Ontogeny of sex differences in the energetics and kinematics of terrestrial locomotion in leghorn chickens (Gallus gallus domesticus)

Sex differences in locomotor performance may precede the onset of sexual maturity and/or arise concomitantly with secondary sex characteristics. Here, we present the first study to quantify the terrestrial locomotor morphology, energetics and kinematics in a species, either side of sexual maturation. In domestic leghorn chickens (Gallus gallus domesticus) sexual maturation brings about permanent female gravidity and increased male hind limb muscle mass. We found that the sexes of a juvenile cohort of leghorns shared similar maximum sustainable speeds, while in a sexually mature cohort maximum sustainable speeds were greater by 67% (males) and 34% (females). Furthermore, relative to that in juveniles of the same sex, the absolute duration of leg swing was longer in mature males and shorter in mature females. Consequently, the proportion of a stride that each limb was in contact with the ground (duty factor) was higher in sexually mature females compared to males. Modulation of the duty factor with the development of secondary sex characteristics may act to minimize mechanical work in males; and minimise mechanical power and/or peak force in females. A greater incremental response of mass-specific metabolic power to speed in males compared to females was common to both age cohorts and, therefore, likely results from physiological sexual dimorphisms that precede sexual maturation.

. Mean (± s.e.m) morphological measurements. l fem , femur length; l tib , tibiotarsus length; l tars , tarsometatarsus length; Σ l segs sum of the hind limb bone lengths. Results of the two-way ANOVAS conducted to test for age and sex affects are in Table 2.
Scientific RepoRts | 6:24292 | DOI: 10.1038/srep24292 of the IC, FL, GL, FCLP and FMT, in which it was greater in J ♀ than in J ♂ (the opposite sexual dimorphism to the mature cohort). Since the females of the two age cohorts did not differ significantly in the absolute masses of their individual muscles, the lower %M b of the muscles in M ♀ relative to J ♀ was due to the greater the M b of the M ♀ relative to J ♀ (attributed to increased mass in the body outside of the pelvic limb) and linked to gravidity. Therefore, M ♂ had greater relative muscle masses than J ♂ associated with the increase in absolute muscle mass that occurs with male sexual maturation. In opposition, M ♀ had lower relative muscle masses than J ♀ , associated with the lack of change in muscle mass but increase in reproductive mass that occurs with female sexual maturation. Standing metabolic rates. Mass-specific P met during quiet standing was similar in the males and females within each cohort but was greater in the juvenile compared to sexually mature cohort by ~2 W kg −1 (Table 4).

Maximum sustainable speed.
Metabolic rates during locomotion. The incremental increase in gross mass-specific P met ( Fig. 2A) with U was greater in males compared to females in both cohorts (Table 4) and also greater in the mature compared to the juvenile cohort (Table 4). Following the subtracting of standing-P met from gross locomotor P met to calculate net-P met (Fig. 2B), the same statistical differences were true. Therefore, faster speeds were more metabolically expensive for males than for females, and more expensive for mature, than for juvenile birds.
CoT tot (Fig. 2C) decreased curvilinearly as a function of U in all four groups. The rate of decrease in CoT tot with U, however, was greater in the juvenile than in the mature cohort ( Table 4). The rate of decrease was also greater in females than in males. CoT net (Fig. 2D) also decreased curvilinearly with U in all but M ♂ in which it was invariant with speed.
Kinematics. Each kinematic parameter responded (increased or decreased) to increases in U at a similar rate in all of the four bird groups, unless otherwise stated below.
Duty factor (DF, the relative contribution of the stance phase to the stride period) decreased linearly with U. A significant age × sex interaction was present in DF ( Fig. 3A) due to DF being greater in males than in females (< 1%) in the juvenile cohort but greater in females than in males in the mature cohort (by 2%) ( Table 4).
Stance duration (t stance ) decreased curvilinearly with U at a similar rate in all bird groups (Fig. 3B, Table 4). t stance was greater in males than in females by 0.03 s in the juveniles and by 0.04 s in the mature birds; however, no significant variety × sex interaction in t stance was identified (Table 4). Much smaller, but significant differences were also observed between the age cohorts, with t stance being slightly greater in the juvenile compared to the mature group.
Swing duration (t swing ) also decreased curvilinearly with U and at the same rate in all birds groups; however, t swing (the intercept) was greater in males compared to females at any given U by 0.02 s in the juveniles, and by 0.04 s in the mature birds (Fig. 3C, Table 4). A significant age × sex interaction was identified in t swing as the sex difference was greater in the mature compared to the juvenile cohort. The intercept was lower in M ♀ relative to J ♀, and higher in M ♂ relative to J ♂. Therefore, the sexes of the mature cohort deviate in t swing from their corresponding sexes in the juvenile cohort in different ways.
Stride frequency (f stride ) increased with U and the trend was best described by a power function (Fig. 3D, Table 4). Sex significantly influenced f stride (Table 4), which was 0.22 Hz and 0.23 Hz faster in females than in males across all U in the juvenile and mature cohorts respectively. Again, smaller but significant differences in f stride were associated with age, which were greater in the mature compared to the juvenile cohort (by 0.06 in females and 0.05 in males).
Stride length (l stride ) increased with U and the trend was also best described by a power function (Fig. 3E, Table 4). Strides were 0.07 m longer in males compared to females, and 0.02 m longer in the juvenile compared to the mature cohort across all U.

