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Identification of reliable reference genes for expression studies in the magnum of laying hens housed in cage and cage-free systems.

ABSTRACT: Stress factors during poultry production can evoke changes in gene transcription and protein synthesis in the hen oviduct and could affect the internal and external egg quality. Studies of relative gene expression require the identification of the most stable reference genes for the quantitative polymerase chain reaction (qPCR) to investigate the reproductive tissues' response in laying hens kept in different production systems. The objective of this study was to determine the most stable reference genes of the magnum tissues of laying hens housed in two different production systems. Hy-Line Brown hens were reared under the same sanitary conditions until 15 weeks of age. Later on, they were transferred into two different production systems, conventional cage (CC) and cage free (CF), up to 82 weeks of age. At 50 and 60 weeks, a total of six hens from each production system were euthanized, and magnum samples were collected. The qPCR was used to determine the RNA transcription level of five reference genes, ACTB, 18S, GAPDH, MSX2 and HMBS. These genes were evaluated for transcript expression in magnum tissues by NormFinder, BestKeeper, geNorm and RefFinder software. The results indicated that the most stable gene in the CF housing system was HMBS in three of the algorithms and in the CC housing system was the 18S, and the best combination of reference genes was HMBS/GAPDH in CF and 18S/HMBS in CC. In conclusion, HMBS, 18S and GAPDH genes could be used together as reference genes for the normalization of the magnum tissues transcript expression of hens in CF and CC housing systems.

SUBMITTER: Rodriguez-Hernandez R

PROVIDER: S-EPMC8464264 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:With the majority of conventional cage (CC) laying facilities transitioning into cage-free (CF) systems in the near future, it is important to characterize biological markers of health in layers housed in commercial housings for sustainable production. The objectives of this study were to compare i) blood markers, that is heterophil:lymphocyte (H:L) ratios and susceptibility to avian pathogenic Escherichia coli (APEC) and ii) lung and ceca microbiome between hens at different maturity stages in commercial CC and CF farms. Laying hens at 3 maturity stages were randomly sampled (N = 20 per maturity and per farm). Blood was tested for H:L ratios and APEC killing ability using microscopy and in vitro assay, respectively. Microbiomes were assessed using 16S rRNA sequencing and QIIME2 analysis. Data show H:L ratios did not differ between maturities in both farms. Avian pathogenic Escherichia coli killing was only different in CC hens, where ?7122 level was higher (P < 0.05) in peak compared with early lay. In both farms, microbiome diversity was consistently different (P < 0.05) in both ceca and lung of early lay compared with peak and late lay. In the ceca and lung, relative abundances of the 3 predominant phyla (Bacteroidetes, Firmicutes, and Proteobacteria) did not significantly change with maturity in both farms. Potential pathogens Campylobacter and Staphylococcus reached greater (P < 0.05) abundances in CC lungs in early lay and in CF lungs in late lay, respectively. Overall, this study showed no differences in the stress marker H:L but identified some differences in resistance to APEC and microbiome composition across maturity stages in CC and CF. The lung and gut microbiomes were highly similar, with both serving as potential reservoirs for Campylobacter and Staphylococcus. Future studies on controllable environments for CF and CC are needed to develop adequate strategies for each housing and maturity stage to reduce pathogens and optimize disease-resistance.

Project description:Piling, a dense cluster of hens, is a behavior of major concern to the cage-free egg industry. It can cause large numbers of mortalities at which point it is considered smothering. The aim of this study was to quantify whether piling can also have consequences on production and non-smothering mortalities, which have not previously been described. Additionally, we aimed to describe characteristics of piling behavior relevant to for management. Video footage from 12 flocks of laying hens was analyzed for piling behavior across 3 wks. Production and mortality data were accessed using an integrated online management system. Bayesian linear mixed-effect models were used for formal statistical testing of the relationships between piling and production. Accounting for some missing data, a total of 252 d amounting to approximately 15,624 h were analyzed for the presence or absence of piling behavior, which we believe constitutes the largest analysis of piling behavior in the scientific literature to date. All flocks observed exhibited piling behavior even if they had no history of smothering. On average, flocks piled more than 4 times per day for around 44 min per event and the peak piling time occurred between 1300 and 1359. We found that the number of piling events was associated with a reduction in the number of eggs produced the next day; based on the average of 4 piles per day this amounted to 7.35 fewer eggs per 1,000 birds per day. Contrary to our hypothesis, we found total piling duration per day was positively associated with fewer Grade B eggs, with a decrease of 0.74 Grade B eggs for every hour of piling per day. No relationship was observed between piling and mortality not attributed to smothering. We discuss possible causes and explanations for these results including birds’ response to stress, crowd dynamics, and daily rhythms. Here we show the potential for piling behavior to have sublethal consequences on production even in the absence of smothering-related deaths.

Project description:Purpose: With the advent of Next-generation sequencing (NGS), several novel genes/proteins and cellular pathways in wide variety of tissues has been discovered. The aim of this study are to perform uterine transcriptome profiling (RNA-seq) to determine differently expressed genes in laying and non-laying hens and to further validate the expression of candidate genes using real-time quantitative reverse transcription polymerase chain reaction (qRT–PCR) in laying, non-laying and molting hens. Methods: Uterine mRNA profiles of 35-60 weeks-old laying and non-laying hens, three each, were generated with NextSeq 500 sequencer in single-end mode with a read length of 1x76 bp. Raw sequencing reads were cleaned and trimmmed with Prinseq tool and good reads were aligned against the chicken reference gemone (Galgal 5.0) in Array Studio. Differential gene expression analysis was performed by the DESeq2 algorithm as implemented in Array Studio. The genes with at least two-fold change (FC) and Benjamini and Hochberg q-value < 0.05 were called differentially expressed. Results: Using an optimized data analysis workflow, we mapped about 32 million reads from layers and 28 million reads from non-layers to the chicken genome. A total of 19,152 gene transcripts were annotated from Ensembl alignment which represents 50.24% of the chicken genome assembly. Differential gene expression analysis showed 616 were differentially expressed between layer and non-layer hens. 229 DEGs were significantly up-regulated and 286 were significantly down-regulated in the laying hens when compared to the non-laying hens. Twelve candiate genes, linked to calcium remodeling, were identified by gene function analysis and validated using qPCR. MEPE, CALCB, OTOP2, STC2 and ATP2C2 were confirmed to be highly expressed in laying hens as compared to molting and non-laying hens. RNA-seq and qPCR data for relative gene expression were highly correlated (R2 =0.99). Conclusions: Our study reports the expression of four novel genes that are speculated to transport calcium ions across the uterine epithellium for eggshell mineralization. These genes can be used as quantitative basis of selecting hens with an improved eggshell quality.

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Identification of reliable reference genes for expression studies in the magnum of laying hens housed in cage and cage-free systems.

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