Nociceptive thresholds in broiler chickens are modulated by lameness of their progenitors and sex category

Abstract

There are increasing concerns about fast-growing broiler’s welfare. The impact of lameness issues of parental hens and cockerels to their immediate broiler’s offspring remains underexplored and was an objective of this study. Additionally, it was added the novelty to investigate nociceptive responses of these broilers in the field. The study was conducted in commercial settings in Brazil including a reproduction farm, an industrial-scale hatchery, and a rearing farm. Parental-birds were gait scored as lame(C) and non-lame(S) and combined into four mating groups: SS, SC, CS, and CC. 371 adult broilers, immediate progeny of these parents, evaluated for lameness and nociceptive thresholds. Although no treatment effects were observed, when comparing sexes, regardless parental conditions, male broilers exhibited higher 0–5 gait scores than females (males 1.74, females 1.20). Males had also higher nociceptive thresholds and this reduced sensitivity was more pronounced in their left legs. The sex-based analysis indicated that male broilers descended from lame females mated with non-lame males, presented higher nociceptive thresholds than those whose parents were both non-lame birds. These findings may guide deeper investigation into phenotypic lameness resilience, nociceptive thresholds, pain processing, and chronic pain-induced analgesia in industrial poultry.

Introduction

Brazil ranks as the world’s leading exporter of chicken meat, with 35.36% of its production shipped abroad in 2024¹ The high consumption of this protein food, production efficiency and consumer awareness of welfare problems have resulted in concerns about changing breeding and housing conditions of the birds². The main component of this production chain is the broiler type of chicken, Gallus gallus domesticus, which has been selectively bred for rapid weight gain³. Among the welfare concerns in broilers, pain and lameness are extensively discussed^2,4–8.

Broiler lameness in Brazil

A comparative evaluation of 11 Brazilian (BR) and 11 Belgian (BE) broiler farms, showed that BR averaged 1.12 lameness, gait score 0–2, in comparison with 1.23 in BE. Nevertheless overall mortality in BR was 5.11% and in BE 3.95%⁹. A study in 11 farms selected in Southern Brazil applied the Welfare Quality^® protocol and indicated that, using the gait score 5 point scale, there was a high percentage (14%) of the animals at the more severe levels 4 and 5 of impaired locomotion¹⁰. In a comparison of 10 farms with a Free Range (FR) production system versus 11 farms with Intensive Indoor (IN) systems in Brazil, there was 2% mortality in the FR ones compared to 4.7% in IN. These findings were similar to those of other studies¹¹. Using the 0 to 100 scale (0-worst to 100-best welfare), the same work revealed less lameness in FR than in IN systems, with lameness level of 97/FR and 19/IN and hock burn level of 93/FR and 37/IN¹¹.

Broiler lameness outside Brazil

Several studies between 1992 and 2017 cited that 14 to 50% of broilers had lameness between levels 3 and 5 by the traditional gait score 0–5 assessment method¹². In a study in the United Kingdom¹³ 51,000 (fifty-one thousand) birds were sampled from a population of 4.8 million broilers from 176 flocks at a mean age of 40 days, which resulted in 27.6% of animals with gait scores higher than 3, out of which 3.3% were unable to walk. The most significant factors were that lameness was worse in older birds, there were differences according to the breeding company of origin and hence genotype, animals fed with whole wheat had improved locomotion (slower digestion, longer satiety, slower growth), companies with longer dark periods had better lameness scores (less feed intake, slower growth), higher densities worsened lameness due to less space to move, higher levels of both ammonia and litter moisture, and antibiotic usage possibly reduced the occurrence of infections exacerbating lameness. 7,500 chickens (150/farm in 50 commercial farms) were described in 2019 with 19% of the broilers with a gait score above 3, including 2.89% with scores 4 and 5¹². An investigation of 66,000 broilers in 2022¹⁴ from two different breeding companies found 2.13% of animals with lameness, which were culled and at necropsy main findings were osteochondrosis and inflamed joints with purulent contents. In general, males were more affected than females¹⁴. Automatic, camera-based research indicated an average gait score of 1.9 in broilers 35 days-old¹⁵. Impact perspective estimates indicated that lameness can generate 2 to 8% losses in the slaughter lines for chicken-meat production¹⁶.