Discussion
Here we report the first comparison of the locomotor energetics, kinematics and morphology of a species just prior to and just after the onset of sexual maturity. In comparison to a cohort of juvenile white leghorn chickens, whose sexes were similar in body form, a sexually mature cohort showed strong male-biased sexual size dimorphism, greater limb length and relative muscle mass in males and greater reproductive mass in females. Despite the large ontogenetic differences in hind limb skeletal muscle mass in males and reproductive mass in females (assumed negligible in males), no age × sex interactions in locomotor energetics were identified. The lower incremental metabolic cost of locomotion in females relative to males, common to both juvenile and sexually mature leghorns must, therefore be due to sexual dimorphisms in physiology than precede the onset of sexual maturity. An age × sex interaction was identified in only U max and two kinematics parameters (t swing and DF). The sex differences in maximum performance can be linked to the measured sex differences in morphology. Maximum running speed scales with positive allometry against M b 31 ; therefore the greater U max in mature, relative to juvenile, birds and in mature males relative to females, is expected simply because of greater body size. However, the lack of sex difference in U max in the juveniles (which were also dimorphic in leg length, but to a lesser degree) suggests a more important role of muscle in determining U max than leg length. Muscle physiological cross sectional area (largely influenced by muscle mass) is directly proportional to the maximum force and power that a muscle can produce 32 . The greater volumes of the hind limb skeletal muscles in M ♂ relative to the other three chicken groups, therefore, likely contributed to their greater U max (which would require greater peak muscle forces).
U max is not only dependent upon the maximum f stride and l stride that the birds can achieve, but also the ability of the birds to sustain the speed aerobically (~5min of locomotion is required for respirometry measurements). A lower U max in juveniles is consistent with previous findings from ontogenetic comparisons, whereby stamina is usually lower in juvenile forms 24 . The greater U max in M ♂ may be indicative of a greater aerobic capacity. In a study on red jungle fowl, maximum rate of oxygen consumption (  V O max 2 ) in mature males exceeded that of mature females, but no sex difference was identified in chicks 33 . Therefore, it seems likely that with the onset of sexual maturity there are physiological changes in males, which increase their capacity for aerobic respiration in the muscles. There are a number of levels at which this physiological difference could manifest. For example, in the red jungle fowl,  V O max 2 correlated with cecum, heart, pectoralis and hind limb skeletal muscle masses as well as pectoralis citrate synthase activity, indicative of system wide (peripheral and central organ) specialisation (symmorphosis) 34 .
Although there is evidence amongst vertebrate species for reductions in locomotor performance with pregnancy/gravidity [35][36][37][38][39][40] , U max was actually greater in M ♀ than in J ♀ . This is despite the two age cohorts of female sharing similar absolute limb lengths and muscle mass, whilst only M ♀ were gravid and were also 1.36-fold heavier than J ♀ . Age differences in U max in female leghorns are, therefore, likely explained by differences in aerobic capacity that are not linked to muscle quantity. Interestingly, in the red jungle fowl,  V O max 2 did not correlate with the mass or enzyme capacities of skeletal muscles (peripheral organs) but was correlated with haematocrit and the mass of the large intestine (a central organ) 34 .
