RESEARCH ARTICLE

Differential sex-specific walking kinematics in leghorn chickens(Gallus gallus domesticus) selectively bred for different body sizeKayleigh A. Rose, Jonathan R. Codd and Robert L. Nudds*

ABSTRACTThe differing limb dynamics and postures of small and largeterrestrial animals may be mechanisms for minimising metaboliccosts under scale-dependent muscle force, work and powerdemands; however, empirical evidence for this is lacking.Leghorn chickens (Gallus gallus domesticus) are highlydimorphic: males have greater body mass and relative musclemass than females, which are permanently gravid and havegreater relative intestinal mass. Furthermore, leghorns are selectedfor standard (large) and bantam (small) varieties and the formerare sexually dimorphic in posture, with females having a moreupright limb. Here, high-speed videography and morphologicalmeasurements were used to examine the walking gaits of leghornchickens of the two varieties and sexes. Hindlimb skeletalelements were geometrically similar among the bird groups, yetthe bird groups did not move with dynamic similarity. In agreementwith the interspecific scaling of relative duty factor (DF, theproportion of a stride period with ground contact for any givenfoot) with body mass, bantams walked with greater DF thanstandards, and females walked with greater DF than males.Greater DF in females than in males was achieved via variety-specific kinematic mechanisms, associated with the presence/absence of postural dimorphism. Females may require greater DFin order to reduce peak muscle forces and minimise powerdemands associated with lower muscle to reproductive tissuemass ratios and smaller body size. Furthermore, a more uprightposture observed in the standard, but not bantam, females, mayrelate to minimising the work demands of being larger and havingproportionally larger reproductive tissue volume. Lower DF inmales relative to females may also be a work-minimising strategyand/or due to greater limb inertia (as a result of greater pelvic limbmuscle mass) prolonging the swing phase.

KEY WORDS: Froude number, Locomotion, Posture,Sexual dimorphism, Duty factor, Dynamic similarity

INTRODUCTIONThe size of an animal influences its walking kinematics. Whenmoving at the same speed (U, m s−1), larger animals generally takelonger and fewer strides per unit time than smaller animals.Comparison of the walking kinematics of different-sized animalscan be conducted at speeds at which the ratios of inertial togravitational forces acting upon the body centre of mass (CoM) are

equal, using either the Froude number (Fr=U2/ghhip) or its squareroot, often termed relative speed:

u ¼ U=ffiffiffiffiffiffiffiffiffiffighhip

p; ð1Þ

where hhip is hip height (m) and g is gravitational acceleration(9.81 m s−2) (Alexander, 1976; Alexander and Jayes, 1983).Dynamic similarity of motion between different-sized animalsrequires geometric similarity in body plan and equal values ofdimensionless kinematic parameters (scaled appropriately to negatethe effects of size) for a given relative speed (Alexander, 1976;Alexander and Jayes, 1983; Hof, 1996).

Animals may move in such a way as to minimise metabolic cost.Themetabolic cost of transport (CoT) is the energy required to movea unit body weight over a unit distance [power/(bodyweight×speed)]. Geometrically similar animals of different sizemoving in a dynamically similar fashion are expected to have equalCoT (Alexander and Jayes, 1983). The dynamic similarityhypothesis of Alexander and Jayes (1983) postulated that differentquadrupedal mammals would locomote with dynamic similarityat equivalent relative speeds. Within non-cursorial (<1 kg) andcursorial (>10 kg) mammalian groups (Jenkins, 1971), thehypothesis was supported. Observed kinematic differencesbetween the two groups, however, were not accounted for(Alexander and Jayes, 1983). Furthermore, between avian speciesof small and large body size, there is considerable deviation fromdynamic similarity of locomotion (Gatesy and Biewener, 1991;Abourachid and Renous, 2000; Abourachid, 2001). However, ageneral pattern exists across these vertebrates, whereby smallerspecies move with greater relative duty factor (DF, the proportion ofa stride with ground contact for any given foot) and relative stridelengths. These deviations from dynamic similarity of locomotionhave been attributed to differences in the relative lengths of the limbsegments and limb posture (Alexander and Jayes, 1983; Gatesy andBiewener, 1991; Abourachid and Renous, 2000; Abourachid,2001). Crouched and upright limb postures are generally adoptedby small and large vertebrate species, respectively, which are cleardepartures from geometric similarity in body form (Biewener, 1989;Gatesy and Biewener, 1991).

The differing gait kinematics and postures of small and largeterrestrial animals may be mechanisms for minimising metaboliccosts under scale-dependent muscle force, work and powerdemands; however, empirical evidence for this is lacking. Bodyweight (∝l3∝v1, where l is length and v is volume) increases at afaster rate with body size than the strength (i.e. ability to resistforces, ∝cross-sectional area ∝l2∝v2/3) of the biological materialsthat must support it (Biewener, 1989). An erect limb aligns bodyweight with each limb bone’s long axis, reducing mechanicalloading on the muscles associated with turning moments about thejoints (Biewener, 1989). The ‘cost of muscle force’ hypothesis forthe scaling of limb posture and gait with body size states that theReceived 2 March 2016; Accepted 8 June 2016

Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

*Author for correspondence (robert.nudds@manchester.ac.uk)

