Splenic Gene Expression Signatures in Slow-Growing Chickens Stimulated in Ovo with Galactooligosaccharides and Challenged with Heat

Abstract

Galactooligosaccharides (GOS) that are delivered in ovo improve intestinal microbiota composition and mitigate the negative effects of heat stress in broiler chickens. Hubbard hybrids are slow-growing chickens with a high resistance to heat. In this paper, we determined the impact of GOS delivered in ovo on slow-growing chickens that are challenged with heat. The experiment was a 2 × 2 × 2 factorial design. On day 12 of incubation, GOS (3.5 mg/egg) was delivered into the egg (n = 300). Controls (C) were mock-injected with physiological saline (n = 300). After hatching, the GOS and C groups were split into thermal groups: thermoneutral (TN) and heat stress (HS). HS (30 °C) lasted for 14 days (days 36-50 post-hatching). The spleen (n = 8) was sampled after acute (8.5 h) and chronic (14 days) HS. The gene expression of immune-related (IL-2, IL-4, IL-6, IL-10, IL-12p40, and IL-17) and stress-related genes (HSP25, HSP90AA1, BAG3, CAT, and SOD) was detected with RT-qPCR. Chronic HS up-regulated the expression of the genes: IL-10, IL-12p40, SOD (p < 0.05), and CAT (p < 0.01). GOS delivered in ovo down-regulated IL-4 (acute p < 0.001; chronic p < 0.01), IL-12p40, CAT and SOD (chronic p < 0.05). The obtained results suggest that slow-growing hybrids are resistant to acute heat and tolerant to chronic heat, which can be supported with in ovo GOS administration.

Keywords: GOS; heat; immune response; in ovo stimulation; prebiotic; spleen.

Figure 1

Relative messenger RNA (mRNA) expression of immune-related and stress-related genes in the spleens of slow-growing chickens that were challenged with chronic heat. Gene panel includes: interleukin: IL-2, IL-4. IL-6, IL-10, IL-12p40, and IL-17 and stress-related genes: CAT, SOD, BAG3, HSP25 and HSP90. The x-axis shows a list of genes. The y-axis indicate the relative mRNA abundance of the genes after heat challenge (n = 8). Gene expression analysis was carried out with RT-qPCR. qPCR reactions were performed in triplicate. The geometric mean of the ACTB and UB reference genes was used to calculate delta cycle threshold (dCt) values. The relative gene expression (FC—fold change) was calculated with the delta delta cycle threshold (ddCt) formula and the fold change (FC) was calculated as follows: FC = 2^−ΔΔCt. FC values were transformed and presented as Log₂FC. The standard error of the means (SEM) shows distribution of the Ct values. Normalized data (dCt values) of thermoneutral control (mock-injected) and heat-stressed control (mock-injected) groups were compared with a Student’s t-test. Significant differences (p < 0.05) are labelled with an asterisk (*). Figures were prepared by using GraphPad Prism 7 (GraphPad, La Jolla, CA, USA).

Figure 2

Relative mRNA expression of immune-related and stress-related genes in the spleens of slow-growing chickens injected in ovo with GOS and challenged with heat on two levels: A—acute (30 °C for 8.5 h); and B—chronic (30 °C for 14 days). Gene panel includes: interleukin: IL-2, IL-4. IL-6, IL-10, IL-12p40, IL-17 and stress-related genes: CAT, SOD, BAG3, HSP25 and HSP90. X-axis shows a list of genes. Y-axis indicate relative mRNA abundance of the genes after heat challenge (n = 8). Gene expression analysis was carried out with RT-qPCR. qPCR reactions were performed in triplicates. Geometric mean of ACTB and UB reference genes was used to calculate dCt values. The relative gene expression was calculated with ddCt formula (FC = 2^−ΔΔCt). FC values were transformed and presented as Log₂FC. Standard error of the means (SEM) shows distribution of the Ct values. Normalized data (dCt values) of heat-challenged (A-acute, B-chronic) control (mock-injected) and heat-stressed GOS treatment groups were compared with Student’s t-test. Significant differences (p < 0.05) were labelled with an asterisk (*). Figures were prepared by using GraphPad Prism 7 (GraphPad, La Jolla, CA, USA).