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. 2022 Mar 11;100(4):skac076. doi: 10.1093/jas/skac076

Keel bone damage affects behavioral and physiological responses related to stress and fear in two strains of laying hens

Haidong Wei 1, Yanru Feng 1, Susu Ding 1, Haoyang Nian 1, Hanlin Yu 1, Qian Zhao 1, Jun Bao 1,2, Runxiang Zhang 1,2,✉
PMCID: PMC9030218  PMID: 35275597

Abstract

Keel bone damage (KBD) is more prevalent in alternative laying hen housing systems than in conventional cages, and its incidence differs from strain to strain. However, the information of KBD in Lindian chickens, a native Chinese strain, is limited. To investigate the effect of KBD on fearfulness and physiological indicators of stress in Lindian chickens and commercial laying hens, a total of two hundred 25-wk-old chickens (100 Hy-line Brown and 100 Lindian chickens) were studied for 7 wk. The birds were housed in furnished cages with 10 birds per cage for each strain. At 32-wk of age, the birds in each strain were divided into normal (NK), deviated (DK), and fractured (FK) hens according to the keel bone status. Ten birds in each keel bone status per strain were subsequently selected to collect blood for the determination of stress and fear-related indicators, including corticosterone, serotonin, interleukin-1β, and interleukin-6, and measure fear responses, including novel object test (NOT), human approach test (HAT), and tonic immobility (TI) test. The results showed that egg production was lower and the incidence of keel bone fractures was higher in Lindian chickens than in Hy-line Brown hens (P < 0.05). Lindian chickens showed a significantly increased whole blood serotonin content, NOT-latency, HAT-score, and TI induction times (P < 0.05) and decreased serum interleukin-6 content and TI-duration (P < 0.05) compared with Hy-line Brown hens. Additionally, FK hens had significantly elevated whole blood corticosterone, serum interleukin-1β and interleukin-6 levels, TI-duration, and NOT-latency (P < 0.05), and a reduced whole blood serotonin content (P < 0.05) compared with NK and DK hens. Our results indicated that KBD affected stress and fear responses, and this impact was mainly reflected by FK hens compared with NK and DK hens. We suggest that keel bone fractures are the main factor impairing hen welfare. Besides, the incidence of keel bone fractures and stress and fear responses of Lindian chickens are more severe than Hy-line Brown laying hens, indicating that the strain type can affect the health and welfare of laying hens.

Keywords: fear, keel bone fracture, laying hen, stress, welfare

Lay Summary

Keel bone damage (KBD) impairs production performance, welfare, and health in laying hens. This study aimed to compare the incidence of KBD and investigate the effects of KBD on stress and fear in two strains of laying hens. The results showed that commercial Hy-line Brown laying hens had high egg production and low incidence of KBD compared with Lindian chickens, a Chinese native breed. Besides, Lindian chickens had higher blood serotonin content and fear responses to human approach test and novel object test than Hy-line Brown laying hens. In addition, laying hens with keel bone fractures had elevated concentrations of blood corticosterone, interleukin-1β, and interleukin-6, and had a longer duration of tonic immobility and latency to approach a novel object, as well as reduced blood serotonin content compared with laying hens with normal and deviated keel bone. Overall, keel bone fractures caused stress and fear responses, impairing hen welfare; and behavioral and physiological responses in relation to stress and fear differed between strains of hens.

Keel bone fractures could induce negative emotion, impairing chicken welfare, and behavioral and physiological responses of laying hens to stress and fear differed between the two strains studied.

Introduction

With the transferring of laying hens from conventional cages to alternative systems, such as furnished cage, aviary, and free-range systems, the occurrence of keel bone damage (KBD, including deviations and fractures) has become a serious health and welfare problem. In recent years, KBD attracted much attention from many researchers because of the prevalence of KBD in layers. Previous studies reported that KBD (especially fractures) altered behavior (Nasr et al., 2012a; Casey-Trott and Widowski, 2016), reduced egg production (Rufener et al., 2019), and egg quality (Wei et al., 2020), caused pain (Nasr et al., 2012b) and physiological stress (Wei et al., 2019) and induced a depressive-like state (Armstrong et al., 2020) in laying hens. In addition, severe keel bone fractures could even lead to the death of laying hens (reviewed by Hardin et al., 2019). Thus, KBD results in economic losses for the laying hen production industry.

