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British Poultry Science

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Physicochemical determinants of pH in pectoralismajor of three strains of laying hens housed inconventional and furnished cages

K. M. Frizzell, M. J. Jendral, I. M. Maclean, W. T. Dixon & C. T. Putman

To cite this article: K. M. Frizzell, M. J. Jendral, I. M. Maclean, W. T. Dixon & C. T. Putman(2018) Physicochemical determinants of pH in pectoralis�major of three strains of laying henshoused in conventional and furnished cages, British Poultry Science, 59:3, 286-300, DOI:10.1080/00071668.2018.1445198

To link to this article: https://doi.org/10.1080/00071668.2018.1445198

Accepted author version posted online: 26Feb 2018.Published online: 23 Mar 2018.

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Physicochemical determinants of pH in pectoralis major of three strains of layinghens housed in conventional and furnished cagesK. M. Frizzella, M. J. Jendrala,b, I. M. Macleana, W. T. Dixonc and C. T. Putmana,d

aFaculty of Physical Education and Recreation, University of Alberta, Exercise Biochemistry Laboratory, Edmonton, Canada; bDepartment ofPlant and Animal Sciences, Dalhousie University Agricultural Campus, Truro, Canada; cDepartment of Agriculture, Food and NutritionalScience, Faculty of Agriculture, Life and Environmental Sciences, University of Alberta, Edmonton, Canada; dFaculty of Medicine & Dentistry,Neuroscience and Mental Health Institute, University of Alberta, Edmonton, Canada

ABSTRACT1. Post-mortem decline in muscle pH has traditionally been attributed to glycogenolysis-inducedlactate accumulation. However, muscle pH ([H+]) is controlled by complex physicochemical relation-ships encapsulated in the Stewart model of acid–base chemistry and is determined by three system-independent variables – strong ion difference ([SID]), total concentration of weak acids ([Atot]) andpartial pressure of CO2 (PCO2).2. This study investigated these system-independent variables in post-mortem pectoralis majormuscles of Shaver White, Lohmann Lite and Lohmann Brown laying hens housed in conventionalcages (CC) or furnished cages (FC) and evaluated the model by comparing calculated [H+] withpreviously measured [H+] values.3. The model accounted for 99.7% of the variation in muscle [H+]. Differences in [SID] accountedfor most or all of the variations in [H+] between strains. Greater PCO2 in FC was counteracted bygreater sequestration of strong base cations. The results demonstrate the accuracy and utility ofthe Stewart model for investigating determinants of meat [H+].4. The housing differences identified in this study suggested that hens housed in FC have improvedmuscle function and overall health due to the increased opportunity for movement. These findingssupport past studies showing improved animal welfare for hens housed in FC compared to CC.Therefore, the Stewart model has been identified as an accurate method to assess changes in themuscle at a cellular level that affect meat quality that also detect differences in the welfare status ofthe research subjects.

ARTICLE HISTORYReceived 3 November 2016Accepted 23 December 2017

KEYWORDS[H+]; conventional cage;furnished cage; laying hens;meat; physicochemicalmodelling

Introduction

Muscle pH is commonly measured to assess meat qualitybecause it influences visual and sensory characteristicssuch as colour (Ahn and Maurer 1990), tenderness(Jeleníková et al. 2008) and water-holding capacity(WHC) (Hughes et al. 2014). Meat pH that is too highis typically associated with darker hues, decreased tender-ness and increased WHC (Ahn and Maurer 1990; Barbutet al. 2005; Berri et al. 2007; Jeleníková et al. 2008),whereas excessive post-mortem pH decline has been asso-ciated with protein denaturation, paleness, extreme ten-derness and lower WHC (Ahn and Maurer 1990; Barbutet al. 2005; Berri et al. 2005; Le Bihan-Duval et al. 2008).Traditionally, the post-mortem decline in muscle pH hasbeen attributed to glycogenolysis resulting in lactate accu-mulation (Bendall 1973; Berri et al. 2007; Wang et al.2013; England et al. 2014, 2016). However, lactate accu-mulation only accounts for a portion of the post-mortempH decline (England et al. 2016), and significant dissocia-tions have been noted between glycolytic potential andpH (Monin and Sellier 1985), between post-mortem gly-cogenolysis and lactate accumulation (Matarneh et al.2015; England et al. 2016), and between lactate accumu-lation and pH (Monin and Sellier 1985; Matarneh et al.2015). The underlying physicochemical bases for thesedissociations have not yet been resolved.

Within biological systems, pH has been shown to becontrolled by complex physicochemical relationships encap-sulated in the Stewart model of acid–base regulation(Stewart 1981, 1983) that has been validated across severalspecies (Putman et al. 2003; Stämpfli and Constable 2003;Lindinger et al. 2005; Stämpfli et al. 2006; Lindinger andHeigenhauser 2011). This model of acid–base regulationstipulates that a change in the dependent variable, [H+], isdetermined by the balance struck by three system-indepen-dent variables acting within the constraints of physicochem-ical laws of electrical neutrality, conservation of mass andmass action equilibrium constants (i.e. dissociation of weakacids, formation of bicarbonate ion, dissociation of water,formation of carbonate ion) (Stewart 1981, 1983). The firstsystem-independent variable, the strong ion difference([SID]), is defined as the sum of fully dissociated strongbase cations (i.e. [Ca2+] + [Mg2+] + [Na+] + [K+]) minus thesum of strong acid anions (i.e. [Cl−] + [Lac]) and has beenshown to vary inversely with muscle [H+] (Stewart 1981,1983). The second variable is the net concentration ofpartially dissociated non-volatile acids including metabo-lites, phosphates and soluble proteins termed total concen-tration of weak acids ([Atot]). Such weak acids (HA)partially dissociate and, therefore, [H+] varies in proportionto increase in muscle [Atot] (Stewart 1983). The third vari-able is the partial pressure of CO2 (PCO2), which exerts its

CONTACT C. T. Putman ted.putman@ualberta.ca Exercise Biochemistry Laboratory, University of Alberta, 4-250 Van Vliet Complex, Edmonton, AB T6G2H9, Canada; Neuroscience and Mental Health Institute, Faculty of Medicine & Dentistry.

BRITISH POULTRY SCIENCE2018, VOL. 59, NO. 3, 286–300https://doi.org/10.1080/00071668.2018.1445198

© 2018 British Poultry Science Ltd

acid properties when hydrated to form carbonic acid(H2CO3) and ultimately bicarbonate (HCO3

–) + [H+](Stewart 1981, 1983).

A fundamental tenet of the Stewart model of acid–baseregulation is that variation in [H+] between neighbouringcompartments does not result from the physical movementof [H+] across membranes. Rather, [H+] is determined bythe net transmembrane movement of strong base cations,strong base anions, weak acids, CO2 and the instantaneousestablishment of new equilibria between the three system-independent variables (Stewart 1983). Although the Stewartmodel of acid–base regulation provides the essential con-ceptual and empirical framework to investigate variations inpost-mortem acidification of muscle, it has not been appliedto meat science.

In a previous study that evaluated meat quality in 80–81-week-old laying hens (Frizzell et al. 2017), it was reported thatpectoralis major pH, colour and tenderness differed betweenthree strains of laying hens housed in an established productionsetting that included both conventional cage (CC) and furn-ished cage (FC) systems (Frizzell et al. 2017). Furthermore, sincehens housed in CC are behaviourally and physically restricted(Appleby et al. 2002) and exhibit weaker bones (Jendral et al.2008; Jendral 2012) and increased aggression (Jendral 2012)than hens housed in FC that have increased space and theamenities to promote natural behaviours such as nesting, fora-ging, dust-bathing and perching, it was expected that henshoused in FC would also exhibit improved meat quality com-pared with conventionally caged hens (Frizzell et al. 2017).However, no differences in meat quality were found betweenhens housed in CC and FC and this was attributed to differingenvironmental stressors in the two housing systems (Frizzellet al. 2017).

