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Different yeast strains benefit postweaning piglets by promoting intestinal health. The objective of this study was to investigate the effect of a yeast mixture containing Kluyveromyces marxianus fragilis, Pichia guilliermondii, and Saccharomyces cerevisiae (Vetoquinol italia s.r.l., Italy) on gut health parameters and growth performance traits of weaned piglets. Forty-eight postweaning castrated male piglets (27 ± 1.7 days, 7.19 ± 0.54 kg) were randomly allocated to two homogeneous experimental groups and involved in a 28-day trial. Both the groups received a basal diet with (yeast mixture, YM) or without (control, CTR) the inclusion of 0.8% yeast mixture during weeks 1 and 2, and 0.6% during weeks 3 and 4. Individual BW and box feed intake were determined on days 0, 14, and 28, and average daily gain and Gain:Feed ratio were subsequently calculated for each administration period (0-14, 14-28). Individual faecal samples were collected for microbiota analysis on days 4, 14, 21, and 28, and faecal score was evaluated on the same days. At the end of the trial, 12 piglets for each group were sacrificed, and ileal tissue was sampled for morphological analysis and the evaluation of mucins profile, using Alcian-Blue/Periodic Acid-Shiff (PAS) staining. On ileum samples, dividing and differentiated epithelial cells were also identified using proliferating cell nuclear antigen and alkaline phosphatase expression, respectively. Differences in the means between the experimental groups were determined by ANOVA, while the metataxonomics analyses were performed by sequencing for V3 and V4 hypervariable regions of the 16S rRNA gene. Growth performance traits were not different among the two experimental groups when considering the whole trial period, while treated animals showed increased faecal consistency on weeks 1 and 4 (P = 0.036 and 0.021, respectively). Yeast mixture administration increased the abundance of Bifidobacterium (P = 0.006) and Coprococcus 2 (P = 0.015), and decreased Clostridium Sensu Stricto 1 (P = 0.019) at all the considered timepoints. Ileum villous height, villous width, and crypt depth were significantly increased by yeast mixture supplementation (P = 0.019; P = 0.013; P = 0.036, respectively), while no differences were observed for the villous:crypt ratio among the groups. The mucin profile showed no differences among experimental groups for acid and neutral glycoconjugates. However, a higher presence of PAS-positive mucins was highlighted in the villi of YM piglets (P < 0.001) compared to CTR. Overall, the administration of a yeast mixture to postweaning piglets showed positive effects on gut health when compared to piglets not receiving the tested product, improving beneficial genera and intestinal morphology., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)