Although the above studies used a variety of measurements (gait 0–2, gait 0–5, or percentages), it is possible to infer that lameness affected at least 19% of fast-growing broilers reared in intensive systems, and nearly 3% are more severe cases, i.e. score 2 on the gait 0–2 or 4 and 5 on the gait 0–5, that is they are unable to walk^10,12–14.

Lameness in broilers

Lameness in broilers have been described as originating from multifactorial reasons^7,13, namely: genotype background associated to rapid weight gain^7,16–19, conditions of mothers during oogenesis⁷, artificial incubation impacts on the embryo development^20–23, metabolic factors⁷ and housing and environment in general^7,13,21–23. There has been described that limb issues have been stabilized and improved by persistent genetic selection¹⁹, nevertheless there are also recent description remarking need of further bone characterization to guide quantitative genetic modelling and phenotyping characterization²⁴. Few studies addressed the epigenetic factors caused by pain in the progenitors on their offspring²⁵ that may shape phenotype through inheritance, genomic influence, and environmental modulation. Addressing such epigenetic factors would demand a shift from production-focused studies to fundamental inquiry, for example responding to which genetic markers are able to indicate the transference of environmental changes to the offspring²⁶.

Nociception thresholds

In the context of lameness and animal welfare, there are also concerns for appropriate measures of pain in chickens⁹. Pain can be acute, characterized by quick nociceptive transmission and protective withdrawal of the affected area, followed by increased blood pressure, heart and respiration rates; or chronic, persisting past an expected recovery time and inducing distress and anxiety²⁷. Both forms of pain experiences may modulate future responses, through lasting changes in neural systems involved in nociceptive thresholds. Nociception, the neural process of detecting and transmitting noxious stimuli, provides a measurable framework for assessing actual or potential pain²⁷. The impact of pain experience in progenitors to nociceptive responses by their offspring have been reported in pigs²⁵ and dairy cattle²⁸.

Understanding that (i) genetic variation is reduced under intensive selection²⁹, (ii) besides the parental influences the chicken embryo is affected by environmental in-ovo not in-utero developments²⁰, (iii) transgenerational germline-dependent effects are considered after three generations in females and two in males^26,30,31 and (iv) intergenerational context-dependency generates effects while the environmental stressors persist and has multigenerational impact³¹, the influence of lameness in parental stock to their immediate broiler offspring under commercial farming conditions remains underexplored. The main hypothesis of this present study was that observable lameness in male and female breeder parents would also manifest in the immediate offspring of fast-growing broilers (i.e. their progeny), and such issues would be associated with nociception thresholds.

Materials and methods

Ethical approval

This study was reviewed and approved by the Committee on Ethics in Animal Use (CEUA) of the Faculty of Veterinary Medicine and Animal Science – FMVZ - University of São Paulo (USP), under the number 9498121222, within the National Council for Control of Animal Experimentation (CONCEA) - Brazil. This approval is placed as Supplementary Information. All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Experimental design

The whole study was carried out within commercial facilities. In a breeding farm 100 female and 20 male parental chickens were selected and mated according to their individual lameness conditions in four combinations of lame and non-lame birds. The fertile eggs of those progenitors were marked and incubated, and from the hatched broilers, immediate progeny of the experimental parents, 374 day-old-chicks were housed in a rearing farm, grouped in four treatments with four replicates per treatment, according to the lameness conditions of their progenitors. At 39 days of age, 371 adult broilers were tested for lameness and nociception thresholds, with data recorded per treatment and sex. There was no anaesthesia, nor euthanasia performed on chickens in the study.