R.L.N., 0000-0002-7627-6324

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more upright limbs of larger species serve to reduce the large forcesthat would otherwise have to be exerted by the limb muscles(Biewener, 1989). An alternative to the cost of muscle forceapproach is that animals of differing size optimise active musclevolume under scale-dependent muscle work and power demands(Usherwood, 2013). A more erect limb requires shorter stance(push-off ) periods, reducing fore–aft speed fluctuations and,consequently, muscle work (J kg−1) requirements (Usherwood,2013). Although the same benefits of an upright limb (in terms ofreducing muscle work) would apply to smaller animals,theoretically, their muscle power (J s−1 kg−1) requirements maybe disproportionately high (Usherwood, 2013). Therefore, a morecrouched limb, requiring a longer push-off period, may act tominimise power requirements in smaller animals (Usherwood,2013). Indeed, for a given relative speed, human (Homo sapiens)toddlers were found to deviate more from work-minimising gaitsthan adults, via longer relative stance periods (Hubel andUsherwood, 2015).Understanding of the gaits and postures of different-sized animals

is compromised because the majority of comparisons are conductedbetween different species (Alexander and Jayes, 1983; Gatesy andBiewener, 1991; Abourachid and Renous, 2000; Abourachid,2001). Intraspecifically, the sexes may differ not only in bodysize (Lislevand et al., 2009; Remes and Szekely, 2010) but also inmorphological proportions, which are likely to influence muscleforce, work and power demands. For example, in many vertebratespecies, the relative proportions of total body mass (Mb) allocated todifferent somatic and reproductive components are usually biasedtowards males and females, respectively (Shine et al., 1998;Hammond et al., 2000; Lourdais et al., 2006). Furthermore, femalereproductive specialisation may even require specific skeletalproportions [e.g. a wider pelvis (Baumel, 1953; Smith et al.,

2002; Cho et al., 2004) or posture, during pregnancy (Franklin andConner-Kerr, 1998) or gravidity (Rose et al., 2015b)]. Most studieson gait kinematics, however, have been conducted using individualsof only one sex (Reilly, 2000); without comparing sexes (Rubensonet al., 2004; Watson et al., 2011); or using individuals whose sexeswere not reported (Gatesy and Biewener, 1991; Abourachid, 2000,2001; Abourachid and Renous, 2000; Griffin et al., 2004; Nuddset al., 2010). Previous studies have identified sex differences inwalking kinematics in humans (Bhambhani and Singh, 1985) andtwo species of bird (Lees et al., 2012; Rose et al., 2014), but whethersize variations alone or both size and additional unidentified sexualdimorphisms were behind the differences in kinematics was notdetermined.

The leghorn chicken, Gallus gallus domesticus (Linnaeus 1758)is highly dimorphic, with males having greater body size andmuscle mass than females (Mitchell et al., 1931; Rose et al., 2016a).Female leghorns have greater digestive organmasses thanmales andremain permanently gravid (Mitchell et al., 1931). Furthermore,leghorns are selectively bred for standard (large) and bantam (small)varieties, and only the standard variety is sexually dimorphic in limbposture, with females possessing a more upright limb than males atmid-stance during a walking gait (Rose et al., 2015b). For a givensex, the two varieties are expected to be closer to geometricsimilarity in anatomical proportions (a prerequisite for dynamicsimilarity of motion). Whilst the males of the two varieties aregeometrically similar in their axial and appendicular skeletons, thebantam males adopt a more upright posture at mid-stance thanthe standards during a walking gait (Rose et al., 2015a). Themorphological variations that have resulted from selective breedingin these leghorns provide a novel opportunity to investigate theeffects of limb posture and differing relative locomotor muscle,digestive and reproductive tissue masses (i.e. varied muscle force,work and power demands) on walking dynamics.

Here, high-speed videography and morphological measurementswere used to test the hypothesis that male and female standard andbantam varieties of leghorn show clear departures from dynamicsimilarity of motion associated with their morphological variations.

MATERIALS AND METHODSAnimalsMale and female bantam brown leghorns (B♂ and B♀) and standard-breed white leghorns (L♂ and L♀) were obtained from localsuppliers and housed in the University of Manchester’s AnimalUnit. All leghorns (>16 weeks, <1 year) had reached sexualmaturity and females were gravid. Sexes and varieties werehoused separately with ad libitum access to food, water andnesting space. Birds were trained daily for a week to sustainlocomotion for ∼5 min within a Perspex® chamber mounted upon aTunturi T60 (Turku, Finland) treadmill. The kinematics of 24 of the28 leghorns used for the simultaneously collected metabolicmeasurements described in Rose et al. (2015b) are presentedhere (B♂: N=9, 1.39±0.03 kg; B♀: N=5, 1.04±0.03 kg; L♂: N=5,1.92±0.13 kg; L♀: N=5, 1.43±0.06 kg; means±s.e.m.). Allexperiments were approved by the University of Manchester’sethics committee, carried out in accordance with the Animals(Scientific procedures) Act (1986) and performed under a UKHome Office Project Licence held by J.R.C. (40/3549).