It was reported that the incidence of KBD was different between cage and cage-free systems, and laying hens housed in cage-free systems had a higher incidence of KBD than in furnished cages (Hardin et al., 2019). Several studies found that the average percentage of laying hens with keel bone fractures was 40.0% at 37 wk of age (Wei et al., 2021), 54.4% at 42 wk of age (Wei et al., 2020), and 62.0% at 60 wk of age (Rodenburg et al., 2008) in furnished cages. Overall, the incidence of KBD of laying hens in furnished cage was around 15% to 55% (Hardin et al., 2019), and keel bone status was affected by age, strain, and nutrients of laying hens. It was also found that the incidence of KBD varied from chicken strain to chicken strain. Some studies reported that brown strains had more severe and frequent keel bone deviations (Habig and Distl, 2013) and more keel bone fractures in commercial laying hens (Eusemann et al., 2018; Fawcett et al., 2020) when compared with white strains. However, other studies reported that white laying hens had more and severe keel deviations or fractures compared with brown hens (Candelotto et al., 2017; Ali et al., 2020). Studies on commercial, noncommercial, and native strains of laying hens found that commercial strains had higher KBD than noncommercial (Candelotto et al., 2017) and native (Kittelsen et al., 2020) strains. Besides, there was a strain difference in the occurrence of KBD in native chickens from different countries or regions (Kittelsen et al., 2020).

Similar to mammals and other birds, chickens have behavioral and physiological responses to various environmental stimulations and stressors (Cockrem, 2013). Previous studies found that KBD could cause psychological stress measured by fear-related tests (Rokavec and Šemrov, 2020) and physiological stress measured by blood corticosterone (Wei et al., 2019). Fear is generally regarded as an undesirable state of suffering of animals in threatening circumstances. It is also considered a psychophysiological (emotional) response to perceived danger or threat (Jones, 1996). Many factors, such as social environment, genetic background, housing system, and health status of animals, induce fear (Uitdehaag et al., 2011; Rokavec and Šemrov, 2020). Different fear responses, including struggling, violent escaping or panicking, avoidance, and immobility, can be measured by behavioral tests. The tonic immobility (TI) is a well-validated and widely used test for measuring fear response in chickens performed by immobility as freezing or crouching. TI duration is a valid indicator for assessing fear (Jones, 1996). A longer TI duration indicates a stronger fear. The novel object test (NOT) is another popular method to measure fear in poultry, and the latency of avoidance or approach of birds to a novel object can directly reflect the fear levels (Hocking et al., 2001). The approach of birds to novel objects suggests a lower fear and a positive emotional state. In contrast, the avoidance of novel objects suggests a higher fear and a negative emotional state. In addition, the human approach test (HAT) is applied to assess the fear level of chickens (Nelson et al., 2020; Yoshidome et al., 2021). The stress response is involved in activating the hypothalamic–pituitary–adrenal (HPA) axis in animals. Corticosterone is an end-product of the activated HPA axis, which is often used to indicate endocrine response to stress in the HPA axis in poultry (Charmandari et al., 2005). Generally, fear and stress can reflect an emotional state. A high concentration of serotonin and low levels of cytokines interleukin-1β and interleukin-6 are associated with positive emotion in humans and animals (Korte, 2001; Stellar et al., 2015).

Lindian chicken, a native breed from Lindian County, Heilongjiang province, China, is famous for the unique texture and flavor of its meat and eggs, and stronger cold resistance and high tolerance to crude feed (Qiu et al., 2015). In addition, the Lindian chicken has a medium body size with light-yellow skin, and its plumage is mainly dark ephedra or light ephedra (Yuan et al., 2019). It is also a national resource protection strain and has been listed on the National Animal Genetic Protection List of China since 2014 due to the decline of its population year by year (Qiu et al., 2015). Owing to the unique characteristics of Lindian chickens and the sharp decline in their population, the percentage of KBD and behavioral and physiological responses of Lindian chickens with different keel bone statuses have not been reported. Thus, the present study aimed to assess the incidence of KBD and measure fear and stress responses related to emotion in Lindian chickens and commercial Hy-line Brown laying hens with and without KBD at the peak period of laying to evaluate the welfare state of two strains of hens housed in furnished cages. Our results may provide some information to improve the welfare of Lindian chickens.

Materials and Methods

Ethics statement

All experimental protocols and the use of animals were approved by and conducted according to the guidelines of the Institutional Animal Care and Use Committee of Northeast Agriculture University (NEAU-[2011]-9).