Animals, facilities and handling

All assessments and data collection were executed in farms and hatchery installations integrated to the production chain of a large Brazilian producer of broiler day-old-chicks. The initial phase was staged in a breeding barn for traceability identified as number 652 with 5072 females and 408 males, of which 100 female and 20 male parents were selected according to lameness gait scores 0–2³² (0 for birds with easy, regular and steady steps, and balanced posture (no lameness issues), 1 for irregular and unsteady steps, appearing unbalanced (moderate lameness), and 2 for birds reluctant to move and unable to walk a few steps before sitting down (severe cases). The higher the score, the worse the lameness of the bird³². Per Table 1 the number of chickens was proportionated per their lameness levels, mostly level 1 and confirmed by two Veterinarians of having no infectious origin. The chickens were classified into “lame” (code “C”) or “non-lame” (code “S”), and four treatment groups were formed with iterations of C/S birds, with every parental bird identified by numbered leg-rings: CC – colour ID blue, lame females and lame males (both gait score 1 or 2); SC – colour ID red, non-lame females (score-0) and lame males (score-1 or 2); SS – colour ID yellow, non-lame females (score-0) and non-lame males (score-0); and CS – colour ID black, lame females (score-1 or 2) and non-lame males score-0. To mitigate interference of residual semen from prior matings, after the groupings the fertilized eggs were not considered for experimental purposes during a 14-day gap^33–37, then the fertile eggs produced were collected and permanent-pen marked with the colours indicative of the parents’ lameness conditions.

Table 1.

Identification in the commercial barn 652 and number of experimental parents initially selected, grouped according to their lameness conditions and fenced inside the same barn for production of fertile eggs.

Treatment (TTO)	Gait-score (0–2)	Female	Male
SS – non-lame female parent and non-lame male parent Average weight (g)	0 1 2	25 - - 3973	5 - - 4400
SC – non-lame female parent and lame male parent Average weight (g)	0 1 2	25 - - 3840	- 5 - 4269
CS – lame female parent and non-lame male parent Average weight (g)	0 1 2	- 24 1 3797	5 - - 4460
CC – lame female parent and lame male parent Average weight (g)	0 1 2	- 24 1 3734	- 5 - 4370
Total Average weight (g)		100 3836	20 4375

In a commercial hatchery the marked eggs were incubated in separate trays, with no egg recovery treatment nor special settings of time, temperature and humidity in the incubation machines. The progeny day-old-chickens were hatched in separate plastic boxes identified with the same pattern of colours as for the parents and 374 birds not sexed, not individually marked were utilized for the experiment. The hatched day-old-chicks were housed in a commercial rearing farm with litter, lighting, ventilation, feed, water and handling similar to other commercial flocks housed in the same barn. The distribution and size of the experimental pens (Table 1) were adapted to the unchanged location of feed and drink dispensing lines at the commercial barn. The day-old-chicks had an average density of 6.44birds/m2 equivalent to 16 kg/m2 final weight as adult broilers. This is within the limits of European organic systems whose maximum density is 20 kg/m²³⁸.

Table 2.

Total numbers of broiler day-old-chicks (DOCs) housed at 31 Dec 2024 for experimental purposes and numbers of adult broilers examined on 07 Feb 2025. The weight refers to the average of the mixed females and males.

Treatment and colour id		31 Dec 2024		07 Feb 2025
		Number of broiler day-old-chicks	Weight (g)	Number of adult broilers	Mortality (%)	Weight (g)
CC	Blue	90	42.69	89	1.11%	2355
SC	Red	99	42.95	98	1.01%	2270
SS	Yellow	75	42.61	75	0.00%	2201
CS	Black	110	42.66	109	0.91%	2174
Total		374	42.73	371	0.80%	2250

The chicks’ placement per pen followed the colour identification of the treatment. The replicate pen placements were proportional to the number of chicks available, with 4 repetitions, resulting in 4 pens per treatment/group as shown in Table 3. For operational reasons the pens of each treatment were adjacent, therefore the evaluation and data collection followed a repeated-randomized sequence starting at the first pen of the group SC, colour ID red, then to the first pen of the group SS, colour ID yellow, then first pen of the group CS, colour ID black, and finishing at the first pen of the group CC, colour ID blue. The same pattern was then followed for the second, third and four pens of each group, till completion of all replicates.

Table 3.

