Assessment of the Effects of Stocking Density on Laying Hens Raised in Colony Cages: Part I—The Effect of Density, Time of Day and Hen Age on Behavior and Aggression

Abstract

The amount of space provided to laying hens has been an animal welfare topic of concern from consumers, special interest groups, and lawmakers. The freedom to perform normal behaviors is one component of animal welfare, and, therefore, the objective of this study was to assess behavior at different stocking densities. Shaver White laying hens were housed in colony cages at five stocking densities. Video recordings of three cages/treatment were taken at 30, 46, and 62 weeks of age in the morning, afternoon, and night. At each age, a 20 min segment of video from each time of day was analyzed. Every minute was annotated to identify the behavior that hens spent the majority of their time performing. Data were averaged to calculate the percentage of hens engaged in each behavior per cage. The total count of aggressive bouts was recorded across the 20 min period. Data were analyzed with a general linear model utilizing the variables time of day, density, age, and the full factorial of interactions. Intermediate stocking densities displayed the lowest percentage of crouching (p = 0.009) and aggressive acts per hen (p < 0.0001). Hens stocked at 1341.93 cm² walked the most (p < 0.0001). Hens were seen standing and preening (p < 0.0001) more in the morning, crouching and sham dust bathing (p < 0.0001) more in the afternoon, stretching less while feeding and drinking, and being more aggressive at night (p < 0.0001). Finally, as hens aged, they began to stand and crouch more (p < 0.0001) and preen (p = 0.013), walk (p < 0.0001), and demonstrate aggressive behaviors (p = 0.007) less. In conclusion, the majority of behaviors assessed were not influenced by stocking density. However, the different amounts of aggression between the treatments could indicate higher stress and frustration, which warrants more investigation.

1. Introduction

Over the past 20 or so years, the demand for more welfare-centered practices in animal agriculture has grown, specifically moving from intensive systems and practices to more extensive systems and practices. This shift in demand has largely been driven by animal rights special interest groups influencing both lawmaker and consumer perceptions, which can be seen in laws and trends surrounding the commercial egg layer market [1,2,3]. The success of these groups is largely attributed to consumer’s lack of knowledge, as many believe that moving towards more extensive practices, such as cage-free environments and lower stocking densities, will improve animal welfare [4]. This trend, however, has been slowed by consumers low willingness to pay for these changes, resulting in most of the changes being driven at the legislative level [2,5,6]. Regardless of the reasons behind the market shift, there is a belief among some consumers and groups that current production systems and practices lead to poor welfare parameters in animal agriculture.

While advancements in methods assessing hen welfare have become more developed over the past several years, accurately determining welfare is still rather difficult. One of the standards of welfare that management practices and environments are measured by, the five freedoms, is still somewhat subjective. As the hens (or other animals, for that matter) cannot directly tell researchers how they are feeling, subjective analyses, such as evaluating the five freedoms, are the best tools to use in some cases. The five freedoms, developed by the UKs animal welfare council, are freedom from hunger and thirst, freedom from discomfort, freedom from pain, injury, and disease, freedom to express normal behavior, and freedom from fear and distress [7,8]. While some of these freedoms are easy to meet and to address, such as providing food, water, shelter, and a clean environment, as well as practicing proper biosecurity, it is difficult to define others, as they are quite complex. Examples of these freedoms are the freedom to express normal behavior and freedom from fear and distress, which denote more complex conditions and have not been specifically defined [9]. Regardless, these five freedoms are considered to be the general guidelines by which welfare is assessed [9,10,11,12].

To assess welfare, researchers have several tools available to them. One way is through analysis of behavioral profiles. Analyzing behaviors can give researchers insight into normal behavior practices and also analyze if the animals are free from distress and pain. Determining a model for natural behaviors can be difficult, as it is known that genetic selection and breed differences can affect behaviors of laying hens, leading to some strains better suited to extensive practices and some better suited to intensive practices [13,14]. For example, using the Red Asian Jungle Fowl, the precursor species to the modern chicken, as a model of natural behaviors would not be applicable as the modern chicken is much different from its ancestor [15]. Researchers have elucidated that natural responses to the environment by a wild animal are responses to adversity not found in captive environments [16,17]. Furthermore, other researchers conclude that an absence of wild behaviors in captivity does not indicate poor welfare or a lack of normal behavior expression [18]. While assessing natural behaviors may not be possible due to a lack of consistent and applicable models, comparisons of behavior between groups can indicate changes in stress, frustration, and aggression. For example, comfort stretching, pecking, foraging, and dust bathing behaviors can all be considered natural behaviors which can be used to compare different groups. Furthermore, aggression can often be caused by frustration and boredom, which can indicate a poorer mental state of the animal [15].

