Housing systems are among the most important non-genetic factors affecting hen production performance and intestinal microbes. With increased interest in animal welfare, cage-free laying hen housing systems have become common, providing behavioral freedom and health benefits. The present study aimed to compare the effects of plastic net housing system (NRS) and floor litter housing system (LRS) on the composition and function of the duodenal and cecal microbiota in Shendan chicken, one of the most popular laying hen strains in China. The associations between the differential microbiota abundance and production traits and intestinal morphological parameters were determined. Compared with the LRS, the NRS improved the laying rate (p < 0.05) and increased the villus height (VH) of the duodenum (p < 0.05) and the VH-to-crypt depth ratio (VCR) of the cecum (p < 0.05). Alpha diversity analysis showed that LRS chickens had a significantly higher diversity and richness than NRS chickens. Beta diversity analysis demonstrated differences in the microbiota composition based on housing systems. Within the cecum, Proteobacteria and Kiritimatiellaeota were significantly more abundant in the LRS than in the NRS (p < 0.05), while Bacteroidetes were significantly less abundant in the LRS (p < 0.05). Phascolarctobacterium and Ruminococcaceae_UCG-005 were significantly less abundant in the LRS (p < 0.05) compare to the NRS. Within the duodenum, Lactobacillus was significantly less abundant in the LRS (p < 0.05) than in the NRS, while Pseudomonas was significantly more abundant in the LRS (p < 0.05). Cecal Phascolarctobacterium and Ruminococcaceae_UCG-005 were significantly positively correlated with eggshell strength (R = 0.608, p < 0.01) and egg weight (R = 0.526, p < 0.05), respectively. Duodenal Lactobacillus was significantly positively correlated with VH and VCR (R = 0.548 and 0.565, p < 0.05), while Pseudomonas was significantly negatively correlated with the Haugh unit (R = −0.550, p < 0.05). In conclusion, there are differences in the cecal and duodenal microbiota compositions of Shendan laying hens reared in different non-cage housing systems, and the NRS was superior to the LRS in improving the laying performance and intestinal morphology and microecological environment.
The microbes in poultry intestines play an essential role in feed conversion (1), nutrient digestion and absorption (2), host protection against pathogens (3), and the maintenance of intestinal physiological balance (4) by affecting the intestinal structure and modulating the function of the digestive and immune systems. The duodenum is crucial for food digestion and absorbs most glucose and other nutrients, with the phyla Proteobacteria and Firmicutes being predominant (5, 6). The cecum mainly ferments complex carbohydrates and has a greater ability to absorb sugars actively at low concentrations, with the phyla Bacteroidetes and Firmicutes being predominant (6, 7). However, the diversity and community structure of gut microbes in chickens are influenced by many factors, such as dietary changes, geographical locations, growth phases, and rearing conditions. Feeding broilers with fructooligosaccharide enhanced the growth of Bifidobacterium and Lactobacillus but inhibited Escherichia coli in the small intestinal and cecal digesta (8). Variations in microbe composition and diversity were found among populations of chicken cecal bacteria from five locations in Tibet (9). The ileum and cecum developed their own unique bacterial community during different growth periods as the broilers matured (10). Dysbiosis of the intestinal microbiota could lead to impaired digestion and immunity, which causes an increase in susceptibility to pathogens and results in reduced growth performance and health status. H Type Battery Cage
Although extensive studies concerning the factors that affect bacterial microbes in the gut of chickens have been conducted, little is known about the effects of housing systems on intestinal bacterial communities and functions in chickens. The abundance of Faecalibacterium, Ruminococcaceae, and Helicobacter in the gut was found to be significantly greater in Lohmann hens raised in cage rearing systems than in those raised in free-range systems (11). A higher abundance of cecal microbiota associated with functions involved in amino acid and glycan metabolic pathways was observed in free-range Dagu chickens than in cage reared chickens (12). With the increased interest in animal welfare, poultry housing systems have been a concern for the last decade, and conventional cages have been gradually replaced by non-cage systems (13). Non-cage housing systems are perceived as being more respectful to animal welfare than cage housing systems which could allow behavioral freedom and promote eco-friendliness (14). There are two primary non-cage housing systems for laying hens in China: the plastic net housing system (NRS) and floor litter housing system (LRS). The NRS comprises perforated plastic nets isolated above the ground, which keep hens away from excreta and could maintain good environmental hygiene. The LRS uses various litters, such as wood shavings, straw or rice husks; it puts hens directly in contact with feces and requires more floor space. Gut microbiota is a good indicator of variations innutrient digestion and absorption capacity of laying hens, which could be affected by environmental condition changes (15). Therefore, it is of great significance to investigate the intestinal microbiota or morphology of hens raised in different non-cage housing systems with the ban on housing hens in conventional cages.
