Effects of strain and sex on the behaviour of free-range slow-growing chickens raised in a hot environment

ABSTRACT Behaviour is a good indicator of the well-being of chickens. The objective of the study was to compare foraging behaviour in males and females of three indigenous chicken strains under generally hot free-range conditions. Behavioural activities were monitored in 21-wk-old Potchefstroom Koekoek (PK), Ovambo (OV) and Naked Neck (NN) chickens. Birds were separated by sex and allocated to four pens of Chloris gayana. Three birds per pen were randomly chosen and marked with paint 20 min before observation. Temperature humidity index (THI) was calculated and main effects analysed using the general linear models procedure. Naked Necks spent more time walking than OV and PK. Strain did not affect other behaviours. Females spent more time foraging, while males dominated standing and walking. There was negative correlation between THI and time spent foraging. There was interaction between strain and sex on time spent standing. Foraging and drinking behaviours were more prominent in the morning (07:00 h) and late afternoon (17:00 h) compared to the 12:00-h period, whereas preening and dust-bathing were dominant around mid-day. Strain, sex and THI influence behaviour in free-range chickens. Breeding programmes should be cognizant of these attributes in order to produce hardier birds in view of worsening climatic uncertainties.


Introduction
Animal welfare activists campaign for the use of natural or nearnatural environments for chickens. This has stimulated an increase in the popularity of free-range systems across the world. Free-range chickens have limited access to feed additives and artificial ingredients and are grown in an environmentally friendly manner (ERS 2002). Free-range or organic systems allow birds access to an outside area, promoting foraging, feed selection and activity, thus theoretically improving their welfare (Ponte et al. 2008). These outdoor production systems could decrease stress and allow the selection of strains that may increase comfort and bird welfare (Wang et al. 2009), particularly in the wake of production conditions that are only likely to worsen with predicted trends in climate change. Conventional systems limit the expression of normal behaviour and have become unpopular. Conventional cage systems for laying hens were banned in the European Union (EU) from January 2012 according to the EU Council Directive 1999/74/ EC on the welfare of laying hens (CEC 1999). In the developed world, free-range and organic livestock production are well defined with products derived from such systems getting certified (ERS 2002). Free-range products are perceived to be safer and healthier and may carry several health benefits to consumers (Midmore et al. 2005).
Meteorologic elements constitute a major variable for outdoor operations (Sossidou et al. 2011), and the concern and emphasis for such elements in recent years are due to the fact that they are not constant, but change continuously (Ayo et al. 2011). Factors such as temperature (Kristensen et al. 2007) and humidity influence life cycles, reproductive ability, growth rates and thus body weights (BWs) of birds. Direct meteorologic factors affecting birds include, especially, high ambient temperature and relative humidity, and may result in severe heat stress (Ayo et al. 2011). High humidity impacts thermoregulation and welfare of chickens (Lin et al. 2005), in that humid conditions reduce the effectiveness of heat dissipation (Warriss et al. 2005). This impairs normal body functions as efficiency is achieved if body temperature is kept constant or maintained within a narrow range (Ayo et al. 2011). The normal body temperature of an adult chicken is 40.6-41.7°C, while the thermo-neutral zone (TNZ) is 18-24°C (Fanatico et al. 2007).
Free-range systems make use of slow-growing strains which are more adapted to these production systems (Castellini et al. 2002;Gordon & Charles 2002). Utilization of the slow-growing indigenous strains enhances the sustainability of chicken production systems. Indigenous strains are preferred to exotic chickens, because of their pigmentation, taste, flavour and leanness (Moreda et al. 2013). Popular strains in Southern Africa include Naked Neck (NN), Ovambo (OV) and Potchefstroom Koekoek (PK) (Nthimo et al. 2004;Grobbelaar et al. 2010) which are dual-purpose strains. The NN and OV are closely associated with rural livelihoods where they are used to meet the nutritional and economic needs of households (Mapiye et al. 2008). They are considered hardy and adaptable to harsh local climatic conditions, which are important attributes since predominant systems often entail exposing birds to adverse environmental conditions. Such exposure influences the behaviour of birds in various ways and behavioural responses are the most pertinent indicators of the well-being of an animal (Moura et al. 2006). In hot weather, birds thermoregulate behaviourally by exposing a larger body surface area to encourage heat loss and body temperature is elevated (Warriss et al. 2005). Thermal stimulation, among other factors, influences behaviours such as dust-bathing (Orsag et al. 2011).