The objectives of this research were to assess how stocking density, hen age, and time of day can affect behavior patterns as well as aggressiveness. Several studies have evaluated how stocking density affects behavioral trends. However, many of these studies were performed in conventional cages. This study aims to evaluate the aforementioned variables utilizing higher bird numbers and colony cages. Furthermore, in a USDA research bulletin, Rexroad et al. [19] indicated the importance of performing similar studies, as new genetic strains can behave differently than their counterparts have in the past. While the current UEP stocking density guidelines state a minimum of 67 in² (432.3 cm²) per bird in any cage environment, this study aims to determine if hens can benefit from more space [20].

When evaluating past behavior studies, Anderson et al. [21] found no difference in appetitive behaviors or aggression by density; however, hens performed more movement behaviors in lower stocking densities. Anderson et al. [21] also found that younger hens were more active, although no difference was found with aggressive behaviors. Zimmerman et al. [22] determined that while stocking density did not affect welfare, incidents of aggressive behaviors increased as stocking density increased and hens in the highest stocking density aged; aggression in that density also increased. It has also been seen that lower feeder space per hen can cause higher amounts of aggressive behaviors as birds fight for a place at the feeder [23]. Unfortunately, research determining how time of day affects hen behavior is lacking. We hypothesize that hens will feed more in the morning and night and, therefore, also show more aggressive patterns during these times. Informed by past research, we also hypothesize that the higher stocking densities will result in higher instances of aggressive behaviors and lower movement activity. Finally, we hypothesize that older hens will be less active than younger hens, and, therefore, older hens will demonstrate less aggressive behaviors.

2. Materials and Methods

2.1. Hen Housing and Care

This study took place at the North Carolina Department of Agriculture Piedmont Research Station in Salisbury, NC, USA. The first recording took place in February 2021 and the last recording was taken in September 2021. The study was approved by the North Carolina State University Institutional Animal Care and Use Committee. The hens were kept in a tri-decked layer colony cage system that measured 66.04 cm × 121.92 cm per cage (Ford Dickinson Inc., Mitchell, Ontario, ON, Canada). The hens were provided ad libitum access to feed and water. Each cage contained 4 nipple drinkers, and the feeder measured 121.92 cm long. No enrichments were provided, and the diet is shown in

Table 1. Ingredient composition and calculated nutrient analysis for the diet fed to all hens.

Ingredient	Diet (%)
Corn	51.84
Soybean Meal	32.24
Calcium Carbonate	9.42
Dicalcium Phosphate	1.81
Salt	0.38
DL-Methionine	0.18
Soybean Oil	3.73
Santoquin	0.05
Choline Chloride	0.05
NCSU Trace Mineral Premix	0.20
NCSU Vitamin Premix	0.05
NCSU Selenium Premix	0.05
Calculated Values
Crude Protein %	19.50
Metabolizable Energy kcal/kg	1328.00
Calcium %	4.14
Available Phosphorus %	0.45
Total Lysine %	1.10
Total Sulfur Amino Acids %	0.825

. The hens were provided with 16 h of light, starting at 7 am and ending at 11 pm, and 8 h of darkness each day. Before the study, the hens were beak-treated at 6 days of age with a hot knife. One hundred and eighty Shaver White hens were randomly assigned to 1 of 3 replicates of 5 density treatments (Hendrix-Genetics, CK Boxmeer, The Netherlands). The treatments were as follows: 1341.93 cm² per hen (6 hens per cage), 896.77 cm² per hen (9 hens per cage), 670.97 cm² per hen (12 hens per cage), 535.48 cm² per hen (15 hens per cage), and 445.16 cm² per hen (18 hens per cage). A total of 15 cages were evaluated. The chicks were reared in a cage brooding system and moved to the lay house at 16 weeks of age (November 2020).

