Simple SummaryIn the agricultural sector, particularly the poultry industry, antibiotics have been used as feed additives to treat and prevent infections or to improve the growth, health, and welfare of chickens. Many nutritional alternative strategies, including natural ingredients, have been investigated for their role in improving gut health without any adverse effect on productivity. The possibility of using microalgae as a new source of nutrients and health additives in animal feed formulations has been increased. Therefore, the aim of this study was to determine the effect of dietaryC. vulgarisorT. obliquuson the laying performance, egg quality, intestinal histology, immune characteristics, antioxidant status, and gut microbiome of the laying hen.AbstractA feeding trial was conducted to investigate the effect of dietary supplementation ofChlorella vulgaris(CV) orTetradesmus obliquus(TO) on laying performance, egg quality, and gut health indicators of laying hens. A total of 144 Hy-Line Brown laying hens aged 21 weeks were randomly assigned to one of three dietary treatments with eight replicates of six hens. Dietary treatments were as follows: CON, basal diet; CV, basal diet + 5 gC. vulgaris/kg of diet; TO, basal diet + 5 gT. obliquus/kg of diet. The results showed that diets supplemented with CV or TO had insignificant effects on laying performance, egg quality (i.e., Haugh unit and eggshell strength and thickness), jejunal histology, cecal short-chain fatty acids, and antioxidant/immune markers in ileal mucosa samples of laying hens. Compared with the control group, the egg yolk color score was higher (p< 0.05) in laying hens fed on diets containing CV and TO, although the former was a more intense yellow than the latter. Small intestinal lamina propria cells were isolated using flow cytometry to examine the percentages of immune cell subpopulations. Dietary microalgae did not affect B cells or monocytes/macrophages but altered the percentage of CD4+ T cells and CD8− TCR γδ T cells. Collectively, diets supplemented withC. vulgarisorT. obliquuscan improve egg yolk color and would modulate host immune development and competence in laying hens.