The purpose of the present study was to investigate thethree system-independent variables in post-mortem pector-alis major muscles of flock 1 birds from the previous study(Frizzell et al. 2017) to further investigate why differences inmuscle pH at 17 min post-mortem (pH17) were observedbetween genetic strains but not between housing environ-ments. Moreover, the model was evaluated by comparingmeasured values of [H+] with values calculated using equa-tions derived by Stewart (1981, 1983). It was hypothesisedthat the three system-independent variables would be sus-ceptible to ante-mortem and post-mortem changes imposedby production conditions that can account for variations inmeat [H+] between bird strains.

Materials and methods

Animals and experimental design

Experiments were conducted in accordance with the guide-lines of the Canadian Council for Animal Care and wereauthorised by the Animal Care and Use Committee of theFaculty of Agriculture at Dalhousie University (formerlyNova Scotia Agricultural College – NSAC) and by theAnimal Care and Use Committee for Livestock at theUniversity of Alberta. A flock comprised 280 ShaverWhite (SH), 280 Lohmann Lite (LL) and 280 LohmannBrown (LB) laying hens was housed at the AtlanticPoultry Research Centre (APRC) of the NSAC. Each henstrain represented a single genetic line. Upon arrival, 19-week-old birds (Clarke’s Chick Hatchery, Burtt’s Corner,

New Brunswick) were randomly assigned to either a CCor FC. Ambient temperature was maintained at 23°C andlighting was gradually increased from 11.5 to 15 h/d by25 weeks of age. Birds received a standard layer diet(National Research Council (NRC), 1994) and freshwaterad libitum.

The CC system was composed of 9 mobile batteries eachcontaining two tiers of 4 cages for a total of 72 CC (Jendral2012). Twenty-four cages were allocated to each bird strain.Each CC (60 cm wide × 55 cm deep × 45 cm high) housed 5hens of the same strain providing 660 cm2 of floor spaceper hen.

The FC system was composed of 12 furnished batterycages (240 cm wide × 110 cm deep × 50 cm high) (Jendral2012). Four cages were allocated to each strain and each FChoused 40 birds, providing 660 cm2 of floor space per hen.Each FC also contained a nest box, perches and a dust bath.Nest boxes contained an artificial turf lining and measured60 cm wide × 55 cm deep, which provided an additional92 cm2 of space per hen. Three semi-circular hardwoodperches measuring 5 cm wide × 2.5 cm deep spanned thelength of the cage. An elevated metal dust bath measuring60 cm wide × 20 cm deep and containing a substrate madeof wood shavings and feed was opened daily 8 h after thebeginning of the daylight cycle and was closed 45 minbefore the end of the daylight cycle to prevent dust bathegg-laying.

Processing and tissue collection

All hens were processed between 80 and 81 weeks of age. Inthe FC system, 25 hens were randomly selected for proces-sing from each of the 12 FC and in the CC system; for eachof the 9 batteries, all hens from one randomly selected cageper strain (i.e. three CC per battery) were designated forprocessing. Hens were gently removed from cages, weighedand scored for feather condition, palpated for bone frac-tures (Jendral 2012) and transported to the processing facil-ity at the APRC. Since muscle samples were collected toassess the effect of genetic strain and housing environmenton meat quality, as determined in part by muscle pH, it wasnecessary to minimise the impact of other factors that alsoaffect meat quality. Therefore, to minimise stress associatedwith transportation and feed restriction, hens received feedand water ad libitum up to the time of transport, and lightswere dimmed during handling. Further, transportationcrates were lined with bedding to prevent injury, mealworms were scattered on the floor of the crates and cratestocking density was limited to 5 hens per crate.

Hens were gently and individually lifted from their trans-portation crates at processing. To minimise the timebetween shackling and stunning and to further minimisestress, only 5 hens were processed at a time. Hens werequickly rendered unconscious by an electrical stun and theright jugular artery was immediately severed. Hens werebled out for 90 s, placed in a scalder at 57°C for 2 minand then in a feather plucker for 45 s. Following thisprocess, hens were re-palpated for post-processing bonefractures (Jendral 2012). At 17 min post-mortem, 2.5 cm3

samples were extracted from the right superior pectoralismajor muscle (temperature, 17°C) and snap frozen in liquidnitrogen. Muscle samples were subsequently transported ondry ice to the University of Alberta and stored at −80°C

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until further analysis. To account for variations in musclesize, average muscle depth of the left pectoralis major wasdetermined (Frizzell et al. 2017).

Muscle analysis

Muscle sample preparation, wet-to-dry weight ratio andtotal tissue waterApproximately 600 mg of each muscle sample was freeze-dried, dissected free of blood and connective tissue, andstored (−80°C) for later analysis of strong base cations andstrong acid anions. The remaining snap frozen portion ofeach sample was also stored at −80°C for later analysis. Aportion of each sample (450–500 mg) was weighed beforeand after lyophilisation and used to calculate the wet-to-dryweight (dw) ratio and total tissue water (TTW).

Glycogen, lactate and glycolytic potentialMuscle glycogen content was determined spectrophotome-trically on snap frozen samples using the phenol–sulphuricacid method (Lo et al. 1970; Morifuji et al. 2005). Musclelactate content was measured according to Putman et al(1993; 1998). Briefly, 100–150 mg of snap frozen musclewas pulverised in liquid nitrogen, then extracted in 6volumes of ice-cold 6% (v/v) perchloric acid using aPolytron (Brinkmann Instruments, Mississauga, ON,Canada), and cleared by centrifugation (3000g) (IECMicromax RF). Lactate concentrations were assayed at340 nm using a 96-well plate reader (BioTek EL808,BioTek Instruments). Muscle lactate concentration([lactate−]) was calculated using TTW. Glycolytic potentialwas calculated using a modified form of the equation pub-lished by Monin and Sellier (1985). The modified equationexcluded glucose and glucose-6-phosphate, to reflect thatglycogen plus lactate account for 90% of the absolute gly-colytic potential, as well as ~90% of the difference in glyco-lytic potential between experimental conditions (Monin andSellier 1985; Matarneh et al. 2015; England et al. 2016).Glycolytic potential was calculated as follows:

glycolytic potential ¼ 2 � glycogenð Þ þ lactate (1)

Total muscle proteinTotal protein was measured using the method of Lowryet al. (1951). Briefly, 100–150 mg of snap frozen musclewas homogenised in 9 vol of Milli-Q ultrapure water using aPolytron (Brinkmann Instruments). Duplicate 10 µl aliquotswere diluted 5-fold with Milli-Q ultrapure water, mixedwith 100 µl of 0.3 M KOH (Sigma-Aldrich, Oakville, ON,Canada) and incubated for 30 min at 37°C. Protein concen-tration was assayed at 750 nm using a 96-well AbsorbanceMicroplate Reader (BioTek EL808, BioTek Instruments,Vermont, USA).

Measurement of muscle Ca2+, Mg2+, Na+ and K+

Muscle samples were prepared according to Wang et al.(1996). Briefly, 20 mg of freeze dried muscle was dissolvedin 2 ml of 1 N nitric acid (HNO3) (TraceSELECT Ultra,Sigma-Aldrich) in a glass-covered 10 ml glass beaker andincubated at 50°C for 48 h for elemental extraction (FisherEconotemp Laboratory Oven Model 15G). Samples werethen filtered (Whatman No. 2 paper 110 mm, FisherScientific, Edmonton, AB, Canada), diluted in Milli-Q and

placed in polypropylene vials with aluminium free caps(Fisher Scientific). Samples were stored overnight at 4°Cprior to analysis by inductively coupled plasma-opticalemission spectrometry (ICP-OES). To avoid exogenous ele-mental contamination, all glassware was acid-washed in 2%HNO3. Positive controls were prepared from freeze-driedhen faecal samples, while negative controls (field reagentblanks) contained only Milli-Q ultrapure water.Additionally, laboratory reagent blanks consisting of pureHNO3 were prepared following the same procedure used formuscle sample preparation. The contents of Ca2+, Mg2+,Na+ and K+ within positive controls were 101.00 ± 2.42,9.07 ± 0.15, 5.55 ± 0.10 and 46.54 ± 0.40 mg/g dw,respectively.

