Differential sex-specific walking kinematics in leghorn chickens (Gallus gallus

domesticus) selectively bred for different body size

Running title: Sexual dimorphisms in chicken gait kinematics

Kayleigh A. Rose, Jonathan R. Codd and Robert L. Nudds*

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

* Address for reprints and other correspondence:

Dr. Robert Nudds

Faculty of Life Sciences

University of Manchester

Manchester M13 9PT, UK

E-mail: robert.nudds@manchester.ac.uk

Tel: +44(0)161 275 5447

KEY WORDS: Froude number; locomotion; posture; sexual dimorphism; walking

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

Summary statement: In two size classes of layer chicken, sexually dimorphic

walking kinematics is linked to the differential muscle force, work and power

demands of varied visceral and muscle proportions

ABSTRACT

The differing limb dynamics and postures of small and large terrestrial animals may

be mechanisms for minimising metabolic costs under scale-dependent muscle force,

work and power demands; however, empirical evidence for this is lacking. Leghorn

chickens (Gallus gallus domesticus) are highly dimorphic: males have greater body

mass and relative muscle mass than females, which are permanently gravid and have

greater relative intestinal mass. Furthermore, leghorns are selected for standard (large)

and bantam (small) varieties and the former are sexually dimorphic in posture, with

females having a more upright limb. Here, high-speed videography and

morphological measurements were used to examine the walking gaits of leghorn

chickens of the two varieties and sexes. Hind limb skeletal elements were

geometrically similar among the bird groups, yet the bird groups did not move with

dynamic similarity. In agreement with the interspecific scaling of relative duty factor

(DF, proportion of a stride period that a foot has ground contact) with body mass,

bantams walked with greater DF than standards and females 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 DF in order to reduce peak muscle forces and minimize

power demands associated with lower muscle to reproductive tissue mass ratios and

smaller body size. Furthermore, a more upright posture observed in the standard, but

not bantam, females, may relate to minimizing the work demands of being larger and

having proportionally larger reproductive volume. Lower DF in males relative to

females may also be a work-minimizing strategy and/or due to greater limb inertia

(due to greater pelvic limb muscle mass) prolonging the swing phase.

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

INTRODUCTION

The size of an animal influences its walking kinematics. When moving at the same

speed (U, m s-1) larger animals generally take longer and fewer strides per unit time

than smaller animals. Comparison of the walking kinematics of different sized

animals can be conducted at speeds at which the ratios of inertial to gravitational

forces acting upon the body centre of mass (CoM) are equal, using either the Froude

number (Fr = U2/ghhip) or its square root, often termed relative speed:

(1),

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 animals requires geometric similarity in body plan and equal values of

dimensionless kinematic parameters (scaled appropriately to negate the 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 minimize metabolic cost. The

metabolic cost of transport is the energy required to move a unit body weight over a

unit distance ([power]/body weight x speed). Geometrically similar animals of

different size moving in a dynamically similar fashion are expected to have equal

metabolic costs of transport (Alexander and Jayes, 1983). The dynamic similarity

hypothesis of Alexander and Jayes (1983) postulated that different quadrupedal

mammals would locomote with dynamic similarity at equivalent relative speeds.

Within non-cursorial (<1 kg) and cursorial (>10 kg) mammalian groups (Jenkins,

1971) the hypothesis was supported. Observed kinematic differences between the two

groups, however, were not accounted for (Alexander and Jayes, 1983). Furthermore,

between avian species of small and large body size, there is considerable deviation

from dynamic similarity of locomotion (Gatesy and Biewener, 1991; Abourachid and

Renous, 2000; Abourachid, 2001). A general pattern, however, exists across these

vertebrates, whereby smaller species move with greater relative duty factors (DF,

proportion of a stride with ground contact for any given foot) and relative stride

lengths. These deviations from dynamic similarity of locomotion have been attributed

to differences in the relative lengths of the limb segments and limb posture

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

(Alexander and Jayes, 1983; Gatesy and Biewener, 1991; Abourachid and Renous,

2000; Abourachid, 2001). Crouched and upright limb postures are generally adopted

by small and large vertebrate species, respectively, which are clear departures from

geometric similarity in body form (Biewener, 1989; Gatesy and Biewener, 1991).

The differing gait kinematics and postures of small and large terrestrial

animals may be mechanisms for minimising metabolic costs under scale-dependent

muscle force, work and power demands; however, empirical evidence for this is

lacking. Body weight (l3 v1) increases at a faster rate with body size than the

strength (i.e. ability to resist forces, cross-sectional area l2 v2/3) of the biological

materials, which must support it (Biewener, 1989). An erect limb aligns body weight

with each limb bone’s long axis reducing mechanical loading on the muscles

associated with turning moments about the joints (Biewener, 1989). The ‘cost of

muscle force’ hypothesis for the scaling of limb posture and gait with body size states

that the more upright limbs of larger species serve to reduce the large forces that

would otherwise have to be exerted by the limb muscles (Biewener, 1989). An

alternative to the ‘cost of muscle force’ approach is that animals of differing size

optimise active muscle volume 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 of reducing muscle work) would apply to smaller animals, theoretically, their

muscle power (J s-1 kg-1) requirements may be disproportionately high (Usherwood,

2013). Therefore, a more crouched limb, requiring a longer push-off period, may act

to minimise 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 gaits than adults, via longer relative stance periods (Hubel and

Usherwood, 2015).