Number of adult broilers chickens on the examination date of 07 Feb 2025 with the distribution of females and males per replicate – experimental pen. The complete association of treatment abbreviations, colours and treatment (replicate) pens was: CC-blue: az1, az2, az3 and az4; SC-red: v1, v2, v3 and v4; SS-yellow: am1, am2, am3 and am4; CS-black: p1, p2, p3 and p4; Abbreviated columns: F=females; M=males; T=total numbers; Each pen size of 3.60 × 1 m. with the equivalent density per square meter (sqm). The density in kg per sqm can be estimated by the total number of birds times the maximum estimated final weight of 2.5 kg per bird.

Treatment	Numbers of adult broilers examined per replicate (pen)															Weight average (g)		Density
	Replicate 1 1 st Pens			Replicate 2 2nd Pens			Replicate 3 3rd Pens			Replicate 4 4th Pens			Total
	F	M	T	F	M	T	F	M	T	F	M	T	F	M	T	F	M	Birds/sqm
CC-Blue	11	10	21	15	8	23	13	11	24	12	9	21	51	38	89	2100	2611	6
SC-Red	15	9	24	14	11	25	19	7	26	11	12	23	59	39	98	1958	2583	7
SS-Yellow	15	5	20	13	7	20	12	6	18	9	8	17	49	26	75	1872	2531	5
CS-Black	22	12	34	14	7	21	22	6	28	16	8	26	76	33	109	1936	2413	8
Total	63	36	99	56	33	89	66	30	96	50	37	87	235	136	371	1966	2534	6

When the birds reached 39 days of age they had an average weight of 2.25 kg and were evaluated by qualified veterinarians and subject-experts unaware of the treatment group being examined. The evaluations here reported are lameness and nociceptive responses to noxious mechanical stimulus. The examination and data collection followed the colours’ IDs, that is, giving evaluators a sequential guidance but blind to the experimental group being assessed. Lameness was evaluated using the validated Welfare Quality^® protocol⁶ and carried out by an independent certified auditor.

Lameness in broilers

Lameness in broilers are part of the Good Health principle which is used as a criterion for the absence of injuries in the Welfare Quality^® protocol⁶. Assessment was performed on all adult broilers individually with data recorded per sex, and per experimental pen of pododermatitis (FPD) score (from 0-absent to 4-severe), hock burn (HB) score (from 0-absent, 1 and 2-superficial, 3-deep and 4-severe), keel cyst/breast blister (BB) score (from 0-absent to 2-present), and gait score 0–5 scale of lameness (From 0-absent to 5-severe), i.e., 0-soft/smooth walking, the feet curve when lifted, and the bird appears to be balanced, 1-irregular walking, the feet may or may not curve when lifted and it is difficult to tell on which side the leg or foot is affected, 2-irregular walking, the feet remain flat when lifted, the stride is short, with little balance, and the bird uses its wings for support, 3-similar to score 2, but bird remains lying down unless gently nudged to move, more likely to use wings for balance and support, and does not stand for more than 15 s (without being disturbed), 4-reluctant to move and uses wings as crutches to walk, takes only a few steps before lying down again, 5-unable to take a step and will drag itself if pushed to move.

Nociception threshold

From the randomized sequence of pens, a random sample of 6 broilers was taken from each experimental pen: three males and then three females were caught and gently placed in open-top plastic boxes (56 × 36 × 31 cm) that allowed them to stand, turn and crouch. The boxes contained only either females or males. Starting with the males, one bird was selected from each three counted, after gathering them with a handling board in the experimental pen. The plastic boxes of each pen were manually hauled off the rearing area to the service area of the barn, which was fitted as an experimental room. A standard approach was adopted to let the sampled birds rest for 5 min, and only proceed to nociceptive threshold tests in absence of signs of agitation by the birds. The average time of the measurement per bird was 10 min. For nociception threshold tests, birds were gently placed on a plain surface by the examiner and three measures of nociceptive thresholds were obtained in the distal portion of the tarsometatarsus of each leg using noxious mechanical stimulation applied by a portable force transducer or algometer (electronic von Frey model EFF 301, Insight max. 10 N), equipped with a polypropylene tip of 1 mm. Birds were only tested when standing calmly at rest, and they responded by lifting the limb. Taking a step was not considered a response. The choice of the limb to be tested first was randomized, sequentially alternating between right and left limb for each bird. Results were immediately recorded by another experimenter for each measure (either the precise threshold as indicated by the algometer upon response, or the maximum possible reading in case of no response by the bird).