2.2. Hen Behavior Collection

Hen behavior was recorded on Zosi 1080p cameras and saved for analysis later (Zosi Technology, Shiqi, Guangdon, China). Recordings took place at 30, 46, and 62 weeks of age. The hens were recorded at 3 different time points in the day, for 1 h each, at 7:30 am–8:30 am, 2:30 pm–3:30 pm, and 9:30 pm–10:30 pm. Within each hour-long recording, one uninterrupted, 20 min chunk was chosen for analysis by a trained annotator. Similarly to Anderson et al. [21], 2 categories of behaviors were analyzed: acts independent of conspecific (AC) and aggressive behaviors [21]. Each 20 min video was split into 20 equal 1 min sections. The AC behaviors used were as follows: standing, crouching, foraging, sham dust bathing, preening, walking, stretching (comfort movement less preening), non-aggressive feather pecking, feeding, drinking, pecking inedible objects, and egg laying. Aggressive behaviors included aggressive pecking, standing upon another hen, and pecking a neighboring hen.

Table 2. Description of behaviors measured using a modified version of the definitions in Hurnik et al.’s ‘Dictionary of Farm Animal Behaviors’ [24].

Behavior	Description	Category
Feeding	Voluntary investment of solids and semi-solids	Appetitive
Drinking	Voluntary oral ingestion of liquids	Appetitive
Peck inedible objects	Making contact with an inedible object with the tip of the beak with a quick forward motion of the head	Appetitive
Sham forage	Behavior used in search of feed, typically scratching at the ground	Appetitive
Standing	To assume and maintain an upright position on extended legs	Movement and Comfort
Preening	An act of integumentary care in birds similar in function to grooming in mammals. Preening is manifested as manipulation of feathers and distribution of secretions from the uropygial gland (pineal gland) using the beak.	Movement and Comfort
Crouching	Lowering body by bending legs	Movement and Comfort
Moving	The activity of an organism moving itself between two points in a space.	Movement and Comfort
Stretching	Muscular activity characterized by brief, forceful extension of limbs or another part of the body	Movement and Comfort
Feather pecking (gentle)	Pecking of the plumage of another bird that does not illicit a response or is in a non-aggressive way	Movement and Comfort
Sham dust bathe	Dust-bathing activity performed without the presence of litter material.	Movement and Comfort
Aggressive pecking	Any pecking of another hen (within the same cage) with the actual or potential result of harming, limiting, or depriving it. Includes injurious feather pecking.	Aggression
Submissive response	Acceptance of the dominance of another hen, usually by assuming a crouching, head down position when aggressed	Aggression
Avoidance and escape	Neutralization of an aggressive hen by means of escape	Aggression
Standing upon hen	Perching on top of or placing full body weight on the top of another hen	Aggression
Peck neighbor	Aggressive pecking on a hen in a neighboring cage, typically at the feeder or through the cage wire.	Aggression

displays the specific definitions of these behaviors according to the Dictionary of Farm Animal Behavior [24]. During each 1 min section, an AC behavior was recorded for each hen. This AC behavior was the behavior that the hen spent the most time performing. All aggressive behaviors were recorded independent of the AC behaviors.

2.3. Statistical Analysis

Statistical analysis and data preparation were performed utilizing Rstudio 4.2.1 with the packages tidyverse, lubridate, agricolae, AICcmodave, and janitor [25,26,27,28,29]. the statistical unit was considered to be each cage per time of day per age, and, therefore, all AC data was averaged over the 20 min period for the percent of birds performing each act, and all aggression data was summed for total aggressive behaviors per 20 min and total aggressive behaviors per hen per 20 min. A general liner model was used, and Tukeys HSD was performed for multiple comparisons, while statistical significance was considered if a p-value was <0.05. The statistical model used was the full factorial of age, density treatment, and time of day, including interactions. Data were tested for normalcy prior to analysis and found to meet the assumption of normalcy. Finally, no outliers were excluded from the analysis.

3. Results

3.1. Appetitive Behaviors

Density was only found to affect inedible object-pecking behaviors (

Table 3. The effect of stocking density, time of day, and age on percent time engaged in appetitive behaviors per minute.

	Feeding	Drinking *	Pecking Inedible Objects *	Sham Forage
Density (cm 2)
1341.93	42.00	5.88	14.17 AB	0.20
896.77	41.47	6.94	15.50 A	0.26
670.97	41.69	7.15	13.23 AB	0.18
535.48	42.93	7.10	14.30 AB	0.07
445.16	41.51	7.09	12.16 B	0.07
Pooled SEM	1.82	0.49	2.10	0.06
p-value	0.947	0.304	0.037	0.151
Time of day
Morning	34.37 C	6.47 AB	6.74 B	0.18
Afternoon	41.11 B	6.33 B	6.74 B	0.12
Night	50.28 A	7.69 A	27.89 A	0.18
Pooled SEM	1.06	0.37	0.63	0.05
p-Value	<0.001	0.024	<0.001	0.526
Age (weeks)
30	41.07	7.01	14.08	0.06 B
46	42.90	6.31	14.75	0.27 A
62	41.79	7.17	12.78	0.14 AB
Pooled SEM	1.44	0.38	1.62	0.05
p-Value	0.497	0.233	0.065	0.007

^A,B,C: Mean values within a column with different letter superscripts are significantly different (p < 0.05). * The interaction between period and time of day was significant (p < 0.05).

