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Colostrum feeding is vital for the development of the immune system and gastrointestinal tract in neonatal calves; however, it is currently unknown whether different colostrum feeding strategies affect their neuroendocrine system and potentially the gut-brain axis. The present study investigated the effect of 3 different colostrum feeding regimens on the expression of neuroendocrine genes in adrenal glands and gastrointestinal tissues and on the abundance of intestinal commensal bacteria. Holstein bull calves were fed colostrum immediately after birth and randomly assigned to 3 groups: whole milk (n = 8), mixture of 50% colostrum and 50% whole milk (n = 8), and colostrum (CF; n = 8) for 72 h with 12-h intervals. Adrenal glands, ileum, and colon tissues were collected at 75 h and were subjected to the expression of 11 targeted neuroendocrine genes and the abundance of tissue mucosa-associated bacteria measurement using quantitative real-time PCR and quantitative PCR, respectively. The expressions of all targeted genes were detected, and the expression of α-adrenergic receptor (ADRA1A) gene was affected by CF in adrenal glands and gut tissues. In addition, CF upregulated the expression of HTR4 (serotonin receptor) and SLC4A4 (serotonin transporter) genes in the ileum and increased the abundance of active Lactobacillus spp. and Escherichia coli (as detected at RNA level) associated with ileum and colon tissue. Furthermore, there were positive correlations between the abundance of active Lactobacillus spp. and E. coli with expression of HTR2B and HTR4 genes in the colon, suggesting that extended colostrum feeding strategies may affect the interaction between gut microbiota and host endocrine functions in neonatal calves., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)