Statistical analysis

Ordinal outcomes (hock burns and gait score) were modelled using cumulative linked mixed models (CLMMs) fitted with the ordinal package³⁹. The fixed effects included treatment, sex, and their interaction, with pen as a random intercept to account for the non-independence of birds within the same housing unit. For gait score analysis, body weight was included as a covariate to adjust for the known relationship between bird size and gait quality. Model selection was guided by Akaike’s Information Criterion (AIC) to identify the most parsimonious model. Estimated marginal means (EMMs) were computed using the emmeans package⁴⁰ on the logit scale and back-transformed to probability scale for interpretation. Pairwise comparisons used Tukey adjustment and were generated with the multcomp package⁴¹ to identify homogeneous subgroups. Mechanical nociceptive thresholds were analysed using linear mixed-effects models from the “nlme” package⁴². The model included treatment, sex and their interaction as fixed effects and pen specified as a random effect. The significance of fixed effects was assessed using Analysis of Deviance from the car package⁴³. For all analyses, significance was set at P < 0.05, and pairwise comparisons were adjusted for multiple testing using Tukey’s method. Model convergence and assumptions were verified using the performance package⁴⁴. All analyses were conducted in the R statistical environment, version 4.5.0⁴⁵.

Results and discussion

Lameness

On the scale 0–5 gait score the overall 1.43 lameness mean of this study is considered mild. Overall, 10% of the studied birds had a gait score above level 3 and 3% had severe lameness, levels 4 and 5, being the last within the findings of other studies in terms of birds with inability to walk. The 3% is considered relevant because it relates only to extreme immobility, subjected to be culled. This is an industry which works 5% losses due for example to mortality. Considering the increases in rapid weight gain a 3% extreme lameness could be arguably indicated as stable, however it was not found evidence of consistent genetic modelling giving assurance to that, as well as in the perspective of animal welfare it indicates a lameness level on which the animals are suffering and need to be humanely euthanised.

The treatment comparisons showed no significant emmeans differences. The sex comparisons indicated male broilers had significantly worse HB and Gait 0–5 scores (lameness) than female broilers. The Tables 4 and 5 show the means of gait scores 0–5 and hock burn (HB) 0–4 points, respectively. Sex and treatment effects were chosen to project the estimated marginal means.

Table 4.

Gait score 0–5 of the adult broilers, immediate progeny of the progenitors grouped by their lameness conditions. Emmean values with no differences per treatment and with significant differences in the Sex comparison.

Treatment (code)	Treatment (TTO) (lameness conditions of the progenitors)	Gait 0–5 scores Lameness of the immediate progeny (adult broilers)
		Males	Females	Means	Emmeans (TTO comparison)
SS	Non-lame female parent; no-lame male parent	1.46	1.38	1.41	−1.81(a)
SC	Non-lame female parent; lame male parent	1.72	1.20	1.41	−1.87(a)
CS	Lame female parent; non-lame male parent	1.82	1.24	1.41	−1.72(a)
CC	Lame female parent; lame male parent	1.95	1.16	1.50	−1.68(a)
Mean		1.74	1.24	1.43
Emmean – SEX comparison p-value: 3.76 × 10− 7		−1.24(b)	−2.3(a)

Different letters denote significant differences (p < 0.05).

Table 5.

Hock burns (HB) injury severity scores in the classification 0–4 points. No significant variation in total Emmean comparison per treatment and significant differences between females and males broilers.

Treatment (code)	Treatment (TTO) (lameness conditions of the progenitors)	Hock burns Injuries of the immediate progeny (grown broilers)
		Males	Females	Means	Emmeans (TTO comparison)
SS	Non-lame female parent; no-lame male parent	0.31	0.14	0.20	−2.75(a)
SC	Non-lame female parent; lame male parent	0.13	0.13	0.13	−3.27(a)
CS	Lame female parent; non-lame male parent	0.39	0.22	0.27	−2.45(a)
CC	Lame female parent; lame male parent	0.39	0.22	0.29	−2.37(a)
Mean		0.30	0.18	0.23
Emmean – SEX comparison p-value: 0.02		−2.40(b)	−3.01(a)

Different letters denote significant differences (p < 0.05).

