454 P=0006) Here the post hoc analysisrevealed that cold-acclimated birds had larger stomachs than

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Fig 1 Comparison of body mass andmetabolic rate in Chinese bulbuls(Pycnonotus sinensis) acclimated todifferent temperature andphotoperiod for 4 weeks Birds wereacclimated to warm (W) or cold (C)temperature with a long (L) or short (S)photoperiod (A) Body mass (Mb)(B) Resting metabolic rate (RMR) andRMR as a function ofMb (CD) RMR asa function of Mb following acclimation toa long (C) or short (D) photoperiod Theallometric equation representing thelinear curve for all birds isRMR=078Mb

084 and RMR=066Mb094

for temperature acclimation in the shortphotoperiod Data are meansplusmnsembars with different letters indicatesignificant differences Plt005

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warm-acclimated birds (Table 1) The mass of both the smallintestine and the total digestive tract was affected by theexperimental treatment (small intestine wet mass F323=5253P=0007 dry mass F323=6900 P=0002 total digestive tract

wet mass F323=6837 P=0002 dry mass F323=7889P=0001) and the post hoc analysis showed that cold-acclimated birds had heavier small intestines and total digestivetracts than warm-acclimated birds (Table 1) Muscle heart lung

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Fig 2 Comparison of gross energy intake feces energy and digestible energy intake in Chinese bulbuls acclimated to different temperature andphotoperiod for 4 weeks (A) Gross energy intake (GEI) (B) Feces energy (FE) (C) Digestible energy intake (DEI) (D) Digestibility Data are meansplusmnsemPlt0001 Treatment abbreviations as for Fig 1

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Fig 3 Correlations between Mb and GEIand RMR and GEI in Chinese bulbulsacclimated to different temperature andphotoperiod for 4 weeks (AB) Mb and GEIin the long (A) and short (B) photoperiod(CD) RMR and GEI in the long (C) andshort (D) photoperiod The allometricequations representing the linear curve for allbirds are Mb=098GEI019 andRMR=107GEI041 RMR=086GEI058 fortemperature acclimation in the longphotoperiod and Mb=095GEI021 andRMR=140GEI027 for temperatureacclimation in the short photoperiodTreatment abbreviations as for Fig 1

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kidney and rectum mass were not affected by temperature andphotoperiod (Table 1) Allometric relationships for the all-birdspooled data indicated that log dry organ mass was positivelycorrelated with log RMR in the case of the heart liver smallintestine and total digestive tract (Table 2) The correlationanalysis between log dry organ mass and log RMR of thetemperature acclimation data for the long photoperiod showed apositive and significant association only for the small intestineand the total digestive tract These analyses also revealed asignificant association between these variables in the lung liversmall intestine and total digestive tract in temperature acclimationdata for the short photoperiod (Table 2) The residuals of dryorgan and RMR against Mb minus wet mass of the organ onlyshowed a positive and significant association in lung and smallintestine in the all-birds pooled data (Table 2)

Protein content mitochondrial respiration and COX activityin liverAlthough mitochondrial protein content of the liver was not affectedby the experimental treatment (F324=0741 P=0538 Fig 5A)state-4 respiration was significantly affected (F324=14378Plt0001 Fig 5B) and the post hoc analysis revealed that CS-acclimated birds had the highest mitochondrial respiration COXactivity was also significantly affected by the experimental treatment(F324=5750 P=0004 Fig 5C) and the post hoc analysis showedthat the cold-acclimated birds had heightened COX activitycompared with warm-acclimated birds When all temperatureacclimation data were pooled for the long photoperiod log RMRshowed a positive and significant correlation with log mitochondrialstate-4 respiration (r2=0482 P=0006 Fig 6A) but not with logCOX activity (r2=0127 P=0210 Fig 6C) During temperatureacclimation in the short photoperiod there was no significantrelationship between log RMR and log mitochondrial state-4respiration (r2=0094 P=0287 Fig 6B) but there was asignificant positive relationship between log RMR and log COXactivity (r2=0457 P=0008 Fig 6D)