). Hens housed under a density of 896.77 cm² per hen were found to participate in sham forage behaviors more often than hens under a density of 445.16 cm² by 3.34% (p = 0.037). Time of day was found to affect feeding, drinking, and pecking inedible objects (Table 3). The hens were found to spend more time feeding at night than in the afternoon by 9.17% and than in the morning by 15.37%, while the hens also spent more time feeding in the afternoon than in the morning by 6.74% (p < 0.001). The hens were also more likely to engage in drinking behaviors at night compared to the afternoon by 1.36% (p = 0.024). Furthermore, the hens were more likely to be found pecking inedible objects at night compared to in the afternoon and morning by 21.15% for both (p < 0.001). Time of day was not found to affect sham forage behaviors. Finally, age was only found to affect sham forage behaviors. On average, 0.22% fewer hens participated in sham foraging behavior at 30 weeks of age than at 46 weeks of age (p = 0.007).

3.2. Movement and Comfort Behaviors

Density treatment was found to influence the percentage of hens crouching, moving, stretching, feather pecking, and sham dust bathing (

Table 4. The effect of stocking density, time of day, and age on percent time engaged in movements and comfort movements per minute.

	Standing *	Preening *	Crouching	Moving **	Stretching †	Gentle Feather Pecking *	Sham Dust Bathing *
Density (cm 2)
1341.93	4.56	15.71	8.37 AB	8.09 A	0.38 A	0.21 B	0.40 AB
896.77	5.27	16.23	7.65 B	4.95 B	0.21 AB	0.18 AB	0.81 A
670.97	5.72	17.73	9.99 AB	2.92 B	0.17 AB	0.69 AB	0.43 AB
535.48	4.90	18.50	7.65 B	2.83 B	0.09 B	1.21 A	0.32 AB
445.16	6.06	16.52	11.74 A	3.01 B	0.08 B	0.71 AB	0.24 B
Pooled SEM	1.04	2.64	1.63	0.78	0.06	0.16	0.16
p-Value	0.402	0.572	0.009	<0.001	0.002	0.004	0.040
Time of day
Morning	10.32 A	31.50 A	5.39 B	4.22	0.25 A	0.45	0.15 B
Afternoon	3.95 B	16.20 B	18.32 A	4.84	0.30 A	0.83	0.83 A
Night	1.65 C	3.09 C	3.51 B	4.02	0.01 B	0.83	0.36 B
Pooled SEM	0.51	0.98	0.81	0.72	0.04	0.13	0.12
p-Value	<0.001	<0.001	<0.001	0.549	<0.001	0.052	<0.001
Age (weeks)
30	3.45 B	18.91 A	6.30 B	7.29 A	0.24	0.67	0.52
46	4.26 B	17.33 AB	9.39 A	3.78 B	0.12	0.54	0.45
62	8.21 A	14.56 B	2.01 B	2.01 B	0.20	0.89	0.35
Pooled SEM	0.71	2.01	0.57	0.57	0.05	0.13	0.13
p-Value	<0.001	0.014	<0.001	<0.001	0.151	0.141	0.493

^A,B,C: Mean values within a column with different letter superscripts are significantly different (p < 0.05). * The interaction between period and time of day was significant (p < 0.05). ** The interaction between period and treatment was significant (p < 0.05). ^† The interaction between time of day and treatment was significant (p < 0.05).

). Hens housed at a density of 445.16 cm² per hen were found to crouch more than hens housed at 535.48 cm² and 896.77 cm² per hen by 4.09% for both (p = 0.009). Hens housed at a density of 1341.93 cm² were found to move more than all other treatments by a range of 3.47–5.26%. Hens in the 1341.93 cm² density treatment were found to partake in stretching more than hens in the 535.48 cm² and 445.16 cm² densities by 0.39% and 0.30%, respectively (p = 0.002). Hens in the 1341.93 cm² density were also found to perform more gentle feather pecking behaviors than hens in the 535.48 cm² treatment by 0.91% (p = 0.004). Finally, hens housed in the 445.16 cm² treatment were found to sham dust bathe less than hens raised in the 896.77 cm² density by 0.56%.

