WANG Lai-di,ZHANG Yang,KONG Ling-ling,WANG Zhi-xiu,BAl Hao,JlANG Yong,Bl Yu-lin,CHANG Guo-bin,CHEN Guo-hong

1 Jiangsu Key Laboratory for Animal Genetic,Breeding and Molecular Design,Yangzhou University,Yangzhou 225009,P.R.China

2 Joint International Research Laboratory of Agriculture and Agri-Product Safety,Ministry of Education/Institutes of Agricultural Science and Technology Development,Yangzhou University,Yangzhou 225009,P.R.China

Abstract This study investigated the effects of floor and cage rearing systems on performance,meat quality,and enteric microorganisms of male and female yellow feather broilers.Two hundred Xueshan chickens (42 days old;100 of each sex) were divided into four groups,according to sex and rearing system (stainless-steel cage or litter floor).Male and cage-reared broilers exhibited better (P＜0.001) performance and higher (P≤0.001) eviscerated percentage than female and floor-reared broilers.The interaction between rearing system and sex had an effect (P＜0.05) on the percentage of breast and thigh muscles.Female floor-reared broilers presented the highest (P＜0.02) percentage of breast and thigh muscles among all the groups.In both sexes,the abdominal fat content in cage-reared broilers was higher (P＜0.01) than that in floor-reared broilers.Female cage-reared broilers exhibited better (P＜0.05) meat quality of breast muscle than other groups.An analysis of welfare observations indicated that the feather quality of floor-reared broilers was better (P＜0.01) than that of cage-reared broilers,whereas the gait scores were not significantly affected (P＞0.05) by rearing system.The enteric microbiota assessment by 16S rRNA amplicon-based sequencing showed that Firmicutes,Proteobacteria,and Bacteroidetes were the most prevalent phyla in both rearing systems,and that the cecum was relatively stable in terms of microbiome composition.Floor-reared broilers exhibited a richer diversity of bacteria in the intestinal tract than cage-reared broilers,while alpha diversity was not significantly different (P＞0.05) among the groups.The ratio of Bacteroidetes to Firmicutes and the abundance of Helicobacter and Romboutsia could potentially affect the production of broilers.These findings indicate that cage rearing improved the production of male Xueshan broilers,which may be due to the difference in enteric microbiota between cage and floor rearing systems.

Keywords:cage rearing,yellow feather broilers,performance,meat quality,microbiome

1.lntroduction

Domestic broilers are a popular protein source for humans.White feather broiler with a fast growth rate accounts for a major portion of the global broiler production.The slowgrowing yellow feather broiler with high meat quality,popular mainly in some Asian countries,accounts for approximately half the market share of chicken in China.Floor,cage,and free-range rearing are the three main methods used for rearing broilers globally.Although the effect of rearing systems on white feather broilers is well understood,there is a lack of information on efficient rearing methods for yellow feather broilers.

The litter floor system is a traditional rearing method used worldwide because of the low production cost and simple operation,although it requires more floor space than other systems.Sawdust,rice husk,bagasse,and wheat straw are often used as litter.In this system,when a chicken rests,its body is in contact with the litter or manure surface(Tamilvananet al.2003).Consequently,floor-reared chickens are subject to unhygienic environmental conditions.Cages of various materials and sizes have been used globally for broiler chicken production for years (Shields and Greger 2013),which helps maximize the use of space and saves energy.It also reduces the occurrence of diseases and parasites,as chickens in cages are not usually in direct contact with their faeces.However,the cage system has not been widely adopted because of animal welfare issues that heavy broiler chickens are prone to leg deformities,breast blisters,and other skin imperfections (Shields and Greger 2013).The free-range system is a healthy rearing system(Stadiget al.2016),and consumers believe that free-range access is important for the welfare of broiler chickens.Although an increase in exercises due to free-range system can increase the feed-to-gain ratio,it also increases feed cost and decreases land use efficiency.Studies on meat tenderness,water-holding capacity and protein content of free-range chickens have reported conflicting results,ranging from superior (Sunet al.2013) to inferior (Castelliniet al.2002) or no difference (Chenet al.2013) as compared to the alternatives.

