Elevated serotonin coordinates mammary metabolism in dairy cows

Abstract

Serotonin plays a diverse role in maternal and mammary metabolism. Recent research in the dairy cow has shown a relationship between serotonin and calcium, with increased serotonin concentrations improving calcium homeostasis in the peri-partum dairy cow. Therefore, the objective was to elucidate how administration of 5-hydroxy-l-tryptophan (5-HTP), the immediate precursor to serotonin, altered serotonin and calcium metabolism in lactating dairy cows. Twelve mid-late lactation multiparous cows were blocked by parity, production and days in milk and allocated to a daily intravenous infusion of (i) 1.5 mg/kg of 5-HTP (n = 6) or (ii) saline (n = 6) for 3 consecutive days. Milk samples were collected daily. Blood samples were collected before and after each infusion with mammary biopsies and blood samples collected at 48, 56, and 72 h relative to termination of first infusion. Infusion of 5-HTP increased (p = 0.001) circulating serotonin concentrations and decreased blood calcium via a transient hypocalcemia immediately after each infusion (p = 0.02). Treatment with 5-HTP increased milk calcium concentrations (p = 0.02) and calcium release-activated channel protein 1 (ORAI1) mRNA at 56 h and protein at 48 h relative to termination of first infusion (p = 0.008 and p = 0.09, respectively). Fifty-six hours from termination of the first infusion mRNA of parathyroid hormone-related protein and mammary serotonin content were increased relative to control (p = 0.03 and p = 0.05, respectively). These findings demonstrate the ability of 5-HTP infusion to increase circulating serotonin concentrations and alter endocrine and mammary autocrine/paracrine calcium and serotonin metabolism in the lactating dairy cow.

Keywords: 5-HTP; calcium; dairy cow; mammary gland; serotonin.

FIGURE 1

Schematic representation of experimental design and sampling timeline. Twelve mid‐late lactation multiparous Holstein dairy cows were blocked by parity, milk production and days in milk in a randomized complete block design and assigned to intravenous infusion of (i) 1 L of 1.5 mg/kg 5‐HTP dissolved in saline (n = 6/treatment) or (ii) 1 L of saline solution (n = 6/treatment) for three consecutive days. Milk samples were collected daily at the morning milking. Day 0 milk samples were taken prior to treatment administration.

FIGURE 2

Mammary gland histological findings and milk yield (kgs/d) in multiparous Holstein dairy cows receiving 1 L of 1.5 mg/kg 5‐HTP dissolved in saline or 1 L of saline solution (Control = Saline) for three consecutive days. Mammary gland tissues were sectioned (5 µm) and stained with hematoxylin and eosin (a) and analyzed with Image J software to determine alveolar quantity and area (c–d) (48 h n = 5; 56 h n = 5; 72 h n = 3). Images were taken at 20x magnification (scale bar: 100 µm). Milk yield of 5‐HTP and control cows across the experimental period (b) (n = 6). Statistics were performed using PROC MIXED procedure in SAS with repeated measures. Data presented as LSMEANS ± SEM. Milk yield: day (p = 0.01) and treatment*day (p = 0.008). Asterisks denote statistical significance (*p < 0.05)

FIGURE 3

Whole blood serotonin concentrations (a) (n = 6), serum total calcium concentrations (b) (n = 6) and plasma parathyroid hormone concentrations (c) (5‐HTP n = 5, Control n = 6) in multiparous Holstein dairy cows receiving 1 L of 1.5 mg/kg 5‐HTP dissolved in saline or 1 L of saline solution (Control = Saline) for three consecutive days. Statistics were performed using PROC MIXED procedure in SAS with repeated measures. Data presented as LSMEANS ± SEM. Whole blood serotonin concentrations: treatment (p = 0.001), time (p = 0.07) and treatment*time (p = 0.004). Serum total calcium concentrations: treatment (p = 0.02), time (p < 0.0001) and treatment*time (p = 0.0002). Asterisks denote statistical significance between groups at each timepoint (***p < 0.001, **p < 0.01, *p < 0.05, and ^#0.10 < p > 0.05)