Muscle contents of Ca2+, Mg2+, K+ and Na+ were mea-sured using ICP-OES (Optima ICP-OES 2100 DV, PerkinElmer, Inc., Waltham, MA, USA) (Ashoka et al. 2009; Sogliaet al., 2015); ICP-OES wavelengths were set to 317.077,279.077, 589.592 and 766.490 nm for measurement of Ca2+,Mg2+, Na+ and K+, respectively. Standard solutions wereprepared in 2% (v/v) HNO3 from commercial stock prepara-tions of Ca2+, Mg2+, Na+ and K+ (TraceSELECT, Sigma-Aldrich). Sample aspiration and drain tubes were placedaround the pump head and clipped into the tubing slots.The plasma flame was then ignited and the system wasflushed with 4% (v/v) HNO3 for 20 min, followed by a 2%(v/v) HNO3 flush for 10 min to minimise tube contamina-tion. Sample analysis was carried out in the following order:laboratory reagent blanks, field reagent blanks, standards,positive controls and finally muscle sample extracts. Theaspiration tube was rinsed in Milli-Q ultrapure water anddried between each sample aspiration. All measures werecompleted in triplicate. Muscle cation contents (mg/g dw)were converted to mEq/l TTW. The concentration sum ofstrong base cations (∑[Ca2+] + [Mg2+] + [Na+] + [K+]) wascalculated.

Measurement of muscle Cl−

Muscle chloride content was determined by titrationaccording to Cotlove (1963, 1968) with modifications(Wang et al. 1996). Briefly, 50 mg of freeze-dried musclesamples were digested in 10 ml 0.1 N HNO3 for 16 h atroom temperature, cleared by centrifugation (1500g) (IECCentra-7R, International Equipment Company, MA, USA),and the pH was adjusted to 7.5 (6.5–8.5) with NaOH(Sigma-Aldrich). The molar quantity of Cl− was measuredin duplicate 2 ml aliquots of the supernatant; samples weretransferred to 20 ml beakers containing 30 µl of 0.25 MK2CrO4 indicator (Fisher Scientific) and titrated using0.1 M AgNO3 (Fisher Scientific). Duplicate blank solutionsconsisted of only 0.1 N HNO3. To avoid exogenous ele-mental contamination, all glassware was acid-washed in 2%HNO3. The concentration of Cl− in extracts (mol/l) andmuscle lactate− content (mmol/kg ww) were converted tomEq/l TTW. The concentration sum of strong acid anions(∑[Cl−] + [lactate−]) was calculated.

Calculation of [SID]

The [SID] was calculated using the following equation(Stewart 1981, 1983):

288 K. M. FRIZZELL ET AL.

SID½ � ¼ Ca2þ� � þ Mg2þ

� � þ Naþ½ � þ Kþ½ �� �

� Cl�½ � þ lactate�½ �ð Þ (2)

where all concentrations are expressed as mEq/l TTW.

CO2 content, PCO2 and CO2 solubility constant

Muscle CO2 was quantified for 7 randomly selected hens fromeach of the 6 groups (n = 42) according to the method ofPörtner et al. (1990) with modifications. Briefly, 100–200 mgof snap frozen muscle tissue was pulverised under liquidnitrogen, weighed, transferred to sealed Vacutainers (BDVacutainer Blood Collection Tubes, Becton DickinsonLabware, Mississauga, ON) and extracted in 2 ml of 0.01 MHCl (Fisher Scientific). Using a Hamilton syringe, 200 µl of thesample headspace was injected into a Scion 456 gas chromato-graph (GC) (Bruker Daltonics, East Milton, ON, Canada)equipped with Compass CDS software (Bruker Daltonics),an HP Plot Q sample column and a thermal conductivitydetector set at 180°C. Samples were manually injected intothe GC split programmed temperature vaporising injector at55°C with a split ratio of 20:1. Helium was the carrier gas witha constant flow of 15ml/min and the oven was held at 35°C forthe total sample run time of 2 min. Molar CO2 content wasdetermined against standards of CO2 gas. Samples and stan-dards were run in triplicate.

The CO2 solubility coefficient (SCO2) was calculated at 17°C, which was the ambient temperature of the post-mortemmuscle processing environment. Using previously reportedSCO2 coefficients at 0°C (0.0895 mmol/(l mmHg)) (Carrollet al. 1991), 10°C (0.0597 mmol/(l mmHg)) (Harrison 1988),15°C (0.0499 mmol/(l mmHg)) (Truchot 1976), 20°C(0.0452 mmol/(l mmHg)) (Harrison 1988) and 37°C(0.0308 mmol/(l mmHg)) (Arthurs and Sudhakar 2005),regression analysis was completed using SigmaPlot Version12.5 (SYSTAT Software Inc., San Jose, CA) to derive SCO2 at atemperature (T) of 17°C. The resulting equation was asfollows:

SCO2 ¼ 4x10�5 T2� �� 0:0032 Tð Þ þ 0:0887;

R2 ¼ 0:995; P < 0:0001 (3)

The derived value for SCO2 at 17°C was 0.04586 mmol/(l mmHg).

Muscle bicarbonate ([HCO3–]), anion gap ([A−]) and

[Atot]

Muscle [HCO3–] was calculated according to Stämpfli et al.

(2006) using the following equation:

HCO3�½ � ¼ SCO2x PCO2x 10ðpH�pK0

1Þ (4)

where SCO2 = 0.04586 mol/(l mmHg) (Equation 3) andpK'1= 6.120.

The anion gap ([A−]) was calculated according to Stämpfliet al. (2006) using the strong ion electroneutrality equation:

SID� HCO3�½ �� A�½ �¼ 0 (5)

[Atot] was then calculated also according to Stämpfli et al.(2006):

Atot½ �¼ A�½ �þð Hþ½ �measured� A�½ �=KAÞ (6)

where KA = 3.0 × 10−7 Eq/l and [A−] and [H+]measured areexpressed as Eq/l.

The contribution of muscle proteins to [Atot] (i.e. [Atot]protein) was calculated from total protein and TTW andusing a net charge of 2.45 per mg/dl, as previously reported(Putman et al. 2003). The contribution of other organicnon-volatile weak acid buffers to [Atot] (i.e. [Atot]weak acids)was calculated as the difference between [Atot] and [Atot]protein since [Atot] represents the sum of [Atot]protein and[Atot]weak acids.

Measurement of muscle pH and conversion to [H+]

Muscle pH was measured using the homogenate method(Santé and Fernandez 2000; Yu et al. 2005; Wang et al.2013). Briefly, 0.5 g of snap frozen muscle tissue was homo-genised in 5 ml of ice-cold buffer containing 5 mM iodoa-cetate (Sigma-Aldrich) and 150 mM KCl (Sigma-Aldrich)using a Polytron (Brinkmann Instruments). Iodoacetate wasincluded in the buffer to arrest glycolytic activity (Englandet al. 2014). After mixing in sealed tubes, pH was measureddirectly in ice-cold homogenates using a calibrated AccumetBasic pH meter (Accumet Basic AB15, Fisher Scientific).Measured muscle pH was converted to [H+] usingEquation 7 and expressed as nEq/l.

Hþ½ �¼ 10�pH�109 (7)

Calculation of [H+]

Muscle hydrogen ion concentration [H+] was calculated for7 randomly selected hens from each of the 6 groups (n = 42)using the equation derived by Stewart (1981, 1983; Jones1987). Equation 8 defines the empirical relationshipbetween the dependent variable [H+] and the three sys-tem-independent variables, [SID], [Atot] and PCO2, withinthe constraints of physicochemical laws of electrical neu-trality, conservation of mass and mass action equilibriumconstants.

Hþ½ �4 þ KA þ SID½ �ð Þ Hþ½ �3þ KA SID½ � � Atot½ �ð Þ � KCPCO2 þ K0

W

� �� �� Hþ½ �2� KA KC PCO2 þ K0

W

� �þ K3KCPCO2ð Þ� �Hþ½ �

� KAK3KCPCO2ð Þ¼ 0 (8)

The mass action equilibrium constants were as follows: (1)dissociation of weak acids, KA = 3.0 × 10−7 Eq/l; (2) forma-tion of bicarbonate ion, KC = 2.58 × 10−11 (Eq/l)2; (3)dissociation of water, KʹW = 4.4 × 10–14 (Eq/l)2; (4) forma-tion of carbonate ion, K3 = 6.0 × 10−11 Eq/l. The corre-sponding pH values were also calculated. Regressionanalyses were completed (SigmaPlot Version 12.5,SYSTAT Software Inc.) to examine the relationshipsbetween measured [H+] and calculated [H+], and betweenmeasured pH and calculated pH.