Cage and floor rearing systems have higher space utilization rate and are therefore more cost-competitive than the free-range system.It was reported that broilers reared on floor were more likely than those in cages to inhale litter dust and noxious fumes which impaired pulmonary gas exchange (Wanget al.2002).Sogunleet al.(2008)showed that Anak titan strains of broiler chicks in the floor system could demonstrate better performance and higher carcass yield than those in the cage system.Only Tonget al.(2015) focused on yellow feather broilers,comparing the effect of the free-range and floor rearing systems on growth performance,carcass yield and meat quality.Besides,it has been proved that sex can impact production performance,slaughter performance and meat quality of yellow feather broilers (Liuet al.2005).Nevertheless,there are limited studies on comparing the effects of sex and rearing systems of yellow feather broilers.Furthermore,the enteric microbiome is closely related to broiler health(Diaz Carrascoet al.2019).This is because variation in the growth rate between sexes may be associated with the difference in the gastrointestinal tract (GIT) microbiome that has a considerable effect on feed digestion,nutrient absorption,and metabolism of chicken (Leeet al.2017).Altogether,it is still not clear about the relationship between the enteric microbiome and broiler production in different rearing systems or sexes.It was hypothesized that boiler production was more effective,under the cage rearing system than the floor rearing system,for male broilers than female broilers,and the difference in enteric microbiota could explain the specific mechanism underlying the changes in performance indicators of different rearing systems (cage and floor) and sexes.For this purpose,we compared the growth performance and meat quality of yellow feather Xueshan broilers of different sexes and in different rearing systems.Besides,differences in enteric microbiome were also analyzed.The results will improve the understanding of the influence of cage and floor rearing systems and provide insights in large-scale production of yellow feather chickens.

2.Materials and methods

2.1.Experimental design and treatments

A completely randomized design involving a 2 (floor rearing system and cage rearing system)×2 (male and female broilers) factorial arrangement of treatments was used in this experiment.Stainless steel cages with size 1 m×1 m were used;they were placed 0.4 m above the concrete.The floor space of 1 m×1 m was allocated,and 6 cm deep fresh wood shavings were used as litter.

2.2.Chickens and feeding

Commercial Xueshan broiler chickens were provided by Jiangsu Lihua Animal Husbandry Co.,Ltd.(Changzhou,China).Male and female Xueshan chickens achieve the body weight (BW) at market size of 1.25–1.5 kg at 91 and 105 d,respectively (Zhang 2012).The chickens were reared for 42 d on a free-range farm.Two hundred yellow feather Xueshan broilers (100 males and 100 females)aged 42 d,with similar BW ((854.38±14.62) g for males and (730.63±10.93) g for females),were randomly selected into four groups (male floor,male cage,female floor,and female cage).Each group had five replicates of 10 chickens each (50 chickens per group).Ten chickens of the same sex were randomly selected to each cage or floor pen.Replicates of all groups were evenly distributed to all parts of the barn to eliminate environmental effects on the groups.The immunization program followed a routine procedure of Xueshan broilers (Liuet al.2020).Lighting was on for 24 h d–1up to day 5 and for 23 h d–1thereafter.

The experiment was conducted in Zhenjiang Tiancheng Agricultural Technology Co.,Ltd.,Jiangsu Province.All chickens were provided the same diet supplied by Jiangsu Lihua Animal Husbandry Co.,Ltd.(Changzhou,China)(Table 1),which met the criteria of the feeding management regulation for yellow feather chicken in China (NY/T 1871-2010).Feed and water were providedad libitum.Throughout the study,the mean daily indoor and outdoor temperature and relative humidity (RH) were similar at 23.5°C (range 19.3–26.0°C) and 60–75%,respectively.

2.3.Response variables

Production performance and carcass traitsThe average BW per replicate was determined at the market age of 91 d for males and 105 d for females.Feed consumption and bird mortality were recorded.After fasting for 12 h,five chickens from each replicate were sacrificed by exsanguination and subjected to a full postmortem examination.The percentage of slaughter,eviscerated weight,breast muscle,thigh muscle,and abdominal fat was calculated for all groups.

Welfare traitsAt market age,all chickens used for welfare observations were assigned feathering and gaiting scores using the score system of 0 to 4 (Table 2) (Joneset al.2005;Mahmoudet al.2015),with zero indicating a good feather or gait condition.