Time of day was found to affect the percentage of hens performing standing, crouching, preening, stretching, and sham dust bathing behaviors, while time of day did not affect the percentage of hens performing moving and gentle feather-pecking behaviors. The hens were found to stand more in the morning than in the afternoon by 6.37% and than at night by 8.67%, while the hens were also found to stand more in the afternoon than at night by 5.60% (p < 0.001). The hens were also found to preen more in the morning compared to the afternoon by 15.30% and at night by 28.41%, while the hens preened more in the afternoon than at night by 19.29% (p < 0.001). Furthermore, the hens were found to crouch more in the afternoon than in the morning and night by 12.93% and 14.81%, respectively (p < 0.001). The hens also stretched less at night compared with the morning and afternoon by 0.24% and 0.29%, respectively (p < 0.001).

More hens were found performing sham dust bathing behaviors in the afternoon than in the morning and night by 0.68% and 0.47%, respectively (p < 0.001). Age was found to affect the number of hens standing, preening, crouching, and moving, while age did not affect stretching, gentle feather pecking, and sham dust bathing. Overall, 62-week-old hens were found standing more often than 40-week-old hens by 3.95% and 32-week-old hens by 4.76% (p < 0.001). The 62-week-old hens were also found to preen less than 32-week-old hens by 4.35% (p = 0.014). Younger hens (32 weeks old) were found to crouch more than 40- and 62-week-old hens by 3.09% and 5.22%, respectively (p < 0.001). Finally, the hens were found to move more early in life, as the 30-week-old hens were found to move more than 40-week-old hens by 3.51% and more than 62-week-old hens by 5.28%, respectively.

3.3. Aggression Behaviors

Density treatment affected aggressive pecking, submissive response, acts of standing on another hen, acts of pecking neighboring hen, total aggressive acts, and aggressive acts per hen (p = 0.007) (

Table 5. The effect of stocking density, time of day, and hen age on aggressive behaviors per hen over 20 min.

	Aggressive Pecking *,†	Submissive Response	Avoidance and Escape	Standing on Hen *	Pecking Neighbor	TotalAggressive Behaviors per 20 min †	Aggressive Behaviors per Hen per 20 min
Density (cm 2)
1341.93	0.83 BC	0.74 BC	0.08	0.08 B	0.17 A	6.44 D	1.09 ABC
896.77	0.67 C	0.57 C	0.08	0.01 B	0.04 B	6.37 D	0.72 C
670.97	0.90 ABC	0.82 ABC	0.08	0.07 B	0.03 B	11.70 C	1.00 BC
535.48	1.65 AB	1.07 AB	0.10	0.09 B	0.04 B	19.33 B	1.30 AB
445.16	1.95 A	1.08 A	0.10	0.26 A	0.02 B	25.81 A	1.47 A
Pooled SEM	0.11	0.11	0.02	0.03	0.02	1.48	0.13
p-Value	<0.001	<0.001	0.877	<0.001	<0.001	<0.001	<0.001
Time of day
Morning	0.64 C	0.52 C	0.11	0.10	0.04 B	9.73 B	0.77 C
Afternoon	0.89 B	0.81 B	0.08	0.08	0.04 B	12.69 B	1.07 B
Night	1.33 A	1.24 A	0.08	0.14	0.10 A	19.38 A	1.51 A
Pooled SEM	0.08	0.07	0.02	0.03	0.02	1.52	0.09
p-Value	<0.001	<0.001	0.482	0.172	0.015	<0.001	<0.001
Age (weeks)
30	0.83 B	0.69 B	0.13 A	0.16 A	0.08	13.50 AB	1.07 AB
46	1.21 A	1.15 A	0.06 B	0.07 B	0.04	16.50 A	1.32 A
62	0.81 B	0.74 B	0.08 AB	0.08 B	0.06	11.80 B	0.95 B
Pooled SEM	0.09	0.08	0.02	0.03	0.02	1.64	0.10
p-Value	<0.001	<0.001	0.034	0.008	0.205	0.003	0.007

^A,B,C: Mean values within a column with different letter superscripts are significantly different (p < 0.05). * The interaction between period and time of day was significant (p < 0.05). ^† The interaction between time of day and treatment was significant (p < 0.05). Footnote: All aggression data in this Table is presented as total acts per hen over the entire 20 min video (except for total aggressive behaviors, which is the sum of behaviors regardless of hen number).