Protein content mitochondrial respiration and COX activityin muscleThe mitochondrial protein content of skeletal muscle was not affectedby the experimental treatment (F324=0550 P=0653 Fig 5A)However state-4 respiration and COX activity in muscle weresignificantly affected by the experimental treatment (state-4respiration F324=4602 P=0011 COX activity F324=3478P=0032 Fig 5BC) and post hoc analysis revealed that cold-acclimated birds had heightened activity of respiratory enzymescompared with warm-acclimated birds An analysis of the temperatureacclimationdata for the longphotoperiod indicated that logRMRhad anon-significant association with log state-4 respiration (r2=0080P=0328 Fig 7A) and logCOXactivity (r2=0136P=0195 Fig 7C)There was a significant positive relationship between log RMR andlog state-4 respiration (r2=0456 P=0008 Fig 7B) but not with logCOX activity (r2=0127 P=0044 Fig 7D) during temperatureacclimation for the short photoperiod

DISCUSSIONTemperature and photoperiod have been shown to affect awide varietyof morphological physiological and behavioral functions in birds(Swansonet al 2014Zhouet al 2016) In thepresent study inChinesebulbuls we found that temperature and photoperiod had significanteffects on the Mb energy budget RMR and organ mass of Chinesebulbuls all of which increased significantly in birds acclimated to acolder ambient temperature and shorter day length These birds alsounderwent a significant increase in mitochondrial respiration and COXactivity in liver and muscle following cold acclimation

Effects of temperature and photoperiod on morphology andphysiology in Chinese bulbulsColder temperatures or shorter photoperiods can cause increasedsurface heat loss in birds (Saarela and Heldmaier 1987 Tielemanet al 2003) Proper adjustment of the morphology physiology andbehavior of small birds helps to ensure their survival in seasonalenvironments (Swanson 2010 Zheng et al 2014a) Changes in

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Fig 4 Correlations between Mb and DEIand RMR and DEI in Chinese bulbulsacclimated to different temperature andphotoperiod for 4 weeks (AB) Mb and DEIin the long (A) and short (B) photoperiod(CD) RMR and DEI in the long (C) andshort (D) photoperiod The allometricequations representing the linear curve for allbirds are Mb=094DEI024 andRMR=092DEI053 RMR=077DEI060 fortemperature acclimation in the longphotoperiod and Mb=084DEI021 andRMR=123DEI038 for temperatureacclimation in the short photoperiodTreatment abbreviations as for Fig 1

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Mb especially in small birds are considered an adaptive strategyessential for survival (Cooper 2000) Mb can be influenced by anumber of environmental factors including temperaturephotoperiod the quality and abundance of food andphysiological status (Chamane and Downs 2009 Ni et al 2010Zheng et al 2014a) Some small birds that live in seasonalenvironments increase their Mb in winter (McKechnie 2008McKechnie and Swanson 2010) by increasing their fat depositsandor lean mass (Swanson 1991a Piersma et al 1996) There isevidence to suggest that seasonal variation in the Mb of Chinesebulbuls is due at least in part to seasonal variation in fat depositsand lean mass (Zheng et al 2014a Wu et al 2014a) Our resultsare consistent with those of previous reports on the response ofChinese bulbuls to seasonal change (Zheng et al 2008a 2014a)and the Mb of bulbuls was higher in cold groups than in warmgroups and in short photoperiod than in long photoperiodIncreased Mb is thought to be associated with cold resistancebecause this reduces heat loss by decreasing an animalrsquos surfacearea to volume ratio (Christians 1999 Zheng et al 2008aChamane and Downs 2009 Swanson 2010) Increased Mb mayinfluence thermogenic demands and contribute to the observedincrease in RMR as indicated by the positive correlation betweenthese two variables (see below) The increase in RMR commonlyobserved under such conditions is thought to be an adaptiveresponse to cold (Swanson 2001 Veacutezina et al 2006 McKechnieet al 2007 Wiersma et al 2007) Although we attempted tominimize the potential confounding effects of circadian rhythms bytesting experimental subjects simultaneously we cannot exclude thepossibility that our results were confounded by variation in the timeof testing which was conducted between 2000 h and 2400 hNonetheless the fact that we detected significant differences amongthe four treatment groups suggests that the effects of temperatureand photoperiod on thermogenesis were unaffected by between-birddifferences in circadian rhythm Our results show that bothtemperature and photoperiod are important environmental cues