KinematicsThe left greater trochanter of the hip of each bird was located byhand and any overlying feathers were removed and replaced with areflective marker. Each leghorn was exercised at a minimum speed

List of symbols and abbreviationsB♀ female bantamsB♂ male bantamsCoM centre of massDF duty factorFr Froude numberfstride stride frequencyfstride relative stride frequencyg gravitational accelerationhback back heighthhip hip heighthhip:Σlsegs posture indexL♀ female standardsL♂ male standardslfem femur lengthlstride stride lengthlstride relative stride lengthltars tarsometatarsus lengthltib tibiotarsus lengthMb body masststance stance durationtstance relative stance durationtswing swing durationtswing relative swing durationu relative speedU speedwfem femur widthwpelv pelvis widthwtars tarsometatarsus widthwtib tibiotarsus widthΣlsegs sum of the hindlimb long bone lengths

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of 0.28 m s−1 and at increasing increments of 0.14 m s−1 (in arandomised order), up to the maximum they could sustain withoutshowing signs of fatigue. The birds were rested between speed trials.All trials were filmed from a lateral view (left of each bird) using avideo camera (HDR-XR520VE, Sony, Japan; 100 frames s−1).All video recordings were analysed using Tracker software (Open

Source Physics). Distance was calibrated for each video recordingusing a known distance from the front to the back of therespirometry chamber. This allowed for the alignment of acalibration tool through the line of travel of each bird (alwayspassing through digit 3), eliminating any error that could be incurredby a bird’s displacement from it (i.e. the bird’s position on thetreadmill/distance from the camera did not affect our distancecalibration). At each speed, the phasing of the sum of the verticalkinetic and gravitational potential energies with the horizontalkinetic energy of the body CoM (approximated by the trochantermarker) was determined using spatial and temporal data. Unlike themales, the female leghorns are either unable or unwilling to usegrounded running gait mechanics (Rose et al., 2015b). Hence, onlydata for speeds at which the birds used walking gait mechanics (outof phase fluctuation of gravitational potential and horizontal kineticenergy) were used in the analyses.The left foot of each bird was tracked across ∼10 continuous

strides (constant speed and position) to obtain the times of toe-on andtoe-off, which were used to calculate DF, stride frequency ( fstride,Hz), stride length (lstride=U/fstride, m), swing duration (tswing, s) andstance duration (tstance, s). A single fixed measure of hip height (hhip)was used per individual (see ‘Morphological measurements’, below)as the characteristic length for normalising their speed and gaitkinematic parameters. U was normalised for size differences as thesquare root of Fr, here termed relative speed ðU ¼ U =

ffiffiffiffiffiffiffiffiffiffighhip

p Þ.Kinematic parameters were normalised based on the Hof (1996)record of non-dimensional forms of mechanical quantities as:relative stride length ðlstride ¼ lstride=hhipÞ, relative stridefrequencyðfstride ¼ fstride=

ffiffiffiffiffiffiffiffiffiffiffiffiffig=hhip

p Þ, relative swing durationðtswing ¼ tswing=

ffiffiffiffiffiffiffiffiffiffiffiffiffihhip=g

p Þ and relative stance durationðtstance ¼ tstance=

ffiffiffiffiffiffiffiffiffiffiffiffiffihhip=g

p Þ.

Morphological measurementsFor each experimental bird, hhip wasmeasured from a video recording(accuracy, ±1 mm) of a slow walking speed (0.28 m s−1) for aminimum of 7 strides. hhip was taken (using the same method as inRose et al., 2015a,b) as the distance from the treadmill belt (wheredigit 1 meets the base of the tarsometatarsus) to the hip marker at90 deg to the direction of travel during mid-stance, when it is at itsgreatest. Back height (hback) was measured in the same way as hhip.Digital Vernier callipers (accuracy, ±0.01 mm) were used to measurehindlimb long bone (femur, tibiotarsus and tarsometatarsus) length(lfem, ltib, ltars) andwidth (wfem,wtib,wtars). Total leg length (Σlsegs) wascalculated as the sum of the three element lengths. Note that Σlsegsdoes not represent the true functional length of the hindlimb, becausethe measurements were taken from dried bones excluding the inter-joint soft tissue. Themeasurements were taken as the shortest distancebetween the most proximal and distal grooves of each element, whichwould further decrease the value of Σlsegs relative to the maximumpotential length of the three leg segments if they were verticallyaligned. Therefore, a posture index near the value of 1.00 at mid-stance in the present study is not indicative of a completely uprightlimb. The width of the pelvis (wpelv, the distance between the left andright acetabula) was also measured.Soft-tissue components from five experimental individuals of

each bird group were dissected and weighed upon electronic scales

(accuracy, ±0.01 g). Thirteen major muscles of the right pelvic limb(m. iliotibialis cranialis, m. iliotibialis lateralis, m. iliofibularis,m. flexor cruris lateralis pars pelvica, m. flexor crurismedialis, m. iliotrochantericus caudalis, m. femerotibialis medialis,m. pubioischiofemoralis pars lateralis, m. pubioischiofemoralis parsmedialis, m. gastrocnemius pars lateralis, m. gastrocnemius parsmedialis, m. fibularis lateralis and m. tibialis cranialis) weredissected for a sister study on variety- and sex-specific musclearchitectural properties (Rose et al., 2016a). The masses of thesemuscles were summed to give a comparable total pelvic limbmusclemass between chicken groups. The right breast muscles(m. pectoralis and m. supracoracoideus) and the intestines (smalland large combined) were also weighed. Reproductive tissue mass(developing eggs, ovaries and oviduct) was measured from femalebirds only and was assumed negligible in males in terms ofinfluencing locomotion dynamics. For the bantam females, four ofthe five reproductive tissue masses measured were from individualswhose experimental data were not included in the present study.These individuals, however, were from the same cohort andunderwent the same training and experimental process as the birdsfor which kinematic data are presented here. All anatomicalcomponents were compared between varieties and sexes as apercentage of dead bird body mass.