Statistical analysis

Data are reported as least square means ± SEM. Responsevariables were analysed using the PROC MIXED procedureof SAS 9.4 (SAS Institute 2013) with pectoralis major muscledepth used as a covariate for all analyses. Statistical analyses

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were performed to assess treatment, strain and treatment bystrain effects. Differences between group means weredetected using the least significant differences test forplanned comparisons. To examine the relationshipsbetween measured [H+] and calculated [H+], and betweenmeasured pH and calculated pH, regression analyses werecompleted (SigmaPlot Version 12.5, SYSTAT SoftwareInc.). The level of significance for all analyses was assessedat P ≤ 0.05. Statistical trends were noted when0.05 < P ≤ 0.1. P-values are reported.

Results

Muscle depth, wet-to-dw ratio and TTW

The average pectoralis major muscle depth for all groupswas 17.22 ± 0.20 cm and did not differ between strains(P > 0.60) or between housing treatments (P > 0.73).Individual group means for muscle depth are reported else-where (Frizzell et al. 2017). The muscle wet-to-dw ratio didnot differ between bird strains (P > 0.84) or between CCand FC systems (P > 0.60) (Table 1). Similarly, muscle TTWdid not differ between bird strains (P > 0.85) or between CCand FC systems (P > 0.59) (Table 1).

Glycogen content, lactate content and glycolyticpotential

Muscle glycogen content varied between 47.1 and54.0 mmol/kg wet weight (ww) (Figure 1(a)) and fell withinthe range of previously reported data (Zhang et al. 2009;Wang et al. 2013). Independent of housing system, therewas a significant strain effect in which glycogen content waselevated by 5.4 mmol/kg ww in SH hens (P < 0.04) and LBhens (P < 0.04), compared with LL hens. Within the CCsystem, glycogen content trended 5.2 mmol/kg ww higherin SH compared with LL hens (P = 0.055). Within the FCsystem, glycogen content trended 6.8 mmol/kg ww higherin LB compared with LL hens (P = 0.068). Muscle glycogencontent did not differ between the CC (51.3 ± 1.3) and FC(50.9 ± 1.4) housing systems (P > 0.80).

Muscle lactate content varied between 37.9 and48.7 mmol/kg ww (Figure 1(b)) and was similar to pre-viously reported values in post-mortem poultry muscles(Debut et al. 2005; Berri et al. 2007; Le Bihan-Duval et al.2008). Strain differences were noted for muscle lactate con-tent that were independent of the housing system; musclelactate was 7.0 mmol/kg ww greater in LB hens comparedwith SH (P < 0.03) and LL (P < 0.03). Within the CChousing system, muscle lactate content was 9.7–10.8 mmol/kg ww greater in LB compared with LL(P < 0.02) and SH (P < 0.007) hens, respectively (Figure 1

(b)). Within the FC system, muscle lactate content wassimilar between all three strains (P > 0.36). Muscle lactatecontent did not differ between CC (41.9 ± 1.5) and FC(43.8 ± 1.9) housing systems (P > 42).

Glycolytic potential of hen pectoralis major variedbetween 135.1 and 153.9 mmol/kg ww (Figure 1(c)) andwas similar to previously reported data (Debut et al. 2005;Le Bihan-Duval et al. 2008). Strain differences were notedfor glycolytic potential that were independent of the hous-ing system; glycolytic potential was elevated by 10.5 and17.5 mmol/kg ww in SH (P < 0.02) and LB hens(P < 0.0001), respectively, compared with LL hens. Withinthe CC system, the glycolytic potential of LB hens waselevated by 17.6 mmol/kg ww compared with LL hens(P < 0.005). Glycolytic potential also trended 10.8 mmol/kg ww higher in SH compared to LL hens (P = 0.058).Similarly, within the FC system, the glycolytic potential ofLB hens was 17.6 mmol/kg ww higher than LL hens(P < 0.005). Glycolytic potential did not differ between CC(144.5 ± 2.4) and FC (145.6 ± 2.5) housing systems(P > 0.76).

Strong base cations and strong acid anions

Muscle concentrations of Ca2+, Mg2+, Na+ and K+

Muscle [Ca2+] varied between 30.8 and 37.7 mEq/l (Figure 2(a)) and did not differ between bird strains (P > 0.67) orbetween CC (33.2 ± 1.7) and FC (36.5 ± 2.5) housingsystems (P > 0.29). Muscle [Ca2+] values fell within therange of previously reported data (Al-Najdawi andAbdullah 2002).

Muscle [Mg2+] varied between 44.2 and 46.5 mEq/l(Figure 2(b)) and did not differ between strains (P > 0.51).In addition, muscle [Mg2+] did not differ between CC(45.3 ± 0.9) and FC (45.6 ± 1.3) housing systems(P > 0.82). Muscle [Mg2+] values were also comparable topreviously reported data (Misra et al. 1980; Sun et al. 2011).

Muscle [Na+] varied between 26.2 and 34.0 mEq/l(Figure 2(c)) which fell between the minimum and max-imum muscle [Na+] values previously reported (Misra et al.1980; Al-Najdawi and Abdullah 2002). Independent of thehousing system, muscle [Na+] was 4.9 and 7.1 mEq/l greaterin LL (P < 0.02) and LB hens (P < 0.0002) compared withSH hens and [Na+] also trended 3.2 mEq/l higher in LBcompared with LL (P = 0.057). In the CC system, [Na+] was4.3 and 6.6 mEq/l greater in LB hens compared with LL(P < 0.03) and SH (P < 0.002) hens, respectively. In contrast,within the FC system, muscle [Na+] was similar for LL andLB hens (P > 0.52); compared to SH, [Na+] was 5.6 and7.6 mEq/l greater in LL (P < 0.05) and LB (P < 0.007) hens,respectively. Muscle [Na+] did not differ between CC(29.1 ± 0.8) and FC (30.8 ± 1.1) housing systems (P > 0.21).

Table 1. Water content in pectoralis major muscles of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown) housed in CC (conventional cages) or FC(furnished cages).

Variable Housing treatment Strains combined SH LL LB

Wet-to-dry weight ratio Treatments combined – 3.8 ± 0.03 3.8 ± 0.03 3.8 ± 0.03CC 3.8 ± 0.02 3.8 ± 0.04 3.9 ± 0.03 3.8 ± 0.03FC 3.8 ± 0.02 3.8 ± 0.04 3.8 ± 0.04 3.8 ± 0.04P – housing 0.60 0.36 0.69 0.73

Total tissue water(ml/g ww)

Treatments combined – 0.74 ± 0.002 0.74 ± 0.002 0.74 ± 0.002CC 0.74 ± 0.001 0.74 ± 0.003 0.74 ± 0.002 0.74 ± 0.002FC 0.74 ± 0.001 0.74 ± 0.002 0.74 ± 0.003 0.74 ± 0.003P – housing 0.60 0.35 0.69 0.72

Data are least square means ± standard error.

290 K. M. FRIZZELL ET AL.

Muscle [K+] varied between 111.7 and 119.3 mEq/l(Figure 2(d)) and was similar to previously reported data(Misra et al. 1980; Al-Najdawi and Abdullah 2002).Independent of the housing system, muscle [K+] was5.3 mEq/l higher for LL compared with SH hens(P < 0.03). Within CC, muscle [K+] was 6.2 mEq/l higherfor LL compared with SH hens (P < 0.02). Within FC,muscle [K+] did not differ between strains (P > 0.24).Muscle [K+] did not differ between CC (114.7 ± 1.0) andFC (117.2 ± 1.5) housing systems (P > 0.18).

