1. Introduction
Chicken meat is distinguished by being a low-cost, high-quality source of protein, and it is considered one of the most environmentally friendly meats to produce. [
1]. In traditional livestock farming, decision-making criteria primarily rely on the producer’s experiential and empirical knowledge [
2]. In the routine management practices of farmers, particular attention is paid to broiler chickens’ behavioral activity and spatial occupancy patterns during visual inspection. This process demands a considerable amount of time.[
3]. In precision livestock farming (PLF), decision-making relies on quantitative data obtained through process engineering principles and techniques. Such an approach enables the automatic monitoring, modeling, and management of animal production. [
4]. Therefore, in precision livestock farming (PLF), technologies enable the continuous collection of behavioral and physiological data at the individual level [
5], allowing for the automated and real-time monitoring of critical indicators of herd behavior.
The substantial collection and storage of data in smart agricultural production have presented a new challenge: how can this large volume of data be processed to derive actionable insights for optimizing animal production? This situation introduces an additional layer of complexity, which can be addressed by applying machine learning and data mining techniques [
6].
Recent technologies for monitoring farm animals rely on computer vision and machine learning algorithms [
2]. The application of digital image processing and machine learning has facilitated a rise in research focused on avian welfare [
7]. The goal of environmental enrichment is to enhance environmental complexity. [
8] providing opportunities for animals to perform natural behaviors [
9], changing broiler behavior and distribution patterns [
10,
11,
12,
13,
14,
15].
Among all environmental stressors, thermal stress is the most harmful, with great emphasis on animal agriculture. [
16]. When exposed to heat stress, broilers exhibit behavioral, immunological, and physiological changes that negatively impact their overall productivity [
17]. Husbandry workers routinely observe animal postural patterns to assess thermal comfort and adjust environmental settings or management. [
18]. Environmental enrichment during early age (1 to 22 days) effectively mitigates fear and thermal stress responses in broiler chickens to unexpected environmental changes without adversely affecting growth performance and stress status [
11]. Computer vision has enabled the detection of the impact of environmental alterations (environmental enrichment and temperature) on the locomotion of broiler chickens housed in a controlled climate chamber [
19]. Behavioral analysis holds significant potential in developing a remote monitoring system for detecting heat stress in poultry reared in non-enriched environments [
20,
21,
22]. Therefore, behavioral analysis represents a promising and non-invasive tool for estimating the level of animal comfort and welfare.
For an extended period, behavioral responses have predominantly been quantified based on their intensity, frequency, or duration. [
23]. The gold standard for video analysis in behavioral research has been the implementation of the ‘scan-sampling’ method by researchers. [
24]. On the other hand, the intricate patterns of behavior encompassing directional, sequential, and temporal organization have been largely disregarded and underestimated. [
23]. Nevertheless, research interests are progressively shifting towards analyzing more intricate behavioral patterns. [
23,
25,
26,
27]. The research by [
28] indicated that the complexity of locomotive sequences diminished under stress, while the complexity of behaviors such as perching, foraging, and resting increased in enriched conditions. [
25] chickens can sustain a consistent behavioral pattern during rearing, characterized by individual rather than flock-based rhythms, as evidenced by their spatial utilization within the barn. Such distinct rhythmic behavior indicates an organism’s health [
29]. Existing literature recognizes that stressors can disrupt behavioral rhythms [
29,
30,
31,
32,
33]. Consequently, detecting recurrent behavioral patterns over time can be crucial for evaluating breeding practices and animal welfare. The Generalized Sequential Pattern (GSP) algorithm is a data mining method that identifies recurring sequences surpassing a user-specified support threshold, initially developed by [
34] to understand customer behavior patterns. The potential of this algorithm for the behavioral profiling of broiler chickens in conditions of thermal comfort and heat stress was investigated by [
26,
27]. However, its applicability for poultry housed in enriched environments remains unexplored.
This study serves as a proof of concept aimed at validating the application of the GSP algorithm for identifying temporal correlations between heat stress and the behavior of broiler chickens in enriched environments.
3. Results and Discussion
Table 3 presents the frequent behavioral patterns for broiler chickens aged 21 days (third week) reared in an unenriched and enriched environment under comfortable temperatures.