Statistical analysesAll statistical analyses were conducted in R (v3.0.2 GUY 1.2 SnowLeopard build 558; R Development Core Team, 2011). The Carpackage (Fox and Weisberg, 2011) was used for all analyses ofvariance (ANOVA) in which variety and sex were included as fixedfactors. Shapiro–Wilk tests were performed on the standardisedresiduals generated by all statistical models to ensure the dataconformed to a normal distribution. Where morphological data(Table 1) did not conform to a normal distribution even after logtransformation, a Kruskal–Wallis test was conducted to compare themeans of the four groups: B♂, B♀, L♂ and L♀. Dunn post hoc testswere used to indicate which groups differed. The relationshipsbetween absolute and relative kinematics variables with U and uwere compared between the bird groups using linear models. U andu were included in the models as covariates and all potentialinteraction terms were considered before a stepwise backwardsdeletion of non-significant interaction terms was conducted tosimplify the models. Outputs from the final models are reported.Best-fit lines were obtained from the coefficients tables of the finalstatistical models and were back-transformed where data had beenlog transformed.

RESULTSMorphological measurementsBody mass (Mb, Fig. 1A) and total leg length (Σlsegs, Fig. 1B) weregreater in the standard than in the bantam variety, and greater inmales than in females (Table 1). Hip height (hhip, Fig. 1C), however,was greater in males than in females in the bantam variety, but,conversely, greater in females than in males in the standard variety(Table 1). Posture index (hhip:Σlsegs; Fig. 1D) did not differ betweenthe sexes of the bantam variety; in contrast, the posture index of L♀was ∼27% greater than that of L♂, indicative of a more erect limb(Table 1).

Each hindlimb segment was a similar proportion of Σlsegs(Table 2) in all of the groups excluding B♀, which had a relativelylonger lfem, and concomitantly shorter ltars, resulting in a small, butnonetheless statistically significant, difference (Table 2). The widthof each limb segment (Table 2) was a similar proportion of its

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respective segment length in all groups (Table 2). The finding of amore erect posture in L♀when compared with the other three groupswas further supported by indices incorporating the height of theback (hback). hhip:hback did not differ between the bird groups, andΣlsegs:hback was lower in L♀ than in the other three groups (Table 2).wpelv, relative to Σlsegs (Table 2), did not differ between the sexes,but was ∼1% greater in the bantams than in the standards (Table 2).Total pelvic limb muscle mass (the sum of the masses of 13

pelvic limb muscles) was a greater percentage of body mass (Fig. 2)in males than in females in both varieties (Table 1). Total pelviclimbmuscle mass was also greater in the bantam than in the standardvariety (Fig. 2, Table 1). The observed differences betweenvarieties, however, were small in comparison to the sexualdimorphisms. The same statistical outcomes obtained for the totalpelvic limb muscle mass were mirrored by the breast muscles,m. pectoralis and m. supracoracoideus (Fig. 2, Table 1). Intestinemass as a proportion of body mass (Fig. 2) was greater in femalesthan in males (Table 1). The two varieties, however, shared similarrelative intestinal masses. The reproductive tissue mass as apercentage of body mass was greater in L♀ (11.49±0.56%) than inB♀ (8.40±0.08%).Therefore, all four groups were similar in their hindlimb skeletal

bone geometry (a prerequisite of dynamic similarity of locomotion).

The sexes, however, differed markedly in each of the measuredanatomical masses when expressed as percentage body mass. Incontrast, with the exception of limb posture and the relative mass ofthe female reproductive system, for a given sex, the two varieties ofleghorn were more similar in their anatomical proportions.

Walking kinematics and dynamicsDF, tswing and tstance were negatively correlated withU, and lstride andfstride were positively correlated with U. The same correlations werealso found for the relationship between size-normalised kinematicsparameters and U . For all four groups of leghorns, the exponentsdescribing the relationships between absolute or size-normalisedparameters and U or U were similar, unless stated otherwise below.

Table 1. Results of the one- and two-way ANOVA that tested for varietyand sex differences in morphological measurements/indices

Measurement/index ANOVA (final model)

Mb (kg) Variety: F1,20=47.34, P<0.001Sex: F1,20=33.96, P<0.001R2=0.76

Σlsegs (mm) Variety: F1,20=125.26, P<0.001Sex: F1,20=84.44, P<0.001R2=0.89

hhip (mm) Variety: F1,19=95.44, P<0.001Sex: F1,19=0.29, P=0.594Variety×sex: F1,19=29.55,P<0.001R2=0.85

Posture index Variety: F1,19=14.42, P=0.001Sex: F1,19=19.44, P<0.001Variety×sex: F1,19=42.30,P<0.001R2=0.75

Total pelvic limb muscle mass (% Mb) Variety: F1,17=9.10, P=0.008Sex: F1,17=161.53, P<0.001R2=0.90

M. pectoralis mass (% Mb) Variety: F1,17=18.50, P<0.001Sex: F1,17=31.00, P<0.001R2=0.71

M. supracoracoideus mass (% Mb) Variety: F1,17=8.35, P=0.010Sex: F1,17=29.01, P<0.001R2=0.65

Intestine mass (% Mb)a Variety: F1,16=0.71, P=0.411

Sex: F1,16=48.09, P<0.001R2=0.73

Reproductive tissue mass (% Mb)b Variety: F1,8=8.74, P=0.018

R2=0.46aN=4 for standard males.bFemales only, as reproductive tissue mass was assumed to be negligible inmales.Bodymass (Mb), total leg length (Σlsegs), hip height (hhip) and posture index (hhip:Σlsegs) were measured from the full sample of experimental birds. Total pelviclimbmusclemass (%Mb) is the sumof themassesof 13pelvicmuscles.With theexception ofmale intestinal mass, themasses for all soft tissues were calculatedfor 5 individuals of each bird group. Breastmusclemass (M. pectoralismass plusM. supracoracoideus mass, % Mb) is for the right side of the body only. Theadjusted R2 values of the final statistical models are reported.