The sex differences in most of the kinematic parameters were common to both cohorts. This is likely due to the fact that the juvenile cohort also exhibited some sexual size dimorphism in hind limb skeletal length. t stance and l stride were greater, and f stride lower, in larger males relative to smaller females, as would be expected when  Table 3. A significant age × sex interaction was identified in all measurements (Table 2). Asterisks denote where the sex differences are the opposite between the two age cohorts. Error bars represent s.e.m.
Scientific RepoRts | 6:24292 | DOI: 10.1038/srep24292 comparing a larger animal with a smaller one (based on interspecific comparison) 41 . The sex difference in DF in the mature cohort (greater in females than in males) differed from what would be expected based on interspecific differences associated with size but can be attributed to mechano-physiological constraints imposed by the measured secondary sex characteristics. The distribution and placement of additional mass on the body have Table 4. Results of the final linear model outputs performed to investigate age and sex related differences in energetics and kinematics. The adjusted R 2 values are reported from the final models. important consequences for locomotion. For example, in manipulative studies in which masses were added to the distal limbs of birds, a corresponding increase in t swing has been reported (e.g. a 5% M b load to the distal limb caused a ~16% increase in t swing in the barnacle goose 42 ). The greater muscle mass on the hind limbs (proximal and distal) and increase in leg length of M ♂ relative to that of J ♂ , may increase limb inertia, which might be expected to increase t swing . Muscle mechanical work requirements are lower, the shorter the duration of the stance [43][44][45] . It is also possible that males decrease their duty factors at sexual maturity in order to minimize muscle mechanical work demands, which would be expected to increase with the rise in muscle and bone mass.
In opposition to the ontogenetic differences in t swing found in males, t swing was faster in M ♀ than in J ♀ . The two groups shared a similar quantity of muscle mass but M ♀ possessed greater reproductive mass and overall body mass to support than J ♀ . Therefore, if all kinematic parameters were identical between M ♀ and J ♀ , peak external forces and muscle mechanical work and power might each be expected to be greater in M ♀ compared to J ♀ . One potential reason for a faster t swing in gravid mature females might be to increase the relative contribution of t stance to the stride period (DF was greater in M ♀ than all other bird groups), which would allow more time for generating sufficient muscle force to support the increase in M b . A greater DF for a given U would decrease peak external forces, which may be important in hens, due to a reduction in bone strength associated with the utilisation of medullary bone calcium in egg-shell formation 46,47 . Furthermore, a greater DF decreases muscle mechanical power requirements 43 . The ontogenetic differences in t swing in female leghorns may, therefore, also represent a power minimizing mechanism. Loads added to the backs of birds, which increase the amount of body weight that the stance muscles must support, are not always associated with changes in kinematics parameters 42,48 . In a study by Marsh et al. 49 , however, guineafowl (Numida meleagris) increased their DF when carrying back loads. The location of the added load to the females during gravidity, however, has not yet been mimicked in any load carrying studies in birds. Pregnant humans 50 and wallabies carrying young in the pouch 51 are, like the leghorns here, known to increase DF with pregnancy.

