Effects of Monochromatic Lighting During Incubation and Vaccination on the Splenic Transcriptome Profiles of Chicken

Abstract

Lighting is a crucial environmental variable in poultry operations, but illumination during incubation is relatively understudied. The ability to stimulate development or immune performance using in ovo lighting is a promising approach for improving poultry health and welfare. This study investigated how monochromatic green light during incubation and vaccination method and timing affected chicken splenic gene expression patterns. We performed this study with 1,728 Hy-Line white layer eggs incubated under two light treatments during incubation: continuous dark and continuous green monochromatic light, over the entire incubation period. Half the eggs in each light treatment received in ovo vaccination, applied on embryonic day 18 (ED18). The remaining half were vaccinated by spraying on hatch day. After hatching, the light treatments followed the industry-standard lighting regimens. The study had six treatment groups with light-dark pairs for non-vaccinated, in ovo vaccinated, and post-hatch vaccinated. We assessed splenic gene expression at ED18 and at 7 days post-hatch (PH) in all the treatments. We isolated and sequenced 24 mRNA libraries on the Illumina platform, followed by bioinformatics and differential gene expression analyses. RNAseq analysis showed between 62 and 6,755 differentially expressed genes (DEGs) between comparisons, with the most prominent differences observed between ED and PH samples, followed by comparisons between vaccination methods. In contrast, light vs. dark treatments at ED showed limited effects on transcriptomic profiles. However, we observed a synergistic effect of lighting during incubation on post-hatch vaccination responses, with differentially expressed genes (DEGs) unique to the light treatment showing stimulation of cell proliferation with significance for immune activity (inferred from gene ontology terms). Gene ontology and pathway analysis indicated biological processes like cellular component organization or biogenesis, rhythmic process, developmental process, response to stimulus, and immune system processes were explained by the DEGs. While lighting is an important source of circadian stimulation, other controlled studies are required to clarify whether in ovo circadian entrainment plays a role in modulating immune responses.

Keywords: Newcastle disease; incubation; monochromatic lighting; poultry; spleen; transcriptome; vaccination.

FIGURE 1

Mean average (MA) plot highlighting the log fold change and average abundance of each gene in pre- vs. post-hatch spleen transcriptome comparison. Significantly up and down DE genes and their counts are highlighted in red and blue, respectively. (A) Shows the DEGs in the embryonic spleen (E18) during green monochromatic light biostimulation pre-hatch in LNV vs. DNV treatment groups. (B) Shows the DEGs in spleen tissue post hatch (D7) in LPHV vs. DPHV treatment groups that received vaccination post hatch on day one. (C) Shows the DEGs in spleen tissue post hatch (D7) in LIV vs. DIV treatment group received in ovo vaccination E18. The Y-axis corresponds to the mean average of log10 count per million (CPM), and the X-axis displays the log2 FC, with significance assessed at FDR < 0.05. LNV, light not vaccinated; DNV, dark not vaccinated; LPHV, light post-hatch vaccinated; DPHV, dark post-hatch vaccinated; LIV, light in ovo vaccinated; DIV, dark in ovo vaccinated.

FIGURE 2

The top three detected gene networks underline the affected genes in green monochromatic light biostimulation comparisons during incubation and their interaction in potentially regulating developmental biological processes pre- and post-hatch generated by QIAGEN’s Ingenuity Pathway Analysis (IPA, QIAGEN Inc.) (Krämer et al., 2014). (A) LNV vs. DNV gene network for cell morphology, digestive system development and function, and organ morphology. (B) LPHV vs. DPHV gene network for carbohydrate metabolism, lipid metabolism, and protein synthesis. (C) LIV vs. DIV gene network for developmental disorder, hematological disease, and hereditary disorder. Differentially expressed genes in the biostimulated comparisons were used in the ingenuity pathway analysis, and significant gene networks based on IPA scores were identified. Genes highlighted in red were upregulated, while those highlighted in green were downregulated in all biostimulated comparisons.

FIGURE 3

The frequently observed pathway terms based on differentially expressed genes in pre- and post-hatch birds incubated in green monochromatic light versus dark. (A) top canonical pathway, (B) upstream regulator, (C) top molecular and cellular functions. All DEGs from pre (E18) and post-hatch (D7) were subjected to IPA analysis to detect molecules across observations.

FIGURE 4

Overlapped Gene Ontology enrichment terms based on DEGs in pre- and post-hatch from illuminated and dark treatments. (A) biological process, (B) cellular component, and (C) Up_Keywords. All DEGs from pre (E18) and post-hatch (D7) were subjected to the DAVID database for Gene Ontology (GO) enrichment analysis. All the GO terms were significant based on a modified Fisher Exact P-value < 0.05.

FIGURE 5

Number and overlapping DEGs in spleen tissues between pre-hatch (LNV vs. DNV) and post-hatch (LPHV vs. DPHV and LIV vs. DIV) treatments, stimulated by green monochromatic light during incubation. The DEGs were determined by statistical algorithms EdgeR. Notably, embryonic spleen samples had a greater number of highly expressed DEGs (FDR < 0.05) compared to post-hatch spleen samples indicating the dilution of biostimulation in the post-hatch environment (shift to standard lighting).

FIGURE 6

Activated and inhibited lipid metabolism networks in the green light biostimulation comparisons underline the role of green monochromatic light biostimulation regulating developmental biological processes pre- and post-hatch. (A) LNV vs. DNV and LPHV vs. DPHV gene network activated lipid metabolism. (B) LIV vs. DIV gene network for inhibited lipid metabolism. Genes highlighted in red were upregulated, while those highlighted in green were downregulated in all biostimulated comparisons.

FIGURE 7

The mean average (MA) plot highlighting the log fold change and average abundance of each gene, comparing the differences between the method of vaccination with or without green monochromatic light on the spleen transcriptomes collected on day 7 post-hatch. Significantly up and down DE genes and their numbers are highlighted in red and blue, respectively. (A) Shows the DEGs in the post hatch spleen tissue (D7) receiving either in ovo or post-hatch vaccination in the unilluminated treatment. (B) Shows the DEGs in the post-hatch spleen tissue (D7) receiving either in ovo or post-hatch vaccination in green light treatments. The y-axis corresponds to the mean average of log counts per million (CPM), and the x-axis displays the log2FC. DIV, dark in ovo vaccinated; LIV, light in ovo vaccinated; DPHV, dark post-hatch vaccinated; LPHV, light post-hatch vaccinated.

FIGURE 8

Similarities of non-canonical pathways induced by the interaction of the method of vaccination and green monochromatic light interaction.

FIGURE 9

Unique and shared DEGs between method of vaccination and the interaction of green monochromatic light biostimulation in spleen tissues in post-hatch comparisons (DIV vs. DPHV and LIV vs. LPHV), stimulated by green monochromatic light during incubation.