Fast and slow-growing breeds: characterisation of broiler caecal microbiota development throughout the growing period

Background: The caecal microbiota and its modulation play an important role in animal health, productivity and disease control in poultry production. In this sense, it could be considered as a biomarker of poultry health. Furthermore, due to the emergence of resistant bacteria and the increasing social pressure to establish animal-friendly management on farms, producers are motivated to select more extensive and antibiotic-free breeds. It is therefore necessary to gain better knowledge on the development of major bacteria in healthy broilers, both in commercial fast-growing and in new slow-growing breeds. Hence, the aim of this study was to characterise caecal microbiota in two genetic poultry breeds throughout the growing period using 16S rRNA sequencing analysis. Results: A total of 50 caecal pools (25 per breed) were sequenced by the 16S rRNA method. The complexity of caecal microbiota composition increased signicantly as animals grew. Furthermore, there were statistical differences between breeds at the end of the growing period. The dominant phyla throughout the production cycle were Firmicutes, Bacteroidetes and Proteobacteria. The predominantly identied genera were Ruminococcus spp., Lactobacillus spp. and Bacteroides spp. Conclusion: The results showed that the main caecal bacteria for both breeds were similar. Thus, these phyla or genera should be considered as biomarkers of poultry health in the evaluation of different treatments applied to animals.

these animals represent an important food resource for humans, while also being a potential reservoir of food-borne pathogens [1,20].
Moreover, due to the emergence of antimicrobial-resistant bacteria, society is pressing for a reduction in antibiotic administration by nding effective alternatives to control infectious diseases at farm level [21][22][23][24]. Some of these alternatives are feed additives (prebiotics, probiotics, symbiotics, organic acids, enzymes, phytogenics and metals), alternative medical treatments (antibacterial vaccines, immunomodulatory agents, antimicrobial peptides and bacteriophages) and, nally, different broiler growth systems [25][26][27][28][29][30][31]. Although the bene cial effects of many of these alternatives have been demonstrated in vitro, the general consensus is that the effect of these products depends on the farm, management and animal characteristics, such as the breed selected [19,23,32].
Historically, producers have looked for breeds adapted to intensive conditions and with the best productive performance. However, in response to social pressure to establish welfare-friendly management systems in poultry production, producers are motivated to choose breeds selected for their ability to cope with the natural environment and with a lower performance [27]. Therefore, in order to make management decisions, it is necessary to have better knowledge of the effect of these alternatives under production conditions, both in commercial fast-growing and slow-growing breeds [6,20,33,34].
In this context, the aim of this study was to characterise the caecal microbiota in two genetic poultry breeds, fast-growing and slow-growing, during the growing period, using 16S rRNA sequencing analysis.

Results
During this study, a total of 50 caecal pools (25 per breed) were collected, processed and sequenced. No clinical signs were observed, and the productive parameters obtained were in accordance with the breed standards. There were no statistical differences between replicates (P-value > 0.05). 16  Assessment of rarefaction curves based on the Chao1 biodiversity index calculated for the six sequence read groups (day-old chicks, mid-period and slaughter day results for fast and slow-growing breeds) indicated that four of the curves tended to reach a plateau. However, samples from groups 1 and 2 (dayold chicks from both breeds) are at the limit of the rarefaction, leaving a rarefaction number of 72 060 reads (Fig. 1). The Chao1 alpha diversity index reveals a notable difference between the caecal microbiota depending on the age of the animals (Table 1). Statistically signi cant differences (P-value < 0.05) were found between these groups; samples from day-old chicks of both breeds (88.3 and 111.9 for the fast and slow-growing breed, respectively) displayed a lower level of complexity of the microbiota compared to that found at mid-period (384.4 and 373.8), and samples from mid-period animals displayed a lower level of complexity than the samples from the end of the growing period (420.3 and 447.2 for the fast and slow-growing breed, respectively). Finally, there were statistically signi cant differences in gut microbiota diversity between both breeds at the end of the growing period (P-value < 0.05).