inducing thermogenesis in Chinese bulbuls Mass-independent andtotal RMR in the cold-acclimated treatment groups were 16 and18 higher respectively than in the warm-acclimated groupsMass-independent and total RMR in the short photoperiodtreatment groups were 6 and 7 higher respectively than inthe long photoperiod groups However the fact that temperature hada more pronounced effect than photoperiod on RMR is consistentwith the documented role of winter temperature as a proximate cuefor regulating thermogenic capacity in small birds includingChinese bulbuls in cold winter climates (Zheng et al 2008a 20102014a) The ability to adjust energy intake to compensate for theenergy expended in thermogenesis is essential for survival(Hegemann et al 2012) Environmental temperature andphotoperiod can however alter birdsrsquo energy intake (Kendeigh1945 Stokkan et al 1986 Lou et al 2013 Wu et al 2014b) Wefound that Chinese bulbuls in the CS group had the highest Mb andthat this was consistent with changes in GEI and DEI These resultssuggest that acclimation to colder temperatures and shorterphotoperiods increases energy consumption because of theincreased energy required to maintain body temperature (Cain1973 Syafwan et al 2012) If different physiological systemscompete for energy this would be expected to affect heatproduction This is exactly what we found Chinese bulbulsacclimated to a colder temperature and shorter photoperiod for4 weeks increased their RMR liver stomach and small intestinemass and liver and muscle mitochondrial state-4 respiration andCOX activity compared with those acclimated to a warmertemperature and longer photoperiod In view of the mass-specificenergy metabolism of these organs andor tissues the observedincreases in GEI and DEI are not surprising

McKechnie (2008) and Swanson (2010) found that metabolicrates are regulated via three major physiological and morphologicalpathways one of which is changes in internal organ and musclemass Organs such as the liver brain heart and kidney collectivelyconsume about 60 of an endothermrsquos total energy expenditure and

Table 1 Effects of temperature and photoperiod acclimation on organ mass in Chinese bulbuls (Pycnonotus sinensis)

Long day Short day

Warm Cold Warm Cold

Sample size 7 7 7 7Organ wet mass (g)Muscle 369plusmn026 357plusmn025 318plusmn025 375plusmn028Brain 090plusmn002 096plusmn002 093plusmn002 088plusmn003Heart 035plusmn002 030plusmn002 033plusmn002 035plusmn002Lung 026plusmn001 024plusmn001 024plusmn001 025plusmn001Liver 095plusmn010a 129plusmn010bc 115plusmn010ab 148plusmn011c

Kidney 030plusmn002 033plusmn002 030plusmn002 037plusmn002Stomach 037plusmn002a 052plusmn002c 041plusmn002ab 047plusmn003bc

Small intestine 085plusmn011a 121plusmn010bc 107plusmn010ab 149plusmn012c

Rectum 011plusmn001a 014plusmn001ab 012plusmn001a 016plusmn001b

Total digestive tract 133plusmn012a 187plusmn012bc 160plusmn012ab 211plusmn013c

Organ dry mass (g)Muscle 101plusmn008 095plusmn008 089plusmn007 104plusmn008Brain 020plusmn001 020plusmn000 020plusmn001 019plusmn001Heart 009plusmn001 008plusmn001 008plusmn001 009plusmn001Lung 006plusmn000 005plusmn000 005plusmn000 006plusmn00Liver 028plusmn003a 038plusmn003ab 037plusmn003b 049plusmn003c

Kidney 008plusmn001 009plusmn001 008plusmn001 009plusmn001Stomach 011plusmn001a 015plusmn001ab 013plusmn001c 014plusmn001bc

Small intestine 019plusmn003a 027plusmn002b 024plusmn002ab 036plusmn003c

Rectum 003plusmn000 004plusmn000 004plusmn000 005plusmn000Total digestive tract 033plusmn003a 045plusmn003b 041plusmn003ab 055plusmn003c

Long day 16 h light8 h dark short day 8 h light16 h dark warm 30degC cold 10degC Organ values were corrected by body mass minus wet organ mass resultingfrom the regression for all birds Different letters indicate significant differences among treatments after ANCOVA atPlt005 Data are presented asmeansplusmnsem

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consequently are major contributors to overall RMR (Daan et al1990 Vermorel et al 2005) The elevation in RMR of the coldgroup was presumably related to metabolic andor morphologicaladjustments including changes in organ mass required to meet theenergy demands of acclimation to colder temperature conditionsThe dry mass of the liver stomach small intestine and totaldigestive tract all increased significantly with cold acclimation but

with the exception of the liver photoperiod did not significantlyinfluence internal organ mass Similar winter increases in the massof the liver stomach small intestine and total digestive tract inChinese bulbuls in temperate parts of their range suggest that winterincrements in internal organ mass are an important and generalmetabolic adjustment to cold in this species (Starck and Rahmaan2003 Zhang et al 2008 Zheng et al 2010 2014a) Moreover