The Shendan chicken, which originated in North China, is one of the most popular local laying hen breeds in China. Shendan chickens are native to Anlu City, Hubei Province, China, and are characterized by its black plumage and blue-colored eggshells. Whether the formation of the gut microflora is affected by different non-cage housing systems and its association with production performance and intestinal health in Shendan chickens are unknown. The present study used the 16S rRNA sequencing approach to analyze the changes in species abundance and diversity of intestinal microbes in Shendan chickens under different non-cage housing systems and to explore their association with production traits and intestinal morphology to help us better master Shendan chicken management practices. The results of this study will provide a better understanding of the effects of non-cage housing systems on the intestinal microbial ecology of Shendan chickens and support poultry production and welfare.
Eggs were collected and counted every day to calculate the laying rate. Twenty eggs were randomly sampled from each replicate for egg quality analysis every 2 weeks. All eggs were kept in the same storage room, and egg quality measurements were completed on the day of collection. Egg weight was measured using an electronic scale with an accuracy of 0.01 g. Shell strength was measured with an eggshell force gauge (EGG-0503, Robotmation Co., Ltd., Tokyo, Japan). Haugh units were measured using an automatic egg multitester (EMT-5200, Robotmation Co., Ltd., Tokyo, Japan).
At 40 weeks of age, ten birds from each group (two from each replicate) were immediately dissected using sterile scissors to aseptically remove the intestines from the abdominal cavity, and the contents of the duodenum and cecum were gently squeezed into 2 mL cryopreservation tubes and stored immediately at −80°C for further analysis.
Microbial DNA was extracted using HiPure Stool DNA Kits (Magen, Guangzhou, China) according to the manufacturer's protocols. The concentration and integrity of the DNA was verified using a NanoDrop™ 2000 spectrophotometer (Thermo Scientific, MA, USA) and agarose gel electrophoresis. The DNA samples were stored at −80°C until processing for amplification. To construct 16S rDNA sequencing libraries, the V3-V4 regions of the 16S rDNA gene was amplified from the DNA samples by PCR using the universal primers 341 F and 806 R (341F: CCTACGGGNGGCWGCAG; 806R: GGACTACHVGGGTATCTAAT). The negative control is water, and the positive control is the sample with stable amplification in the previous experiment. PCR reaction was carried out in a 50 μL reaction volume with TransGen High-Fidelity PCR SuperMix (TransGen Biotech, Beijing, China), 0.2 μM forward and reverse primers, and 5 ng template DNA. The PCR condition was as follows: 95°C for 2 min, followed by 35 cycles of 95°C for 30 s, 60°C for 45 s, and 72°C for 90 s, with a final extension of 72°C for 10 min. Amplicons were evaluated with 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions. Sequencing libraries were generated using the SMRTbell TM Template Prep Kit (PacBio, Menlo Park, CA, USA) following the manufacturer's recommendation. The library quality was assessed with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) and FEMTO pulse system (Agilent Technologies, Santa Clara, CA, USA). The libraries were sequenced on an Illumina HiSeq 2500 platform.