FIGURE 4

Milk calcium concentrations (a) (n = 6), mammary gland mRNA expression of PMCA2 (b) (5‐HTP n = 6, n = 6, n = 5; Control n = 6, n = 6, n = 6), PTHLH (c) (5‐HTP n = 6, n = 5, n = 6; Control n = 4, n = 4, n = 5), CASR (d) (5‐HTP n = 6, n = 5, n = 5; Control n = 3, n = 4, n = 3), ORAI1 (e) (5‐HTP n = 6, n = 6, n = 6; Control n = 5, n = 6, n = 6), quantification and western blot of mammary gland ORAI1 (f–g) (5‐HTP n = 4, n = 4, n = 3; Control n = 4, n = 4, n = 3), mammary mRNA expression of NCX1 (h) (5‐HTP n = 6, n = 6, n = 6; Control n = 6, n = 5, n = 5), SPCA1 (i) (5‐HTP n = 6, n = 6, n = 6; Control n = 5, n = 6, n = 5), and SERCA2 (j) (5‐HTP n = 6, n = 6, n = 6; Control n = 5, n = 4, n = 3) in multiparous Holstein dairy cows receiving 1 L of 1.5 mg/kg 5‐HTP dissolved in saline or 1 L of saline solution (Saline = Control) for three consecutive days. Statistics were performed using PROC MIXED procedure with repeated measures in SAS. Transformed data presented as mean ± SEM. Normally distributed data presented as LSMEANS ± SEM. Milk calcium: treatment (p = 0.02). ORAI1 mRNA: treatment (p = 0.09) and treatment*time (p = 0.06). PTHLH mRNA: time (p = 0.04) and treatment*time (p = 0.03). Asterisks denote statistical significance between groups (**p < 0.01, *p < 0.05 and #0.10 < p > 0.05)

FIGURE 5

Milk serotonin concentrations (a) (n = 6) and mammary gland mRNA expression of 5HTR2b (b) (5‐HTP n = 6, n = 6, n = 5; Control n = 5, n = 4, n = 4), 5HTR7 (c) (5‐HTP n = 6, n = 6, n = 5; Control n = 4, n = 4, n = 3), TPH1 (d) (5‐HTP n = 6, n = 5, n = 5; Control n = 6, n = 4, n = 3), MAO‐A (e) (5‐HTP n = 6, n = 6, n = 6; Control n = 5, n = 5, n = 5) and SERT (f) (5‐HTP n = 6, n = 6, n = 5; Control n = 4, n = 6, n = 4) in multiparous Holstein dairy cows receiving 1 L of 1.5 mg/kg 5‐HTP dissolved in saline or 1 L of saline solution (Control = Saline) for three consecutive days. Statistics were performed using PROC MIXED procedure with repeated measures. Transformed data presented as means ± SEM. Milk serotonin concentrations: day (p < 0.0001). 5HTR2b mRNA: time (p = 0.01) and treatment*time (p = 0.04). SERT mRNA: time (p = 0.08) and treatment*time (p = 0.08). Asterisks denote statistical significance between groups (*p < 0.05 and #0.10 < p > 0.05)

FIGURE 6

Mammary tissue serotonin concentrations (a) (n = 6) and mammary tissue calcium concentrations (b) (5‐HTP n = 5, n = 6, n = 4; Control n = 6, n = 5, n = 5) in multiparous Holstein dairy cows receiving 1 L of 1.5 mg/kg 5‐HTP dissolved in saline or 1 L of saline solution (Control = Saline) for three consecutive days. Statistics were performed using PROC MIXED procedure with repeated measures. Transformed data presented as mean ± SEM. Mammary tissue serotonin concentrations: treatment (p = 0.07). Mammary tissue calcium concentrations: time (p = 0.004). Asterisks denote statistical significance between groups (*p < 0.05 and ^#0.10 < p > 0.05)