WL WSMuscle

CL CS

b a a a

b ab a a

WL WSLiver

CL CS

b ba a

cb

ab a

005

10

15

20 C

CO

X a

ctiv

ity(micro

mol

O2

min

ndash1 g

ndash1 ti

ssue

)

0

04

08

12

16 B

S4R

(microm

ol O

2 m

inndash1

gndash1

tiss

ue)

0

10

20

30

40 A

Mito

chon

dria

l pro

tein

(mg

gndash1 )

Fig 5 Differences in mitochondrial protein state-4respiration and cytochrome c oxidase activity in the liverand pectoral muscle of Chinese bulbuls acclimated todifferent temperature and photoperiod for 4 weeks(A) Mitochondrial protein (B) State-4 respiration (S4R)(C) Cytochrome c oxidase (COX) activity Data are meansplusmnsem bars with different letters indicate significant differencesTreatment abbreviations as for Fig 1

Table 2 Allometric correlation and residual correlation for RMR versus dry organmass (controlled forMbminus wet organmass) in Chinese bulbul

Correlation Muscle Brain Heart Lung Liver Kidney GizzardSmallintestine Rectum

Digestivetract

AllometricR2 All birds 0063 0004 0185 0018 0306 0132 0106 0496 0081 0446

Temperature LP 0001 0028 0008 0003 0225 0146 0208 0473 0002 0383Temperature SP 0233 0025 0258 0447 0312 0204 0003 0416 0114 0365

P All birds 0199 0760 0025 0489 0002 0057 0091 0000 0143 0000Temperature LP 0942 0572 0760 0860 0087 0177 0101 0007 0893 0018Temperature SP 0080 0590 0064 0006 0047 0105 0852 0013 0238 0022

Slope All birds 0137 0172 0452 0218 0367 0398 0339 0409 0181 0514Temperature LP 0017 0524 0112 0091 0323 0687 0449 0514 0029 0556Temperature SP 0162 -0116 0347 0814 0387 0289 0048 0275 0165 0363

ResidualR2 All birds 0001 0001 0101 0242 0007 0002 0051 0189 0001 0097



Slope All birds 010 3714 26779 14453 1344 3999 15018 8821 2346 5333Temperature LP 191 13731 28834 95214 1139 2394 21504 17795 4853 5769Temperature SP 364 17612 28756 135936 715 12413 20074 6359 973 3837

RMR resting metabolic rate Mb body mass LP long photoperiod SP short photoperiodP-values in bold type are statistically significant

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these results suggest that temperature is the main factor influencingseasonal variation in liver stomach and small intestine mass inbirds and that increasing the mass of these organs increases thethermogenic capacity of small birds in winter

Effects of temperature and photoperiod on biochemicalresponses in liver and muscleIn addition to RMR cold-acclimated bulbuls in the present studyalso differed in several physiological and biochemical markers

indicative of differences in thermogenic capacity The dominantrole of the liver in RMR is well established (Villarin et al 2003Else et al 2004 Zheng et al 2008b 2010) Cellular metabolicintensity in the liver inferred from mitochondrial respiration orCOX activity is higher in winter than in summer in several birdspecies (Zheng et al 2008b 2013b 2014ab) The liver cangenerate heat by uncoupling oxidative phosphorylation futilecycling of substrates and high mass-specific metabolic intensity(Else et al 2004 Zheng et al 2008b 2014a Zhou et al 2016)

log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23

log COX activity (micromol O2 minndash1 gndash1 tissue)

WarmCold

ndash01 0

D

17

18

19

20

21

22

23

A

17

18

19

20

21

22

23

log S4R (micromol O2 minndash1 gndash1 tissue)ndash16 ndash14 ndash12 ndash10 ndash08 ndash06 ndash04