Muscle concentrations of Cl− and lactate−

Muscle [Cl−] (Figure 3(a)) varied between 8.1 and 9.0 mEq/land was similar to previous values reported in rodent and

poultry muscles (Misra et al. 1980; Lindinger et al., 1987).Muscle [Cl−] did not differ between hen strains (P > 0.62)or between the CC (8.7 ± 0.3) and FC (8.5 ± 0.4) housingsystems (P > 0.69).

Muscle [lactate−] (Figure 3(b)) varied between 51.6 and65.9 mEq/l and was similar to previously reported values inpoultry muscles (Debut et al. 2005; Berri et al. 2007; LeBihan-Duval et al. 2008). Muscle [lactate−] was 10.1–10.4 mEq/l higher in LB hens compared with LL(P < 0.03) and SH (P < 0.03) hens, respectively, independentof the housing system. In the CC system, muscle [lactate−]was 13.1 and 14.3 mEq/l higher in LB hens compared withLL (P < 0.02) and SH (P < 0.007) hens. The strain effect wasabsent in the FC system (P > 0.36). Muscle [lactate−] did notdiffer between CC (56.8 ± 2.1) and FC (58.8 ± 2.5) housingsystems (P > 0.54).

Sums of strong base cations and strong acid anionsStrain differences were noted for ∑[Ca2+] + [Mg2+] +[Na+] + [K+] (Figure 4(a)). Independent of the housing system,∑[Ca2+] + [Mg2+] + [Na+] + [K+] was 10.1 mEq/l greater in LLhens compared with SH hens (P < 0.03) and also trended8.6 mEq/l higher in LB compared with SH hens (P = 0.059).Housing treatment differences were observed in which the∑[Ca2+] + [Mg2+] + [Na+] + [K+] was 7.7 mEq/l greater in theFC system compared with the CC system (P < 0.04).

Strain differences were also noted for ∑[Cl−] + [lactate−](Figure 4(b)). When housing treatments were combined, the∑[Cl−] + [lactate−] was 9.3 mEq/l greater in LB hens comparedwith SH (P < 0.05) and LL (P < 0.05) hens. Within CC, the∑[Cl−] + [lactate−] was 12.8 and 13.9 mEq/l greater in LB henscompared with LL (P < 0.03) and SH (P < 0.02) hens, respec-tively. In FC, this strain effect was absent (P > 0.38). The∑[Cl−] + [lactate−] did not differ between CC (65.4 ± 2.2)and FC (67.5 ± 2.8) housing systems (P > 0.57).

[SID], [A−] and [HCO3−]

The [SID] (Figure 5(a)) varied between 150.0 and 170.0 mEq/land was not significantly different between strains (P > 0.16)or between CC and FC housing systems (P > 0.28). However,when housing treatments were combined, [SID] trended11.3 mEq/l higher for LL hens compared with LB hens(P = 0.091). Within the CC system, the [SID] trended14.0 mEq/l higher in LL hens compared with LB hens(P = 0.077). In FC, this strain effect was absent (P > 0.21).Muscle [SID] did not differ between CC (156.9 ± 3.1) and FC(162.7 ± 4.2) housing systems (P > 0.28).

The [A−] (Figure 5(b)) varied between 138.1 and161.2 mEq/l and was not significantly different betweenstrains (P > 0.33) or between housing systems(P > 0.84). However, within the FC system, [A−] trended23.0 mEq/l higher in LL compared with SH hens(P = 0.087).

Muscle bicarbonate ([HCO3−]) varied between 11.4 and

21.0 mEq/l (Figure 5(c)) and trended 4.2 mEq/l higher forLL compared to LB hens (P = 0.057). Housing differenceswere noted for muscle [HCO3

−]. When all strains werecombined, muscle [HCO3

−] was 5.0 mEq/l higher in theFC compared with CC system (P < 0.009).

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Figure 1. Concentrations of (a) glycogen and (b) lactate, and (c) glycolyticpotential in pectoralis major muscles of SH (Shaver White), LL (Lohmann Lite)and LB (Lohmann Brown) housed in conventional cages (CC) or furnishedcages (FC). Data are least square means ± standard error. a,bDifferent lettersindicate a significant difference (P ≤ 0.05) between hen strains or within ahousing treatment. *Denotes a trend (0.05 < P < 0.1).

BRITISH POULTRY SCIENCE 291

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Figure 2. Concentrations of (a) calcium, (b) magnesium, (c) sodium and (d) potassium in pectoralis major muscles of SH (Shaver White), LL (Lohmann Lite) andLB (Lohmann Brown) housed in conventional cages (CC) or furnished cages (FC). Data are least square means ± standard error. a,bDifferent letters indicate asignificant difference (P ≤ 0.05) between hen strains or within a housing treatment. *Denotes a trend (0.05 < P < 0.1).

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Figure 3. Concentrations of (a) chloride and (b) lactate in pectoralis majormuscles of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown)housed in conventional cages (CC) or furnished cages (FC). Data are leastsquare means ± standard error. a,bDifferent letters indicate a significantdifference (P ≤ 0.05) between hen strains or within a housing treatment.

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Figure 4. The sum of (a) strong base cations and (b) sum of strong acidanions in pectoralis major muscles of SH (Shaver White), LL (Lohmann Lite)and LB (Lohmann Brown) housed in conventional cages (CC) or furnishedcages (FC). Data are least square means ± standard error. a,bDifferent lettersindicate a significant difference (P ≤ 0.05) between hen strains or within ahousing treatment. *Denotes a trend (0.05 < P < 0.1).

292 K. M. FRIZZELL ET AL.

[Atot] and PCO2

Total muscle protein (P > 0.68), [Atot]protein (P > 0.68) and [Atot]weak acids (P > 0.96) (Table 2) did not differ between strains orbetween housing systems (P > 0.54). Muscle [Atot] variedbetween 247 and 281 mEq/l in hen pectoralis major (Figure 6(a)) and did not differ between strains (P > 0.86) or betweenhousing systems (P > 0.84). [Atot]protein and [Atot]weak acids

accounted for 19.5 ± 1.2% and 80.5 ± 1.2% of [Atot], respectively.Muscle PCO2 varied between 86.1 and 134.6 mmHg

(Figure 6(b)) and did not differ between hen strains(P > 0.85). However, housing differences were noted for

muscle PCO2. When all strains were combined, musclePCO2 was 33.7 mmHg higher in the FC system comparedwith the CC system (P < 0.006). Additionally, within the LBstrain, muscle PCO2 was elevated by 48.5 mmHg in the FCsystem compared with the CC system (P < 0.02).

Muscle pH and [H+]

Measured muscle pH and [H+]Measured pH (pHmeasured) varied between 6.551 and 6.689(Figure 7(a)), while the corresponding [H+] ([H+]measured) var-ied between 208 and 291 nEq/l (Figure 7(b)). There was a straineffect for muscle pHmeasured and [H+]measured. When housingtreatments were combined, muscle pHmeasured was 0.103 lowerfor LB compared with LL hens (P < 0.005) (Figure 7(a)) and thecorresponding [H+]measured was 56 nEq/l higher in LB com-pared with LL hens (P < 0.005) (Figure 7(b)). Within the FCsystem, pHmeasured was 0.138 lower (Figure 7(a)) and [H+]measured was 81 nEq/l higher (Figure 7(b)) (P < 0.04) in LBcompared with LL hens. Neither muscle pHmeasured nor [H+]measured differed between the CC and FC systems (P > 0.34).

Calculated muscle pH and [H+]Muscle pHcalculated and [H+]calculated varied between 6.573 and6.719 (Figure 8(a)) and 192 and 266 nEq/l (Figure 8(b)),respectively. There was a strain effect for muscle pHcalculated

and [H+]calculated. When housing treatments were combined,muscle pHcalculated was 0.111 lower (P < 0.005) (Figure 8(a))and [H+]calculated was 56 nEq/l higher for LB as compared withLL hens (P < 0.005) (Figure 8(b)). In the FC system, pHcalculated

was 0.146 lower (Figure 8(a)) and [H+]calculated was 74 nEq/lhigher (Figure 8(b)) (P < 0.04) for LB compared with LL hens.Neither pHcalculated nor [H

+]calculated differed between the CCand FC systems (P > 0.81).