The understanding of the gaits and postures of different sized animals is

compromised because the majority of comparisons are conducted between different

species (Alexander and Jayes, 1983; Gatesy and Biewener, 1991; Abourachid and

Renous, 2000; Abourachid, 2001). Intraspecifically, the sexes may differ not only in

body size (Lislevand et al., 2009; Remes and Szekely, 2010), but also in

morphological proportions, which are likely to influence muscle force, work and

power demands. For example, in many vertebrate species, the relative proportions of

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

total body mass (Mb) allocated to different somatic and reproductive components are

usually biased towards males and females, respectively (Shine et al., 1998; Hammond

et al., 2000; Lourdais et al., 2006). Furthermore, female reproductive specialisation

may even require specific skeletal proportions (e.g. a wider pelvis (Baumel, 1953;

Smith et al., 2002; Cho et al., 2004)), or posture, during pregnancy (Franklin and

Conner-Kerr, 1998) or gravidity (Rose et al., 2015b). Most studies on gait kinematics,

however, have been conducted using either individuals of only one sex (Reilly, 2000);

without comparing sexes (Rubenson et al., 2004; Watson et al., 2011); or using

individuals whose sexes were not reported (Gatesy and Biewener, 1991; Abourachid,

2000; Abourachid and Renous, 2000; Abourachid, 2001; Griffin et al., 2004; Nudds et

al., 2010). Previous studies have identified sex differences in walking kinematics in

humans (Bhambhani and Singh, 1985) and two species of bird (Lees et al., 2012; Rose

et al., 2014), but whether size variations alone, or both size, and additional

unidentified sexual dimorphisms were behind the differences in kinematics was not

determined.

The leghorn chicken (Gallus gallus domesticus) is highly dimorphic, with

males having greater body size and muscle mass than females (Mitchell et al., 1931;

Rose et al., 2016a). Female leghorns have greater digestive organ masses than males

and remain 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 limb posture, with females possessing a

more upright limb than males at mid stance during a walking gait (Rose et al., 2015b).

For a given sex, the two varieties are expected to be closer to geometric similarity in

anatomical proportions (a prerequisite for dynamic similarity of motion). Whilst the

males of the two varieties are geometrically similar in their axial and appendicular

skeletons, the bantam males adopt a more upright posture at mid stance than the

standards during a walking gait (Rose et al., 2015a). The morphological variations to

have resulted from selective breeding in these leghorns provide a novel opportunity to

investigate the effects 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 measurements were used to

test the hypothesis that male and female standard and bantam varieties of leghorn

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

would show clear departures from dynamic similarity of motion associated with their

morphological variations.

MATERIALS AND METHODS

Animals

Male and female bantam brown leghorns (B♂ and B♀) and standard breed white

leghorns (L♂ and L♀) were obtained from local suppliers and housed in the University

of Manchester’s Animal Unit. All leghorns (> 16 weeks < 1 year) had reached sexual

maturity and females were gravid. Sexes and varieties were housed separately with ad

libitum access to food, water and nesting space. Birds were trained daily for a week to

sustain locomotion for ~5 min within a Perspex® chamber mounted upon a Tunturi

T60 (Turku, Finland) treadmill. The kinematics of twenty-four of the twenty-eight

leghorns used for the simultaneously collected metabolic measurements described in

(Rose et al., 2015b) are presented here (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, mean ± s.e.m). All

experiments were approved by the University of Manchester’s ethics committee,

carried out in accordance with the Animals (Scientific procedures) Act (1986) and

performed under a UK Home Office Project Licence held by Dr Codd (40/3549).

Kinematics

The left greater trochanter of the hip of each bird was located by hand and any

overlying feathers were removed and replaced with a reflective marker. Each leghorn

was exercised at a minimum speed of 0.28 m s -1 and at increasing increments of 0.14

(in a randomised order), up to the maximum they could sustain without showing signs

of fatigue. The birds were rested between speed trials. All trials were filmed from a

lateral view (left of each bird) using a video 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 recording using a known distance

from the front to the back of the respirometry chamber. This allowed for the

alignment of a calibration tool through the line of travel of each bird (always passing

through digit 3), eliminating any error that could be incurred by a bird’s displacement

from it (i.e. the bird’s position on the treadmill/distance from the camera did not affect

our distance calibration). At each speed, the phasing of the sum of the vertical kinetic

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

and gravitational potential energies with the horizontal kinetic energy of the body

centre of mass (approximated by the trochanter marker) was determined using spatial

and temporal data. Unlike the males, the female leghorns are either incapable or

unwilling to use grounded running gait mechanics (Rose et al., 2015b). Hence, only

data for speeds at which the birds used walking gait mechanics (out of phase

fluctuation of gravitational potential and horizontal kinetic energy) 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 and toe–off, which were used to

calculate DF, stride frequency (fstride, Hz), stride length (lstride= U/fstride, m), swing

duration (tswing, s) and stance duration (tstance, s). A single fixed measure of hip height

(hhip) was used per individual (see the following morphological measurements section)

as the characteristic length for normalising their speed and gait kinematic parameters.

U was normalised for size differences as the square root of Froude, here termed

relative speed ( ). Kinematic parameters were normalised based on the

Hof (1996) record of non-dimensional forms of mechanical quantities as: relative

stride length ( = lstride/hhip); relative stride frequency ( ); relative

swing duration ( ); and relative stance duration (

).

Morphological measurements

For each experimental bird, hhip was measured from a video recording (accuracy, ± 1

mm) of a slow walking speed (0.28 m s-1) for a minimum of 7 strides. hhip was taken

(using the same method as Rose et al (2015a,b)) as the distance from the treadmill belt

(where digit one meets the base of the tarsometatarsus) to the hip marker at 90° to the

direction of travel during mid stance, when at its greatest. Back height (hback) was

measured the same way as hhip. Digital vernier callipers (accuracy, ± 0.01 mm) were

used to measure hind limb long bone (femur, tibiotarsus and tarsometatarsus) lengths

(lfem, ltib, ltars) and widths (wfem, wtib, wtars). Total leg length (Σlsegs) was calculated as the

sum of the three element lengths. Note that Σlsegs does not represent the true functional

length of the hindlimb, because the measurements were taken from dried bones

excluding the inter-joint soft tissue. The measurements were taken as the shortest

203

204

205

206

207

208

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

distance between the most proximal and distal grooves of each element, which would

further decrease the values of Σlsegs relative to the maximum potential length of the

three leg segments if they were vertically aligned. Therefore, a posture index near the

value of 1.00 at mid stance in the present study is not indicative of a completely

upright limb. The width of the pelvis (wpelv = the distance between the left and right

acetabula) was also measured.