Table 4 inserts the frequent behavioral patterns for broiler chickens at 22 days reared in an unenriched and enriched environment under heat stress. As the MinSupport value is reduced, new sequences are found, and the results (sequences) of the previous supports are excluded from the tables because they are identical subsets at the higher level. This pruning reduces repetitiveness and facilitates the reader’s analysis.
Of all the behavioral possibilities described and considered in the ethogram of
Table 1, four out of the total of 13 behaviors were not observed (frequent) at the 20% support (Run, Dust bathing, Explore, and Wing flap). This result may be rooted in some relevant aspects, either in isolation or combination. Factors that explain our results include specificities involving the tool (the operational aspect of the GSP algorithm that requires the behavior frequency to occur in the chronological order in which they appeared) [
34,
50]; particularities inherent to the behavior itself such as low frequency (rare events); characteristics related to the individual; and also the bias of chance (“error” of uncontrolled origin).
The comfort temperature resulted in a more significant number of behavioral patterns compared to heat stress. The number of patterns found between the treatments was similar under thermoneutral conditions (27 non-enriched vs. 29 enriched) and heat stress conditions (7 non-enriched vs. 8 enriched). Sequence sizes ranging from 1 to 4 were identified in both rearing environments under thermoneutrality. The size 4 sequence in the non-enriched environment shows that the broiler performed the behaviors in chronological order according to the analysis criterion (during 5 continuous minutes) <{P,F,P} – 1 step {Ld,P} – 2 step {Ld,F}- 3 step {Ld,P}- 4 step>. In the enriched environment, the size 4 sequence involved the same behaviors but in a different chronological order <{Ld,P}- 1 step {Ld,P,F,P}- 2 step {Ld,F}- 3 step {Ld,F}- 4 step>. However, the ambient temperature above the comfort zone restricted the sequence sizes to a maximum of 2. Heat stress drastically reduced the number of pattern sequences of broilers in both tested situations, reducing the level of activity and movement [
17,
19,
20,
21,
22,
26,
27,
44]. [
28] studied broilers in an enriched environment and established that the fractal complexity of the behavior sequence decreased with increased heat stress due to insufficient energy to perform complex behaviors.
The sequence <{Ld,P}> was the first behavioral pattern found in both treatments (non-enriched and enriched) under comfort temperature and heat stress only in broilers housed in a non-enriched environment. Branco et al. [
26] also observed this same sequential pattern in broilers of the same age (21 days) in non-enriched and thermoneutral situations. The most frequent behavior in the sequential patterns was Ld (Lying down), mainly under comfort temperature. Broilers spend an average of 76-80% of their time lying down [
53], which predisposes them to locomotor problems (lesions and fractures) due to poor movement [
15,
54]. Lameness, long periods of lying down, and other health problems will likely interfere with the broilers’ ability to perform high-priority behaviors [
55], such as foraging, comfort, and dust bathing [
56]. Preening is a natural broiler behavior, highly motivated and indicative of comfort [
53,
54], although it may be more present in thermal stress environments [
26]. Specifically, for this research, comfort behaviors considered were preening (P), dust bathing (Db), shaking feathers (Sf), and stretching (Sf) [
55]. Foraging (F) and exploring (Ex) behaviors are classified as exploratory behavior [
55], being essential and considered healthy for the broiler. Our sequence pattern results identified a high prevalence of “Ld”, “P”, and “F” in the sequence formation in both treatments, indicating strong motivation and priority from the animal’s perspective. Broilers in comfort temperature have high motivation to perform these last mentioned behaviors, whose sequences <{Ld,P}>; <{Ld,F}>; <{P,F,P}>; <{Ld,P,F}> and <{E,W,F}> were the only ones observed in both treatments. All other sequential patterns (comfort and stress) were distinct, suggesting that environmental enrichment alters the behavioral pattern of broilers.
