TABLE 5.

CD8⁺/CD4⁺ ratio and CD4⁺CD25⁺ cell percentages at 24 and 48 h post-LPS injection in thymus and spleen (Experiment II).

	0-hole-Control	2-holes-Control	0-hole-LPS challenge	2-holes-LPS challenge	Main effects		Interaction	SEM
					Hole P-value	LPS challenge P-value	Hole × LPS challenge P-value
24 h
Thymus
CD8/CD4	2.5c	2.8c	4.0b	5.7a	0.01	0.04	0.01	0.2
Tregs	0.5c	1.6b	2.0b	3.6a	0.05	0.01	0.01	0.2
Spleen
CD8/CD4	1.1c	0.9c	2.2b	3.1a	0.13	0.05	0.04	0.3
Tregs	3.1b	3.5b	4.0b	5.0a	0.26	0.07	0.05	0.3
48 h
Thymus
CD8/CD4	2.8	2.0	4.6	4.4	0.18	0.03	0.32	0.1
Tregs	2.2b	2.8b	3.7b	5.8a	0.34	0.05	0.05	0.1
Spleen
CD8/CD4	1.6b	1.6b	4.1a	2.5b	0.48	0.05	0.05	0.3
Tregs	2.3c	3.8bc	4.3ab	5.9a	0.05	0.07	0.05	0.4

Fertile broiler eggs were incubated at standard (37.5°C) incubation temperature from embryonic day (ED) 1–8. At ED8, the eggs incubated at 37.5°C were drilled either 0-hole or 2-holes in the eggshell above the air cell and continued to incubate until ED18. The embryos from each treatment group were transferred to the hatcher and maintained at 37.0°C and 70% relative humidity until hatch. At D1, hatchlings from each treatment group were challenged intraperitoneally with either 0 or 500 μg lipopolysaccharide (LPS)/kg BW in a 2 (0-hole, 2-holes) × 2 (control, LPS challenge) factorial design. At 24 and 48 h post-LPS injection, thymus, and spleen from all the treatment groups were collected and analyzed for CD4⁺_/CD8⁺ ratio and CD4⁺/CD25⁺by flow cytometry after staining with fluorescent-linked anti-chicken CD4, CD8, and CD25 antibodies. Means with no common superscripts differ significantly (P ≤ 0.05). n = 6.