The literature shows that there are strain differences in response to heat stress and that slower growing strains range more widely (Altan et al. 2003;Nielsen et al. 2003) compared to fast-growing birds. Even among slow-growing strains, thermoregulatory capabilities vary. It is thought that the thermoregulatory ability of NN chickens at high temperature is slightly better than that of normally feathered birds (Yahav et al. 1998). Naked Necks are a light-weight multicoloured strain with white, red and black feather combinations. They reach sexual maturity at 155 d of age, with males weighing about 2.0 kg and females 1.4 kg (Chikumba & Chimonyo 2014). The reduced feather cover in the NN strain may be of advantage in thermoregulation at high ambient temperature (Eberhart & Washburn 1993). The strain carries a gene which results in reduced overall plumage cover (Rajkumar et al. 2010;Fathi et al. 2013). The OV is a predominantly dark coloured fairly heavy strain that attains sexual maturity at average weights of 2.2 kg for males and 1.5 kg for females about 140 d of age (Nthimo et al. 2004). It is generally regarded as adapted to high ambient temperatures though the degree of thermal tolerance does not match NN owing to darker plumage colour and fairly heavier BW. The PK is a composite strain developed by crossing Black Australorp cockerels with White Leghorn hens and the Plymouth Rock (Grobbelaar et al. 2010). It is a heavy strain with an average adult BW varying from 3.0 to 4.0 kg for cocks and 2.5 to 3.5 kg for hens (Joubert 1996). The strain reaches sexual maturity at 130 d. Though the birds are known to be adaptive and to survive under low input conditions, little is known about adaptability to high ambient temperature, particularly differences between sexes which exhibit clearly defined sexual dimorphism in plumage colour intensity. The strain has a characteristic black and white speckled colour pattern described as barred. The barred appearance is darker in females. The pattern is a sex-linked character that is useful for colour sexing in breeding for egg-producing hens suitable for medium input production systems (Grobbelaar et al. 2010). This is vital as it may have a direct influence on the fate of male PK after the females are selected for egg production.
Despite being adapted to harsh environmental conditions, the productivity of free-range indigenous chickens is low. Given that numerous factors affect the behaviour of birds under free-range conditions, investigating the influence of environmental factors and their interaction with bird factors on the behaviour of birds is essential. This would inform the designing of efficient management techniques aimed at improving productivity. Comparison of foraging habits and behaviour of indigenous chicken strains, and sexes of the same, is useful in view of the increasing importance of outdoor systems across the world. The objective of the study was to establish and compare the foraging behaviour of PK, OV and NN chicken strains under free-range conditions.
It was anticipated that birds would adjust their behaviour in order to cope with changes in environmental conditions (Bertin et al. 2013) and that the degrees of adaptability would vary with strain and sex. Based on plumage colour and BW, better behavioural adaptation to high ambient temperature and humidity would be anticipated in NN, more so in females which are lighter than males. It was, thus, expected that NN would be the least affected by heat stress, hence more time spent on feeding-related behaviours and consequently higher BW in comparison to other strains. It was rather difficult to predict the relative adaptabilities of the OV and PK strains due to darker plumage colour in one strain and higher BW in the other; thus, this study may allow to disentangle the respective importance of the two factors.

Animal ethics
Bird management, care and use were compliant with internationally accepted standards for the welfare and ethics of research animals (Austin et al. 2004) and were specifically approved by the University of KwaZulu-Natal Animal Ethics Research Committee (Reference Number: 039/15/Animal).

Study site description
This study was conducted between January and March 2015 at Cedara College of Agriculture in Pietermaritzburg, South Africa (SA). Cedara is located in an upland Savanna zone on latitude 29.53°S and longitude 30.3°E at altitude 613.0 m. The study area has a varied yet verdant climate owing to its diverse and complex topographic characteristics. It is characterized by very warm summers and cold winters. The mean temperature and humidity recorded over the trial period were 25°C (range: 17-40°C) and 61%, respectively. Mean, minimum and maximum humidity recorded were 61%, 35% and 87%, respectively. Ambient humidity was highly variable, particularly in wk 1 (SD = 22.8). The weekly average, minimum and maximum temperature and humidity experienced over the trial period are given in supplementary Table S1. Temperature humidity index (THI) means ranged from 68 to 79.2. The overall mean THI value for the observation period was 73.2.

