Background: A prebiotic is defined as an indigestible feed substance that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the large intestine, thereby improving host health and products. Objectives: This study was conducted to determine the effects of supplementing prebiotic fructooligosaccharide (FOS) to the diets of Hy-Line W-36 laying hens. Methods: A total of 168 Hy-LineW-36 laying hens were allocated to four dietary levels of FOS (0, 1.0, 2.0, 3.0 g/kg diet), 6 replicates of 7 birds each during 63–74. The experiment was performed using a completely randomized design. Results: Productive performance was not significantly affected by the FOS supplementation. Body weight gain was linearly decreased with increasing FOS levels in the diet (p < 0.01). However, eggshell strength, shell thickness and Ca and p percentages were not significantly affected, as were anti-sheep red blood cell titres, blood parameters and blood metabolites. In the first period of the experiment (63–65 weeks), shape index and Haugh unit at the dose of 3.0 g/kg FOS were significantly increased and decreased in comparison with control, respectively (p < 0.05). In the third and fourth periods (69–71 and 72–74 weeks of age), the FOS had no significant effect on the internal egg quality traits. Furthermore, FOS had a linear decrease in the most saturated fatty acids (SFAs), includingmyristic, palmitic, margaric and stearic fatty acids; some of the mono-un-SFA (MUFA; palmitoleic and ginkgolic acids), and poly-unsaturated fatty acids (γ-linolenic and eicosatrienoic). Conclusions: Supplementing different levels of FOS to the diet of commercial laying hens had no significant effect on the layers’ performance, immune response and blood parameters, whereas there was a significant effect on some of the internal egg quality traits and egg yolk fatty acid contents.

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