Despite the mild levels of lameness and hock burns (HB), the regression coefficients of lameness and hock burn (HB) indicated that for all treatments the male broilers were likely to have more lameness issues than the females. There was no significant mean variance among the treatment groups, however the probability estimation resulted in slightly lower lameness and hock burn (HB) occurrences in the SC group (broiler offspring from non-lame hens mated with lame males). Figure 1 illustrates both lameness and hock burn (HB) distributions.

Fig. 1.

Lameness and hock burns (HB) estimated means confirming statistically significant variations between female and male broilers, and no relevant effects from treatment.

The lower lameness and hock burn injuries in the SC treatment (progeny of non-lame females and lame-males) related also to a larger proportion of broiler females in this group: at the moment of the evaluation there were 59 females with a rather mild mean gait score of 1.20, which contrasted with 39 males with gait 1.72. The overall better female gait score was also reconfirmed in the group SC. This finding that males have worse lameness was confirmed in the works of GUEVARA-TORRES et al.¹⁴ and ERENSOY et al.⁴⁶. The 1.5 worse gait score mean in the whole CC group had no statistical significance, nevertheless breaking it down by sex, the males worse lameness with a score of 1.95, contrasted with 1.16 of the females in the some group.

Previous research supports our findings, whereas males’ showed worse lameness as described in the work of ERENSOY et al.⁴⁶, who studied tonic immobility (TI) inhibitory impact in the rapid growth trait of broilers. In their work they did not find genotype differentiation impacting the duration of TI, but concluded from the direct correlation between genotype and sex to gait score. They found more lameness in males, as we reported in the current study. In their view, genetic selection for resistance to stressors and gait problems, especially for males, might provide permanent solutions for more profitable and sustainable broilers. BOLUWATIFE et al.⁴⁷ concluded that different levels of fear measured through tonic immobility tests, varied according to the genotype, and was influenced by different breeding programs.

Nociception threshold

The results for the nociceptive signal assessment were validated using both mean and median data of three measures obtained in each limb of the sampled birds. The results of the right limbs showed no significant statistic variation. The repeated measures comparisons showed that males had higher nociceptive thresholds than females (see Table 6). Our data also identified differences between the treatments CS (progeny of lame females and non-lame males) versus SS (progeny of non-lame females and non-lame males): for the left limb, a borderline difference was observed in the pairwise group comparison, marginally exceeding the conventional significance threshold (P < 0.05), which however and through estimated marginal means, reconfirmed a sex-related deviation in nociceptive thresholds (P = 0.01). The SS progeny, with both sound parents, demonstrated higher sensitivity to the noxious stimulus than the other treatments. Related also to the left limb, the progeny of CS parents, showed nociception at higher pressure than the other groups, indicating lesser sensitivity than the progeny of other treatments (Table 6).

Table 6.

Nociception means and emmeans per treatment and per sex. Significant differences in treatments and sex at the left limb.

Treatment (code)	Treatment (TTO) (lameness conditions of the progenitors)	Nociception thresholds (gf/cm2 – gram-force per square centimetre)
		Means Right Limb			Means Left Limb
		Males	Females	Emmeans	Males	Females	Emmeans
SS	Non-lame female parent; non-lame male parent	730.6	744.2	737.4(a)	746.6	739.3	743.0(a)
SC	Non-lame female parent; lame male parent	796.2	757.4	776.8(a)	779.3	788.6	784.0(ab)
CS	Lame female parent; non-lame male parent	853.3	768.0	810.7(a)	948.2	820.3	884.2(b)
CC	Lame female parent; lame male parent	868.5	817.7	843.1(a)	906.9	741.9	824.4(ab)
Mean		812.1(a)	771.8(a)	792.0	845.3(b)	772.5(a)	808.9

Pairwise comparisons with difference at the left limb between treatment CS-SS, P < 0.05.

Emmeans p-value left limb nociception sex comparison: P = 0.01.

Different letters denote significant differences (p < 0.05).