Housing, feeding and health management prior to observation
Day-old chicks of OV, NN and PK strains were obtained from the parent flock kept at the Agricultural Research Council (ARC), Irene, Pretoria, SA. At 1 d old, BW of chicks across the three strains ranged from 39.7 to 49.8 g. From d 1 to d 49 chicks of each strain were reared in 2 × 1.5 m pens in a closed well-ventilated poultry house which was 4 × 10 m. The house floors were covered with a 10-cm-thick layer of wood shavings. Heat and light were provided using 75 W infrared lamps. The day-old chicks were maintained at a temperature of 32°C which was gradually reduced to 21°C by 21 d old. A thermometer was kept in the house just above the level of the birds and was used to monitor changes in temperature. A foot bath drenched with a disinfectant (virukill ® ) was placed at the entrance to the brooding house.
A broiler starter diet was offered ad libitum from standard tube feeders. Potable tap water was offered ad libitum through 4 L plastic fonts. Chicks were vaccinated against Newcastle disease at 10 and 35 d of age. From d 50, birds were given a grower meal. Both feeds were supplied by Meadow feeds, SA. The nutritional composition of the feeds is shown in supplementary Table S2.
At 20 wk of age, selected birds were moved from the poultry house and assigned to four pens under free-range conditions. The pens, where Chloris gayana (Katambora Rhodes grass) was the dominant grass spp, were located side by side and separated by galvanized steel mesh fencing. During establishment, the pens were watered regularly and were rain-fed once established. Weeds and other invader grass spp were hand-picked and eliminated from the pens. Cattle manure was used to fertilize the pens. Wooden cages measuring 2.5 × 2.0 m were placed in one corner of each pen to provide shelter for the birds overnight and shade during the day. The cages, with slatted floors elevated 1.0 m above the ground surface, were fitted with wire mesh doors to deter predators.
Doors were left open during the day and closed at night after all birds had voluntarily climbed into the cages. A standard plastic drinker was placed under shade near each cage to provide cool clean water. The drinkers were inspected, washed and replenished at least twice a day to ensure ad libitum access to clean water.

Treatments and experimental design
A total of 144, 20-wk-old PK, OV and NN chickens were used in this study. Birds were separated by sex and allocated to 4 freerange pens such that there were 12 males × 3 strains on each of 2 pens and 12 females × 3 strains on each of 2 separate pens. Strains were mixed to enable comparison of their responses under exactly the same management conditions. The pens, measuring 900 m 2 each (Figure 1), were demarcated by 2.2 m high wire mesh reinforced by wooden and steel poles. The birds were weighed individually on a digital crane scale, model UME CCS-150 K, S/N: NXC 100020, to determine initial BWs.

Observations and data collection
After placement onto the pens, the birds were allowed a 7-d adaptation period before commencement of data collection. For behavioural observations, three birds, one of each strain, in each of the four paddocks were randomly chosen and marked with paint on the tip of the tail 20 min before being let out of the cage at 07:00 h. Paint of a different colour was used each time such that the same bird was not observed more than once. Two trained observers recorded the activities of one bird each, simultaneously, in two pens purposively chosen to represent males and females for each observation session. As a result, two pens of males and females were observed simultaneously for 30 min as a result. Birds in the other two pens were observed immediately after. The observers switched from pens with males to females and vice versa.
Birds in each pen were observed three times a day, once a wk, for a total of 3 wk (Wk 1, 3 and 5) from 07:00 to 08:00 h; 12:00 to 13:00 h and 16:00 to 17:00 h. During behaviour observation, a distance long enough to avoid disrupting the expression of normal behaviour by the birds was maintained. The time spent on each of the following behaviours was recorded: (1) Drinking behaviour (standing over a drinker with the head towards the drinker) (2) Foraging (pecking on vegetation in the paddock or scratching the ground) (3) Preening (cleaning of feathers) (4) Dust-bathing (the act of moving around in dirt) (5) Hunting (chasing after insects) (6) Standing (remaining still in inactivity) (7) Other activities (e.g. lying down and crouching) Other general behavioural activities such as panting were also noted. A stopwatch, model 870A Century clock-timer, was used to time and record specific time intervals devoted to a particular activity. A thermo hydrometer was used to record ambient temperature, in degree Celsius (°C), and relative humidity as a percentage. Ambient temperature and relative humidity values used were recorded on the same days that behaviour observations were made. As a result, meteorological data reported in the current study correspond to measurements made on the day of observation. The recorded ambient temperature and relative humidity (RH) values were used to estimate the THI as follows: where T d is the ambient temperature (Spencer 1995). In addition to these observations, BW was measured weekly using a digital scale (±1 g sensitivity) as part of a separate experiment.