The walking behavior of broilers in thermoneutrality was more directly related to feeding (eating and drinking) and exploration activities (foraging), as observed in the sequences <{E,W,F}>; <{W,F,P}>; <{D,W,E}>; <{F,P,W,E}>. Broilers explore the environment in search of food and water even under thermal stress situations, as seen in non-enriched housing <{E,W,P,W,F}>; <{Ld,P,St,Ll}> <{Ld,P,W,D,W}> and enriched housing <{E,W,F}>; <{Ld,W,D}>; <{E,W,D}>; <{P,F,St,P}> <{E,W,F}>. In a conventional statistical approach, [
57] observed frequencies ≥20% for the behaviors of lying down, walking, eating, drinking, and preening, reinforcing from another perspective the probable connection of locomotor activity behavior related to feeding. Broilers housed in non-enriched environments under heat-stress conditions tend to walk and congregate near the drinker to benefit from the microclimate near the water [
19,
21,
58]. However, in the same situations but in enriched environments, they gather around enrichment objects, indicating that environmental enrichment can minimize the negative effect of thermal stress on the broilers [
19]. Rufener et al. [
25] investigated behavioral time series in laying hens, revealing that the birds exhibited consistent daily results specific to each individual’s movement and location patterns. In other words, the hens can maintain a relatively stable behavioral rhythm under rearing conditions, following an individual rhythm rather than a flock rhythm, which can be reflected in the use of areas in the barn [
25].
In the thermally comfortable and enriched environment, the broiler exhibited the sequence <{Ld,P,Sf,P}>, indicating probable positive well-being, as the behavior “Sf” was not observed in any pattern for broilers in a sterile environment. It should be noted that the more frequent a behavior is, the higher the probability of the GSP algorithm finding a pattern according to chronology and threshold criteria.
The most prominent sequential pattern for broilers raised in environments devoid of environmental enrichment when thermally challenged were: <{Ld,P,St,Ll}> and <{Ld,P,St,Ll} {Ld,P,W,D,W}>. Specifically for the sequence “St,Ll” some critical points should be evaluated. Firstly, the behavior “St” is classified as a comfort behavior for broilers and could be used to assess animal welfare [
59]. However, measuring its occurrence in isolation may not be the best way to estimate the welfare status of this animal, and it should be correlated with climatic factors. Thus, an individual signature in temporal behavior patterns could provide a new opportunity to assess an animal’s state [
23]. Therefore, the chronological context should be specially considered, as the subsequent behavior observed in the sequence of “St” is lying laterally “Ll”,
which is a strong indicator of heat stress [
26]
and [
27]
. In other words, chronologically speaking, if the broiler performs “St” and then immediately lies laterally, the goal of this broiler is to increase the body surface area for heat exchange (dissipation) for thermal relief [
26,
27,
60]
. Conversely, we did not observe this “St,Ll” pattern for broilers housed in enriched environments, which may suggest that environmental enrichment provides the animal with better opportunities for adaptation to face stress-inducing challenges, such as heat stress, corroborating [
10]
and [
19]
.
However, under heat stress in an enriched environment, there was a tendency for a lower prevalence of the Ld behavior. The sequences observed in broilers housed in enriched environments indicated a more active repertoire involving activities such as the sequences: <{Ld,W,D}> (n=2); <{Ld,P,W,P}> (n=2); <{E,W,D}> (n=2); <{P,F,St,P}> (n=2); <{E,F,E}> (n=2); <{P,F,St,P} {E,W,F}>. Various authors reported more significant movement of broilers in environments provided with environmental enrichment [
11,
15,
19,
61,
62], correlating with a broader range of usual and motivated behaviors [
38,
63,
64] and, therefore, with potentially fewer health and welfare impairments [
65].
Author Contributions
Conceptualization, J.M.M, and D.J.M; methodology, J.M.M, D.J.M, and T.B.; software, S.R.M.O; validation, S.R.M.O, T.B.; formal analysis, J.M.M, S.R.M.O.; data curation, D.F.P., J.S.G.B; writing—original draft preparation, J.M.M., and D.J.M., and D.F.P.; writing—review and editing, I.A.N., D.F.P., J.S.G.B., and D.J.M.; visualization, I.A.N., D.F.P., and D.J.M.; supervision, D.J.M., D.F.P., and I.A.N.