Animals and management

At 17 wk of age, 120 commercial Hy-line Brown laying hens were purchased from Yinong Poultry Industry Corporation Ltd., Harbin, China, and 120 Lindian chickens were obtained from the Lindian chickens core breeding population of the National Production Farm in the Lindian county, Heilongjiang, China. All birds were kept in rearing battery until 17 wk of age and thereafter they were housed in furnished cages (Figure 1) with 10 birds per cage. The dimension of each cage was 150 cm length × 70 cm width × 70 cm height, and it was enriched with two quadrate wood perches (20 cm and 40 cm above the wire-mesh floor, and 45 cm and 25 cm away from the front wire-mesh sidewall, respectively; the horizontal distance of both perches was 20 cm), an elevated enclosed red nest box with a size of 40 cm length × 70 cm width × 25 cm height (installed right wire-mesh sidewall and 45 cm above the wire-mesh floor), a plastic-mesh (60 cm length × 20 cm width, installed left wire-mesh floor), a rectangular feed trough and waterline with four nipple drinkers (installed outside of front wire-mesh sidewall). At 25 wk of age, a total of 100 Hy-line Brown commercial laying hens and 100 Lindian chickens with normal keel (NK) bone were selected from obtained birds and used in this study. Laying hens were then randomly assigned to 10 replicated cages with 10 birds per cage for each strain. Furnished cages per strain were separately allocated at a semi-enclosed hen house with natural ventilation, and the size of two hen houses and the arrangement of cages in each hen house were totally the same. A lighting program combining natural light and artificial light was provided. Artificial light was provided for 16 h from 0500 to 2100 hours, and light intensity was 20 to 24 lux. The ambient temperature and relative humidity of the hen house were 18 to 21 °C and 50% to 70%, respectively. All laying hens were fed a commercial corn–soybean meal layer diet with 16.08% crude protein and 2,800 kcal/kg metabolic energy. They were given ad libitum access to feed and water throughout the entire experimental period from 25 to 34 wk of age.

Figure 1.

Figure 1.

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A front view of furnished cage used in this study.

Determination of egg production

The number of eggs from Hy-line Brown laying hens and Lindian chickens was recorded daily from 25 to 34 wk of age. The average weekly egg production rate of two laying hen strains was calculated based on the total number of eggs per day divided by the number of birds, respectively.

Assessment of keel bone status

Keel bone status of all laying hens was assessed through palpation and X-ray methods. Palpation mainly assessed whether keel bone deviation was present, while X-ray primarily checked whether keel bone fractures were present in laying hens. Briefly, palpation of live birds was first performed by one experienced worker, and spinal or ventral and lateral surfaces of keel bone were palpated by running the thumb and forefinger up and down by feeling the bone deformities, such as S-deviations, bumps, sharp bends, fragmented sections, callus materials, and others (Casey-Trott et al., 2015). After the palpation, the keel bone of these birds was assessed by a portable X-ray instrument (WAT-LES100D, Shenzhen, China) according to the method of Eusemann et al. (2018) to test the reliability of the palpation. Keel bone status can be classified into three main categories: NK, deviated keel (DK), and fractured keel (FK) bones based on the presence or absence of deviations and fractures. If a laying hen had both DK and FK bones, the keel bone was recognized as FK (Scholz et al., 2008). Meanwhile, a NK means that it is neither DK nor FK. After the assessment of KBD, all selected birds (25 wk of age) with NK were housed in furnished cages for 7 wk. Chickens of two strains (32 wk of age) were assessed, and the birds were divided into NK, DK, and FK hens according to the keel bone status. In the present study, keel bone fractures were mainly located in nearly one-third of the caudal border of the keel. Finally, all birds were housed in original cages.

Focal animal selection and sample collection

After the assessment of KBD in 32-wk-old laying hens, 10 birds in each keel bone status were randomly selected from 10 furnished cages with 1 bird per cage and marked individually with different numbered leg tags in their original cages so that they could be easily found for later sampling. In our study, a total of 10 NK, 10 DK, and 10 FK laying hens for each strain were used as focal animals to collect blood samples and measure fear and stress responses according to the experimental design and operational process (Figure 2). For example, 10 NK Hy-line Brown laying hens were removed from 10 furnished cages in turn with 1 bird per cage for sampling, and only after the blood sample collection or fear test of the current NK hen was finished (from removal to return to the cage), the next NK hen was removed from another cage for sampling. The DK and FK laying hens began sampling after all NK laying hens had completed the blood sample collection and fear tests. The sequences of blood sample collection and various fear tests on the focal animals from each keel bone status were consistent. A total of 3 mL of blood from each focal bird was collected from the wing vein by venipuncture within 2 min from initiating capture to total extraction. About 1 mL of blood was collected in 5-mL ethylenediaminetetraacetic acid tubes to determine corticosterone and serotonin, and the remaining 2 mL blood was centrifugated at 3,000 rpm for 15 min at 4 °C. Then, serum samples were obtained and stored in the freezer at −20 °C for the determination of emotion-related cytokines.

Figure 2.

Figure 2.

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Experimental operation timeline in the present study. HAT, human approach test; KBD, keel bone damage; NOT, novel object test; TI, tonic immobility.

Determination of physiological indicators in blood

Concentrations of corticosterone and serotonin in whole blood of two strains in each keel bone status (n = 10 per bone status) were measured by commercially available enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s instructions (Shanghai Jinma Laboratory Equipment Corporation Ltd., Shanghai, China). Levels of interleukin-1β and interleukin-6 in serum of each group were measured by commercially available ELISA kits (Shanghai Xinle Biotechnology Co., Ltd, Shanghai, China) in accordance with the manufacturer’s instructions. Briefly, 50 μL whole blood or serum of each experimental sample was added into the micropore for determining physiological indicators. A standard curve was obtained with standard solutions of different concentrations of each indicator to quantify the concentrations of experimental samples. The optical density values were measured at a wavelength of 450 nm by a microplate reader (Biotek Instrument Inc., Winooski, VT, USA).