0.5

1.0

1.5

2.0

2.5

Mb

(kg)

0.5 0.6 0.7 0.8 0.9 1.0 1.1

L� L� B� B�

Pos

ture

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x

V***, S***, I***

150

200

250

300

350

h hip

(mm

) V***, I***

150

200

250

300

350

Σlle

gs (m

m)

V***, S***

V***, S***A

D

C

B

Fig. 1. Morphological measurements for the variety and sexcombinations of leghorn chicken. (A) Body mass (Mb). (B) Total leglength (Σlsegs, sum of the three skeletal element lengths). (C) Hip height (hhip).(D) Posture index (hip height:total leg length). Bars represent means±s.e.m.for standard males (L♂, grey), standard females (L♀, purple), bantam males(B♂, blue) and bantam females (B♀, red). Significant variety, sex andvariety×sex interaction terms are denoted by V, S and I, respectively(***P<0.001). Results of the two-way ANOVA conducted to test for variety andsex differences are in Table 1. These morphological differences have beenreported previously (Rose et al., 2015b) for a different sample size.

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Across all speeds, DF (Fig. 3A) was greater in females than inmales (∼2%) and greater in the bantam than in the standard varietyby ∼2% (Table 3). At comparable U , DF was, similarly, greater infemales than in males in both varieties and this sex difference wasgreater in the bantams. In addition, DF was greater in the bantamthan in the standard variety (Fig. 3B, Table 3).fstride (Fig. 3C) was greater in females than in males at any givenU

(0.11 Hz; Table 3). Absolute fstride and the rate of increase in fstridewith U were significantly greater in the bantam than in the standardvariety. At comparable U , fstride was greater in females than in males

in the standard variety but, contrastingly, greater in males than infemales in the bantam variety (Fig. 3D, Table 3).

At any givenU, lstride was greater in males than in females, and bya larger amount in the standard variety (70 mm) than in the bantamvariety (20 mm) (Fig. 3E, Table 3), and was associated with agreater difference between the males of the two varieties thanbetween the females of the two varieties. At any given U , lstride(Fig. 3F), was greater in females than in males in the bantam variety,but, contrastingly, it was greater in males than in females in thestandard variety (Table 3).

Table 2. Mean (±s.e.m.) morphometric indices and results of the statistical tests conducted to investigate whether the indices differ betweenvarieties and sexes

Index B♂ B♀ L♂ L♀ Statistical results

lfem:Σlsegs 0.28 0.29 0.28 0.28 Kruskal–Wallis: χ2=19.6, d.f.=3, P≤0.001Dunn test: B♂ vs B♀: Z=3.71, P<0.001;B♂ vs L♂: Z=−0.61, P=0.272; B♂ vs L♀: Z=0.00, P=0.500;B♀ vs L♂: Z=−3.89, P<0.001; B♀ vs L♀: Z=−3.35, P<0.001;L♂ vs L♀: Z=0.55, P=0.292

ltib:Σlsegs 0.42 0.42 0.42 0.42 Kruskal–Wallis: χ2=0.13, d.f.=3, P=0.988ltars:Σlsegs 0.30 0.29 0.30 0.30 Kruskal–Wallis: χ2=12.47, P=0.006

Dunn test: B♂ vs B♀: Z=−2.48, P=0.007;B♂ vs L♂: Z=0.90, P=0.18; B♂ vs L♀: Z=0.90, P=0.18;B♀ vs L♂: Z=3.05, P=0.001; B♀ vs L♀: Z=3.05, P=0.001;L♂ vs L♀: Z=0.00, P=0.500

wfem:lfem 0.11 0.11 0.11 0.11 Kruskal–Wallis: χ2=0.46, d.f.=3, P=0.929wtib:ltib 0.07 0.06 0.07 0.07 Kruskal–Wallis: χ2=7.20, d.f.=3, P=0.066wtars:ltars 0.10 0.10 0.10 0.10 Kruskal–Wallis: χ2=0.36, d.f.=3, P=0.948hhip:hback 0.78±0.01 0.77±0.02 0.76±0.00 0.77±0.02 Variety: F1,20=0.45, P=0.512

Sex: F1,20=0.00, P=0.948R2=0.00

Σlsegs:hback 1.01±0.03 1.02±0.02 1.03±0.01 0.76±0.02 Variety: F1,19=20.58, P<0.001Sex: F1,19=22.16, P<0.001Variety×sex: F1,19=37.91, P<0.001R2=0.78

wpelv:Σlsegs 0.17 (N=6) 0.18 0.16 0.17 Variety: F1,18=11.368, P=0.003Sex: F1,18=0.347, P=0.079R2=0.39

lfem, femur length; Σlsegs, sum of the hindlimb long bone lengths; ltib, tibiotarsus length; ltars, tarsometatarsus length;wfem, femur width;wtib, tibiotarsus width;wtars,tarsometatarsus width; hhip, hip height; hback, back height; wpelv, pelvis width.The adjusted R2 values of the final models are reported; only the results of the final two-way ANOVA are reported; s.e.m. is not presented where it was 0.00 to 2decimal places.The sample size is indicated in parentheses if lower than the total sample size of the leghorn group.