Soft tissue components from 5 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 cruris medialis, m.

iliotrochantericus caudalis, m. femerotibialis medialis, m. pubioischiofemoralis pars

lateralis, m. pubioischiofemoralis pars medialis, m. gastrocnemius pars lateralis, m.

gastrocnemius pars medialis, m. fibularis lateralis and m. tibialis cranialis) were

dissected for a sister study on variety- and sex-specific muscle architectural properties

(Rose et al., 2016a). The masses of these muscles were summed to give a comparable

total pelvic limb muscle mass between chicken groups. The right breast muscles (m.

pectoralis and m. supracoracoideus) and the intestines (small and large combined)

were also weighed. Reproductive mass (developing eggs, ovaries and oviduct) was

measured from female birds only and was assumed negligible in males in terms of

influencing locomotion dynamics. For the bantam females, 4 of the 5 reproductive

masses measured were from individuals whose experimental data were not included in

this present study. These individuals, however, were from the same cohort and

underwent the same training and experimental process as the birds for which

kinematic data are presented here. All anatomical components were compared

between varieties and sexes as a percentage of dead bird body mass.

Statistical analyses

All statistical analyses were conducted in R (v. 3.0.2 GUY 1.2 Snow Leopard build

558) (Team, 2011). The Car package (Fox and Weisberg, 2011) was used for all

analyses of variance (ANOVA) in which variety and sex were included as fixed

factors. Shapiro-Wilk tests were performed on the standardised residuals generated by

all statistical models to ensure the data conformed to a normal distribution. Where

morphological data (Table 1) did not conform to a normal distribution even after log

transformation, a Kruskal Wallis test was conducted to compare the means of the four

235

236

237

238

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

groups: B♂, B♀, L♂, and L♀. Dunn post-hoc tests were used to indicate which groups

differed. The relationships between absolute and relative kinematics variables with U

and were compared between the bird groups using linear models. U and were

included in the models as covariates and all potential interaction terms were

considered before a stepwise backwards deletion of non-significant interaction terms

was conducted to simplify the models. Outputs from the final models are reported.

Best-fit lines were obtained from the coefficients tables of the final statistical models

and were back transformed where data had been log transformed.

RESULTS

Morphological measurements

Body mass (Fig 1A) and total leg length (Σlsegs, Fig 1B) were greater in the standard

than in the bantam variety and greater in males than 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 between the 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 hind limb segment was a similar proportion of Σlsegs (Table 2) in all of

the leghorn groups excluding B♀, which had a relatively longer lfem, and concomitantly

shorter ltars, resulting in a small, but, nonetheless statistically significant difference

(Table 2). The width of each limb segment (Table 2) was a similar proportion of its

respective segment length in all groups (Table 2). The finding of a more erect posture

in L♀ when compared to the other three groups was further supported by indices

incorporating the height of the back (hback). hhip:hback did not differ between the bird

groups, and Σlsegs:hback was lower in L♀ than in the other three leghorn 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 thirteen pelvic limb

muscles) was a greater % of body mass (Fig 2) in males than in females in both

varieties (Table 1). Total pelvic limb muscle mass was also greater in the bantam than

in the standard variety (Fig 2, Table 1). The observed differences between varieties,

however, were small in comparison to the sexual dimorphisms. The same statistical

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

outcomes obtained for the total pelvic limb muscle mass were mirrored by the breast

muscles, m. pectoralis and m. supracoracoideus (Fig 2, Table 1). Intestine mass as a

proportion of body mass (Fig 2) was greater in females than in males (Table 1). The

two varieties, however, shared similar relative intestinal masses. The reproductive

tissue mass as a percentage of body mass was greater in L♀ (11.49 ± 0.56 %) than in

the B♀ (8.40 ± 0.08 %).

Therefore, all four leghorn groups were similar in their hind limb skeletal bone

geometry (a prerequisite of dynamic similarity of locomotion). The sexes, however,

differed markedly in each of the measured anatomical masses when expressed as %

body mass. In contrast, with the exceptions of limb posture and the relative mass of

the female reproductive system, for a given sex, the two varieties of leghorn were

more similar in their anatomical proportions.

Walking kinematics and dynamics

DF, tswing and tstance were negatively correlated, and lstride and fstride were positively

correlated with U. The same correlations were also found for the relationship between

size-normalised kinematics parameters and . For all four groups of leghorns, the

exponents describing the relationships between absolute or size-normalised

parameters and U or were similar, unless otherwise stated below.

Across all speeds, DF (Fig 3A) was greater in females than in males (~ 2 %)

and greater in the bantam than in the standard variety by ~ 2 % (Table 3). At

comparable , DF was, similarly, greater in females than in males in both varieties

and this sex difference was greater in the bantams. In addition, DF was greater in the

bantam than in the standard variety (Fig 3B; Table 3).

fstride (Fig 3C) was greater in females compared to males at any given U (0.11

Hz) (Table 3). Absolute fstride and the rate of increase in fstride with U were significantly

greater in the bantam than in the standard variety. At comparable , , was greater

in females compared to males in the standard variety but, contrastingly, greater in

males compared to females in the bantam variety (Fig 3D; Table 3).