Determination of fear response

Measurement of fear response in two strains of chickens with NK, DK, and FK was performed at 32 and 33 wk of age. Fear measurement included NOT, HAT, and TI test. The specific measured time of each fear test was shown in Figure 2.

Novel object test

At 32 wk of age, the NOT was carried out as described by Fraisse and Cockrem (2006). These tested chickens were brought from their cages and placed individually in a test cage with a size of 50 cm length × 70 cm width × 70 cm height, where chickens could not contact the conspecifics. When the bird was placed in the test cage, a polyvinyl chloride pipe painted with multicolors was used as a novel object and placed into the feed trough. Behavioral responses of the bird during each NOT period (180 s) were recorded using a digital video recorder (Hikvision DS-IT5, Hangzhou, China). Pecking latency per bird was recorded during the experimental period.

Human approach test

A HAT was carried out for each focal animal at 33 wk of age. The bird was placed in the same test cage while an experimenter stood in front of the cage. Once the bird faced and looked at the experimenter, the experimenter started to move his hand toward the bird and into the cage to approach it, recorded the bird’s reaction using a digital video recorder (Hikvision DS-IT5, Hangzhou, China), and subsequently scored by a scoring system between 0 and 3 as described previously (Nelson et al., 2020). Score 0 represented that the bird did not move and the experimenter’s hand could touch it. Score 1 represented that the bird was crouched and immobile; score 2 represented that the bird turned away from the direction of the experimenter’s hand and calmly moved to the side of the cage; score 3 represented that the bird jumped and impetuously tried to escape the experimenter’s approach. A higher score indicated a greater tendency toward active avoidance as a fear response.

TI test

A TI test was performed at 33 wk of age according to the method described by Jones and Faure (1981). In brief, the selected chickens from each keel bone status were taken out from their cages and tested individually in a testing room. The chicken was laid on its back on a U-shaped wooden groove and restrained for 15 s by a tester. The body of the chicken was gently pressed by one hand, and another hand was pressed over the head to induce TI. At the end of 15 s of restraint, the tester slowly removed the hands from the bird’s body. The immobility of the bird indicated successful TI induction, and the TI test duration of the bird kept in the lying position was recorded until it turned over and stood up. If the bird moved or stood up indicated the induction had failed, another TI induction of 15 s was then started, up to a maximum of three inductions. The bird was excluded after three unsuccessful inductions in TI test. The number of inductions and duration of the first successful TI test were recorded. If the bird remained in TI over 600 s, a maximum TI-duration of 600 s was recorded in this study.

Statistical analysis

Statistical analysis was performed using Statistical Package for the Social Science, SPSS ver. 22 (SPSS Inc., Chicago, IL, USA). The differences in the percentage of keel bone status between two chicken strains were analyzed by proportions by Pearson Chi-Square test. The data on egg production was analyzed by a repeated measures analysis under general linear models, and each replicated cage was used as an experimental unit. Serum stress and fear-related physiological indicators and behavioral responses were first tested for normality with the Shapiro–Wilk test. The analysis of multivariate with nested models under general linear models was applied to compare the differences among keel bone status, strain, cage, and their interactions for each physiological and behavioral indicator. The main factors in this statistical model included strain, keel bone status and their interaction, and cage (strain). Cage (strain) represented the cage was nested into the strain. Comparisons among means were performed by the Tukey test. The results were expressed as mean ± standard error of the mean (SEM), and the difference was considered as statistically significant when P ≤ 0.05.

Results

Determination of egg production

The egg production in two strains of laying hens is shown in Figure 3. The effects of strain, time (week of age), and their interaction had a significant influence on egg production of laying hens (P < 0.05). The egg production of laying hens was significantly increased with the age of birds 
(P < 0.05). Additionally, the average laying rates of Hy-line Brown hens and Lindian chickens were 92.1% and 43.5%, respectively, thus the Hy-line Brown laying hens had significantly high egg production compared with that in Lindian chickens throughout the experimental period (P < 0.05).

Figure 3.

Figure 3.

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The result of egg production in two strains of laying hens. Data are expressed as mean ± SD, n = 100 each strain.

Percentage of KBD

As shown in Figure 4, at 32 wk of age, the percentages of NK, DK, and FK bones in Hy-line Brown laying hens were 52.0%, 27.0%, and 21.0%, respectively, and those in Lindian chickens were 44.0%, 22.0%, and 34.0%, respectively. There were no significant differences in the percentages of NK (P = 0.26) and DK (P = 0.41) between Hy-line Brown laying hens and Lindian chickens. The percentage of FK in Lindian chickens was significantly higher than that in Hy-line Brown laying hens (P = 0.04) at 32 wk of age.