Statistical analyses
Data on the daily activities and BW of the free-range chickens were analysed using the general linear models procedure of the Statistical Analysis System (SAS 2010). Least square means (LsM) were generated by the least significant means and separated using the probability of differences option of SAS (2010). The model Y ijklmn = µ + B i + S j + W k + TD l + THI m + (S × B) ij + ε ijklmn was used for free-range-related activities, where Y ijklmn = the response variable (time spent on a particular activity); µ = overall mean common to all observations; B i = effect of the ith strain (i = NN, OV, PK); S j = effect of the jth sex ( j = male, female); W k = effect of the kth wk (k = 1, 3, 5); TD l = effect of the lth time of day (l = 07:00 h, 12:00 h, 16:00 h); THI m = combined effect due to ambient temperature and humidity; (B × S) ij = effect of the interaction between strain and sex of bird and ε ijklmn is the random residual error. A linear regression model was fitted to test the relationship between time spent on feeding-related activities and THI. Significance was considered at the 5% level of probability in all cases.
Data on BW were subjected to analysis of variance using the model Y ijkl = µ + B i + S j + W k + (B × S) ij + ε ijkl , where Y ijkl = the response variable (BW); µ = overall mean common to all observations; B i = effect of the ith strain (i = NN, OV, PK); S j = effect of the jth sex ( j = male, female); W k = effect of the kth wk (k = 1, 3, 5); (S × B) ij = effect of the interaction between sex and strain of bird and Y ijkl is the random residual error. All interactions that had no effect at the 5% level of probability were dropped from the model.

Body weights
BW was influenced (P ≤ .01) by strain, sex and wk of observation. Significant interactions were observed between the strain and sex of bird on this parameter. Of the three strains studied, OV chickens were the heaviest, followed by PK and the NN chickens. Sexual dimorphism was observed in BW for NN and OV chickens. Males were heavier in the NN and OV strains, while male and female PK did not differ in BW ( Figure  2). Marginal weight losses were observed in birds across all three strains over the period of observation.

Time spent foraging and drinking water
Significance levels for time spent on activities studied are presented in Table 1. Week, sex of bird and time of day all influenced (Tables 1 and 2, Figure 3) time spent foraging. Birds spent the most time foraging in the third week. Females spent more time foraging than their male counterparts. In addition, birds spent the most time foraging at 07:00 h followed by 16:00 h (Figure 3). There was a significant negative correlation (t (100) = −2.7, P ≤ .01) between time spent foraging and THI. It was estimated that for a unit increase in THI, time spent foraging would decrease by 48 s. Strain did not influence time spent foraging ( Table 2). Time of day had an effect (P ≤ .01) on time spent drinking water (Figure 3), while strain, sex, week and THI had no effect (P ≥ .05). Chickens spent more time drinking water in the morning than at mid-day and in the evening (Figure 3; Tables 1 and 2). No interactions were observed between THI and strain in this study.

Time spent preening and dust-bathing
Time of day and THI influenced time spent preening, while all other factors had no effect (Table 1). Birds across the three strains were observed preening more at mid-day (12:00 h) than at any other period. Generally, more time was spent preening in the first week relative to wk 3 and 5 (Figure 3). Similarly, the most time spent preening (102 s) coincides with the highest THI recorded. None of the factors studied influenced (Tables 1 and 2) time spent dust-bathing, although time of day approached significance (P = 0.0782). Similar patterns were observed among time spent standing, drinking water, preening and dust-bathing (Figure 4). The chickens dust-bathed mostly during the 12:00 h to mid-afternoon period.