Further, the repeated measure analysis of the nociceptive thresholds (Fig. 2 – emmeans), also indicated that males were less sensitive to the noxious mechanical stimulus than females (P = 0.01). Additional repeated measures analysis indicated significant differences (P = 0.02) when contrasting the treatment CS versus the SS, where SS presented an overall lower nociceptive threshold detected in the left limb.

Fig. 2.

Nociception thresholds emmeans per sex showing that females broilers had more sensitivity than males and broilers progeny of non-lame females and non-lame males were more sensitive than the other groups.

For the left limbs, considering all treatments, the lower mean values of algometer pressure on the female broilers seem to indicate that female broilers had more sensitivity to the stimulus than the males.

The treatment pairwise comparisons seem to indicate by the means of the left limbs, a larger variation of nociception between the treatments CS and SS. The repeated measurements (left limb, all treatments), seem to confirm female broilers were more sensitive than males.

Lateralization in pain perception arises from functional asymmetries in the organization of the nociceptive system in mammals and may extend, at least in part, to other vertebrate taxa. Painful stimuli applied to the right side of the body project predominantly to the left hemisphere, and vice-versa, yet these hemispheres differ markedly in their specializations: the right hemisphere exhibits greater responsivity to the affective–motivational aspects of pain, whereas the left hemisphere is more involved in cognitive appraisal and modulatory control of nociception^48,49. These asymmetries manifest in variations in pain threshold and tolerance - often lower on the non-dominant side - likely reflecting differences in cortical motor oversight, attentional engagement, and hemispheric contributions to salience detection and spatial-attentional processing⁵⁰. Sensorimotor integration, body-schema representation, and parietal cortex lateralization further contribute to inter-hemibody discrepancies, which may become accentuated in chronic pain states such as complex regional pain syndrome (CRPS), migraine, and musculoskeletal disorders, where central sensitization and somatosensory reorganization are prominent⁴⁸.

In birds, evidence demonstrates that they possess the neuroanatomical and physiological substrates required for nociception and likely for the conscious experience of pain, paralleling aspects of mammalian systems⁵¹. Although direct studies on hemispheric lateralization of pain in avian species are scarce, brain lateralization is well established in a wide range of avian behaviours and sensory modalities, and it is plausible that avian species might also exhibit some degree of lateralization in pain processing, with the right hemisphere potentially contributing more to the emotional or defensive dimensions of nociception⁵². However, current evidence remains insufficient to confirm this hypothesis, as in avian species is still lacking and represents an important area for future investigation^51,53,54.

We did not find in the literature studies that indicate how brain lateralization may account for the higher sensitivity observed in the left limb of the birds^54,55. Side differences in nociceptive thresholds were reported in piglets^25,56, corroborating laterality differences in nociception. The work in pigs²⁵ identified that the offspring of non-lame sows had lower nociceptive threshold than the offspring of the lame ones in the total mean and left side measurements. Also in piglets, nociceptive differences in both legs in all treatments have been described⁵⁶.

In 2011 Gentle⁵¹ referred to the repetitive feather removals mimicking feather pecking in egglayer hens which resulted in catatonic defensive immobility response: the electroencephalogram (EEG) pattern was similar to stress induced analgesia in other species, highlighting the hypothesis that in the latter repeated pecking traumas the animal was no longer feeling the pain.

Thermal nociceptive measurements in broilers have already revealed unexpected higher threshold in lame birds than in non-lame birds, that is, lame animals presented less nociceptive signals than the healthy ones: the nociceptive threshold of lame chickens was 42.4 °C against 41.6 of the others, i.e. 2.66% higher⁵⁷. Although relatively small difference as well as unclear mechanisms behind it, the finding was repeatedly observed and with inferred indication by the authors that “stress response to chronic pain may have reduced lame birds’ responsiveness to temperature, resulting in higher threshold”⁵⁷. The thermal nociceptive finding was irrespective of weight, flock uniformity, and sample size⁵⁷. It also ruled out other variables that could have impacted those findings such as secondary thermal hyperalgesia/hypoalgesia, reduced responsiveness to thermal stimulation, reluctance to move or weight bearing on the lame limb⁵⁷. Chronic pain is understood as any form of persistent pain⁵⁸.