B

17

18

19

20

21

22

23

ndash08 ndash06 ndash04 ndash02 ndash01 0

ndash16 ndash14 ndash12 ndash10 ndash08 ndash06 ndash04

ndash08 ndash06 ndash04 ndash02

Fig 7 Correlations between RMR and S4Rand RMR and COX activity in the pectoralmuscle of Chinese bulbuls acclimated todifferent temperature and photoperiod for4 weeks (AB) RMR and S4R in the long (A)and short (B) photoperiod (CD) RMR andCOX activity in the long (C) and short (D)photoperiod The allometric equationsrepresenting the linear curve for all birds areRMR=210S4R018 and RMR=205COX020and RMR=220S4R027 for temperatureacclimation in the short photoperiodTreatment abbreviations as for Fig 1

log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold

ndash03 ndash02 ndash01 0 01 02 03

D

17

18

19

20

21

22

23


A

17

18

19

20

21

22

23

log S4R (micromol O2 minndash1 gndash1 tissue)ndash08 ndash06 ndash04 ndash02 0 02 ndash08 ndash06 ndash04 ndash02 0 02

B

17

18

19

20

21

22

23 Fig 6 Correlations between RMR and S4Rand RMR and COX activity in the liver ofChinese bulbuls acclimated to differenttemperature and photoperiod for 4 weeks(AB) RMR and S4R in the long (A) andshort (B) photoperiod (CD) RMR and COXactivity in the long (C) and short (D)photoperiod The allometric equationsrepresenting the linear curve for all birds areRMR=202S4R033 and RMR=198COX034RMR=203S4R036 for temperatureacclimation in the long photoperiod andRMR=201COX035 for temperatureacclimation in the short photoperiodTreatment abbreviations as for Fig 1

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Muscle is a major organ involved in thermogenesis (Weber andPiersma 1996 Petit and Veacutezina 2014 Swanson et al 2014) andthe observed increase in state-4 respiration and COX activity inmuscle indicate enhanced basal thermogenic capacity (Zheng et al2008b 2014a) Previous studies have shown that acclimation to coldtemperatures or short photoperiods can induce an increase inmitochondrial state-4 respiration and citrate synthase and COXactivity in muscle accompanied by enhanced thermogenic capacityin several passerine species (Veacutezina andWilliams 2005 Liknes andSwanson 2011 Zheng et al 2013a Swanson et al 2014)Mitochondrial respiration and enzyme activity in this study weresignificantly correlated with RMR in either liver or pectoral musclewhich suggests that variation in cellular oxidative phosphorylationcapacity (state-4 respiration and COX activity) is a prominentmediator of RMR variation in Chinese bulbuls Some studies haveexamined correlations of cellular metabolic capacity with metabolicoutput in birds and our results are consistent with data fromEurasian tree sparrow (Passer montanus) where variation in state-4respiration and COX activity was correlated with variation in RMR(Zheng et al 2008b 2014b) In the present study cold temperatureinduced an increase in liver and muscle state-4 respiration and COXactivity in Chinese bulbuls which also displayed enhancedthermogenic capacity Consistent with the results of previousstudies cold-acclimated birds had higher levels of state-4 respirationand COX activity in liver and muscle compared with thoseacclimated to 30degC Many small birds are known to adjust theircellular metabolic capacity during seasonal activities such asreproduction migration and winter acclimatization (Swanson2010) For example winter-acclimatized Chinese bulbuls increasetheir state-4 respiration and COX activity in liver and muscle tissuewhich suggests that these adjustments could play an important rolein winter thermogenesis (Villarin et al 2003 Zheng et al 2014a)State-4 respiration and COX activity in the liver and muscle of birdsmay either increase or remain seasonally stable during winteracclimatization or migration (Swanson 2010 Zheng et al 2008b2013b 2014b) In hwamei cold acclimation increased the oxidativecapacity of the pectoral muscles and liver which collectively couldmake a significant contribution to non-shivering thermogenesis(Zhou et al 2016) High levels of state-4 respiration and COXactivity are related to elevated RMR (Zheng et al 2013ab) afinding that is supported by the significant correlations betweenstate-4 respiration COX activity and RMR in this study Thustemperature is an important factor affecting thermogenesis inChinese bulbuls These results are also consistent with thoseobtained for other avian species including the Eurasian tree sparrow(Zheng et al 2008b 2014b) and little buntings (Emberiza pusilla)(Zheng et al 2013b) It would be interesting to examine in furtherstudies whether increased state-4 respiration and COX activity inbulbuls is also due to their responses to chronic shivering (Bicudoet al 2001 Zhou et al 2016) Although we expected state-4respiration and COX activity in bulbuls to increase with shortphotoperiod acclimation in this study this was not the case Wetherefore found no evidence to support the hypothesis thatphotoperiod influences the biochemical markers we measured inthis study