Time spent standing and walking
Strain and time of day did not affect time spent standing (Tables 1, 2 and 3). Week, sex of bird and THI influenced (P ≤ .001) time spent standing by the birds. There was a significant positive correlation between THI (t (105) = 3.2, P = .0016) and time spent standing. For every unit increase in THI, time spent standing increased by approximately 39.6 s. The effect of week on time spent standing is shown in Figure 4. There was interaction (P ≤ .001) between strain and sex of bird on time spent standing (Table 3). Strain and sex of bird were the only factors that affected (P ≤ .05) time spent walking. Males of all strains spent more time walking than females, while the NN spent the most time walking relative to the other strains (Table 2).

Other observations
It was rather interesting to note that male OV and PK dominated in standing, while males of all three strains spent more time walking than females (Figure 3). A lot of time was also spent lying down in inactivity, particularly during the hottest times of the day. Four PK females and two NN males were attacked by a hawk; as a result six birds were lost over the observation period.

Discussion
Bird behaviour frequently switched among the major activity categories, namely foraging, drinking, preening, dust-bathing, standing, walking and lying down. Similar observations were made in broilers (Merlet et al. 2005). The literature reports strain differences in response to free-ranging behaviour (Nielsen et al. 2003), heat stress (Altan et al. 2003) and BW (Nthimo et al. 2004) in chickens. In this study, we anticipated strain differences in foraging behaviour under heat stress conditions. Our expectation was that the NN would forage for longer than the other strains. The NN chickens possess better post-weaning heat tolerance than OV and PK due to the reduced plumage cover which is effective in minimizing heat stress where birds have to dissipate excess heat (Cahaner et al. 1993;Deeb & Cahaner 2001;Raju et al. 2004;Fathi et al. 2013). The NN carry an autosomal incompletely dominant gene (Na) which results in a 30% reduction in overall plumage cover for heterozygotes and 40% for homozygotes, which are associated with increased thermal tolerance (Raju et al. 2004;Rajkumar et al. 2010;Fathi et al. 2013). In the current study, one behaviour that perhaps demonstrates the marginal advantage of the NN, over other strains, is the more time spent walking by chickens in this group, perhaps suggesting greater adaptability. This presumably shows greater thermal tolerance, thus flexibility in terms of time spent on otherwise heat-intense activities by the strain. OV chickens, which were the heaviest in the study, an observation consistent with earlier studies (Nthimo et al. 2004;Chikumba & Chimonyo 2014) but not Grobbelaar et al. (2010), are predominantly dark coloured. As such, it was anticipated that OV would be most affected by high THI. Strain differences observed in BW in this study are consistent with previous observations (Chikumba & Chimonyo 2014) where differences were recorded in 16-wk BW of OV and NN chickens. Similar BW observations were made by Nthimo et al. (2004).
Generally, females dominated most activities in terms of the time dedicated to a particular behaviour. They spent more time on most activities with the exception of standing and walking. Less time spent walking by females is probably an effort to reduce energy expenditure, particularly because birds were at point of lay. Time spent lying down in inactivity is perhaps yet another energy-conserving effort shown by birds. The fact that this coincided with high THI values shows that birds  refrain from feeding as a means of minimizing the heat burden. Feeding generates heat and, during hot times, would result in increased heat loads, therefore exposing birds to thermal stress. The observation on females spending more time foraging than males is not consistent with previous research (Nthimo et al. 2004). The females, which were at point of lay, probably had greater nutrient demands to meet egg production requirements. The males were expected to forage for a longer time to meet greater nutrient requirements; conversely, they have higher BW which might mean potential susceptibility to heat stress. Sexual dimorphism observed in the BW of NN and OV strains is in agreement with Nthimo et al. (2004) who noted differences in 26-wk BW of OV, PK and NN chickens. Birds did not seem to forage much in the first week probably as a result of the dramatic change in conditions including the transition from indoor to an outdoor environment. The first week of observation had the highest average maximum temperature (35°C) and humidity (87%), hence a high THI. It was shown in this study that increases in THI lead to a reduction in time spent foraging. Time spent standing, however, increased with increasing THI. The time spent standing in the current study is higher than 20 ± 30.8 s reported by Spencer (2013). The discrepancy could be a reflection of the differences in strains and rearing