At any given U, lstride was greater in males than in females by a greater amount

in the standard variety (70 mm) than in the bantam variety (20 mm) (Fig 1C; Table 2)

and was associated with a greater difference between the males of the two varieties

than between the females of the two varieties. At any given , (Fig 1D), was

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

greater in females than in males in the bantam variety, but, contrastingly, was greater

in males than in females in the standard variety (Table 3).

At each U, tswing (Fig 1G) was greater in males than in females and this

difference was lower in the bantam (0.02 s) than in the standard (0.04 s) variety

(Table 2). In the bantams, swing (Fig 1H), was similar in the two sexes, whilst in the

standard variety, it was significantly greater in males compared to females at a given

.

tstance (Fig 1I) was similar in B♂ and B♀, but was greater in L♂ compared to L♀

by 0.03s at all U (Table 2). Across all , stance (Fig 1J; Table 2) was greater in B♀

than in B♂, yet lower in L♀ than in L♂.

Therefore, none of the sex or variety differences in gait kinematics were

accounted for correcting for body size differences. The two varieties differed in the

mechanisms by which females had elevated DF relative to males. In the bantams,

females had relatively longer stance durations than males (Fig 1J) and the sexes

shared similar swing dynamics (Fig 1H). In the standard variety, females had

relatively shorter swing and stance durations than males, but the sex difference in

swing was much greater than that in stance (Fig 1H and J).

DISCUSSION

This study represents the first detailed comparison of the gait of the sexes in any

species. Leghorn chickens, although all similar in their hind limb segment geometry,

show considerable variation in limb posture and the relative contributions of

anatomical components (skeletal muscle, digestive organs and reproductive tissues) to

total body mass. In association with these morphological differences, and in

agreement with our hypothesis, none of the leghorn groups walked with dynamic

similarity.

Incremental responses of absolute kinematics parameters to increasing U are

generally greater in smaller species (Gatesy and Biewener, 1991). With the exception

of fstride, which increased at a faster rate in bantams than in the standard variety, all

birds in the present study, however, showed similar incremental kinematic responses

to U, despite the size differences associated with sex and variety. Most of the sex

differences in absolute kinematic parameters paralleled inter-species differences,

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

355

356

357

358

359

360

361

362

363

364

365

366

associated with body size (Gatesy and Biewener, 1991). In females of the two

varieties, fstride was greater, and lstride, tswing and tstance, smaller, at any given U compared

to that in their conspecific males, which had greater body size (except for tstance in the

bantams, which was similar between the sexes). Similarly, fstride was greater, and lstride,

tswing and tstance shorter at any given U in the bantams compared to the standards. The

only parameter that was not comparable to inter-species patterns associated with body

size for a given absolute speed was DF. Interspecific scaling patterns, would predict

the heavier and longer-legged, animal to have a greater DF than the lighter, shorter-

legged one, at the same U (Gatesy and Biewener, 1991). In contrast, here, females

walked with greater DF than males, and bantams walked with greater DF than

standards.

In agreement with body size dependent inter-species differences in DF

measured at equivalent relative speeds (Alexander and Jayes, 1983; Gatesy and

Biewener, 1991), at any given relative speed, DF here, was still higher in the smaller

bantam relative to the standard variety, and in females relative to males. Deviations

from dynamic similarity of motion with increasing body mass are usually associated

with increasing limb erectness, i.e., an increasing hhip to Σlseg ratio (Biewener, 1987,

1989; Gatesy and Biewener, 1991). Smaller, more crouched, species can achieve

greater lstride relative to their hhip because they can extend the crouched limb, which in

turn, allows a greater DF (Gatesy and Biewener, 1991). In contrast, a more erect limb

is constrained in terms of the range of lstride and DF it can achieve, relative to a given

hhip (Gatesy and Biewener, 1991). The similar pelvic limb skeletal geometry of the

birds in the present study provides a control for the potential effects of limb segment

proportions on walking dynamics and allows investigation into the potential

influences of additional morphological characteristics including limb posture. Despite

L♀ having the most upright limbs, and the lowest relative stride lengths (Fig 1 E),

however, they still produced a greater DF relative to hhip than did the L♂, whose limbs

were more crouched. Furthermore, since sexual dimorphism in limb posture was

exclusive to the standards, limb posture cannot explain the similar sex difference in

DF in the two varieties. The sexual dimorphism in posture in the standard variety only

was reflected in the opposite sex-specific dynamics of , , swing and stance at any

given between the two varieties (i.e the sex differences in gait were variety-

specific, yet ultimately led to greater DF in females than males). The lower in L♀

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

than in B♀ and higher in L♀ than in B♀ are consistent with the general consensus

that a more upright limb limits the length of a step relative to hhip (Gatesy and

Biewener, 1991).

By adopting a more upright limb posture, larger animals reduce the forces that

the muscles must exert and that the bones must resist to counteract joint moments,

which would otherwise scale geometrically ( Mb1/3) (Biewener, 1989). Until recently,

this has been considered the principal reason for the scaling of limb posture and gaits

in vertebrates (Biewener, 1989). Why smaller animals do not also have upright limbs

so that they could have relatively more gracile and lighter bones, however, is not

accounted for by this explanation. Explanations proposed to account for a more

crouched limb include that it may improve manoeuvrability (Biewener, 1989) stability

(Gatesy and Biewener, 1991) or minimize the cost of work associated with bouncing

viscera (Daley and Usherwood, 2010). Another potential explanation, however, is that

animals optimise muscle mechanical work and power demands during push-off

(which are scale-dependent) in order to minimise the volume of active muscle for a

given size (Usherwood, 2013). In this case, a crouched limb at mid stance (allowing

longer DF) for small animals may serve to reduce power demands (which are high

because at any given U shorter legs require quicker stances), whilst a more upright

limb suits the work demands of being large (which are high because a

disproportionate amount of body weight must be supported).