Differential gut microbiota composition
Inspection of predicted taxonomic pro les at phylum level for all samples is summarised in Table 2 and represented in Fig. 2. This analysis exhibited that Firmicutes represented the dominant phylum of the caecal community in both breeds at all sampling times in the production cycle (P-value < 0.05). At the onset of the growing period, Proteobacteria was the second prevalent phylum for fast and slow-growing breeds, outnumbering the Bacteroidetes phylum. However, during the rest of the production cycle, Bacteroidetes phylum was more abundant than Proteobacteria in both breeds. The longitudinal study showed that there were no statistically differences between breeds throughout the growing period (Pvalue > 0.05).
For the fast-growing breed, there were statistically signi cant differences depending on the time of sampling. Proteobacteria and Bacteroidetes phyla were more abundant at the arrival day (36.4% and 5%, respectively) and at the slaughter day (1.5% and 5.7%, respectively), though the high Firmicutes percentage was observed at mid-period (95.1%).
For the slow-growing breed, Bacteroidetes (5.7% and 9.3% at arrival and slaughter days, respectively) and Firmicutes (95.2% at mid-period) showed the same pattern as in the fast-growing breed. However, statistically signi cant differences were shown between day-old chicks and mid-period percentage of Proteobacteria (32.8% and 1.2%, respectively), which subsequently remained stable until the end of the cycle (1.7%). In the longitudinal study, the only statistical differences were between breeds in Lactobacillus spp. at the end of the growing period (2.9% and 2.8% in fast and slow-growing breed, respectively). In order to further identify differences in microbiota composition between breeds, we focused on 33 genera, which were shown to be present at an average relative abundance of more than 0.5% in at least one sample group [35].
For the fast-growing breed, the results for the genera analysis are shown in Table 3   Finally, in order to assess differences in microbiota between breeds, we analysed the beta diversity based on unweighted UniFrac for these groups, after which the UniFrac distance matrix was represented through Principal Coordinate Analysis (PCA) (Additional le 1). Statistically signi cant differences only appeared between breeds at the end of the growing period (P-value < 0.05). Moreover, the comparisons of beta diversity and genera presence between both breeds in the different sampling times are represented in Fig. 4, and genera data details are summarised in Additional le 2.

Discussion
The present study assessed the development of gut microbiota composition in fast and slow-growing breeds throughout the growing period under commercial farm conditions. To our best knowledge, this is the rst study in the scienti c literature to evaluate the relationship between both breeds on commensal bacteria evolution in caeca under the same production conditions.
As described previously, microbiota plays an important role in animal health. Moreover, its alteration (dysbacteriosis) is associated with reduced physiological functions, which represents its importance as biomarker [36][37][38]. It is well demonstrated that a greater complexity of the gut microbiota is observed as animals grow. However, in accordance with other studies, our ndings showed that the bacterial richness became relatively stable at mid-period for both breeds under the same production conditions [19,[39][40][41][42][43][44][45]. This fact evidences the importance of ock management during the production cycle in terms of the microbiota status, as the transmission between environmental and intestinal bacteria is proven [2,7,9,19,42,[46][47][48]. It is important to highlight that bacterial diversity in the intestinal tract is higher in birds with high feed e ciency [2,9,[49][50][51]. Nevertheless, in this study there were only statistically signi cant differences between breeds at slaughter day, probably due to the age difference of the experimental groups, but not to the breed, intestinal health or feed conversion index. Furthermore, a low diversity and evenness of the microbiota could be also a health status indicator, as it is constantly associated with poor intestinal health and, therefore, with lower performance parameters [18,52,53].
Regarding gut microbiota composition, the predominant phyla obtained in this study were Firmicutes and Bacteroidetes, followed by Proteobacteria [7,9,42,45,47,54]. Thus, any alteration in the microbiota balance could lead to an alteration on the health or productivity of the breed. Firmicutes constitutes a heterogeneous phylum containing bacterial groups with different metabolic activities, and several studies have shown that a high level of this phylum is correlated with good intestinal health [18,55]. Conversely, an increment of Proteobacteria is associated with dysbiosis and, consequently, with an increase in the presence of zoonotic bacteria belonging to this phylum, such as Salmonella or Campylobacter. For this reason, it is important to ensure strict biosecurity and management control at the beginning of the growing period, when Proteobacteria presents its higher levels, as any stress that could produce an increment in this phylum may result in higher shedding of pathogenic bacteria and environmental contamination throughout rearing [2,9,18,56,57]. Finally, the Bacteroidetes phylum plays an important role in converting fermentable starch to simple sugars and these, in turn, to volatile fatty acids to meet the energy demand of the host, so their presence could be especially affected by diet components [40,54]. It is important that any antibiotic alternative introduced in farms, such as feed additives or management practices, should not disturb microbiota balance, particularly for phyla related to pathogenic bacteria.
At genus level, the most predominant genera were Ruminococcus spp., Lactobacillus spp. and Bacteroides spp., in line with data reported by other authors [14,45,47]. In this experiment, statistically signi cant differences between breeds were found only for Lactobacillus spp. at the end of the growing period, being higher in fast-growing animals. This is an important probiotic in promoting healthy gut, as these bacteria are believed to be responsible for starch decomposition and lactate fermentation [6,14,42]. Ruminococcus spp. is known for its ability to degrade complex carbohydrates and thus may have contributed to an improved degradation of dietary bre [58,59]. In turn, Bacteroidetes spp. plays an important role in breaking down complex molecules to simpler compounds which are also essential for growth of the host and gut microbiota development. These functions are associated with higher production rates, so it might be said that high levels of these genera are indicators of adequate intestinal health in poultry [5,6,14,42]. Thus, it is important to maintain these genera at higher levels throughout the growing period.