Regression analysisMuscle [H+]calculated was linearly related to [H+]measured

(P < 0.0001) (Figure 9(a)). Muscle [H+]calculated accountedfor 99.7% of the variation in [H+]measured. Muscle pHcalculated

was also linearly related to pHmeasured, accounting for 99.6%of the variation (P < 0.0001) (Figure 9(b)).

Contributions of [SID], PCO2 and [Atot] to muscle pH

Muscle [SID] contributed positively to muscle pH by 47.1–47.7% regardless of housing system or laying hen strain(Table 3). In contrast, muscle PCO2 negatively contributedto muscle pH by between −3.65 and −5.80% regardless ofhousing system or laying hen strain (Table 1). Furthermore,the relative contribution of muscle [Atot] to muscle pH wasbetween −69.7% and −72.0% regardless of housing systemor laying hen strain (Table 3).

Contributions of strong base cations and strong acidanions to muscle [SID]

Strong base cations contributed positively to muscle [SID]regardless of housing system or genetic strain (Table 4). Thecontribution of [Ca2+] to muscle [SID] ranged from 20.5%to 23.5% and the contribution of [Mg2+] to muscle [SID]varied from 27.0% to 31.0% (Table 4). Additionally, thecontribution of [Na+] to muscle [SID] ranged from 16.7%

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Figure 5. The (a) [SID], (b) anion gap [A−] and (c) [HCO3-] in pectoralis majormuscles of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown)housed in conventional cages (CC) or furnished cages (FC). Data are leastsquare means ± standard error. a,b – Different letters indicate a significantdifference (P ≤ 0.05) between hen strains or within a housing treatment.*Denotes a trend (0.05 < P < 0.1).

BRITISH POULTRY SCIENCE 293

and 21.8% and the contribution of [K+] to muscle [SID]varied from 70.3% and 76.2% (Table 4).

Strong acid anions negatively contributed to muscle[SID] regardless of housing system or laying hen strain(Table 5). The contribution of [Cl−] to muscle [SID]ranged from −4.8 to −5.7% and the contribution of[Lac−] to muscle [SID] varied from −32.2 to −43.9%(Table 5).

Discussion

The present study investigated post-mortem poultry pec-toralis major [H+] within the conceptual and empirical

framework of the physicochemical model derived byStewart (Stewart 1981, 1983). The Stewart model ofacid–base regulation proved to be accurate, accountingfor >99% of the variation in pectoralis major [H+] andits corresponding log transformation, pH (Figure 9). Thenovel findings of this study are twofold. First, variationsin the [SID] system-independent variable accounted formost or all of the observed differences in [H+] betweenbird strains. Second, although [H+] did not differbetween CC and FC systems, the relative contributions

Table 2. Total protein [Atot]protein and [Atot]weak acids in pectoralis major muscles of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown) housed in CC(conventional cages) or FC (furnished cages).

Variable Housing treatment Strains combined SH LL LB

Total protein(g/dl TTW)n = 42

Treatments combined – 21.9 ± 2.22 20.1 ± 2.20 19.7 ± 2.13CC 21.1 ± 1.67 22.6 ± 2.94 21.7 ± 2.93 18.9 ± 2.80FC 20.0 ± 1.88 21.1 ± 3.30 18.6 ± 3.30 20.5 ± 3.23P – housing 0.68 0.72 0.49 0.72

[Atot]protein(mEq/l)n = 42

Treatments combined – 53.5 ± 5.43 49.3 ± 5.40 48.2 ± 5.21CC 51.6 ± 4.10 55.5 ± 7.19 53.1 ± 7.18 46.3 ± 6.87FC 49.1 ± 4.60 51.6 ± 8.07 45.5 ± 8.08 50.1 ± 7.92P – housing 0.68 0.72 0.49 0.72

[Atot]weak acids

(mEq/l)n = 42

Treatments combined – 210 ± 15.7 216 ± 15.9 226 ± 15.0CC 217 ± 11.8 224 ± 20.7 205 ± 20.7 223 ± 19.5FC 218 ± 13.5 196 ± 23.3 228 ± 24.2 230 ± 23.0P – housing 0.96 0.38 0.48 0.80

Data are least square means ± standard error.

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Figure 6. The (a) [Atot] and (b) PCO2 in pectoralis major muscles of SH (ShaverWhite), LL (Lohmann Lite) and LB (Lohmann Brown) housed in conventionalcages (CC) or furnished cages (FC). Data are least square means ± standard error.

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Figure 7. Measured (a) pH and (b) [H+] in pectoralis major muscles of SH(Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown) housed inconventional cages (CC) or furnished cages (FC). Data are least squaremeans ± standard error. a,bDifferent letters indicate a significant difference(P ≤ 0.05) between hen strains or within a housing treatment.

294 K. M. FRIZZELL ET AL.

of the three system-independent variables differedbetween housing systems.

Application of the Stewart model to evaluatedifferences in meat pH

Although glycolytic potential has been correlated with lac-tate accumulation (El Rammouz et al. 2004; Zhang et al.2009), and lactate accumulation undoubtedly accounts for asignificant portion of post-mortem acidification of meat(Monin and Sellier 1985; El Rammouz et al. 2004; Berriet al. 2007; Le Bihan-Duval et al. 2008; England et al. 2014,2016), significant dissociations have been reported indicat-ing the involvement of other factors. Examination of areport by Monin and Sellier (1985) reveals that althoughlactate was ~11 mEq/l lower in a variety of muscles inHampshire compared to Pietrain pigs, the correspondingultimate pH (pHu) was lower. Similar dissociations werereported in breast and thigh muscles of broilers exposedto prolonged stress (Zhang et al. 2009). Zhang et al. (2009)showed, for example, that transport stress caused lactate toincrease by 9 mEq/l without a corresponding change inpHu. In a more recent study by Matarneh et al. (2015),initial pH (pHi) was the same within longissimus lumborumof wild-type and AMPKγ3R200Q pigs even though lactateconcentration was 10 mEq/l higher in wild-type pigs.Lower pHu reported for AMPKγ3R200Q pigs amounted toa net increase in [H+] on the order of 1300 nEq/l butoccurred in the absence of differences in lactateconcentration.

A comparison of hen strains examined in the presentstudy also revealed dissociations between glycolytic poten-tial and lactate accumulation, and between lactate and [H+]accumulation that can be reconciled within the context ofthe Stewart model. Whereas greater glycolytic potential ofLB, compared with LL (Figure 1(c)), corresponded to theaccumulation of lactate (Figures 1(b) and 3(b)) and [H+](Figure 7(b)), the same comparison between SH and LLrevealed a moderate dissociation between these variables.Greater glycolytic potential within SH, compared with LL,did not induce a significant increase in lactate content, yet[H+] still demonstrated an absolute increase of 35 nEq/l(Figure 7(b)). Using the Stewart equation (Equation 8), itwas possible to calculate that in this instance, 100% of the35-nEq/l rise in [H+] in SH was attributed to a reduction inthe [SID] that resulted solely from a 10-mEq/l reduction inthe ∑[Ca2+] + [Mg2+] + [Na+] + [K+] (Figure 4(a)). Incontrast, only 70% of the 56 nEq/l increase in [H+] withinLB, compared with LL hens (Figure 7(b)), was attributed toan 11 mEq/l difference in the SID (Figure 5(a)); in thisinstance, all of the decline in the [SID] was attributed tothe greater lactate accumulation in the LB (Figures 3(b) and4(b)). Slight increases in Atot and PCO2 accounted for theremaining 2% and 28% of elevated [H+] in LB hens.

Another major finding of the present study was that henshoused in FC systems achieved acid–base regulation in amanner that differed from hens housed in the CC system.The fact that PCO2 was 33.7 mmHg higher within henshoused in the FC system (Figure 6(b)) but did not demon-strate correspondingly higher [H+] could be explained bycompensatory adaptive changes in the other two system-independent variables. Using Equation 8 and the meanvalues of the three system-independent variables for the

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Figure 8. Calculated (a) pH and (b) [H+] in pectoralis major muscles of SH(Shaver White), LL (Lohmann Lite), and LB (Lohmann Brown) housed inconventional cages (CC) or furnished cages (FC). Data are least squaremeans ± standard error. a,bDifferent letters indicate a significant difference(P ≤ 0.05) between hen strains or within a housing treatment.