Meat qualityBreast and thigh muscles were collected for meat quality evaluation.The pH of the upper,middle,and lower portions of the left breast and thigh muscles was measured in triplicate 24 h postmortem,using a glass electronic pH meter (DELTA 320;Mettler-Toledo,Swiss)inserted 10 mm into the muscles.After cutting,crushing,and removing the fascia,the meat was homogenized and prepared for water,fat,protein,and collagen analyses,using the FoodScan Meat Product Analyzer (Foss,Denmark)(Anderson 2007).The measurements were taken using the mixture of three individual broilers from the same replicate group,and the analysis was repeated three times.

lntestinal microbiological analysis(1) Library construction and sequencing.The contents in the duodenum,jejunum,ileum,cecum,and colorectum regions of one randomly selected broiler from each replicate were collected immediately after slaughter and frozen at–80°C for meta-genomic analysis.The total genomic DNA of 100 samples was extracted using the QIAamp DNA Stool Mini Kit (QIAGEN,Valencia,CA).The quality and concentration of DNA was estimated by agarose gel electrophoresis (Liuyi,China) using a NanoDrop 2000 Spectrophotometer (Thermo Scientific,USA).The PCR amplifications were conducted with specific primers,338F (5´-ACTCCTACGGGAGGCAGCAG-3´) and 806R(5´-GGACTACHVGGGTWTCTAAT-3´) (Leeet al.2012),which were used to amplify the V3–V4 region of the 16S rRNA gene.Sequencing was performed on the Illumina HiSeq 2500 platform by Novogene (Beijing,China),which generated 250 bp paired-end reads.

Table 1 Ingredients of diet and nutrition level of Xueshan chicken

Table 2 Feathering and gaiting score systems for broilers at market age

(2) Data analysis.The paired-end reads were merged using fast length adjustment (FLASH) (V1.2.7) (Magoc and Salzberg 2011),and the raw tags were filtered by quality using quantitative insights into microbial ecology(QIIME) (V1.7.0) (Caporasoet al.2010).Chimera sequences in the acquired sequences were filtered using undetected chimeras (UCHIME) algorithm (Edgaret al.2011).Sequences analysis was performed using Uparse Software (v7.0.100) (Edgar 2013).Effective tags were clustered into operational taxonomic units (OTUs) at 97%identity using Uparse Software,after Ribosomal Database Project (RDP) classification (version 2.2) (Wanget al.2007).The representative sequence of each OTU was used to annotate the taxonomic data based on the GreenGene Database (DeSantiset al.2006).OTU abundance data were normalized using a standard sequence corresponding to the sample with the least number of sequences.

(3) Alpha and beta diversity analyses.Alpha diversity was determined by analyzing the complexity of species diversity for a sample using four indices,namely,Chao1,Shannon,Simpson and ACE.All these indices were calculated using QIIME (version 1.7.0),and displayed with R Software (version 2.15.3).Beta diversity was analyzed by principal coordinate analysis (PCoA) using weighted and unweighted UniFrac distance metrics.The cluster analysis was preceded by the principal component analysis that reduced the dimensionality of the original variables,using FactoMineR package and ggplot2 package in R Software(version 2.15.3).

(4) Function analysis of microbial populations.The linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed to rank abundant modules in cage-and floorreared broiler samples (Segataet al.2011).A size-effect threshold of 4.0 on the logarithmic LDA score was used as a discriminative functional marker.Spearman’s correlation analysis was performed to assess the pivotal functions of the bacterial taxa,irrespective of statistical threshold.Functional Annotation of Prokaryotic Taxa (FAPROTAX),a database that extrapolates the functions of bacteria to estimate metabolic or other ecologically relevant functions,was used to evaluate the difference between the cage and floor rearing systems (Loucaet al.2016).

2.4.Statistical analysis

Statistical analyses of all traits were performed using the generalized linear models (GLM) in SPSS 17.0 (SPSS Inc.,Chicago,IL,USA),with a 2×2 factorial arrangement(two sexes and two rearing systems).Each replicate was the experimental unit for growth performance analysis and each chicken was the experimental unit for other index analyses.An arcsine transformation was applied to the percentage data before statistical analysis.The statistical models included the main effects of rearing system (cage or floor),sex (male or female),and their interaction (rearing system×sex).Differences among means were tested by the least significant difference (LSD) method,and Duncan’s multiple-range test was conducted to separate means when the interactive effects differed significantly.All data were presented as mean±standard error (SE) and a level ofP＜0.05 was used as the criterion for statistical significance.GraphPad Prism 5 (GraphPad Software Inc.,San Diego,CA) was used for graphical representations.