The females in the present study may, therefore, benefit from greater DF,

which would decrease the elevated power demands associated with having small

limbs, yet greater body weight to support per unit of muscle mass because of

gravidity. The L♀ in the present study may have adopted kinematic and postural

mechanisms for reducing both the elevated work demands due to gravity (via upright

limbs) and the power demands of being small (via longer DF, achieved without a

crouched posture). It is possible that L♀ may require a more upright limb than B♀

because of their greater relative reproductive tissue mass. In B♀ minimising power via

a greater relative DF (exceeding that in L♀) appears to be more important. In guinea

fowl, Numida meleagris, adding trunk loads equivalent to 23% of body mass did not

affect tswing but led to a 4% increase in tstance (Marsh et al., 2006). In several additional

avian species, however, no changes in gait kinematics were associated with the

application of trunk loads (McGowan et al., 2006; Tickle et al., 2010; Tickle et al.,

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

2013). These experiments, however, involve unnatural loads applied in backpacks and

may not represent a true gravid loading condition. The hypothesis that the kinematics

of leghorn hens are influenced by muscle mechanical demands associated with

gravidity is further supported by the finding than DF increases with the onset of egg

laying during sexual maturation in leghorn hens (Rose et al., 2016b). Equally, males

may benefit from lower DF via the minimization of work demands associated with

changes in fore-aft acceleration and deceleration, because of being larger.

The hypothesis of Usherwood (2013), that animals adopt kinematic and

postural mechanisms to minimise active muscle volume (according to work and

power demands) in order to minimize metabolic costs is supported by the present

findings together with previously published data on locomotor energy metabolism

collected simultaneously from the same birds (Rose et al., 2015a,b). The metabolic

cost of transport in gravid female leghorns is in fact lower than allometric predictions

based on body mass (Vankampen, 1976; Rose et al., 2015b) and also lower than that

of male leghorns (Rose et 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 and more

upright limb posture may contribute to a lower than expected metabolic cost of

transport in B♂ for their body mass and the lack of scaling in the metabolic cost of

transport between 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 in supporting body weight, which may

again be particularly important when carrying proportionally more weight (due to

greater digestive/reproductive tissue volume) with proportionally less muscle volume.

Furthermore, chickens artificially selected for egg production are well known to suffer

from osteoporosis associated with the utilisation of calcium from limb bone medullary

in order to form egg-shells (Dacke et al., 1993; Whitehead, 2004). A greater

proportion 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♀ due to the fact that the pelvic limb skeletal element diameters of the

two varieties conformed to geometric, and not elastic (positive allometry), scaling as

is found between species (Doube et al., 2012). The bones of L♀ are, therefore, not

expected to be any more robust than those of B♀ to assist with supporting

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462

463

464

465

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 in muscle

physiology or morphology, are linked to the sex differences in dynamics. For

example, simply the distribution of mass across the limb segments may be

responsible. In a recent study in which the swing phase kinematics of different

charadriiform birds were compared, Northern lapwings (Vanellus vanellus) and

Eurasian oystercatchers (Haematopus), which share similar hind limb long bone

proportions, shared similar tswing at any given speed despite oystercatchers having

longer and heavier limbs overall (Kilbourne et al., 2016). In comparison to these two

species, pied avocets (Recurvirostra avosetta) moved with longer swing durations at

higher speeds linked to a more distal concentration of hind limb mass (Kilbourne et

al., 2016). The greater relative pelvic limb muscle mass in males, relative to females

(Mitchell et al., 1931; Rose et al., 2016a), may similarly increase limb moment of

inertia and prolong the swing phase of the limb, increasing its contribution to the

stride period.

In summary, this study represents the first detailed comparison of male and

female gait in a bird. Clear departures from dynamic similarity of motion were

evident between the sexes in standard and bantam varieties of leghorn chicken.

Females walked with greater DF than males at any given relative speed, but this sex

difference was achieved through alternative kinematic mechanisms in each variety

and linked to variety differences in sex-specific posture. L♀ carry a greater relative

reproductive mass than B♀ and potentially represent the first documented example of

an animal adopting mechanisms for minimising the demands of both work (via an

upright limb, relative to B♀) and power (via a longer DF than their heavier, more

crouched male conspecifics).

List of abbreviations

B♀ female bantams

B♂ male bantams

DF duty factor

fstride stride frequency

Fr Froude number

466

467

468

469

470

471

472

473

474

475

476

477

478

479

480

481

482

483

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

normalised stride frequency

lfem femur length

lstride stride length

hhip hip height

hhip: Σlsegs posture index

L♀ female standards

L♂ male standards

normalised stride length

ltib tibiotarsus length

ltars tarsometatarsus length

tstance stance duration

stance normalised stance duration

tswing swing duration

swing normalised swing duration

U speed

relative speed

wfem femur width

wtib tibiotarsus width

wtars tarsometatarsus width

Σlsegs sum of the hind limb long bone lengths

Competing interests

The authors have no competing interests

Author’s contributions

R.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.

Funding

This research was funded by the BBSRC (G01138/1 and 10021116/1 to J.R.C).

K.A.R was supported by a NERC DTA stipend and CASE partnership with the

Manchester Museum.