). Hens housed at a density of 445.16 cm² demonstrated more acts of aggressive pecking and submissive behaviors than hens housed at densities of 1341.93 cm² and 896.77 cm² by 0.37 and 0.53 acts of aggressive pecking per hen and 0.34 and 0.51 submissive acts per hen, respectively (p < 0.001). Hens housed at a density of 535.48 cm² demonstrated more acts of aggressive pecking and submissive acts than hens housed at a density of 896.77 cm² by 0.50 aggressive pecking acts and 0.492 submissive acts per hen (p < 0.001). Hens housed at a density of 445.16 cm² were observed standing on each other at a higher rate than all other densities at a range of 0.16–0.25 acts per hen (p < 0.001). Conversely, hens housed at 1341.93 cm² demonstrated more instances of pecking hens in neighboring cages than all other treatments by a range of 0.24-0.03 acts per hen (p < 0.001). Hens housed at a density of 445.16 cm² demonstrated more total aggressive behaviors than all other densities by a range of 6.48–19.44 acts, while hens housed at a stocking density of 535.48 cm² exhibited more aggressive behaviors total than the lower density treatments by a range of 7.63–12.96 total acts. Hens kept in the 670.97 cm² stocking density demonstrated more aggressive acts than hens kept in the 896.77 cm² and 1341.93 cm² densities by 5.33 acts and 5.26 acts, respectively (p < 0.001). Finally, hens raised in the 445.16 cm² density demonstrated more total aggressive acts per hen than hens in the 670.97 cm² and the 896.77 cm² densities by 0.47 acts per hen and 0.75 acts per hen, respectively, while hens reared under the 535.48 cm² density had higher total aggressive acts than hens from the 896.77 cm² density by 0.58 acts per hen.

Time of day was found to have an effect on the number of aggressive pecking acts, submissive acts, avoidance acts, acts of pecking a neighboring hen, total aggressive behaviors, and aggressive behaviors per hen. At night, the hens exhibited the highest number of aggressive pecking acts compared to the afternoon by 0.44 acts per hen and in the morning by 0.61, while, in the afternoon, the hens exhibited more aggressive pecking than in the morning by 0.25 acts per hen (p < 0.001). The submissive responses followed the same trend as aggressive pecking, with hens at night exhibiting more submissive acts than hens in the afternoon by 0.44 acts per hen and hens in the morning by 0.73 acts per hen, while hens in the afternoon exhibited more submissive acts than hens in the morning by 0.29 acts per hen (p < 0.001). Hens at night pecked neighbors at a greater rate than hens in the afternoon and morning by 0.06 and 0.07 acts per hen, respectively (p = 0.015). At night, the hens demonstrated more total aggressive behaviors compared to the morning, by 9.65 acts and 6.69 acts respectively (p < 0.001). The hens at night demonstrated more aggressive acts than hens in the morning by 0.77 acts per hen and the afternoon by 1.40 acts, while hens in the afternoon also performed more aggressive acts than hens in the morning by 0.30 acts (p < 0.001). Time of day did not affect avoidance and escape behaviors, nor acts of standing on another hen.

Age affected aggressive feather pecking, submissive acts, avoidance and escape acts, acts of standing on another hen, total aggressive behaviors, and aggressive behaviors per hen, while age did not have an effect on pecking neighbors. Aggressive pecking was found to be higher at 46 weeks of age by 0.38 acts per hen compared to 30-week-old hens and 0.48 acts per hen compared to 64-week-old hens (p < 0.001). Submissive acts per hens followed the same trend, where 46-week-old hens exhibited more submissive acts than 30-week-old hens and 64-week-old hens by 0.46 and 0.411 acts per hen, respectively (p < 0.001). However, hens 30 weeks of age performed more avoidance and escape behaviors than 46-week-old hens by 0.07 acts per hen. The 30-week-old hens stood on each other at a greater rate than 46- and 64-week-old hens by 0.09 and 0.08 acts per hen, respectively (p < 0.001). At 46 weeks of age, hens demonstrated more total aggressive behaviors and aggressive behaviors per hen than 64-week-old hens by 4.70 total acts and 0.37 acts per hen, respectively (p = 0.003).