0

2

4

6

8

10

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14

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Pectoralismass

Supracoracoideusmass

Intestinemass

Reproductivemass

% M

b

V**, S*** V***, S***

V**, S***

S***

V*

Fig. 2. Anatomical components as a percentage of bodymass for the variety and sex combinations of leghornchicken. Bars represent means±s.e.m. for L♂ (grey),L♀ (purple), B♂ (blue) and B♀ (red). Limb muscle mass wascalculated as the sum of 13 major pelvic limb muscles onthe right limb. M. pectoralis and M. supracoracoideus massexpressed as a percentage of body mass (Mb) is for theright side of the body only. Reproductive tissue mass wasassumed negligible in males in terms of influencing gait.Significant variety and sex effects are denoted by V and S,respectively. Significance levels: *P<0.05, **P<0.01 and***P<0.001. Results of the one- and two-way ANOVAconducted to test for variety and sex differences are inTable 1.

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At each U, tswing (Fig. 3G) was greater in males than in femalesand this difference was smaller in the bantam (0.02 s) than inthe standard (0.04 s) variety (Table 3). In the bantams, tswing(Fig. 3H), was similar in the two sexes, whilst in the standardvariety, it was significantly greater in males compared with femalesat a given U .tstance (Fig. 3I) was similar in B♂ and B♀, but was greater in L♂

than in L♀ by 0.03 s at all U (Table 3). Across all U , tstance (Fig. 3J,Table 3) was greater in B♀ than in B♂, yet lower in L♀ than in L♂.

Therefore, none of the sex or variety differences in gaitkinematics were accounted for by correcting for body sizedifferences. The two varieties differed in the mechanisms bywhich females had elevated DF relative to males. In the bantams,females had relatively longer stance durations than males (Fig. 3J)and the sexes shared similar swing dynamics (Fig. 3H). In thestandard variety, females had shorter swing and stance durationsthan males, but the sex difference in tswing was much greater thanthat in tstance (Fig. 3H,J).

0.55

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Fig. 3. Absolute gait kinematics parametersversus speed and relative gait kinematicsparameters versus relative speed. Duty factor(DF; A,B), stride frequency ( fstride, C), relativestride frequency (fstride, D), stride length (lstride,E), relative stride length (lstride, F), swing duration(tswing, G), relative swing duration (tswing, H),stance duration (tstance, I) and relative stanceduration (tstance, J) are shown versus speed(U, left) and relative speed (U , right). Each datapoint (L♂, grey circles; L♀, purple crosses; B♂,blue circles; and B♀, red crosses) represents asingle trial for an individual bird. Best-fit lineequations and the results of the linear modelsconducted to test for variety and sex differencesare in Table 3.

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DISCUSSIONThis study represents the first detailed comparison of the gait of thesexes in any species. Leghorn chickens, although all similar in theirhindlimb segment geometry, show considerable variation in limbposture and the relative contributions of anatomical components(skeletal muscle, digestive organs and reproductive tissues) to totalbodymass. In association with these morphological differences, andin agreement with our hypothesis, none of the leghorn groupswalked with dynamic similarity.

Incremental responses of absolute kinematics parameters toincreasing U are generally greater in smaller species (Gatesy andBiewener, 1991). However, with the exception of fstride, whichincreased at a faster rate in bantams than in the standard variety, allbirds in the present study showed similar incremental kinematicresponses to U, despite the size differences associated with sexand variety. Most of the sex differences in absolute kinematicparameters paralleled interspecies differences, associated with bodysize (Gatesy and Biewener, 1991). In females of the two varieties,fstride was greater, and lstride, tswing and tstance smaller, at any given Ucompared with that in their conspecific males, which had greaterbody size (except for tstance in the bantams, which was similarbetween the sexes). Similarly, fstride was greater, and lstride, tswing andtstance shorter, at any given U in the bantams compared with thestandards. The only parameter that was not comparable tointerspecies patterns associated with body size for a givenabsolute speed was DF. Interspecific scaling patterns wouldpredict the heavier and longer-legged animal to have a greater DFthan the lighter, shorter-legged one, at the same U (Gatesy andBiewener, 1991). In contrast, here, females walked with greater DFthan males, and bantams walked with greater DF than standards.

In agreement with body size-dependent interspecies differencesin DF measured at equivalent relative speeds (Alexander and Jayes,1983; Gatesy and Biewener, 1991), we found that at any givenrelative speed, DF was still higher in the smaller bantam relative tothe standard variety, and in females relative to males. Deviationsfrom dynamic similarity of motion with increasing body mass areusually associated with increasing limb erectness, i.e. an increasinghhip to Σlseg ratio (Biewener, 1987, 1989; Gatesy and Biewener,1991). Smaller, more crouched, species can achieve greater lstriderelative to their hhip because they can extend the crouched limb,which in turn allows a greater DF (Gatesy and Biewener, 1991). Incontrast, a more erect limb is constrained in terms of the range oflstride and DF it can achieve, relative to a given hhip (Gatesy andBiewener, 1991). The similar pelvic limb skeletal geometry of thebirds in the present study provides a control for the potentialeffects of limb segment proportions on walking dynamics andallows investigation into the potential influences of additionalmorphological characteristics, including limb posture. Despite L♀having the most-upright limbs, and the lowest relative stride lengths(Fig. 3F), they still produced a greater DF relative to hhip than did theL♂, whose limbs were more crouched. Furthermore, as sexualdimorphism in limb posture was exclusive to the standards, limbposture cannot explain the similar sex difference in DF in the twovarieties. The sexual dimorphism in posture in the standard varietyonly was reflected in the opposite sex-specific dynamics of lstride,tswing, tswing and tstance at any given U between the two varieties (i.e.the sex differences in gait were variety specific, yet ultimately led togreater DF in females than in males). The lower lstride in L♀ than inB♀ and higher fstride in L♀ than in B♀ are consistent with the generalconsensus that a more upright limb limits the length of a step relativeto hhip (Gatesy and Biewener, 1991).