conditions. Regression analyses showed that there was no relationship between THI and time spent on all other activities. The observation that birds foraged and drank water more in the cooler hours of the day agrees with previous reports (Dawkins et al. 2003;Horsted et al. 2007;Spencer 2013). Contrary to the same researchers, birds foraged much longer in the morning than other periods in our study. Dawkins et al. (2003) observed chickens to be most active right before sunset. Chickens forage more during the cooler hours of the day as they are less likely to struggle with thermoregulation. Higher temperatures that are commonly experienced around mid-day to early afternoon also depress appetite, thus compromising feed intake (Dawkins et al.    2003). The THI range of 68-79.2 shows that, at some point, birds were exposed to some degree of heat stress. At THI values of 72-79, mild heat stress occurs, while THI values of 80-89 indicate heat stress (Pennington & Van Devender 2004). The current findings suggest that foraging, hence dry matter intake (DMI), is perhaps more important in influencing drinking behaviour than other factors since invariably more time was spent drinking water during periods when birds foraged more actively. The literature shows that chickens go off-feed if water intake is restricted (Chikumba & Chimonyo 2014) perhaps agreeing with our view, although the direct effect of DMI on drinking behaviour should be tested. Feed consumption in NN chickens given ad libitum water access was 52% and 8% higher than that of birds given water at 40% and 70% of ad libitum, respectively (Chikumba & Chimonyo 2014). The mean time spent drinking water is similar to that reported by Murphy and Preston (1988). The strains used in the two studies differ, perhaps suggesting that the important factor driving drinking behaviour is DMI. The weight loss experienced by the birds in this study is probably a consequence of the change in rearing conditions. The same might have been worsened by the high THI which discouraged foraging. Literature reports reduced feed intakes at high temperatures so as to preserve body water by reducing faecal water loss and body heat increment (Mashaly et al. 2004;Chikumba & Chimonyo 2014). Reduced feed intake by birds is an adaptive strategy to survive under hot environmental conditions. Reduced BW was recorded in broilers exposed to high temperatures and humidity, perhaps indicating depressed feed intake (Lin et al. 2005). Spending the most time preening during the 12:00-h period perhaps indicates more than just a simple trade-off between preening and foraging by birds. It is during the hottest period that birds clean their feathers and wad external parasites (Clayton et al. 2010). A previous study noted that the preferred time of dust-bathing, a similar behaviour driven by thermal stimulation, is the middle of the day (Wichman & Keeling 2009;Orsag et al. 2011). Our observation on dust-bathing contradicts the report by Murphy and Preston (1988) where absence of dust-bathing was noted in broilers. Preening and dust-bathing are important parts of normal bird behaviour (Orsag et al. 2011). These behaviours help to remove stale oil from feathers and are particularly important in free-range chickens which are often exposed to various edaphic and biotic factors, hence parasite infestation. The maximum environmental temperature recorded in the current study was much higher than 18-24°C, which is the TNZ for chickens (Fanatico et al. 2007). At ambient temperatures within the TNZ, chickens are able to maintain their body temperature. Any increase in temperature above this zone initiates heat dissipation mechanisms. At high THI values, heat production decreases while heat dissipation increases (Lin et al. 2005). High THI values experienced in the current study discouraged foraging due to increased heat load (Lin et al. 2005). The high THI positively influenced standing and preening behaviours as birds normally thermoregulate by behavioural changes. High ambient humidity exacerbates the effects of high temperatures by reducing the effectiveness of panting to induce evaporative cooling from the respiratory tract (Warriss et al. 2005). This largely agrees with behavioural trends observed in this study.
It can be concluded that strain and sex influenced the foraging behaviour, and NN chickens and females, in general, spent more time foraging. Sex of bird influenced walking and standing behaviours. There was a negative correlation between THI and time spent foraging, while time spent standing and preening increased with increasing THI. Foraging and drinking behaviours were more prominent in cooler hours of day (morning), while preening and dust-bathing occurred mostly when THI was high (mid-day to afternoon). Ambient temperature and relative humidity are important factors influencing freeranging behaviour and hence overall performance of slowgrowing chickens. Breeding programmes should be cognizant of these attributes in order to produce hardier birds in view of the worsening climatic uncertainties.