ConclusionsChinese bulbuls mainly live in habitats with marked seasonalvariation in temperature and photoperiod (Zheng et al 2008a2014a) In winter these animals show enhanced thermogeniccapacity that is considered to be an important adaptation for theirsurvival (Zheng et al 2008a 2014a) Bulbuls acclimated to a cold

temperature and short day length attained higherMb internal organmass thermogenic capacity and energy intake than those acclimatedto a warmer temperature and longer day length These resultssupport our hypothesis that temperature and photoperiod areimportant environmental cues for adaptive adjustments in Mbenergy metabolism and thermogenesis in birds The activation ofliver andmuscle mitochondrial respiration and the elevation of COXactivity appear to be cellular mechanisms underlying the elevationof RMR The observed physiological and biochemical changesobserved under different temperature and photoperiod treatmentswould allow Chinese bulbuls to overcome the physiologicalchallenges of the extreme seasonal variation in temperature thatthey encounter in much of their natural range (Zhang et al 2008Zheng et al 2010 2014a) The morphological physiological andbiochemical changes induced by cold temperature and shortphotoperiod may lead to energy expenditure thereby enhancingthe survival of Chinese bulbuls in winter the most energeticallychallenging time of the year Further work is required to determinethe maximum metabolic rate and to resolve apparent anomalies inthe geographical distribution of Chinese bulbuls (Swanson andGarland 2009 Zheng et al 2014a) In addition better moleculardata are required to improve our understanding of the complex trade-offs between competing physiological processes that take placeduring seasonal acclimatization (Teulier et al 2010)

AcknowledgementsWe are grateful to Dr David L Swanson for providing several references We thank DrRon Moorhouse of Biological Science Editing New Zealand for reading thismanuscript correcting the English expression and giving some constructivesuggestions We also thank the anonymous reviewers for their helpful comments andsuggestions Thanks to all the members of the Animal Physiological Ecology GroupInstitute of Applied Ecology of Wenzhou University for their helpful suggestions

Competing interestsThe authors declare no competing or financial interests

Author contributionsW-HZ and J-SL conceived the study and designed the experiments S-NHY-YZ and LL collected the data S-NH Y-YZ and J-SL analyzed the dataS-NH LL and W-HZ wrote the manuscript S-NH W-HZ and J-SLinterpreted data and revised the manuscript All authors assume responsibility forthe content of the paper

FundingThis study was financially supported by grants from the National Natural ScienceFoundation of China (no 31470472) and the Zhejiang Province Natural ScienceFoundation (no LY13C030005)

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Zheng W-H Liu J-S and Swanson D L (2014a) Seasonal phenotypicflexibility of body mass organ masses and tissue oxidative capacity and theirrelationship to resting metabolic rate in Chinese bulbuls Physiol Biochem Zool87 432-444

Zheng W-H Li M Liu J-S Shao S-L and Xu X-J (2014b) Seasonalvariation of metabolic thermogenesis in Eurasian tree sparrows (Passermontanus) over a latitudinal gradient Physiol Biochem Zool 87 704-718

Zhou L-M Xia S-S Chen QWang R-M ZhengW-H and Liu J-S (2016)Phenotypic flexibility of thermogenesis in the Hwamei (Garrulax canorus)responses to cold acclimation Am J Physiol 310 R330-R336

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and Swanson 2011 Swanson et al 2014 Zheng et al 2013b2014a) Variation in cellular metabolic intensity is often measuredby examining variation in state-4 respiration (reflecting oxidativephosphorylation capacity) or cytochrome c oxidase (COX) activity(a key regulatory enzyme of oxidative phosphorylation) (Zhenget al 2008b 2014ab)Chinese bulbuls Pycnonotus sinensis (Gmelin 1789) are small