Figure 4.

Figure 4.

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Percentage of normal keel, deviated keel, and fractured keel bones in two chicken strains. Asterisks indicate significant differences between the strains at P < 0.05.

Measurement of physiological indicators

The concentrations of whole blood corticosterone and serotonin, and serum interleukin-1β and interleukin-6 in two chicken strains with different keel bone status are shown in Table 1. The interaction of chicken’s strain and keel bone status had significant effects on the concentration of interleukin-1β (P = 0.01). However, no significant effects were observed on the concentrations of corticosterone (P = 0.30), serotonin 
(P = 0.37), and interleukin-6 (P = 0.61).

Table 1.

Levels of stress and emotion-related indicators in blood of two chicken strains with different keel bone status1

Main effect Treatment Corticosterone, ng/L Serotonin, ng/L Interleukin-1β, ng/L Interleukin-6, ng/L
Strain
Hy-line Brown 479.66 459.47y 173.42 48.29x
Lindian 487.21 482.08x 176.06 46.16y
SEM 3.98 4.70 1.46 0.52
Keel bone status
NK 465.24b 483.39a 169.25b 45.08b
DK 477.58b 472.01ab 171.18b 46.55b
FK 507.50a 456.93b 183.81a 50.06a
SEM 4.88 5.75 1.79 0.64
Cage (Strain)
CH1 481.38 447.56 166.91 49.77
CH2 483.46 422.95 167.91 49.82
CH3 420.45 476.62 189.35 49.37
CH4 425.42 474.43 184.16 48.25
CH5 511.14 471.97 164.23 49.86
CH6 498.31 455.59 167.34 49.00
CH7 514.74 447.25 173.32 46.23
CH8 504.61 480.40 174.31 44.52
CH9 466.93 450.56 173.32 48.20
CH10 490.13 467.38 173.39 47.91
CL1 494.82 469.83 179.80 46.16
CL2 479.40 484.71 179.08 44.98
CL3 476.07 492.76 167.34 45.64
CL4 471.42 490.58 169.34 46.23
CL5 504.36 485.08 182.52 47.40
CL6 497.31 482.90 183.51 47.83
CL7 488.61 497.61 176.93 45.29
CL8 486.38 491.88 171.90 46.53
CL9 480.74 459.27 174.63 43.99
CL10 492.98 466.18 175.58 47.56
SEM 12.61 14.85 4.61 1.64
P-value
Strain 0.191 0.002 0.262 0.006
Keel bone status <0.001 0.009 <0.001 <0.001
Cage (Strain) 0.001 0.392 0.062 0.646
Strain × keel bone status 0.301 0.370 0.005 0.606
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NK, normal keel bone; DK, deviated keel bone; FK, fractured keel bone; CH, cage for Hy-line Brown laying hens; CL, cage for Lindian chickens.

Means between keel bone status in the same column with different superscripts are significantly different at P < 0.05. Means with same or no superscripts represent no significant differences at P > 0.05.

Means between strains in the same column with different superscripts are significantly different at P < 0.05.

As shown in Table 1, the concentration of whole blood corticosterone was affected by keel bone status (P < 0.01) but not by strain (P = 0.19), and FK birds had an elevated corticosterone concentration compared with NK and DK birds. The concentration of whole blood serotonin was affected by both strains (P < 0.01) and keel bone status (P < 0.01). 
The serotonin concentration significantly increased in Lindian chickens compared with Hy-line Brown hens 
(P < 0.01), and the serotonin concentration in NK birds was significantly higher than that in FK birds (P < 0.05). The concentration of serum interleukin-1β in FK birds was significantly increased compared with NK and DK birds 
(P < 0.01), but no significant difference was found between strains (P = 0.28). Furthermore, the concentration of serum interleukin-6 was affected by both strains (P < 0.01) and keel bone status (P < 0.01). The interleukin-6 concentration in Hy-line Brown hens was higher than that in Lindian chickens (P = 0.01). The interleukin-6 concentration in FK birds was higher than that in NK and DK birds (P < 0.01). Additionally, the cage had a significant effect on the concentration of whole blood corticosterone (P < 0.05) but had no effects on the concentrations of whole blood serotonin and serum interleukin-1β and interleukin-6 in laying hens (P > 0.05).

Measurement of fear response

The results of fear responses of chickens to different tests are shown in Table 2. The interaction between chicken strain and keel bone status had no significant effect on the NOT-latency (P = 0.06), HAT-score (P = 0.72), TI-duration (P = 0.19), and the number of TI inductions (P = 0.90).

Table 2.