3.Results

3.1.Growth performance

To determine the effect of the rearing systems on growth performance,the daily feed intake,daily weight gain,and mortality rate were recorded and analyzed (Table 3).Rearing system or sex affected (P＜0.001) the body weight and daily weight gain of broilers between day 42 and the market age.Cage-reared broilers and male broilers had higher (P＜0.001) body weight and daily weight gain than floor-reared broilers and female broilers,respectively.Rearing system,sex and their interaction affected (P＜0.001)the daily feed intake.Cage-reared male broilers had higher(P＜0.001) daily feed intake than others.However,male broilers had lower (P＜0.001) feed-to-gain ratio than female broilers.No significant differences (P＞0.57) were observed in mortality among the four groups.

3.2.Carcass traits

Rearing system,sex and their interaction affected (P＜0.001)breast muscle percentage and abdominal fat percentage of broilers (Table 4).Floor-reared female broilers had the highest breast muscle percentage,while cage-reared female broilers had the highest abdominal fat percentage,among the four groups.Rearing system or sex affected (P＜0.001)eviscerated percentage of broilers.The cage-reared broilers and male broilers had higher (P＜0.001) eviscerated percentage than floor-reared broilers and female broilers,respectively.A significant effect (P＜0.05) of the interaction between rearing system and sex was observed on thigh muscle percentage.Compared with other groups,thigh muscle percentage was significantly increased (P＜0.05) infloor-reared female broilers.The slaughter percentage did not vary significantly (P＞0.24) by rearing system or sex.

Table 3 Effects of rearing system and sex on daily weight gain,daily feed intake,feed/gain,and mortality of broilers from 42 d of age to market age

Table 4 Effects of rearing system and sex on slaughter index of Xueshan chickens at market age (91 d for males and 105 d for females)

3.3.Meat quality

Rearing system or sex affected (P＜0.05) the pH value of breast and thigh muscles,protein content of breast muscles,and fat content of thigh muscles of broilers (Table 5).The pH value of breast muscles of male broilers was lower than that of female broilers,which was consistent with the result about thigh muscles.Moreover,the pH value of breast muscles of cage-reared broilers was higher than that of floor-reared broilers,which was contrary to the result of thigh muscles.Sex and the interaction between rearing system and sex had effects (P＜0.05) on the collagen content of breast and thigh muscles,and the water content of thigh muscles of broilers.Rearing system,sex and their interaction affected(P＜0.05) water content,fat content of breast muscles,and protein content of thigh muscles.Cage-reared female broilers had lower (P＜0.001) water content of breast muscles and higher (P＜0.001) protein content of breast muscles than other groups,and floor-reared male broilers had the highest (P＜0.05) protein content of thigh muscles among all the four groups.The fat content of thigh muscles was higher (P＜0.001) in cage-reared broilers and female broilers than in floor-reared broilers and male broilers,respectively.

3.4.Welfare assessment

Sex and its interaction with rearing system did not significantly affect (P＞0.05) feather scores of broilers (Table 6).Only rearing system significantly affected (P＜0.01) the proportion of 0 and 1 scores for feather assessment.The proportion of 0 scores for floorreared broilers was higher (P＜0.001) than that for cage-reared broilers,which was contrary to the result about the proportion of 1 scores.Rearing system,sex,and their interaction did not significanlty affect(P＞0.17) gait scores of broilers.These results showed that floor-reared chickens had better feather quality than cage-reared ones.There was no significant difference in chickens’ walking ability between the two rearing systems.

3.5.Microbial complexity

On an average,84 600 sequences were obtained from the five intestinal regions (duodenum,jejunum,ileum,cecum,and colorectum) of each of the 20 examined broilers.The sequences were assigned to 6 567 OTUs based on 97% species similarity.All acquired OTUs were mapped to 50 phyla,66 classes,148 orders,296 families,and 847 genera.The rarefaction curves tended to attain the saturation plateau,showing that the microbiota of the 20 samples was large enough to estimate the phenotype richness and microbial community diversity at the 97% similarity threshold (Fig.1-A and B).The top 10 phyla in the intestine are presented in Fig.1-C (males) and D (females).Firmicutes,Proteobacteria,Bacteroidetes,Cyanobacteria,andFusobacteriamembers were the most prevalent phyla in both rearing systems,and they made up more than 90% of the microbiome.In the colorectum region of caged broilers,Firmicutesmembers were lower andProteobacteriamembers were higher than those of floor-reared ones.Compared with other intestinal segments,cecum was relatively stable in terms of the percentage of bacteria at the phylum level.