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

REFERENCES

Abourachid, A. (2000). Bipedal locomotion in birds: the importance of functional parameters in terrestrial adaptation in Anatidae. Can J Zool 78, 1994-1998.Abourachid, A. (2001). Kinematic parameters of terrestrial locomotion in cursorial (ratites), swimming (ducks), and striding birds (quail and guinea fowl). Comparative Biochemistry and Physiology a-Molecular and Integrative Physiology 131, 113-119.Abourachid, A. and Renous, S. (2000). Bipedal locomotion in ratites (Paleognatiform): examples of cursorial birds. Ibis 142, 538-549.Alexander, R. M. (1976). Estimates of Speeds of Dinosaurs. Nature 261, 129-130.Alexander, R. M. and Jayes, A. S. (1983). A dynamic similarity hypothesis for the gaits of quadrupedal mammals. J Zool 201, 135-152.Baumel, J. J. (1953). Individual variation in the white-necked raven. Condor 55, 26-32.Bhambhani, Y. and Singh, M. (1985). Metabolic and Cinematographic Analysis of Walking and Running in Men and Women. Med Sci Sport Exer 17, 131-137.Biewener, A. A. (1987). Scaling body support in sammals - limb posture and the relative mechanical advantage of limb muscles. Am Zool 27, A25-A25.Biewener, A. A. (1989). Scaling body support in mammals - limb posture and muscle mechanics. Science 245, 45-48.Cho, S. H., Park, J. M. and Kwon, O. Y. (2004). Gender differences in three dimensional gait analysis data from 98 healthy Korean adults. Clin Biomech 19, 145-152.Dacke, C. G., Arkle, S., Cook, D. J., Wormstone, I. M., Jones, S., Zaidi, M. and Bascal, Z. A. (1993). Medullary Bone and Avian Calcium Regulation. J Exp Biol 184, 63-88.Daley, M. A. and Usherwood, J. R. (2010). Two explanations for the compliant running paradox: reduced work of bouncing viscera and increased stability in uneven terrain. Biol Lett 6, 418-421.Doube, M., Yen, S. C. W., Klosowski, M. M., Farke, A. A., Hutchinson, J. R. and Shefelbine, S. J. (2012). Whole-bone scaling of the avian pelvic limb. J Anat 221, 21-29.Fox, J. and Weisberg, S. (2011). An {R} companion to applied regression, second edition. Thousand Oaks CA: sage.Franklin, M. E. and Conner-Kerr, T. (1998). An analysis of posture and back pain in the first and third trimesters of pregnancy. J. Orthop. Sports Phys. Ther. 28, 133-138.Gatesy, S. M. and Biewener, A. A. (1991). Bipedal locomotion - effects of speed, size and limb posture in birds and humans. J Zool 224, 127-147.Griffin, T. M., Kram, R., Wickler, S. J. and Hoyt, D. F. (2004). Biomechanical and energetic determinants of the walk-trot transition in horses. J Exp Biol 207, 4215-4223.

531

532

533

534535536537538539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571572573574575576

Hammond, K. A., Chappell, M. A., Cardullo, R. A., Lin, R. S. and Johnsen, T. S. (2000). The mechanistic basis of aerobic performance variation in red junglefowl. J Exp Biol 203, 2053-2064.Hof, A. L. (1996). Scaling gait data to body size. Gait Posture 4, 222-223.Hubel, T. Y. and Usherwood, J. R. (2015). Children and adults minimise activated muscle volume by selecting gait parameters that balance gross mechanical power and work demands. J Exp Biol 218, 2830-2839.Jenkins, F. A. (1971). Limb Posture and Locomotion in Virginia Opossum (Didelphis-Marsupialis) and in Other Non-Cursorial Mammals. J Zool 165, 303-&.Kilbourne, B. M., Andrada, E., Fischer, M. S. and Nyakatura, J. A. (2016). Morphology and motion: hindlimb proportions and swing phase kinematics in terrestrially locomoting charadriiform birds. The Journal of experimental biology 219, 1405-1416.Lees, J. J., Nudds, R. L., Folkow, L. P., Stokkan, K. A. and Codd, J. R. (2012). Understanding sex differences in the cost of terrestrial locomotion. Proc R Soc B 279, 826-832.Lislevand, T., Figuerola, J. and Szekely, T. (2009). Evolution of sexual size dimorphism in grouse and allies (Aves: Phasianidae) in relation to mating competition, fecundity demands and resource division. J Evolution Biol 22, 1895-1905.Lourdais, O., Shine, R., Bonnet, X. and Brischoux, F. (2006). Sex differences in body composition, performance and behaviour in the Colombian rainbow boa (Epicrates cenchria maurus, Boidae). J Zool 269, 175-182.Marsh, R. L., Ellerby, D. J., Henry, H. T. and Rubenson, J. (2006). The energetic costs of trunk and distal-limb loading during walking and running in guinea fowl Numida meleagris: I. Organismal metabolism and biomechanics. The Journal of experimental biology 209, 2050-2063.McGowan, C. P., Duarte, H. A., Main, J. B. and Biewener, A. A. (2006). Effects of load carrying on metabolic cost and hindlimb muscle dynamics in guinea fowl (Numida meleagris). J Appl Physiol 101, 1060-1069.Mitchell, H. H., Card, L. E. and Hamilton, T. S. (1931). A technical study of the growth of White Leghorn chickens. Illinois agricultural experiment station bulletin 367, 81-139.Nudds, R. L., Gardiner, J. D., Tickle, P. G. and Codd, J. R. (2010). Energetics and kinematics of walking in the barnacle goose (Branta leucopsis). Comp Biochem Physiol A Mol Integr Physiol 156, 318-324.Reilly, S. M. (2000). Locomotion in the quail (Coturnix japonica): The kinematics of walking and increasing speed. J Morphol 243, 173-185.Remes, V. and Szekely, T. (2010). Domestic chickens defy Rensch's rule: sexual size dimorphism in chicken breeds. J Evolution Biol 23, 2754-2759.Rose, K. A., Nudds, R. L. and Codd, J. R. (2015a). Intraspecific scaling of the minimum metabolic cost of transport in leghorn chickens (Gallus gallus domesticus): links with limb kinematics, morphometrics and posture. The Journal of experimental biology 218, 1028-1034.Rose, K. A., Nudds, R. L. and Codd, J. R. (2016a). Variety, sex and ontogenetic differences in the pelvic limb muscle architectural properties of leghorn chickens (Gallus gallus domesticus) and their links with locomotor performance. J Anat 228, 952-964.