Behavioral responses of laying hens with different keel bone status to fear tests1

Main effect Treatment NOT-latency, s HAT-score, 0–3 TI-duration, s TI-induction, number
Strain
Hy-line Brown 73.73y 0.93y 340.77x 1.13y
Lindian 170.37x 2.10x 227.27y 1.53x
SEM 2.22 0.16 13.95 0.08
Keel bone status
NK 116.85b 1.25 238.80b 1.40
DK 118.55b 1.85 251.70b 1.25
FK 130.75a 1.45 361.55a 1.35
SEM 2.72 0.19 17.09 0.08
Cage (Strain)
CH1 82.00 0.33 291.00 1.00
CH2 70.67 1.00 399.00 1.00
CH3 71.67 1.00 337.67 1.00
CH4 73.00 1.00 286.67 1.00
CH5 61.67 1.00 348.66 1.33
CH6 83.00 1.00 289.00 1.00
CH7 65.00 1.00 353.00 1.33
CH8 74.00 1.33 384.33 1.00
CH9 66.33 1.00 393.67 1.00
CH10 90.00 0.67 324.67 1.67
CL1 173.33 3.00 156.33 2.00
CL2 173.33 2.00 232.33 1.00
CL3 171.33 2.67 203.67 1.67
CL4 174.00 2.67 264.33 1.33
CL5 180.00 1.33 294.33 1.67
CL6 159.00 1.67 210.33 1.33
CL7 173.33 2.33 227.33 1.33
CL8 162.67 1.67 208.00 2.00
CL9 172.33 2.00 227.67 2.00
CL10 164.33 1.67 248.33 1.00
SEM 7.03 0.13 13.99 0.07
P-value
Strain <0.001 <0.001 <0.001 0.030
Keel bone status 0.002 0.108 <0.001 0.782
Cage (Strain) 0.308 0.791 0.651 0.827
Strain × keel bone status 0.055 0.715 0.190 0.900
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NK, normal keel bone; DK, deviated keel bone; FK, fractured keel bone; CH, cage for Hy-line Brown laying hens; CL, cage for Lindian chickens; HAT, human approach test; NOT, novel object test; TI, tonic immobility.

Means between keel bone status in the same column with different superscripts are significantly different at P < 0.05. Means with same or no superscripts represent no significant differences at P > 0.05.

Means between strains in the same column with different superscripts are significantly different at P < 0.05.

The latency of the NOT was affected by both strains (P < 0.01) and keel bone status (P < 0.01), and Lindian chickens had significantly higher NOT-latency than Hy-line Brown hens (P < 0.01). The latency of the NOT in FK chickens was elevated compared with NK and DK chickens (P < 0.01). The score of the HAT was affected by strain (P < 0.01) but not by keel bone status (P = 0.11). Lindian chickens had a significantly higher score in HAT compared with Hy-line Brown hens (P < 0.01). Besides, the TI duration was significantly affected by both strains (P < 0.01) and keel bone status (P < 0.01). The duration of TI in Hy-line Brown hens was significantly longer than that in Lindian chickens (P < 0.01), and TI duration in FK birds was significantly increased compared with NK and DK birds (P < 0.01). The number of TI inductions was affected by strain (P < 0.05) but not by keel bone status (P = 0.78), and Lindian chickens had a significantly increased number of TI inductions when compared with Hy-line Brown laying hens (P < 0.05). Additionally, the cage had no significant effects on NOT-latency, HAT-score, TI-duration, and the number of TI inductions in laying hens (P > 0.05).

Discussion

Keel bone fractures negatively influence welfare, health, behavior, egg production, and egg quality in all laying hen housing systems (Nasr et al., 2012b; Riber et al., 2018). The percentage of KBD between chicken strains was evaluated, and the effects of KBD on fear and stress levels were investigated during the laying period. Previous studies found that the prevalence of KBD was different between white and brown commercial laying hens and within noncommercial chickens (Eusemann et al., 2018; Kittelsen et al., 2020), suggesting that KBD was influenced by strain. The strain difference in KBD was also found in the present study, which showed that the incidences of keel bone fractures in Lindian chickens were higher than that in Hy-line Brown laying hens at 32 wk of age. Thøfner et al. (2021) reported that the heavier hens had fewer keel bone fractures that were similar to the findings of this study. In this study, the average body weight of Lindian chickens (1.58 ± 0.03 kg) was lighter than Hy-line Brown laying hens (2.05 ± 0.02 kg) throughout the experimental period, but Lindian chickens had more keel bone fractures than Hy-line Brown laying hens, indicating body weight affects keel bone health. Additionally, studies also found that laying hens with the younger age at the onset of lay and with high egg production had a higher incidence of keel bone fractures (Eusemann et al., 2018; Thøfner et al., 2021). However, in this study, the older age (Lindian: 21.4 ± 0.3 wk, Hy-line Brown: 18.2 ± 0.2 wk) at the onset of lay, the lower egg production (Lindian: 44.1 ± 1.12%, Hy-line Brown: 92.4 ± 0.31%), and the higher keel bone fractures were found in Lindian chickens when compared with Hy-line Brown laying hens, which was inconsistent with the findings of Eusemann et al. (2018) and Thøfner et al. (2021). Thus, a possible reason for the high occurrence of fractures in Lindian chickens is that this strain has an excitable personality with more locomotory and jumping activities between perches, which can increase the risk of KBD due to collisions.