3.6.Shared and unique microbial populations

To investigate the enteric microbial community in the two rearing systems,a comparison of microbial similarity was performed.We also analyzed the common and unique OTUs as shown in the Venn and Petal diagram in Fig.2-A–D.The cage and floor rearing systems shared 49.8% of the microbial populations in male broilers and 53.4%in female broilers.Male broilers had more diverse intestinal flora than female broilers.In both rearing systems,there was a high variation in OTUs in the duodenum,jejunum,and ileum.There were fewer unique OTUs in the posterior segment,that is,the cecum and colorectum,than in other segments.It was found that 27.3% of unique microbial populations were identified in floor-reared chickens,and male broilers showed higher microbial diversity than female broilers (Fig.2-E).

3.7.Similarity and diversity in microbial community composition

Rearing system,sex and their interaction did not affect (P＞0.06) the alpha diversity of the enteric microbiome (Table 7) according to the Shannon,Simpson,Chao 1,and ACE values.The beta diversity analysis showed that the PCoA plot with PC1 and PC2 accounted for 47.06 and 18.33% of the total variation,respectively (Fig.3-A).The results revealed that the microbial communities in cecum formed clusters and that they were more similar,but there were overlaps among the duodenum,jejunum,ileum,and colorectum.The top 15 bacterial phyla in terms of relative abundance were different between the two rearing systems (Fig.3-B).The abundance of some phyla in males and females differed (Fig.3-C and D).

3.8.Microbial functional characteristics

The LDA distribution histogram showed 17 kinds of bacteria in male broilers (Fig.4-A) and 15 kinds of bacteria in female broilers (Fig.4-B),which could be potential marker microorganisms between the cage and floor rearing systems.Female broilers showed increased abundance ofFirmicutesmembers andLactobacillus,and male broilers showed increased abundance ofProteobacteria,Stenotrophomonas,Xanthomonadaceae,Xanthomonadales,andGammaproteobacteriamembers (Fig.4-C).FAPROTAX was used for microbial function analysis.The ecological functions of bacteria and archaea in the intestinal sections were classified and sorted using the FAPROTAX Database.Chemoheterotrophy,fermentation,and aerobicchemoheterotrophy were the three main functions of the bacteria in both male and female broilers (Fig.4-D and E).

4.Discussion

Rearing system and sex are two key factors affecting production efficiency and physical condition of poultry.Thus,it is highly beneficial for broiler producers to identify the effect of rearing system and sex on broiler production performance,meat quality and enteric microorganism,and identify the effect of microflora on production performance of yellow feather broilers.Results from the present study supported the hypotheses that cage-reared broilers and male broilers exhibited a higher level of production performance than floor-reared broilers and female broilers,respectively.However,floor-reared broilers presented better welfare outcomes and enteric microbiome diversity than cage-reared ones.It is concluded that the ratio ofBacteroidetestoFirmicutes,the abundance ofHelicobacterandRomboutsiacould potentially affect the production performance of broilers.

It has been reported that the market size BW and daily weight gain of cage-reared broilers were higher than those of floor-reared broilers (LiJet al.2017),which is consistent with our study.The eviscerated percentage ranged between 60 and 66% in this study,which is in agreement with the results of a previous study (Jianget al.2018).Cage-reared broilers and male broilers exhibited higher eviscerated percentage than floor-reared broilers and female broilers,respectively.The cage rearing system has been reported to increase meat production due to the physical restraint and limited space (Chenet al.2019).Compared with floor-reared broilers,the abdominal fat percentage was increased in cage-reared broilers,in both sexes.Similar results have been reported by a previous study (Sogunleet al.2008).

Fig.1 Rarefaction curve and phylum-level analysis (n=5).A,rarefaction curve sequences showing the microbial community complexities in the five gut sections of male broilers from the two rearing systems.B,rarefaction curve sequences showing the microbial community complexities in the five gut sections of female broilers from the two rearing systems.C,relative abundance of male broiler intestinal microbes at the phylum level.D,relative abundance of female broiler intestinal microbes at the phylum level.CCe,cage cecum;CCo,cage colorectum;CDu,cage duodenum;CIl,cage ileum;CJe,cage jejunum;FCe,floor cecum;FCo,floor colorectum;FDu,floor duodenum;FIl,floor ileum;FJe,floor jejunum.

