Amplifying local serotonin signaling prior to dry-off regulates mammary gland involution and redevelopment in dairy cows - Supplementary Data
Interpretive Summary Serotonin is a bioactive monoamine that regulates mammary gland homeostasis during lactation and stimulates mammary involution after milk stasis. We intramammary infused 5-Hydroxy-L-tryptophan, a modified tryptophan and serotonin precursor, via the teat canal for 5 d prior to dry off to measure key cellular processes in the mammary gland during the dry period of dairy cows. Increasing local mammary serotonin concentrations prior to dry-off downregulated milk protein and tight junction genes and increased cell death in early involution, while it increased mammary epithelial cell number and alveoli lumen area, upregulated milk protein genes, and increased proliferating cells as parturition approached. Amplifying serotonin signaling hastens key cellular events leading to a more efficient mammary involution and redevelopment. Supplemental Figure 1: Holstein dairy cows receiving intramammary infusions of 5-Hydroxy-L-Tryptophan (5-HTP) or sterile water (CON) for 5 d prior to dry-off (d0). (A) No difference in milk yield in 5-HTP cows compared to CON (P = 0.70) however, there was a main effect of day (P = 0.0003). (B) No difference in dry matter intake in pre-dry-off (P = 0.23) and post-dry-off (P =0 .12) in 5-HTP cows compared to CON. Significance declared at (*) P ≤ 0.05 and (#) denotes a statistical tendency at 0.05 < P ≤ 0.10. Supplemental Table 1: Nutrient composition of total mixed rations fed to lactating and non-lactating multiparous Holstein dairy cows adapted from Cumberland Valley Analytical Services (Waynesboro, PA). Supplemental Table 2: Supplemental Table 2 Primer sequences utilized for real time PCR analysis of genes involved in tight junctions, milk proteins, serotonin synthesis and metabolism, apoptosis, cell signaling, extra cellular matrix remodeling genes in the mammary gland of Holstein dairy cows. All primer sequences were designed to span exon-exon junctions, to minimize the potential of amplifying genomic DNA, using Primer3 software with sequences obtained from GenBank (http://www.ncbi.nlm.nih.gov/). All primer pairs displayed melting curves with a single peak, indicative of a pure, single amplicon, confirmed the specificity of the primers.