In this present study males exhibited higher gait scores, indicating poorer locomotor health compared with females. This compromised condition may have rendered males less responsive to nociception than females. It is arguable that lame animals, being more accustomed to chronic discomfort, may exhibit reduced sensitivity to acute stimuli. Interestingly, for reasons not yet fully understood, nociceptive responses were more pronounced in the left limbs. Perhaps and similar to what is reported in humans affected by chronic pain, the habit of living in pain might disguise, or perhaps extend, the nociception to acute pain. The 2014 work of thermal induced pain⁵⁷ also concluded that lame birds had more impaired nociception than the non-lame ones, perhaps associated with chronicity. However none of the previous work had investigated the impact on the offspring of lame birds.

We demonstrated for the first time that lame parents reduced pain sensitivity in their offspring. The study was carried out in farming conditions, where the treatment groups were unknown to the experimenters. The impact of lameness on parent birds increasing nociceptive threshold of the offspring, indicates a transgenerational effect that could affect the welfare of the birds. Pain perception is a very important mechanism to maintain homeostasis.

Concerning the higher nociception of the left limb, no description was found that it could be associated with chicken brain lateralized dominance or other mechanism of pain sensitivity. The 2011 thermal threshold work noted some numerically and slightly higher left leg threshold with no statistical significance⁵⁹. SARMIENTO et al.²⁵ identified that non-lame piglets had lower nociceptive threshold than the lame ones in the total mean and left side measurements. Considering the lack of bird studies on brain lateralization of pain processing we explored studies in other species, anticipating that there are similarities between chickens and mammals (humans included), in central pain processing mechanisms. Previous studies suggested that pain perception involves both hemispheres of the brain with functional lateralization, that is right hemisphere (left eye side) amygdala tends to be pro-nociceptive and the left may have anti-nociceptive effects⁵⁰. Sensory signals, including pain, from one side of the body are processed in the opposite hemisphere of the brain, but a shift in lateralization becomes evident in chronic pain: there is higher ipsilateral (same side) activity related to the pain. Thus, a chicken left leg nociceptive reflex would be processed in the right side of the brain and right leg nociceptive reflex in the left side of the brain, however in the event of chronic pain, a shift would occur, i.e., left limb-left hemisphere, right limb-right hemisphere.

Nevertheless, those nociceptive findings indicated an opening for further confirmation about pain lateralization, pain chronicity induced by lameness and sex related pain resilience in broilers.

The evaluation of this experiment indicated some limitations, which could be improved in future studies and would have contributed to strengthen the findings and conclusions: logistics limitation to have researchers present every day in the facilities, individual traceability of the broilers via wing banding, assessment of the lameness impact on the nociceptive response, exploratory necropsy of progenitors and progeny, and laboratory exams of lymphocytes, glucocorticoids, interleukins, and epigenetic markers in the offspring.

Final considerations

The bird model explored in the current study offers a very elegant and novel approach to understand the impact of pain experienced by progenitors on welfare indicators, particularly nociceptive thresholds in the offspring. The time associated with the modulation of nociceptive thresholds, in the offspring, by the pain experience of their progenitors, appear not to be dependent from direct pain experiences of progenitors during the embryo development, as reported in mammalian species.

This study data and conclusions indicated a viable starting point to correlate the influence of parents chickens modulating pain responses on their progeny of broilers, and associated to lameness. Repetition and expansion of the design would create opportunity towards knowledge gaps on epigenetic influences and pain processing in chickens.

Author contributions

M.A.P.A. contributed the idea conception, design and setup of the work, data collection and interpretation, writing of the manuscript and preparation of tables.A.J.Z. contributed with the idea conception, design of the work, data collection and interpretation, supervision and review of the manuscript.C.C. contributed to data processing, interpretation, discussion of results, and preparation of figures.V.A.L. contributed to the data acquisition using the Welfare Quality^® protocol and discussion of results.A.C.D.M. contributed to the experiment setup and nociception data acquisitionC.S.D. contributed to the concepts, and guidance related to nociception.All the authors contributed to the revision of the final version.

Funding

A.J.Z. would like to acknowledge the help received by the Brazilian Research Council, CNPQ, Process 316457/2021-3.

Data availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.