Conclusion
Contrary to our hypothesis that sex differences in locomotor energy metabolism would be associated with sexual maturation in white leghorns; lower incremental metabolic costs of locomotion in females relative to males, were also found in juveniles. Sexual maturation in white leghorns is associated with large increases in hind limb skeletal muscle mass in males and reproductive tissue mass in females. Differences in the location of the additional tissues on the body following sexual maturity differentially impact upon the duration of the swing phase of the limb of the sexes. We suggest that the birds modulate the swing, and hence duty factor, in order to minimize muscle mechanical work (males) and mechanical power and/or peak force (females). A role of secondary sex characteristics in influencing maximum performance in males was indicated by maximum sustainable speeds 67% greater in the mature compared to the juvenile cohort. Furthermore, no evidence was found in females for a constraint of gravidity on maximum sustainable speed. Unlike broiler chickens, which experience locomotor difficulties as they develop the muscle mass for which they were artificially selected, leghorns show a greater capacity for sustained locomotion with the onset of egg-laying.

Animals.
Metabolic measurements from M ♂ and M ♀ (≥ 20-week-old) were taken from Rose et al. 30 .
Juvenile (14-16 weeks-old) white leghorns (J ♂ : N = 5; M b = 1.10 ± 0.10 kg, mean ± s.e.m) and J ♀ (N = 7: M b = 1.05 ± 0.03 kg, mean ± s.e.m) were obtained from the same local suppliers and housed under the same conditions in the University of Manchester's animal unit with the same feeding regimes as the birds in Rose et al. 30 . None of the J ♂ were crowing or exhibiting aggressive behaviour and J ♀ were not gravid when examined post mortem (whilst the opposite was true for the mature cohort 30 ), confirming that the birds had not reached sexual maturity. Experimental procedures were carried out under ethical approval from the University of Manchester Ethics Committee and in accordance with the Animals (Scientific Procedures) Act 1986, covered by a UK Home Office project licence (40/3549) held by Dr Codd.

Respirometry.
Metabolic rates were measured from the birds at rest (standing) and during locomotion on a motorised treadmill (Tunturi T60, Turku, Finland). An open-flow respirometry set up (described in Rose et al. 30,52 ) was used to measure rates of O 2 consumption (  V O 2 , ml min −1 ) and CO 2 production (  V CO 2 , ml min −1 ). The chamber (97.5 × 53.5 × 48.0 cm) within which the birds exercised and the main flow rate (250 L min −1 ) directed through it were identical to those for the sexually mature leghorns in Rose et al. 30 . Juvenile respiratory exchange ratios (RERs:  V CO 2 :  V O 2 ) were similar to those reported for the mature cohort in Rose et al. 30 . Thermal equivalents 53 of the RERs and body mass were used to convert  V O 2 into mass-specific P met (W kg −1 ). Metabolic rates during quiet standing were subtracted from locomotor metabolic rates to determine the net metabolic cost of locomotion surplus to maintenance and postural costs. The cost of transport (CoT tot , J kg −1 m −1 ) was calculated as P met /U. The juveniles were exercised at speed intervals, up to the maximum that they could sustain: 0.28, 0.42, 0.56, 0.69 and 0.83 m s −1 . Each bird completed two trials, composed of 2-3 speeds in a random order, interrupted by 5 min resting periods during which the birds stood quietly and gas levels plateaued. Resting (standing) metabolic rate was taken from the final rest trace in each trial.
Kinematics. Kinematics parameters were obtained using the exact protocol used in Rose et al. 30,52 . All trials were filmed with a high-speed (100 frames s −1 ) video camera (HDR-XR520VE, Sony, Japan) from the side. The tip of digit 3 on the foot closest to the camera was tracked over ~10 strides using Tracker software (v. 4.05, Open Source Physics). Temporal data were used to calculate f stride , l stride (U/f stride ), t swing , t stance and DF. Hip heights were not measured.
Morphological measurements. Juvenile carcasses were scanned using computed tomography (CT) at the Small Animal Teaching Hospital at the University of Liverpool. Three-dimensional reconstruction of full skeletons was carried out by image segmentation and meshing in 3D Slicer (www.slicer.org). MeshLab (www.meshlab. sourceforge.net) was subsequently used to measure lengths of the femur, tibiotarsus and tarsometatarsus from the 3D skeletal models. Five frozen carcasses from each of the bird groups, excluding J ♂ (N = 4), were defrosted for 24 hours prior to dissection. Thirteen major skeletal muscles (Table 3) were identified based on a description by Paxton et al. 9 and dissected from the right pelvic limb and weighed using electronic scales (± 0.01 g).

Statistical analyses.
Only data from the range of speeds utilised by all birds (i.e. up to the U max of the juvenile birds, 0.83 m s −1 ) were included in statistical analyses. All data from the mature birds are presented in the graphs, however, to show their capacities for U max and associated kinematics.
All statistical analyses were performed using the car package version 2.0-12 54 on R 2.14.0 GUI 1.42 Leopard build 64-bit 55 . Shapiro-wilk tests were performed on the standardised residuals of the models to ensure that the data approximated a normal distribution. Where the data did not conform to a normal distribution, data were log transformed. Data were also log transformed if it improved the Akaike's information criterion of the models. Age-cohort and sex were included as fixed factors in all models. Two-way analyses of variance (ANOVAs) were used to test for differences in morphological measurements. Linear models were conducted to test for differences in the relationships between energetic or kinematic variables and U. U was included as a covariate in the models. All potential interaction terms were considered in all primary models before a step-wise backward deletion of non-significant interaction terms was conducted. The final model outputs are reported. Best-fit lines were obtained using the effect sizes from the coefficients tables output by the statistical models and were back-transformed where data had been log-transformed.