Studies on laying hens found that keel bone fractures induced pain (Nasr et al., 2012a), stress, and inflammation (Wei et al., 2019). A recent study reported that keel bone fractures caused a depression-like state in laying hens (Armstrong et al., 2020). These findings indicated that keel bone fractures might affect the layer’s physiology and emotional state. Emotional states, such as fear and anxiety, can be reflected via some physiological and behavioral parameters (Désiré et al., 2006). For instance, the levels of corticosterone, serotonin, and interleukin-6 and behavioral reaction to NOT, HAT, and TI test can be used as predictive indicators to assess stress and fear in animals (de Haas et al., 2012; Kozak et al., 2019). We measured several physiological indicators (corticosterone, serotonin, interleukin-1β, and interleukin-6) and did behavioral tests (TI, NOT, and HAT) in relation to stress and fear to evaluate the effects of KBD on emotion and welfare in two chicken strains.

At the physiological level, corticosterone is a common indicator used to measure endocrine response to stress in the activated HPA axis, and an elevated level of blood corticosterone represents the stress response of the organism (Post et al., 2003). Our previous study showed that keel bone fractures increased the concentration of serum corticosterone in caged laying hens (Wei et al., 2019). Similarly, this study found that KBD had a significant impact on whole blood corticosterone content, and FK hens elevated blood corticosterone content compared with NK and DK hens, indicating that keel bone fractures could induce stress. Besides, this study showed that the concentration of whole blood corticosterone was different among cages, suggesting the cage effect also had a significant impact on corticosterone in laying hens. Under stress and stimulation conditions, the sympathetic–adrenal–medullary axis (a neurohormonal system) regulates the physiological homeostasis in humans and animals. In chickens, the functional changes in the sympathetic–adrenal–medullary axis caused by stress are often accompanied by an unbalanced serotonin level (Bolhuis et al., 2009; Cheng and Fahey, 2009; van der Eijk et al., 2019). Blood serotonin content is related to the degree of fearfulness and stress in laying hens and broilers, and the birds with lower fearfulness and stress had a higher blood serotonin content (Metzger et al., 2002; Uitdehaag et al., 2011; van der Eijk et al., 2019). Thus, serotonin plays an important role in regulating fear and stress responses. Our results showed that FK hens decreased blood serotonin content compared with NK hens, suggesting that FK hens showed high fear responses than NK hens. It was reported that serotonin also plays an essential role in bone remodeling and a low level of serotonin can cause bone mass loss (Yadav et al., 2009), increasing the risk of bone fractures. Thus, keel bone fractures could be induced by low serotonin level in laying hens. Besides, this study found that the content of blood serotonin in Lindian chickens was higher than that in Hy-line Brown hens, which could be associated with more feather pecking by severe feather loss in Lindian chickens. According to a previous study, the hens with a high level of serotonin are more likely to show severe feather pecking (van der Eijk et al., 2019). However, laying hens are likely to exhibit more chasing, escaping, and aggressive behaviors during feather pecking, and these behaviors can increase the risk of collisions, resulting in a higher incidence of keel bone fractures. Proinflammatory cytokines, such as interleukin-1β and interleukin-6, are involved in the reaction of emotions, such as anxiety and fear in humans and animals, and positive emotion is related to decreased levels of proinflammatory cytokines (Anisman et al., 2008; Song and Wang, 2011). This study showed that the concentrations of cytokines (interleukin-1β and interleukin-6) in the serum of FK hens were significantly higher than those in NK and DK hens, indicating that keel bone fractures could induce a negative emotion in laying hens. Besides, the interaction of strain and keel bone status had a significant effect on the concentration of interleukin-1β in serum, suggesting that the emotion of laying hens was not solely influenced by keel bone status, but also by the interaction of strain and keel bone status.

Fear is an emotional response of animals to danger or threat, which stimulates the occurrence of some adaptive behaviors, such as active escape or defense (Steimer, 2002), while a persistent or intense fear response leads to injury, stress, pain, or even the death of birds (Jones, 1996). Therefore, fear is recognized as a negative emotion and welfare state in animals. In poultry, TI is a common test for assessing fear. The experimenter simulates a predator to induce an antipredator response. The prey feigns death and escapes when the predator relaxes its vigilance (Forkman et al., 2007). The TI test has good validity, and the TI duration represents the level of fear. Besides, NOT and HAT are applied to measure fear of hens in cages. In the NOT, a polyvinyl chloride pipe painted with multicolors is generally placed in the front of the cage, and the behavioral reaction of the hen is observed. In brief, HAT assessed the fear of a test animal by observing its behavior when humans gradually approach it (Nelson et al., 2020). Fear response of birds has active and passive behavioral performances that are assessed by these tests. In chickens, active behavior responses include struggling in the physical restraint test (Nelson et al., 2020), distress vocalization in the isolation test (Yoshidome et al., 2021) and escaping or avoidance in HAT and novel object stimulation test (Jones, 1996; Graml et al., 2008). Passive fear behaviors include immobility as freezing or crouching in TI test (Jones, 1996; Forkman et al., 2007). A longer TI-duration and NOT-latency for fear tests indicate a higher fear level in poultry. The present study showed that the fear response of chickens to NOT and TI tests was affected by bird strain and keel bone status.