By adopting a more upright limb posture, larger animals reduce theforces per unit area that themusclesmust exert and that the bonesmustresist to counteract joint moments, which would otherwise scalegeometrically (stress∝Mb

1/3) (Biewener, 1989). Until recently, thishas been considered the principal reason for the scaling of limbposture and gait in vertebrates (Biewener, 1989).Whysmaller animalsdo not also have upright limbs so that they could have relatively moregracile and lighter bones, however, is not accounted for by thisexplanation. Proposed reasons for amore crouched limb include that itmay improve manoeuvrability (Biewener, 1989) and stability (Gatesy

Table 3. Results of the linear models that tested for sex differences inabsolute/normalised kinematics with speed

Parameter Final model Lines of best fit

DF U (F1,111=315.80, P<0.001)Variety (F1,111=7.38, P=0.008)Sex (F1,111=27.79, P<0.001)R2=0.79

B♂: −0.16U+0.79B♀: −0.16U+0.81L♂: −0.16U+0.77L♀: −0.16U+0.79

DF U (F1,110=276.78, P<0.001)Variety (F1,110=30.71, P<0.001)Sex (F1,110=25.30, P<0.001)Variety×sex (F1,110=5.58, P=0.020)R2=0.77

B♂: −0.23U+0.79B♀: −0.23U+0.82L♂: −0.23U+0.77L♀: −0.23U+0.78

fstride U (F1,110=706.18, P<0.001)Variety (F1,110=204.13, P<0.001)Sex (F1,110=24.64, P<0.001)U×variety (F1,110=16.74, P=<0.001)R2=0.87

B♂: 1.76U+0.69B♀: 1.76U+0.80L♂: 1.24U+0.61L♀: 1.24U+0.72

fstride U (F1,110=615.06, P<0.001)Variety (F1,110=1.32, P=0.253)Sex (F1,110=26.84, P<0.001)Variety×sex (F1,110=80.66, P<0.001)R2=0.87

B♂: 0.30Û+0.12B♀: 0.30Û+0.10L♂: 0.30Û+0.08L♀: 0.30Û+0.13

log lstride logU (F1,110=662.71, P<0.001)Variety (F1,110=222.07, P<0.001)Sex (F1,110=40.77, P<0.001)Variety×sex (F1,110=10.93, P=0.001)R2=0.92

B♂: 0.43U0.46

B♀: 0.41U0.46

L♂: 0.56U0.46

L♀: 0.49U0.46

log lstride logU (F1,110=663.00, P<0.001)Variety (F1,110=0.03, P=0.862)Sex (F1,110=52.48, P<0.001)Variety×sex (F1,110=151.57, P<0.001)R2=0.88

B♂: 2.50U0.23

B♀: 2.78U0.23

L♂: 2.95U0.23

L♀: 2.30U0.23

log tswing logU (F1,110=94.88, P<0.001)Variety (F1,110=225.41, P<0.001)Sex (F1,110=71.69, P<0.001)Variety×sex (F1,110=4.51, P=0.036)R2=0.73

B♂: 0.16U−0.22

B♀: 0.14U−0.22

L♂: 0.22U−0.22

L♀: 0.18U−0.22

log tswing logU (F1,110=97.77, P<0.001)Variety (F1,110=40.02, P<0.001)Sex (F1,110=77.88, P<0.001)Variety×sex (F1,110=57.83, P<0.001)R2=0.69

B♂: 1.04U−0.11

B♀: 1.04U−0.11

L♂: 1.31U−0.11

L♀: 1.00U−0.11

log tstance logU (F1,110=1431.42, P<0.001)Variety (F1,110=214.12, P=0.079)Sex (F1,110=22.35, P<0.001)Variety×sex (F1,110=13.76, P<0.001)R2=0.93

B♂: 0.28U−0.64

B♀: 0.28U−0.64

L♂: 0.36U−0.64

L♀: 0.33U−0.64

log tstance logU (F1,110=1057.41, P<0.001)Variety (F1,110=1.85, P=0.177)Sex (F1,110=17.74, P<0.001)Variety×sex (F1,110=126.77, P<0.001)R2=0.92

B♂: 1.60U−0.32

B♀: 1.85U−0.32

L♂: 1.85U−0.32

L♀: 0.47U−0.32

DF, duty factor; fstride, stride frequency; fstride, relative stride frequency; lstride,stride length; lstride, relative stride length; tswing, swing duration; tswing,relative swing duration; tstance, stance duration; tstance, relative stanceduration.The adjusted R2 values of the final statistical models are reported.