4. Discussion

Determining welfare from behavior is not an exact science. The aim of this study was to use behavior analysis as a tool to determine welfare across several different stocking densities, as well as to identify any differences in behavior across different ages or times of day. Currently, proper welfare is defined as following the five freedoms, which is considered the gold standard of animal welfare [7,8,9,30,31,32]. These five freedoms, however, are more akin to ethical guidelines for raising animals. From a research perspective, finding quantitative parameters associated with behavior to measure the five freedoms can be difficult. The main freedom that presents a problem to research is the freedom to express normal behavior, as normal behavior is not well defined. Domesticated chickens are no longer close enough to their wild counterparts to use the Asian Jungle Fowl as a basis of normalcy [15]. Moreover, many wild behaviors are responses to external stimuli not experienced within cages, which means that the absence of wild behaviors does not exactly mean poor welfare [16,17,18]. For the purposes of this paper, comparing levels of acts independent of conspecific behaviors can serve to determine if a density is causing behavior to deviate from normalcy. For example, if one treatment is different than the others, this could indicate a pressure on the hens to behave differently than they otherwise would. The freedom from hunger and thirst can be measured indirectly by time spent at the feeder and drinker. If hens spend all their time at the feeder and drinker, then there is a high likelihood that some hens may not be able to access the feeder and drinker. The freedom from discomfort is slightly more challenging to measure. Discomfort is more of a “catch-all” term that describes more complex mental states. Furthermore, freedom from fear and distress fall close to discomfort as well. These can be measured by measuring fearfulness as well as stress hormones. Aggressive and submissive behaviors can also be used to identify discomfort as well as distress. Finally, the freedom from pain, injury, and disease can be measured not only by behavioral observations but also by submissive behaviors in response to aggressive behaviors, as aggression can cause the submissive birds pain or distress. While other factors influence this freedom, aggressive pecking is an observable behavior that can cause these issues.

4.1. Time of Day

Time of day was found to play a role in almost all independent of conspecific behaviors and several aggressive actions. The birds spent more time engaging in feeding, drinking, and pecking inedible objects at night than other times during the day. Furthermore, as the hens were engaged in more eating behaviors at night, they also spent less time standing, preening, and stretching at night. In the afternoon, the hens exhibited the highest levels of crouching and sham dust bathing. In the morning, the hens demonstrated the least amount of feeding and the highest amount of standing and preening. From the data gathered, the hens appeared most concerned with filling their crops at night before they slept. In the morning, the hens seemed most concerned with comfort behaviors such as stretching and preening. Dixon et al. [33] reported time of day results close to opposite of those reported in this paper, demonstrating that feeding behaviors did not change across timepoints. However, the hens in Dixon’s study were feed-restricted broiler breeders and housed in a cage-free environment. This elaborates the different stimuli that hens in separate environments and on different feeding programs respond to. Other, older research has indicated that laying hens demonstrate a diurnal feeding pattern, performing more feeding behaviors in the morning and night and less in the afternoon [34]. Evaluating time of day’s effect on behavior has not been studied widely and further research is needed, not only by evaluating different environments but also different strains, and by validating the research that has been performed.

Several past studies have demonstrated that diurnal corticosterone levels peak with egg oviposition, which typically occurs early in the day [35,36]. Further studies have also linked corticosterone levels to aggressive behaviors in laying hens [37,38]. Unfortunately, this study did not evaluate daily levels of corticosterone and, furthermore, aggression among laying hens did not follow the assumed daily corticosterone levels. However, aggression appeared to follow feeding behaviors very closely. As feeding behaviors increase the demand for feeder space also increases. Many studies have identified the inverse relationship between feeder space and aggressive behaviors as hens jostle for position at the feeder [23,39]. Therefore, as demand for the feeder increases at night, as hens are filling their crop for nighttime digestion, aggressive behaviors increase due to the higher jostling for position at the feeder.

4.2. Hen Age

Age was not particularly influential on appetitive behaviors except for sham foraging: the hens spent less time sham foraging at 30 weeks than at 46 weeks. Furthermore, crouching behaviors seemed to peak in the middle of the hen’s life. Many aggression behaviors peaked at 46 weeks of age, indicating that as hens age, they become more aggressive with each other and then become less aggressive at the end of the laying cycle. When the hens encountered another aggressive hen, 46-week-old hens were more likely to engage in submissive behaviors, while they were the least likely to engage in avoidance behaviors. Zimmerman et al. [22] further confirms the findings in the present study, as their hens exhibited a peak of aggressive acts at 48 weeks of age compared to hens aged 32 and 60 weeks. Neither study reported any difference in aggressive feather pecking by age. Zimmerman further reported no difference in preening behavior by age, which disagrees with the results of this study [22]. In an earlier study, Anderson et al. [21] similarly evaluated commercial laying hens in colony cage environments and found that during the first cycle, the hens spent more time standing and less time moving and performed less aggressive acts as they aged. Anderson also found no difference between ages for stretching, which agrees with the findings of the present study. There were, however, several key differences in behaviors between the two studies. The hens in the present study exhibited a change in preening behaviors as they aged, while the hens from Anderson et al. [20] did not. These differences in behaviors between studies can perhaps be attributed to differences in strains as well as methods of analysis. Anderson et al. were also unable to collect behaviors from all cages at the same time, as there was not access to cameras that could simultaneously record every cage, and, as can be seen above as well as in other studies, the time of day has an effect on the behaviors of laying hens [21,33,34].