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Figure 9. Correlations between (a) calculated and measured [H+] and between(b) calculated and measured pH in pectoralis major muscles of SH (ShaverWhite), LL (Lohmann Lite), and LB (Lohmann Brown) housed in conventionalcages (CC) or furnished cages (FC).

BRITISH POULTRY SCIENCE 295

CC and FC housing systems, it is possible to calculate thatthe potential for PCO2-induced elevation of the [H+] inhens housed in the FC system was counteracted by the netcumulative effect of a 4-mEq/l decrease in Atot plus a 7-mEq/l rise in the [SID]. The latter having resulted fromgreater intramuscular sequestration of strong base cations(Figure 4(a)) that was independent of variations in lactateaccumulation or glycolytic potential (Figures 1 and 3(b)).This comparison highlights the importance of consideringall three system-independent variables when interpretingthe underlying cause of a change in [H+].

[SID]

In the present study, variations in the [SID] between henstrains accounted for most or all of the observed differencesin [H+]. This indicates that post-mortem muscle [H+] is sig-nificantly affected by heritable differences in ion transportcapacity expressed during the ante-mortem period. The factthat strain differences in the ∑[Ca2+] + [Mg2+] + [Na+] + [K+]

were attributed to variations in muscle [Na+] and [K+] sug-gests differences in the content or specific activities of the Na+/Ca2+ exchanger (NCX) and Na+/K+-ATPase pump (Na+/K+-pump). In this regard, it is interesting to consider the potentialimpact of stress-induced elevation of glucocorticoids, espe-cially in light of the fact that White Leghorns demonstrate agreater stress-induced increase in glucocorticoids comparedwith Brown layer strains (Fraisse and Cockrem 2006). Smithand Smith (1994) showed that expression of NCX was down-regulated by glucocorticoids, while Ravn and Dorup (1997)reported muscle K+ loss was associated with prolonged eleva-tion of glucocorticoid levels. Thus, lower NCX expression inSH hens probably accounted for both lower [Na+] and lower[K+] in those birds. This hypothesis is supported by reportsthat the Na+/K+-pump is dependent on [Na+] and ATP foractivity (Therien and Blostein 2000) and the Km for Na+ isestimated to be 12–33 mM (Zahler et al. 1997) where it isoptimally positioned to regulate the Na+/K+-pump activity indirect proportion to [Na+] in the current study.

Ante-mortem accumulation of muscle lactate is the netresult of an imbalance between production (i.e.

Table 3. Contributions of [SID], PCO2 and Atot to pectoralis major muscle pH of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown) housed in CC(conventional cages) or FC (furnished cages).

[SID] (mEq/l) PCO2 (mmHg) Atot (mEq/l) [H+] (mEq/l)Strain Treatment Contribution (%) Mean ± SEM Contribution (%) Mean ± SEM Contribution (%) Mean ± SEM Mean ± SEM

SH All +47.3 157 ± 4.5 −4.7 110 ± 9.2 −71.1 264 ± 14.7 231 ± 16.9LL All +47.7 167 ± 4.5 −4.3 108 ± 9.3 −71.1 266 ± 14.6 197 ± 11.3LB All +47.3 156 ± 4.6 −4.7 110 ± 8.9 −71.4 274 ± 14.0 253 ± 14.5SH CC +47.3 157 ± 5.3 −4.0 98 ± 12.3 −72.0 281 ± 19.4 249 ± 30.2LL CC +47.7 164 ± 5.4 −3.7 94 ± 12.3 −71.3 259 ± 19.3 203 ± 16.9LB CC +47.1 150 ± 5.5 −3.7 86 ± 11.7 −71.9 269 ± 18.4 241 ± 20.4SH FC +47.4 157 ± 7.3 −5.5 122 ± 13.8 −69.8 247 ± 21.9 215 ± 13.5LL FC +47.7 170 ± 7.2 −4.9 122 ± 13.8 −71.1 272 ± 21.8 192 ± 16.1LB FC +47.3 161 ± 7.3 −5.8 135 ± 13.3 −69.7 280 ± 21.4 266 ± 21.1

Table 4. Contributions of [Ca2+], [Mg2+], [Na+] and [K+] to pectoralis major muscle [SID] of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown)housed in CC (conventional cages) or FC (furnished cages).

[Ca2+] (mEq/l) [Mg2+] (mEq/l) [Na+] (mEq/l) [K+] (mEq/l)

Strain TreatmentContribution

(%) Mean ± SEMContribution

(%) Mean ± SEMContribution

(%) Mean ± SEMContribution

(%) Mean ± SEM[SID] (mEq/l)Mean ± SEM

SH All 23.0 36 ± 2.6 28.3 44 ± 1.4 16.8 26 ± 1.1 72.5 113 ± 1.6 156 ± 4.5LL All 21.3 36 ± 2.6 27.4 46 ± 1.4 18.1 30 ± 1.1 71.1 119 ± 1.6 167 ± 4.5LB All 21.2 33 ± 2.6 29.9 46 ± 1.4 21.5 33 ± 1.1 74.5 116 ± 1.6 155 ± 4.6SH CC 22.6 35 ± 3.0 28.1 44 ± 1.6 16.7 26 ± 1.3 71.4 112 ± 1.8 157 ± 5.3LL CC 20.5 34 ± 3.0 27.7 45 ± 1.6 17.4 29 ± 1.3 71.9 118 ± 1.8 164 ± 5.4LB CC 20.5 31 ± 3.0 31.0 47 ± 1.6 21.8 33 ± 1.3 76.2 114 ± 1.8 150 ± 5.5SH FC 23.5 37 ± 4.4 28.5 45 ± 2.3 16.9 26 ± 1.8 73.6 115 ± 2.6 156 ± 7.3LL FC 22.2 38 ± 4.4 27.0 46 ± 2.3 18.8 32 ± 1.8 70.3 119 ± 2.6 170 ± 7.2LB FC 21.6 35 ± 4.4 28.6 46 ± 2.3 21.0 34 ± 1.8 72.1 117 ± 2.6 162 ± 7.3

Table 5. Contributions of [Cl−] and [lactate−] to pectoralis major muscle [SID] of SH (Shaver White), LL (Lohmann Lite) and LB (Lohmann Brown) housed in CC(conventional cages) or FC (furnished cages).

[Cl−] (mEq/l) [Lac−] (mEq/l) [SID] (mEq/l)Strain Treatment Contribution (%) Mean ± SEM Contribution (%) Mean ± SEM Mean ± SEM

SH All −5.7 9.0 ± 0.47 −34.9 55 ± 2.8 156 ± 4.5LL All −5.0 8.4 ± 0.47 −32.9 55 ± 2.8 167 ± 4.5LB All −5.4 8.4 ± 0.48 −41.8 65 ± 2.9 155 ± 4.6SH CC −5.7 9.0 ± 0.55 −33.0 52 ± 3.5 157 ± 5.3LL CC −5.3 8.7 ± 0.55 −32.2 53 ± 3.5 164 ± 5.4LB CC −5.6 8.4 ± 0.56 −43.9 66 ± 3.7 150 ± 5.5SH FC −5.7 8.9 ± 0.76 −36.8 57 ± 4.4 156 ± 7.3LL FC −4.8 8.1 ± 0.76 −33.5 57 ± 4.4 170 ± 7.2LB FC −5.1 8.3 ± 0.76 −38.1 62 ± 4.4 162 ± 7.3

296 K. M. FRIZZELL ET AL.

glycogenolysis) and removal (Putman et al. 1995) by eithermonocarboxylic lactate transporter (MCT)-dependent extru-sion into the venous blood (Bonen et al. 1998) or metabolismwithin muscle fibres (Putman et al. 1998, 2003). In contrast, inthe absence of blood flow and oxygen, post-mortem lactateaccumulation is only attributed to continued glycogenolysis(Pösö and Puolanne 2005) and must be proportional to theinitial glycogen content (Newsholme and Start 1973). Thestandardised handling procedures used in the present studyshould have minimised ante-mortem glycogenolysis, and thus,it seems reasonable to conclude that greater lactate accumula-tion in LB probably resulted, at least in part, from greater post-mortem glycogen breakdown. However, it is also possible thatthe greater lactate accumulation in LB hens resulted fromlower rates of ante-mortem lactate extrusion secondary tolower sarcolemmal MCT content. Indeed, expression ofMCT has been shown to differ between breeds of variousspecies (Mykkänen et al. 2010; Parkunan et al. 2015) andMCT expression is reportedly elevated in response to stress(Parkunan et al. 2015). It is also possible that greater stressresponses by SH and LL hens accelerated ante-mortem lactateoxidation (Gleeson et al. 1993).