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and Biewener, 1991) or minimise the cost of work associated withbouncing viscera (Daley and Usherwood, 2010). Another potentialexplanation, however, is that animals optimise muscle mechanicalwork and power demands during push-off (which are scaledependent) in order to minimise the volume of active muscle for agiven size (Usherwood, 2013). In this case, a crouched limb at mid-stance (allowing longer DF) for small animals may serve to reducepower demands (which are high because at any given U, shorter legsrequire quicker stances), whilst a more upright limb suits the workdemands of being large (which are high because a disproportionateamount of body weight must be supported).The females in the present study may, therefore, benefit from a

greater DF, which would decrease the elevated power demandsassociated with having small limbs, yet greater body weight tosupport per unit of muscle mass because of gravidity. The L♀ in thepresent study may have adopted kinematic and postural mechanismsfor reducing both the elevated work demands due to gravity (viaupright limbs) and the power demands of being small (via longerDF, achieved without a crouched posture). It is possible that L♀mayrequire a more upright limb than B♀ because of their greater relativereproductive tissue mass. In B♀, minimising power via a greaterrelative DF (exceeding that in L♀) appears to be more important. Inguinea fowl, Numida meleagris, adding trunk loads equivalent to23% of Mb did not affect tswing but led to a 4% increase in tstance(Marsh et al., 2006). In contrast, in several additional avian species,no changes in gait kinematics were associated with the applicationof trunk loads (McGowan et al., 2006; Tickle et al., 2010, 2013).These experiments, however, involve unnatural loads applied inbackpacks and may not represent a true gravid loading condition.The hypothesis that the kinematics of leghorn hens are influencedby muscle mechanical demands associated with gravidity is furthersupported by the finding than DF increases with the onset of egglaying during sexual maturation in leghorn hens (Rose et al., 2016b).Equally, males may benefit from lower DF via the minimisation ofwork demands associated with changes in fore–aft acceleration anddeceleration, because of being larger.The hypothesis of Usherwood (2013) that animals adopt

kinematic and postural mechanisms to minimise active musclevolume (according towork and power demands) in order tominimisemetabolic costs is supported by the present findings together withpreviously published data on locomotor energymetabolism collectedsimultaneously from the same birds (Rose et al., 2015a,b). Themetabolic cost of transport in gravid female leghorns is in fact lowerthan allometric predictions based on body mass (Van kampen, 1976;Rose et al., 2015b) and also lower than that of male leghorns (Roseet al., 2015b), which can be linked to their comparatively greater DF.A lower metabolic cost of transport in L♀ than in B♀ (Rose et al.,2015b) can also be linked to more-upright limbs. Additionally,greater relative DF andmore upright limb posture may contribute to alower than expected metabolic cost of transport in B♂ for their bodymass and the lack of scaling in the metabolic cost of transportbetween males of the two varieties (Rose et al., 2015a).Alternatively/additionally, the greater DF in females relative to

males may be a mechanism for reducing peak muscle forces insupporting body weight, which may again be particularly importantwhen carrying proportionally more weight (because of the greaterdigestive/reproductive tissue volume) with proportionally lessmuscle volume. Furthermore, chickens artificially selected for eggproduction are well known to suffer from osteoporosis associatedwith the utilisation of calcium from medullary limb bone in order toform egg shells (Dacke et al., 1993; Whitehead, 2004). A greaterproportion of ground contact throughout the stride to reduce peak

forces exerted on the bones may reduce the risk of bone fracture.This argument may be particularly pertinent to L♀ because the pelviclimb skeletal element diameter of the two varieties conformed togeometric, and not elastic (positive allometry), scaling as is foundbetween species (Doube et al., 2012). The bones of L♀ are, therefore,not expected to be any more robust than those of B♀ to assist withsupporting proportionally more weight of the reproductive system.The same reasoning may also explain the upright limbs of L♀.

It is also possible that additional sexual dimorphisms, perhaps inmuscle physiology or morphology, are linked to the sex differencesin dynamics. For example, simply the distribution of mass across thelimb segments may be responsible. In a recent study in which theswing phase kinematics of different charadriiform birds werecompared, Northern lapwings (Vanellus vanellus) and Eurasianoystercatchers (Haematopus), which share similar hindlimb longbone proportions, shared similar tswing at any given speed despiteoystercatchers having longer and heavier limbs overall (Kilbourneet al., 2016). In comparison to these two species, pied avocets(Recurvirostra avosetta) moved with longer swing durations athigher speeds linked to a more distal concentration of hindlimbmass (Kilbourne et al., 2016). The greater relative pelvic limbmuscle mass in males, relative to females (Mitchell et al., 1931;Rose et al., 2016a), may similarly increase limb moment of inertiaand prolong the swing phase of the limb, increasing its contributionto the stride period.

In summary, this study represents the first detailed comparison ofmale and female gait in a bird. Clear departures from dynamicsimilarity of motion were evident between the sexes in standard andbantam varieties of leghorn chicken. Females walked with greaterDF than males at any given relative speed, but this sex differencewas achieved through alternative kinematic mechanisms in eachvariety and linked to variety differences in sex-specific posture. L♀carry a greater relative reproductive tissue mass than B♀ andpotentially represent the first documented example of an animaladopting mechanisms for minimising the demands of both work(via an upright limb, relative to B♀) and power (via a longer DF thantheir heavier, more crouched male conspecifics).

AcknowledgementsWe thank two anonymous reviewers for their helpful comments on an earlier versionof this manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsR.L.N., J.R.C. and K.A.R. designed the study and contributed to the manuscript.K.A.R. conducted the experiments and statistical analyses with advice from R.L.N.and J.R.C.

FundingThis research was funded by the Biotechnology and Biological Sciences ResearchCouncil (G01138/1 and 10021116/1 to J.R.C.). K.A.R. was supported by a NaturalEnvironment Research Council DTA stipend and CASE partnership with theManchester Museum.

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