4.3. Stocking Density

Hen behavior was found to change based on stocking density. In this study, in order to change stocking density, group number also had to change. While the change in the group number may also cause an effect, we believed that this method of changing stocking density would be the most applicable to the poultry industry. If a lower stocking density is recommended to producers, the cheapest option to adopt a lower stocking density is to simply reduce bird numbers per cage instead of changing the cage size. In previous studies, stocking density did not affect any feeding behaviors, which is similar to the results found in the present study [21,40,41]. However, hens housed in conventional cages show an inverse correlation between hen number and feeding behaviors, particularly at extreme densities such as those housed at 417 cm²/bird, like those in Khumput and Thiengtham’s research [42]. The highest density of the present study equates to the highest density that laying hens are typically housed at in the USA, which would explain why Khumput and Thiengtham found results contrary to the present study. Unsurprisingly, the hens in the lowest density spent more time moving than other densities [42]. One study also found that hens in lower densities spent more time moving about the pen; however, more research is needed to evaluate hen locomotion over various stocking densities, particularly at greater bird numbers [21]. Stocking density did not affect preening behaviors, although the highest two densities were found to exhibit lower stretching behaviors than the lowest density. These results agree with other studies, which also found no difference in preening behaviors [21,43]. Finally, hens raised in higher stocking densities demonstrated higher levels of aggression against their cage-mates, which is consistent with other studies performed on stocking density [22,44]. Like other parameters, more research is needed to validate these claims.

Part of the purpose of this study was to use behavior as a tool to analyze welfare across different stocking densities. As discussed in the introduction, proper welfare is commonly linked to the concept of the five freedoms. These guidelines do not lend themselves to easily measurable parameters as they are more moral guidelines and not scientific research parameters [9,11]. The final freedom, the freedom to express normal behavior, is the most applicable freedom in this research. Determining normal behavior can be quite difficult, as many behaviors are simply responses to the environment [16]. There was only a single behavior that showed a linear decrease as stocking density increased, which was stretching behavior. It can be argued that because incidences of stretching fell as more hens were placed in a cage, these hens did not have the personal space needed to perform these behaviors and, therefore, this could indicate that as stocking density increases, the ability to perform normal behaviors falls. If the lowest density is removed, a linear increase in aggressive acts per hen as stocking density increases is seen. Looking at levels of aggression can give an indication about the mental state of animals. It is well know that frustration and boredom lead to higher levels of aggression in laying hens [39,45]. It is also known that less access to feed can cause aggression as hens fight for a place at the feeder, which is seen when stocking density increases [46,47]. Therefore, since higher levels of aggression were seen in higher densities, we can hypothesize that greater levels of distress and frustration are experienced by hens in higher densities. Finally, in general, the data appear to demonstrate that increasing space past 896.77 cm² per hen does not provide much of a benefit for behaviors except for the movement behavior, as a density of 1341.93 cm² is not statistically different from 896.77 cm².

5. Conclusions

The aim of this study was to evaluate the effects of stocking density, age, and time of day on behavior parameters of laying hens in colony cage environments utilizing new camera technology so as to take measurements simultaneously. This study found that all three parameters measured influenced hen behavior. Time of day was shown to be very influential in almost all behaviors measured, demonstrating the importance of consistency when measuring behavior for research purposes. Furthermore, this study demonstrated higher appetitive activities at night, when hens are filling their crops to digest during the dark period. Hen age was also found to affect behavior, with the hens performing more active behaviors at younger ages. Finally, stocking density was found to affect behavior as well. This study found that hens housed at high stocking densities exhibited less stretching and more aggression than other densities, indicating that higher densities may cause more frustration in hens, as the hens may not have enough space to perform normal behaviors. The data from this study indicate that past 896.77 cm², minimal benefits are realized from the increase in bird space.