The quantifiable properties of meat such as pH,collagen content,protein solubility,and fat-binding capacity are indispensable features for poultry meat (Miret al.2017).Especially,the intramuscular fat content (IMFC) has a positive effect on meat characteristics such as tenderness,flavor,and juiciness (Yeet al.2014).In this study,the higher fat content in the breast muscles of cage-reared broilers could have enhanced the meat flavor.Female broilers and floor-reared broilers presented a higher fat content in the thigh muscles than male broilers and cage-reared broilers,respectively.The abdominal fat weight (AFW) and fat content in the breast and thigh muscles of cage-reared broilers were higher than those of floor-reared broilers.Chenet al(2008)reported a strong correlation between IMFC and AFW in Chinese Beijing-You chickens (a Chinese local breed).Here,the pH of female broilers was higher than male broilers,and the change trend of the pH in breast muscles was contrary to that in thigh muscles in both rearing systems.Exercise is an important factor affecting muscle pH and meat tenderness.In chicken meat,increased muscle pH reflects decreased glycogen reserve (Le Bihan-Duvalet al.2008).Therefore,differences in exercise between males and females,or between different rearing systems,may affect the physical activities of breast and thigh muscles.

Feather characteristics are basic indicators of environmental conditions and crowdedness in a broiler house (Savory and Hughes 2010).Gait scores are used to indicate walking ability,leg problems,and lameness(LiHet al.2017).The welfare assessment indicated that broilers in the floor group showed better feather quality than those in the cage group,but there was no apparent difference in the gait score between the two rearing groups.Rearing system has a direct influence on leg deformities and feather problems (Shields and Greger 2013).Studies on the welfare of yellow feather broilers in cage and floor systems are limited,except for one study that demonstrated the netting system improved the cleanliness of feathers by separating chickens from litter (LiHet al.2017).The welfare outcomes can be explained by different evaluation criteria.Furthermore,the active feature of yellow feather broilers and limited cage space may reduce feather quality.

Fig.2 Venn diagram and petal diagram of operational taxonomic units (shared or unshared).A,the Venn diagram OTUs shared or unshared by male broiler in the floor and cage rearing systems.B,the Venn diagram OTUs shared or unshared by female broilers in the floor and cage rearing systems.C,the petal diagram OTUs shared or unshared in five gut sections in male broilers in the two rearing systems.D,the petal diagram OTUs shared or unshared in five gut sections of female broilers in the two rearing systems.E,the Venn diagram of OTUs shared or unshared by the four groups.FJe,floor jejunum;CIl,cage ileum;CDu,cage duodenum;CCo,cage colorectum;FDu,floor duodenum;FCe,floor cecum;CCe,cage cecum;CJe,cage jejunum;FIl,floor ileum;FCo,floor colorectum.CM,cage male;FF,floor female;FM,floor male;CF,cage female.

The enteric microbiome has been extensively studied in a wide range of species,including humans (Daliriet al.2017),pigs (Isaacson and Kim 2012),and chickens (Stanleyet al.2014),due to its association with health and productivity.Rearing system influnces the microbiome,which in turn affects animal health.Of the 6 567 OTUs,at phylum level,Firmicutes,Proteobacteria,Bacteroidetes,Cyanobacteria,andFusobacteriamembers made up more than 90% of allbacteria in both cage and floor rearing systems.This is consistent with the result of a previous study (Maet al.2018).The abundance of intestinal flora was different in different segments.FirmicutesandProteobacteriamembers were the dominant bacteria in duodenum,jejunum,and ileum,whereasFirmicutesandBacteroidetesmembers were dominant in cecum and colorectum.This is consistent with the results of a previous study (Xiaoet al.2017).Microbial communities in cecum were more stable than those in other intestinal segments.