The effects of keel bone status on fear response in chickens showed that FK hens significantly increased TI-duration and NOT-latency compared with NK and DK hens, while these indicators were not significantly different between NK and DK hens. This result indicated that keel bone fractures caused a fear response reflected by NOT and TI test in laying hens. From the results of strain, we found that TI-duration was increased but NOT-latency and HAT-score were decreased in Hy-line Brown laying hens in comparison to Lindian chickens, indicating that strain style affected behavioral responses of laying hens to fear tests. In the present study, the results from the TI test indicated that Hy-line Brown hens with a longer TI duration had a higher fearfulness than Lindian chickens, but the results of NOT showed that Lindian chickens had a higher fearfulness than Hy-line Brown hens. On the one hand, these inconsistent results from the TI test and other tests, such as HAT and NOT, in Hy-line Brown hens and Lindian chickens could be associated with the emotional excitability of poultry. Some studies found that excited strains exhibited high mobility and active behavioral activities (Rozempolska-Rucińska et al., 2020), and the TI test was difficult to achieve for them (Kozak et al., 2019). Behavioral performance of birds to TI test is a passive fear response, while behavioral performance to HAT and NOT is an active fear response. In this study, Lindian chickens showed a high active response to NOT and HAT and a low passive response to TI test, as well as an increased number of TI inductions, which suggests that Lindian chickens are excited. Thus, a shorter TI duration is possibly found for Lindian chickens. But it does not mean that Lindian chickens have a lower fear level because the excited birds usually have higher stress levels (Rozempolska-Rucińska et al., 2020). Otherwise, previous studies showed that fear of hens was associated with egg production, and hens with relatively higher fear and stress responses had lower production performance (de Haas et al., 2013; Rozempolska-Rucińska et al., 2017). We found that Lindian chickens had a lower laying rate (approximately 44%) than commercial hens (approximately 92%) during the experiment period. Thus, Lindian chickens showed a high fear response related to lower egg production, as these were not selected for decades for egg production. Therefore, the results of behavioral responses of two strains of laying hens to fear tests may indicate that Lindian chickens can perform an active behavioral response to fear, but commercial Hy-line Brown hens tended to perform passive fear behaviors. Overall, our results indicated that the behavioral response of chickens in the fear test varied from strain to strain. However, in addition to strain and keel bone status individually affected behavior responses of laying hens to fear tests, the interaction of strain and keel bone status had a very nearly significant effect on the latency of birds to approach a novel object in this study, indicating that certain fear responses could be influenced by this interaction.

Additionally, although this study suggested that keel bone fractures could lead to stress and fear responses in two strains of laying hens, this study had some limitations, such as studied hens with the younger age, a shorter experimental period of 7 wk, and the lack of the analysis of bone mineralization. Besides, more importantly, the present study also lacked a detailed description of bone damage, including the severity of keel bone deviation and the style, number and location of keel bone fractures, as these factors may closely affect the behavioral performances of laying hens. Therefore, further study regarding this should be considered for these limitations to strengthen their impacts on KBD and associated behaviors in laying hens.

Conclusions

This study found that the incidence of keel bone fractures in Lindian chickens was higher than that in Hy-line Brown hens. Laying hens with FK bones had significantly elevated whole blood corticosterone, serum interleukin-1β, and interleukin-6 levels, a longer TI-duration and NOT-latency, and a reduced whole blood serotonin content in comparison to hens with NK and DK bones, suggesting that keel bone fractures could lead to stress and fear and damage emotion and welfare state of the hen. Our results also showed that Lindian chickens had a higher whole blood serotonin content, NOT-latency, HAT-score, and TI induction times and decreased egg production, serum interleukin-6 content, and TI-duration compared with Hy-line Brown hens, indicating that the strain of laying hens had a significant effect on physiological and behavioral responses of hens to stress and fear.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (grant number 31972608).

Glossary

Abbreviations

DK

deviated keel bone

ELISA

enzyme-linked immunosorbent assay

FK

fractured keel bone

HAT

human approach test

HPA

hypothalamic–pituitary–adrenal

KBD

keel bone damage

NK

normal keel bone

NOT

novel object test

TI

tonic immobility

Conflict of interest statement

The authors confirm that there are no conflicts of interest.

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