577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624

Rose, K. A., Nudds, R. L., Butler, P. J. and Codd, J. R. (2015b). Sex differences in gait utilization and energy metabolism during terrestrial locomotion in two varieties of chicken (Gallus gallus domesticus) selected for different body size. Biol Open 4, 1306-1315.Rose, K. A., Bates, K. T., Nudds, R. L. and Codd, J. R. (2016b). Ontogeny of sex differences in the energetics and kinematics of terrestrial locomotion in leghorn chickens (Gallus gallus domesticus). Sci Rep-Uk 6, 24292.Rose, K. A., Tickle, P. G., Lees, J. J., Stokkan, K. A. and Codd, J. R. (2014). Neither season nor sex affects the cost of terrestrial locomotion in a circumpolar diving duck: the common eider (Somateria mollissima). Polar Biol 37, 879-889.Rubenson, J., Heliams, D. B., Lloyd, D. G. and Fournier, P. A. (2004). Gait selection in the ostrich: mechanical and metabolic characteristics of walking and running with and without an aerial phase. Proc R Soc B 271, 1091-1099.Shine, R., Keogh, S., Doughty, P. and Giragossyan, H. (1998). Costs of reproduction and the evolution of sexual dimorphism in a 'flying lizard' Draco melanopogon (Agamidae). J Zool 246, 203-213.Smith, L. K., Lelas, J. L. and Kerrigan, D. C. (2002). Gender differences in pelvic motions and center of mass displacement during walking: Stereotypes quantified. J Women Health Gen-B 11, 453-458.Team, R. D. C. (2011). R: A language and environment for statistical computing: Vienna, Austria: R Foundation for Statistical Computing.Tickle, P. G., Richardson, M. F. and Codd, J. R. (2010). Load carrying during locomotion in the barnacle goose (Branta leucopsis): The effect of load placement and size. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 156, 309-317.Tickle, P. G., Lean, S. C., Rose, K. A., Wadugodapitiya, A. P. and Codd, J. R. (2013). The influence of load carrying on the energetics and kinematics of terrestrial locomotion in a diving bird. Biol Open 2, 1239-1244.Usherwood, J. R. (2013). Constraints on muscle performance provide a novel explanation for the scaling of posture in terrestrial animals. Biol Lett 9.Vankampen, M. (1976). Activity and Energy-Expenditure in Laying Hens .2. Energy-Cost of Exercise. J Agr Sci 87, 81-84.Watson, R. R., Rubenson, J., Coder, L., Hoyt, D. F., Propert, M. W. and Marsh, R. L. (2011). Gait-specific energetics contributes to economical walking and running in emus and ostriches. Proc R Soc B 278, 2040-2046.Whitehead, C. C. (2004). Overview of bone biology in the egg-laying hen. Poultry Sci 83, 193-199.

625626627628629630631632633634635636637638639640641642643644645646647648649650651652653654655656657658659660661662663

Figure Legends

Figure 1. Morphological measurements for the variety and sex combinations of

leghorn chicken. (a) Body mass. (b) Total leg length (sum of the three skeletal

element lengths). (c) Hip height. (d) Posture index (hip height: total leg length). Bars

represent means ± s.e.m for standard males (grey), standard females (purple), bantam

males (blue) and bantam females (red). Significant variety, sex and variety x sex

interaction terms are denoted by V, S and I, respectively. Significance levels of 0.05,

0.01 and 0.001 are denoted by *, ** and ***, respectively. Results of the two way

ANOVAs conducted to test for variety and sex differences are in Table 1. These

morphological differences have been reported previously in (Rose et al., 2015b) for a

different sample size.

Figure 2. Anatomical components as a % of body mass for the variety and sex

combinations of leghorn chicken. Bars represent means ± s.e.m for standard males

(grey), standard females (purple), bantam males (blue) and bantam females (red).

Limb muscle mass was calculated as the sum of thirteen major pelvic limb muscles on

the right limb. M. pectoralis and M. supracoracoideus expressed as a % of body mass

are for the right side of the body only. Reproductive mass was assumed negligible in

the males in terms of influencing gait. Significant variety and sex effects are denoted

by V and S, respectively. Significance levels of 0.05, 0.01 and 0.001 are denoted by

*, ** and ***, respectively. Results of the one- and two-way ANOVAs conducted to

test for variety and sex differences are in Table 1.

Figure 3. Absolute gait kinematics parameters versus speed (left column) and

relative gait kinematics parameters versus relative speed (right column). Duty

factor (a-b), stride length (c-d), stride frequency (e-f), swing duration (g-h) and stance

duration (i-j). Each data point (standard males = grey circles, standard females =

purple crosses, bantam males = blue circles and bantam females = red crosses)

represents a single trial for an individual bird. Best-fit line equations and the results of

the linear models conducted to test for variety and sex differences are in Table 3.