Conclusions
In conclusion, despite the demands from society for slow-growing breeds in a welfare-friendly production management and the fact that there are many possibilities of microbiota composition due to the variations between different farms and ocks, the results revealed that the main caecal bacteria in both breeds are the same, so the bene ts derived thereof are also similar. For this reason, it is important to consider some important phylum or genera levels as biomarkers of gut health, controlling their development throughout the growing period to be able to evaluate the different treatments applied to animals.

Methods
In this experiment, all animals were handled according to the principles of animal care published by Spanish Royal Decree 53/2013 [60].

Experiment design
The study was performed in an experimental poultry house in the Centre for Animal Research and Technology (CITA, in its Spanish acronym (Valencian Institute for Agrarian Research, IVIA, Segorbe, Spain)). To this end, 576 broilers (males and females) provided from the same hatchery were randomly housed in two identical poultry rooms (replicates A and B) and 288 animals were housed in each room (144 fast-growing and 144 slow-growing breed). In addition, animals were distributed in 24 pens (12 pens for each breed) of 1.3 m 2 in a nal stocking density of 35 kg/m 2 , with wood shavings as bedding material. The two commercial breeds used were one fast-growing (Ross®) and one slow-growing (Hubbard®). The fast-growing breed is characterised by e cient feed conversion and a good meat yield [61]. In contrast, the slow-growing breed is focused on the criteria of animal welfare and absence of antibiotics [62].
To simulate the real conditions of poultry production, the houses were supplied with programmable electric lighting, automated electric heating and forced ventilation. The environmental temperature was gradually decreased from 32ºC (arrival day) to 19ºC (slaughter day).
The birds received drinking water and were fed ad libitum. Nutritional and product analysis were assessed before the arrival of animals. Two different age commercial diets were offered to the animals: starter (1 day to 21 days) and grower (21 days to 42/63 days). Only one batch of feed per age (starter and grower) was manufactured. The starter diet was the same for both breeds, while the grower feed was the standard diet speci c for each one. Mortality rates and diarrhoea presence were recorded daily. Finally, animals were weighed at weekly intervals and feed consumption per pen was recorded.

Sample collection
To assess the development of microbiota composition of broilers throughout the growing period, 30 animals from each experimental group were randomly selected and caecal samples were collected at different times of the growing period: on arrival (day-old chicks), at the mid-period (21 days old) and before slaughter (42 days of age in fast-growing, and 63 days in slow-growing). Caecal samples were taken individually and placed in sterile jars. The samples were processed immediately after collection.

DNA extraction
Caecal content was removed and homogenised. Afterwards, pools of six animals of each breed from each room were prepared (5 pools/experimental group) and the DNA of pools content was extracted according to the manufacturer's instructions (QIAamp Power Fecal DNA kit, Qiagen, Hilden, Germany) and frozen at -80ºC for shipment to the Centre for Biomedical Research of La Rioja (CIBIR, in its Spanish acronym, Logroño, Spain).

16S rRNA sequencing analysis
First, all samples received were analysed in a Fragment Analyzer (Genomic DNA 50Kb kit, AATI) to ensure their integrity. Additionally, the initial DNA concentration was measured by means of a Qubit uorometer (dsDNA HS Assay kit, Invitrogen). From 12.5 ng of DNA (evaluated in Qubit) of each sample, the library was prepared following the instructions of the 16S rRNA Metagenomic Sequencing Library Preparation (Illumina) protocol. The sequencing run was performed in a MiSeq (Illumina) system in 2 × 300 bp format.
The quality of the raw unprocessed reads was evaluated using the FastQC software Authors' contributions LMD contributed to the acquisition of data, data analysis and manuscript writing. AV and CM contributed to the conception and design of the study. MT contributed to the data analysis. MTPG and SV contributed to the acquisition of data. All authors revised the paper critically and approved the nal version of the manuscript.

Figure 1
Evaluation of alpha diversity in fast and slow-growing breeds.

Figure 2
Taxonomic analysis at phylum level throughout the growing period. A: Phyla evolution throughout the growing period for the fast-growing breed (AD: arrival day, MP: mid-period, E: end). B: Phyla evolution throughout the growing period for the slow-growing breed (AD: arrival day, MP: mid-period, E: end).

Figure 3
Taxonomic analysis at genus level throughout the growing period. A: Evolution of genera throughout the growing period for fast-growing breed (AD: arrival day, MP: mid-period, E: end). B: Evolution of genera throughout the growing period for slow-growing breed (AD: arrival day, MP: mid-period, E: end).