The evaluation of differences between the cloacal microbiota of indoor- and outdoor-layers in commercial flocks may contribute to an increased understanding of interactions between gut microbiota, housing conditions, and other environmental factors, and help to determine whether the microbiota composition might be used as an indicator of the risk of potential pathogen exposure from the farms’ outdoor environments. Furthermore, understanding the dynamics in microbiota community composition of adult layers in a field setting is relevant, as it may contribute to the insights needed to develop ways to modulate the chickens’ microbiota in favour of health and increased production performance. Although we previously found limited change in the hens fecal microbiota after a single oral inoculation with wild duck feces [16], we hypothesized that continued exposure of laying hens to an outdoor environment would be more likely to result in detectable alterations in the fecal microbiota of outdoor-layers.
In this study we found that access to an outdoor range only explained a small proportion (0.2% R2adj) of the total variation in the cloacal microbiota of layers. Instead, poultry house was found to be the most important driver of community composition (20.9% R2adj). When poultry house was excluded from further analysis to more precisely estimate the effect of the outdoor range, the farm where the poultry houses were located (6.5% R2adj), the rearing farm the chickens originated from (1.9% R2adj), and the interaction between these two factors (12.6% R2adj) explained most of the variation. The relatively high R2 for this interaction is to be expected, considering the overlap between the factors rearing farm and layer farm in our study design (Fig. 6). We also found that the diversity and evenness in indoor-layers were slightly higher compared to outdoor-layers, suggesting the presence of more dominant species in outdoor-layers. This contrasts with previous studies, which found higher microbial diversity in outdoor-layers [10,11,12,13]. Differences in diversity and community composition in previous studies has been related to greater substrate diversity and intake of fibrous feedstuff [12], as well as exposure of the chickens to more abundant microbiota in the outdoor environment [11]. Our findings may deviate from those of previous studies due to several reasons.
In the first place, we selected indoor and outdoor poultry flocks of the same breed (Dekalb White) and based on the rearing farm of origin to minimize variation due to host genetics and rearing conditions. Also, the in-house environment of both indoor- and outdoor-layers was similar in our study. Chickens were housed in cage-free aviary systems, with the same stocking density, feed, minimum number of perches, similar litter etc. This is in contrast with previous research where a comparison was made between either free-range poultry with access to an outdoor range and caged layers [11,12,13], between fast- and slow-growing broilers [10], or in a semi-experimental set-up [12]. Effects found in these previous studies are likely confounded by the effects of caged vs. non-caged chickens or the breed of the chicken, and not truly measure the effect of the access to the range. Additionally, two studies were performed on either broiler chicks of 42 days of age [10] or indigenous Chinese Dagu chickens, a dual purpose breed which produces both meat and eggs, of only 12 to 18 weeks of age [12]. The microbiota of adult layers develops over time to a stable equilibrium [17], which is less sensitive to external perturbations [16] and hence, may explain the unanticipated limited effect of the outdoor environment in our field study. The timing of access to the range may also be of importance. In the study by Xu et al. [12], Dagu chickens had access to the outdoor range from the beginning of the experiment when the chickens were 6 weeks of age. In contrast, when access to the outdoor range occurred in the last 4 weeks of the cycle in broiler chicks, no change in the richness of the broiler intestinal microbiota was found [18]. The hens in our study were only able to access the range from 19 to 20 weeks of age, after transport from the rearing farm, which means that their microbiotas were almost fully developed and had likely reached a stable equilibrium prior to given access to the range. It is known that a well-developed normal gut microbiota protects the host through creating gastrointestinal resistant environments, which prevent external (pathogenic) bacteria from colonizing the gut [19, 20].