passerine birds that inhabit vast areas of eastern and southern Asiaincluding central southern and eastern China (Zheng and Zhang2002) Elevated winter RMR in bulbuls is associated with elevatednutritional and exercise organ masses and heightened respiratoryenzyme activity in liver and muscle (Zheng et al 2008a 20102014a) We selected Chinese bulbuls as a study species becausethey are resident in Zhejiang Province where we are based becauseglobal warming appears to have allowed the species to colonizenortheastern and northwestern China and because previous studies(Zhang et al 2008 Zheng et al 2008a 2010 Ni et al 2010)provide critical background information required for our researchHowever the Chinese bulbul commonly encounters coldtemperatures and short photoperiods but the physiological andbiochemical mechanisms are unknown In this study we comparedselected morphological physiological and biochemical indices ofwild-caught Chinese bulbuls to different combinations oftemperature (warm and cold) and photoperiod (long day and shortday) We hypothesized that cold exposure and short day are keyfactors driving metabolic flexibility in Chinese bulbuls We alsohypothesized that there would be an interaction betweentemperature and photoperiod such that the greatest treatmenteffects would be observed in birds acclimated to both coldtemperatures and short days We predicted that Chinese bulbulsexposed to cold temperatures and short days would increase theirRMR and respiratory enzyme activity by and quantifying theseadjustments we aimed to improve understanding of themorphological physiological and biochemical responses of smallbird species to cold temperatures and short photoperiods

MATERIALS AND METHODSAnimalsThe Chinese bulbul is a common resident bird in Zhejiang Province(MacKinnon and Phillipps 2000) The Chinese bulbuls used in theexperiments were captured with mist nets in Wenzhou cityZhejiang Province China in June 2011 The climate in Wenzhouis warmndashtemperate with an average annual rainfall of 1700 mmacross all months with slightly more precipitation during winter andspring Mean daily maximum temperature ranges from 39degC in Julyto 8degC in January The mean temperature from June to August is32degC (Zheng et al 2008a) We determined bulbulMb to the nearest01 g immediately upon capture with a Sartorius balance (modelBT25S) We transported birds to the laboratory on the day of capture

and kept them in outdoor cages (50times30times20 cm) for 1 or 2 daysunder natural ambient temperature (28plusmn1degC) and photoperiodconditions before measurements began Food and water weresupplied ad libitum Birds were then kept in individual cages for atleast 2 weeks after which 28 birds were randomly assigned to one offour treatment groups (1) warm and long photoperiod (WL 30degC16 h light8 h dark) (2) warm and short photoperiod (WS 30degC 8 hlight16 h dark) (3) cold and long photoperiod (CL 10degC 16 hlight8 h dark) and (4) cold and short photoperiod (CS 10degC 8 hlight16 h dark) Each group of birds was acclimated to its respectivetreatment for 4 weeks (Ni et al 2010 Zheng et al 2013a) Allexperimental procedures were approved by the Animal Care andUse Committee of Wenzhou City Zhejiang Province China

Measurement of metabolic rateMetabolic rate of the birds was estimated by measuring their oxygenconsumption in an open-circuit respirometry system (S-3AI AEITechnologies Pittsburgh PA USA) Metabolic chambers were15 l in volume and made of plastic A perch was provided for thebird to stand on (Smit and McKechnie 2010 Zheng et al 2014a)Chamber temperature was regulated by a temperature-controlledcabinet (BIC-300 artificial climate incubator Shanghai BoxunMedical Biological Instrument Corp China) capable of regulatingtemperature to plusmn05degC Water vapor and CO2 were scrubbed fromthe air passing through the chamber in a silica gelsoda limesilicacolumn before passing through the oxygen analyzer We measuredthe oxygen content of excurrent gas from metabolic chambers withan oxygen sensor (N-22M AEI Technologies) We used a flowcontrol system (R-1 AEI Technologies) to set the flow of excurrentgas to 300 ml minminus1 during metabolic rate measurements Thismaintained a fractional concentration of O2 in the respirometrychamber of about 20 calibrated to plusmn1 accuracy with a generalpurpose thermal mass flow-meter (TSI Series 4100 TSI IncShoreview MN USA) (McNab 2006) Oxygen consumption rateswere measured at 30plusmn05degC which is within the thermal neutralzone of Chinese bulbuls (Zheng et al 2008a) Baseline O2