Variations in the ∑[Ca2+] + [Mg2+] + [Na+] + [K+] thatwere observed between housing systems indicate that post-mortem muscle [H+] has the potential to be affected by ante-mortem conditions that includes the opportunity for move-ment within production facilities. Greater sequestration ofstrong base cations within hens housed in the FC systemindicates that those hens possessed a greater capacity to defendagainst post-mortem acidification. It is suggested that thegreater ∑[Ca2+] + [Mg2+] + [Na+] + [K+] observed in the FChousing system probably resulted from increased activity ofhens that expressed natural behaviours on a daily basis. This issupported by previous reports showing that activity increasedthe expression of Ca2+-binding proteins (Ohlendieck et al.1999), Ca2+ storage volume (Murphy et al. 2009; Fryer andStephenson 1996), Mg2+-ATP content (Green 2000), K+

uptake (Green 2000), NCX expression (Bueno et al. 2010)and capillary volume (Škorjanc et al. 1998).

[Atot]

Muscle [Atot] represents the total concentration of undisso-ciated plus dissociated non-volatile organic weak acids thatbehave as classical weak acid buffers (Stewart 1981, 1983). Amajor component of [Atot] is formed by proteins that pos-sess a net negative charge of 2.45 g/dl within the pH rangeobserved in the current study (Jones 1987). Within complexstructural and metabolic proteins, the ionising amino acidside chain of histidine (pKa = 6.04) is ideally positioned tobuffer H+ within the pH range typically observed in ante-and post-mortem muscle; additional contributors may alsoinclude free amino acids with isolelectric points (pI) withinthe same range. The second major component of [Atot]includes an as yet undetermined number of phosphorylatedmetabolites (pKa ~ 6.1), as well as ATP, ADP (pKa ~ 6.5)and AMP (pKa ~ 6.1) (Jones 1987; Lindinger andHeigenhauser; 1991; Lindinger 1995). In the present study,the first component, total protein, was measured but thephosphorylated metabolites were not determined becausethey would have encompassed an unreasonably large num-ber of weak acids and associated KA values. It also proved to

be unnecessary because the Stewart model allowed for thederivation of single values for KA and [Atot].

[Atot] was determined by calculating [A−] using thestrong ion electroneutrality equation (Equation 5) andexperimentally determined values of [SID] and [HCO3

−].It was then possible to calculate [Atot] (Equation 6) based onmeasured [H+] and the dissociation constant for weak acids,KA, which is equal to 3.0 × 10−7 Eq/l (i.e. pKa = 6.6)(Stewart 1981, 1983). This approach is supported by afundamental principle of the Stewart model, which statesthat within biological systems, the value of KA remainsconstant even when the [SID] and [Atot] system-indepen-dent variables change (Stewart 1981, 1983). It is also sup-ported by the findings of Stämpfli et al. (2006) who usedCO2 tonometry to estimate that the KA of avian plasma wasbetween 2.15 × 10−7 and 3.25 × 10−7 Eq/l (i.e. pKa ~ 6.5–6.7). This approach proved to be reliable when the resulting[Atot] values were used to calculate [H+] (Figure 9(a)).

An important finding of the present study was thatmuscle protein concentration represented only 20% of[Atot] and was remarkably constant across hen strains andbetween housing conditions. In our study, the remaining80% of [Atot] was attributed to organic weak acids and wasalso constant across strains and between housing condi-tions. Because the [Atot] systems independent variable isconsiderably more constant than [SID] and PCO2, its quan-titative significance is often overlooked. Using the Stewartequation and empirically determined values of [Atot], [SID]and PCO2 for the three hen strains, it is possible to calculatethat reducing [Atot] by half would result in a 75% decreasein [H+], thus highlighting the importance of applying rig-orous quantitative modelling to determine the contributionsof [Atot]. Such considerations may prove important when,for example, attempting to establish the cause of Pale SoftExudative (PSE) meat. It is plausible that it is not theincrease in [H+] that induces protein denaturation andlower WHC within PSE meat (Ahn and Maurer 1990;Barbut et al. 2005; Berri et al. 2005; Le Bihan-Duval et al.2008), but rather that excessive protein breakdown andwater loss cause [Atot]protein and [Atot]weak acids to risewhich is the actual cause of increased [H+] in PSE meat.

PCO2

Greater muscle PCO2 observed within the pectoralis majorof hens housed in the FC system of the present study isconsistent with ante-mortem adaptive changes havingoccurred that enhanced the capacity for oxidative CO2

production and elevated myocellular oxygen storage capa-city. Branciari et al. (2009) showed that the greater activityof White Leghorns that were allowed daily access to a grasspaddock was sufficient to induce the transition of fast-glycolytic to slower more oxidative fibres within the pector-alis major, while Castellini et al. (2002) reported that thesame production conditions induced a 20% increase inhaeme iron. Thus, most of the post-mortem CO2 accumula-tion observed in hens housed in the FC system probablyresulted from new ante-mortem steady-state CO2 produc-tion. It is also plausible that greater Mb–O2 continued tostimulate Kreb’s cycle flux, further contributing to CO2

accumulation early in the post-mortem period. In the pre-sent study, the corresponding increase of [HCO3

−] within

BRITISH POULTRY SCIENCE 297

the pectoralis major muscle must be considered obligatorygiven the rapidly reversible nature of carbonic anhydrase(Geers and Gros 2000).

Conclusion

The results of this study demonstrate the utility of compre-hensive physicochemical modelling to investigate the factorsresponsible for variations in poultry meat [H+] within anagricultural production setting. The Stewart model allowedcalculation of [H+] with a very high degree of accuracywhen compared with measured values of [H+]. It also pro-vided the conceptual framework to identify factors which,acting through the [SID], were responsible for differences in[H+] between hen strains that included sequestration ofstrong base cations and excess lactate accumulation.Application of the model to evaluate the experimental effectof CC versus FC housing systems demonstrated thatalthough hens housed in the FC system generated higherlevels of PCO2, post-mortem acidification was countered bygreater sequestration of strong base cations in these hens. Itis suggested that housing treatment differences resultedfrom activity-induced adaptations within hens housed inthe FC system.

The results of this study suggest that hens housed in FChave improvedmuscle function and overall health due to theirincreased opportunity for movement compared to conven-tionally housed hens. These findings were not apparent fromthe traditional meat quality measures conducted in the pre-vious study (Frizzell et al. 2017). Past studies have shown thatthe increased opportunity for movement and ability to expresshighly motivated nesting, perching, foraging and dust-bathingbehaviours for hens housed in FC has contributed to strongerbones (Jendral et al. 2008; Jendral 2012) and decreased aggres-sion (Jendral 2012) compared to hens housed in CC. Thesedifferences indicate improved welfare for hens housed in FCcompared to those housed in CC, a conclusion that is alsosupported by the housing differences observed in the currentresearch. The Stewart model has therefore not only beenshown to be an accurate tool to determine muscle [H+] butcan also identify changes in muscle cell physiology that stan-dard meat quality measures could not. The model has there-fore been identified as a novel tool to assess animal welfare aswell as changes in muscle quality.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by the Natural Sciences and EngineeringResearch Council of Canada,Technology Development 2000, NovaScotia Agricultural College, Atlantic Poultry Research Institute, EggFarmers of Alberta, Egg Farmers of Canada, Poultry Industry Counciland University of Alberta.

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