Table 7 Alpha diversity index of the intestinal microbiome of broilers at market age (91 d for males and 105 d for females)

The enteric microbiota is a very dynamic entity influenced by environment and nutritional behaviors (Paoliet al.2019).The floor rearing system exhibited a richer diversity of bacteria than the cage rearing system.This could be because the broilers reared in the floor group had access to their feces,which is beneficial for short-term intestinal micro-flora building and gut development.The alpha diversity of the microbiome in cage-reared broilers did not differ from that in floor-rared ones.A previous study reported that the pathogenic bacteriaEscherichia coliincreased andBifidobacteriadecreased in the floor group compared with the cage group (Li Jet al.2017).Furthermore,the proportion ofFirmicutesmembers was lower and that ofProteobacteriawas higher in the colorectum of the cage group than in that of the floor group.The highBacteroidetesmembers and lowFirmicutesmembers have been associated with obesity in humans (Leyet al.2006).In the present study,although the diversity of bacterial communities was similar between the two rearing systems,the abundance of some microbes varied.For example,the cage group had lower abundance ofFirmicutesmembers than the floor group,which might be because the former had higher BW and abdominal fat content than the latter.Helicobacteris a bacterial genus living mostly in the upper gastrointestinal tract which was often considered to be infectious and pathogenic (Huaet al.1999).Helicobactercould be potential marker microorganisms for floor-reared male broilers,andHelicobactermay have negative effect on the production.Romboutsiawas potential marker microorganism for cage-reared male broilers and floorreared female broilers.Romboutsiathat were positively correlated with lipid metabolism indicators (Liet al.2019)might have positive effect on the production.

Sex is an important factor affecting the intestinal bacterial communities (Lumpkinset al.2008;Zhaoet al.2013).Here,male broilers presented more diverse intestinal flora than female broilers.Thus,the depletion of enteric microbiota may lead to the loss of the“metabolic buffering”function of the microbiota (Díaz-Sánchezet al.2019),causing weight loss in female broilers.Female broilers showed increased relative abundance ofFirmicutesmembers andLactobacillus,and male broilers showed increased abundance ofProteobacteria,Stenotrophomonas,Xanthomonadaceae,Xanthomonadales,andGammaproteobacteriamembers.A previous studyreported that manyFirmicutesandTenericutesmembers were observed in female broilers,whereasBacteroidetesmembers were observed in male broilers (Leeet al.2017).The differences in bacterial community in the intestinal tract between female and male broilers are possibly related to different biological processes (Lumpkinset al.2008) such as sex hormone secretion.

5.Conclusion

Results of the present study indicate that rearing system,sex and their interaction affected a number of production indices to different degrees.In general,the selection of cage rearing system or male yellow feather broilers can achieve a more efficient production performance than the alternatives,and the selection of floor rearing system could obtain good feather quality and diverse enteric microbiome for broilers.

Fig.3 Alpha and beta diversity analyses in microbial community composition.A,weighted principal co-ordinates analysis of dissimilarity among male broiler microbial samples in each group.B,weighted PCoA of the dissimilarity between among female broiler microbial samples in each group.C,Heatmap of the top 15 most abundant phyla in male broiler microbial samples in each group.D,Heatmap of the top 15 most abundant phyla in female broiler microbial samples in each group.The phyla with higher abundance are shown in red,whereas those with lower abundance are shown in blue.FDu,floor duodenum;CDu,cage duodenum;FJe,floor jejunum;CJe,cage jejunum;FIl,floor ileum;CIl,cage ileum;FCe,floor cecum;CCe,cage cecum;FCo,floor colorectum;CCo,cage colorectum;M,male;F,female.

Fig.4 Potential marker microorganisms and functional characteristics between the cage and floor rearing systems.A,potential marker microorganisms in male broilers in the cage and floor rearing systems.B,potential marker microorganisms in female broilers in the cage and floor rearing systems.C,potential marker microorganisms in male and female broilers.D,the ecological functions of microorganisms in the gut of male broilers from the two rearing systems,sorted using the FAPROTAX database.E,the ecological functions of microorganisms in the gut of female broilers from the two rearing systems,sorted using the FAPROTAX Database.F,female;M,male;CCe,cage cecum;CCo,cage colorectum;CDu,cage duodenum;CIl,cage ileum;CJe,cage jejunum;FCe,floor cecum;FCo,floor colorectum;FDu,floor duodenum;FIl,floor ileum;FJe,floor jejunum.

Acknowledgements

This work was supported by the National Science &Technology Pillar Program of China (2015BAD03B03),the National Natural Science Youth Fund of China (31802057),and the priority academic program development of Jiangsu Higher Education Institutions,China.

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

All animal experimental procedures were approved and guided by the Institutional Animal Care and Use Committee of Yangzhou University,China (approval number:182-2019).