concentrations were obtained before and after each test (Li et al2010) All measurements of gas exchange were obtained during theresting phase of the birdsrsquo circadian cycles (between 2000 h and2400 h) in darkened chambers when individual birds couldreasonably be expected to be postabsorptive Resting metabolic rate(RMR) is the energy required to perform vital body functions whilethe body is at rest Because it is doubtful that true RMRs can beachieved in the laboratory the term RMR is often used to refer tosuch measurements even when the standard conditions for RMRhave been met (Swanson 2010) Food was removed 4 h before eachmeasurement to minimize the heat increment associated withfeeding We first ensured that birds were perching calmly in thechamber and started recording oxygen consumption at least 1 hlater Each animal was generally in the metabolic chamber for atleast 2 h The data obtained were used to calculate 5 min runningmeans of instantaneous oxygen consumption over the entire testperiod using eqn 2 of Hill (1972) The lowest 5 min mean recordedover the test period was considered the RMR (Smit andMcKechnie2010) All values for oxygen consumption were expressed asml O2 hminus1 and corrected to STPD conditions (Schmidt-Nielsen1997) Body temperature was measured during metabolicmeasurements using a lubricated thermocouple inserted into thecloaca to a depth of 1ndash2 cm a depth at which a slight withdrawal ofthe thermocouple did not cause a change in the readingThermocouple outputs were digitized using a thermocouple meter(Beijing Normal University Instruments Co) Mb was measured to

List of symbols and abbreviationsCL cold and long photoperiodCOX cytochrome c oxidaseCS cold and short photoperiodDEI digestible energy intakeFE feces energyGEI gross energy intakeMb body massRMR resting metabolic rateWL warm and long photoperiodWS warm and short photoperiod

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WLndash5 0 WS CL CS

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WL WSMuscle

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Digestivetract










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WLndash5 0 WS CL CS

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WLndash5 0 WS CL CS

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A WLWSCLCS

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WL WSMuscle

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ol O

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Digestivetract










851


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WarmCold

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WL WSMuscle

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ofEx

perim

entalB

iology





Long day Short day

Warm Cold Warm Cold











850


Journal

ofEx

perim

entalB

iology



WL WSMuscle

CL CS

b a a a

b ab a a

WL WSLiver

CL CS

b ba a

cb

ab a

005

10

15

20 C

CO

X a

ctiv

ity(micro

mol

O2

min

ndash1 g

ndash1 ti

ssue

)

0

04

08

12

16 B

S4R

(microm

ol O

2 m

inndash1

gndash1

tiss

ue)

0

10

20

30

40 A

Mito

chon

dria

l pro

tein

(mg

gndash1 )




Digestivetract










851


Journal

ofEx

perim

entalB

iology




log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold

ndash01 0

D

17

18

19

20

21

22

23

A

17

18

19

20

21

22

23


B

17

18

19

20

21

22

23





log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold


D

17

18

19

20

21

22

23


A

17

18

19

20

21

22

23


B

17

18

19

20

21

22


852


Journal

ofEx

perim

entalB

iology


















853


Journal

ofEx

perim

entalB

iology






















































854


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology



WL WSMuscle

CL CS

b a a a

b ab a a

WL WSLiver

CL CS

b ba a

cb

ab a

005

10

15

20 C

CO

X a

ctiv

ity(micro

mol

O2

min

ndash1 g

ndash1 ti

ssue

)

0

04

08

12

16 B

S4R

(microm

ol O

2 m

inndash1

gndash1

tiss

ue)

0

10

20

30

40 A

Mito

chon

dria

l pro

tein

(mg

gndash1 )




Digestivetract










851


Journal

ofEx

perim

entalB

iology




log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold

ndash01 0

D

17

18

19

20

21

22

23

A

17

18

19

20

21

22

23


B

17

18

19

20

21

22

23





log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold


D

17

18

19

20

21

22

23


A

17

18

19

20

21

22

23


B

17

18

19

20

21

22


852


Journal

ofEx

perim

entalB

iology


















853


Journal

ofEx

perim

entalB

iology






















































854


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology




log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold

ndash01 0

D

17

18

19

20

21

22

23

A

17

18

19

20

21

22

23


B

17

18

19

20

21

22

23





log

RM

R (m

l O2

hndash1 )

C

17

18

19

20

21

22

23


WarmCold


D

17

18

19

20

21

22

23


A

17

18

19

20

21

22

23


B

17

18

19

20

21

22


852


Journal

ofEx

perim

entalB

iology


















853


Journal

ofEx

perim

entalB

iology






















































854


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology


















853


Journal

ofEx

perim

entalB

iology






















































854


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology






















































854


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology












855


Journal

ofEx

perim

entalB

iology

Documents

Temperature and photoperiod as environmental cues affect ... · Temperature and photoperiod as environmental cues affect body mass and thermoregulation in Chinese bulbuls, Pycnonotus