Moreover, it is likely that only a small proportion of the hens in the outdoor flocks of our study visited the outdoor range. Although limited information is available about actual range usage of layers, in the Netherlands it has been estimated that in large flocks (> 10.000 layers) only 3–15% of the hens use the outdoor range at a certain timepoint which is partly dependent on the degree of cover provided by trees or artificial structures in the range [1, 21]. This is supported by Hegelund et al. [22] who found that in commercial layer flocks with access to a range, on average only 9% of the chickens used the range area. In contrast, Gebhardt-Henrich et al. [23] reported that 47–90% of chickens in outdoor flocks were registered in the outdoor range at least once over a period of approximately 3 weeks; the individual hens used the range differently, and many of them did not enter the free-range every day. Furthermore, chickens tended to only use the area immediately outside the hen house [22], which has also been observed in the Netherlands, resulting in trampled vegetation closer to the hen house and hence lower availability of fibrous feedstuff for the hens [24]. The outdoor ranges in the Netherlands in general consist of open fields with some tree coverage and bare soil close to the poultry house [21]. Both the limited use of the outdoor range by the hens, together with the low availability of fibrous feedstuff in the range, may explain why we only found limited effects of the outdoor range on the microbial community composition of layers.
We hypothesized that the microbiota of outdoor-layers would be more variable due to their exposure to the outdoor environment and the fact that not all layers use the outdoor range. However, we found more variability in microbiota of indoor-layers compared to the outdoor-layers. Previous research has shown that microbiota of free-range layers contained a greater variability of bacterial species compared to caged layers [25, 26]. The greater variability in the bacterial community composition of the chickens with only indoor housing in our study could be a result of the spatial distribution of the hens within the poultry house. In all flocks in this study, compartments were present in the indoor area of both the flocks with indoor housing and those with access to the outdoor range. This is according to Dutch regulations, that stipulate that poultry houses need to be divided into compartments which contain no more than 6000 hens [27]. Although outdoor poultry houses also have these compartments, layers are able to move freely between these compartments because they have access to the range and can enter another compartment from the outdoor range. This means that the outdoor-layers are more evenly distributed across the poultry house, whereas strictly indoor-layers stay in the same separate compartment within the poultry house all the time. Consequently, this increased level of compartmentalization in indoor-layers can cause a so called cage-effect, which has been reported in several animal studies [26, 28], and could explain the higher variation between layers from indoor houses. Unfortunately, we were not able to adequately measure this effect, because we did not take the compartmentalization into account when sampling the flocks. In future studies where different housing types are being compared with regard to microbiota composition, potential impact of differences in compartmentalisation should be taken into account in the study design.
In order to sample commercial flocks, we opted for cloacal swabs, which served as a rapid and accurate sampling methodology that did not entail sacrificing the birds. The fecal microbiota of chickens are qualitatively similar to the cecal microbiota [29], but more variable [30]. While our sampling technique may explain why we found a high degree of variation between individual chickens, it does not explain the differences in variability in the microbiota of layers of different housing types, as the same sampling technique was used across the study. We found one specific ASV, Dietzia maris, that was only found in outdoor-layers and is related to soil [15]. However, this was a lowly abundant taxon, and we did not detect differences between indoor- and outdoor-layers when we looked at the all ASVs in the genus Dietzia jointly. Moreover, no other genera were found to be differentially abundant across all indoor and outdoor poultry houses. This suggests that although the chickens can pick up some specific taxa from the range, access to an outdoor range does not cause a distinct shift in the microbial community of layers. Therefore, we cannot use community-wide microbiota assessments as a measure for biosecurity or exposure to pathogens from the outdoor environment of a farm.
This study furthermore emphasises the importance of the environment of the poultry house, and the likely influence of daily management on the fecal microbiota, which was also found in broilers [31] and several murine models [32]. In the study by Kers et al. [31], broiler chicks were raised in different housing environments, and were given two diets. These feed interventions alone explained 10% (R2) of the variation in microbiota composition between the broilers, whereas housing condition alone explained 28% (R2). The effect of the poultry house environment explained a similar amount of variation in our study. Future research should aim at better understanding the interactions between the gut microbiota in layers and environmental factors at the level of the poultry house over time. This may shed light on important drivers of microbiota community composition in commercial layers and could contribute to better understanding of ways to modulate the microbiota in favour of chicken health and production.