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

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

Measurement/index ANOVA (final model)Body mass(kg)

variety: F1,20= 47.34, P<0.001,sex: F1,20=33.96, P<0.001R2= 0.76

Leg length(mm)

variety: F1,20=125.26, P<0.001,sex: F1,20=84.44, P<0.001R2= 0.89

Hip height(mm)

variety: F1,19=95.44 P<0.001,sex: F1,19=0.29, P=0.594variety x sex: F1,19=29.55, P<0.001,R2= 0.85

Posture index variety: F1,19=14.42, P=0.001,sex: F1,19=19.44, P<0.001variety x sex: F1,19=42.30, P<0.001,R2=0.75

13 pelvic limb muscles(%body mas)

variety: F1,17=9.10, P=0.008,sex: F1,17=161.53, P<0.001, R2=0.90

M. pectoralis(% body mass)

variety: F1,17=18.50, P<0.001sex: F1,17=31.00, P<0.001, R2=0.71

M. supracoracoideus(%body mass)

variety: F1,17=8.35, P=0.010sex: F1,17=29.01, P<0.001, R2=0.65

Intestines a

(%body mass)variety: F1,16=0.71, P=0.411,sex: F1,16=48.09, P<0.001, R2=0.73

Reproductive(%body mass) b

variety: F1,8= 8.74, P=0.018,R2 = 0.46

a N=4 for standard malesb Females only as reproductive mass was assumed negligible in malesBody mass, leg length, hip height and posture index were measured from the full sample of experimental birds Soft tissue masses (% body mass) were calculated for 5 individuals of each bird groupBreast muscle %body masses are for the right side of the body onlyThe adjusted R2 values of the final statistical models are reported

698699700

701

702703704705706707708709

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

Index B♂ B♀ L♂ L♀ Statistical resultslfem:Σlsegs 0.28 0.29 0.28 0.28 Kruskal Wallis: X2= 19.6, df=3, P=<0.001

Dunn test: B♂ v B♀: Z=3.71, P<0.001, B♂ v L♂: Z=-0.61, P=0.272, B♂ v L♀: Z=0.00, P=0.500, B♀ v L♂: Z=-3.89, P<0.001, B♀ v L♀: Z=-3.35, P<0.001, L♂ v L♀: Z=0.55, P=0.292

ltib:Σlsegs 0.42 0.42 0.42 0.42 Kruskal Wallis: X2= 0.13, df=3, P=0.988ltars:Σlsegs 0.30 0.29 0.30 0.30 Kruskal Wallis: X2= 12.47, P=0.006

Dunn test B♂ v B♀: Z=-2.48, P=0.007, B♂ v L♂: Z=0.90, P=0.18 B♂ v L♀: Z=0.90, P=0.18, B♀ v L♂: Z=3.05, P=0.001, B♀ v L♀: Z=3.05, P=0.001, L♂ v L♀: Z=0.00, P=0.500

wfem:lfem 0.11 0.11 0.11 0.11 Kruskal Wallis: X2= 0.46, df=3, P=0.929wtib:ltib 0.07 0.06 0.07 0.07 Kruskal Wallis: X2= 7.20, df=3, P=0.066wtars:ltars 0.10 0.10 0.10 0.10 Kruskal Wallis: X2= 0.36, df=3, P=0.948hhip:hback 0.78 ±

0.010.77 ± 0.02

0.76 ± 0.00

0.77 ± 0.02

variety: F1,20=0.45, P=0.512, sex: F1,20=0.00, P=0.948, R2=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<0.001, sex: F1,19=22.16, P<0.001, variety x sex: F1,19=37.91, P<0.001, R2=0.78

wpelv:Σlsegs 0.17 (N=6)

0.18 0.16 0.17 variety: F1,18=11.368, P=0.003, sex: F1,18=0.347, P=0.079, R2=0.39

The adjusted R2 values of the final models are reported.Only the results of the final two-way ANOVAs are reportedStandard errors are not presented where they were 0.00 to 2 decimal placesThe sample size is indicated in brackets if lower than the total sample size of the leghorn group

710711712713

714715716717718719

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

The adjusted R2 values of the final statistical models are reporte

Parameter Final model Lines of best fitDF 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 (F1,110=276.78, P<0.001),variety (F1,110=30.71, P<0.001),sex (F1,110=25.30, P<0.001),variety x sex (F1,110=5.58, P=0.020),R2= 0.77

B♂ : = -0.23 + 0.79

B♀: = -0.23 + 0.82

L♂: = -0.23 + 0.77

L♀: = -0.23 + 0.78fstride 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 x 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

(F1,110=615.06, P<0.001),variety (F1,110=1.32, P=0.253),sex (F1,110=26.84, P<0.001),variety x sex (F1,110=80.66, P<0.001),R2= 0.87

B♂ : = 0.30 + 0.12

B♀: = 0.30 + 0.10

L♂: = 0.30 + 0.08

L♀: = 0.30 + 0.13log 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 x 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 log (F1,110=663.00, P<0.001),variety (F1,110=0.03, P=0.862),sex (F1,110=52.48, P<0.001),variety x sex (F1,110=151.57 P<0.001),R2= 0.88

B♂ : = 2.50 0.23

B♀: = 2.78 0.23

L♂: = 2.95 0.23

L♀: = 2.30 0.23

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 x 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 log (F1,110=97.77, P<0.001),variety (F1,110=40.02, P<0.001),sex (F1,110=77.88, P<0.001),variety x sex (F1,110=57.83, P<0.001),R2= 0.69

B♂ : = 1.04 -0.11

B♀: = 1.04 -0.11

L♂: = 1.31 -0.11

L♀: = 1.00 -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 x 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 log (F1,110=1057.41, P<0.001),variety (F1,110=1.85, P=0.177),sex (F1,110=17.74, P<0.001),variety x sex (F1,110=126.77, P<0.001),R2= 0.92

B♂ : = 1.60 -0.32

B♀: = 1.85 -0.32

L♂: = 1.85 -0.32

L♀: = 0.47 -0.32

720721722723

725

726