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. 2022 Mar 2;100(3):skac065. doi: 10.1093/jas/skac065

Increasing serotonin concentrations alter calcium metabolism in periparturient dairy goats

ZhiFei Zhang 1, Wei Du 1, WenYi Liu 1, Braden T Wong 2, HuiLing Zheng 1,✉
PMCID: PMC9030229  PMID: 35235945

Abstract

Due to the large amounts of calcium transferred to milk from mammary glands, periparturient dairy goats face challenges with calcium metabolism disorder and hypocalcemia. Serotonin (5-hydroxytryptamine, 5-HT), the product of 5-hydroxy-l-tryptophan (5-HTP) catalyzed by tryptophan hydroxylase 1, is a multifunctional monoamine thought to be a homeostatic regulator of the animal. The objective of the current study was to investigate the effects and underlying mechanisms of intramuscular 5-HTP injections on calcium homeostasis in the goat mammary glands. In the in vivo experiment, 30 multiparous Guanzhong dairy goats were randomly assigned to 2 groups, one group was injected with 5-HTP intramuscularly and the other group was injected with normal saline. From the first 10 d of the expected date for delivery, 5-HTP or saline was injected into goats through the shoulder muscle every morning before feeding, with a dose of 1 mg/kg per body weight. In the in vitro experiment, goat mammary epithelial cells (GMEC) were treated with 100 μM 5-HT for the evaluation of 5-HT in calcium transportation. The results demonstrated that 5-HTP treatment had no effect on the basic composition of colostrum (P > 0.05) but increased the serum 5-HT concentrations on days −5, −4, −3, and 5 relative to parturition (P < 0.05). The 5-HTP injection group had greater serum calcium concentration on day 4 and greater serum parathyroid hormone-related protein (PTHrP) on days −5, −4, −1, 3, 4, and 5 compared with the saline injection group (P < 0.05). It was further confirmed that 5-HT could increase intracellular calcium levels by increasing PTHrP and decreasing plasma membrane Ca2+-ATPases1 (PMCA1) in GMEC (P < 0.05). In conclusion, 5-HTP treatment in multiparous goats during the transition period from pregnancy to lactation is a feasible way to protect goats from calcium metabolism disorder.

Keywords: calcium metabolism, dairy goat, mammary gland, serotonin, transition period

Lay Summary

The monoamine serotonin (5-HT) is thought to be a homeostatic regulator of the mammary gland, especially during the periparturient period of mammals. Periparturient dairy goats face challenges involving calcium metabolism disorder that may lead to incidences of clinical or subclinical hypocalcemia. Increasing the concentrations of serum 5-HT of dairy goats before parturition could help goats better cope with the challenges of calcium metabolism disorder.

Increasing the concentrations of serum monoamine serotonin of dairy goats before parturition could help goats better cope with the challenges of calcium metabolism disorder.

Introduction

Goats (Capra hircus) are important providers of milk and other dairy products. Calcium is one of the main nutrients in goat milk and the most abundant mineral element in mammals, which is of great importance on forming bones, maintaining excitability of muscles, conducting nerve signals, and participating in normal activities of cells (Shingfield et al., 2009). During lactation, large quantities of calcium are transferred to milk through the mammary gland. Because of the formation of colostrum and the contraction of the birth canal (Levine et al., 2014), it is expected that periparturient dairy goats face any challenges regarding calcium metabolism or hypocalcemia.

Mammary glands are the irreplaceable lactation organ of mammals. Improvements toward calcium metabolism in dairy animals are critical for improving animal health, milk yield, and milk component yields (Sklan et al., 1994; Daniel et al., 2021). In mammary epithelial cells, parathyroid hormone-related protein (PTHrP) induces the binding of calcium and calcium-sensitive receptor (CasR) and promotes calcium to enter the cells (Ratcliffe et al., 1992). Sarcoplasmic reticulum Ca2+-ATPase2 is responsible for storing calcium in the endoplasmic reticulum (Liantonio et al., 2007), followed by secretory-pathway Ca2+-ATPases1 (SPCA1) and SPCA2 pumping calcium in and out of the Golgi. Plasma membrane Ca2+-ATPases1 (PMCA1) and PMCA2 then mediate calcium transfer into milk (Cross et al., 2014; Wang et al., 2015). When the concentration of calcium in the circulatory system increases, CasR inhibits the secretion of PTHrP (Ardeshirpour et al., 2006). This negative feedback mechanism is responsible for maintaining the appropriate concentrations of calcium in bone, blood, and milk. Therefore, modulating calcium homeostasis in goat mammary epithelial cells (GMEC) may have therapeutic effects on hypocalcemia.

Serotonin (5-hydroxytryptamine, 5-HT), a multifunctional monoamine, is derived from l-tryptophan (Hery et al., 1977). l-tryptophan is converted to 5-hydroxy-l-tryptophan (5-HTP) and then subsequently converted to 5-HT. The first step is catalysis by tryptophan hydroxylase 1. Besides being a neurotransmitter, 5-HT synthesized in the mammary gland is also considered as a local regulator of mammary physiology (Matsuda et al., 2004; Pai and Horseman, 2008; Berger et al., 2009), playing important roles in inducing tissue development, maintaining calcium homeostasis, and regulating the expression of lactoprotein synthesis genes (Stull et al., 2007; Horseman and Collier, 2014; Laporta et al., 2015a). During the time from pregnancy to lactation, 5-HT has been shown to increase tight junction permeability of the mammary gland (Kessler et al., 2019), promote energy metabolism (Laporta et al., 2013b), regulate PTHrP-related signals (Weaver et al., 2018), enhance calcium mobilization from bone (Laporta et al., 2013c), and improve parturient calcium homeostasis (Hernandez-Castellano et al., 2017). However, the calcium homeostasis regulatory effects of 5-HT on GMEC remain unknown. Thus, it is important to elucidate the regulatory effects of 5-HT on dairy goats for the clinical therapeutic application.

Our previous studies have confirmed the effect of 5-HT on promoting PTHrP production in GMEC (Zang et al., 2018). Here, we further explored the role of 5-HT on calcium homeostasis of periparturient dairy goats. The results showed that prepartum injection of 1 mg/kg 5-HTP can significantly increase serum 5-HT concentration, serum calcium concentration, and serum PTHrP concentration of goats. Furthermore, 5-HT increased intracellular calcium by inducing PTHrP and decreasing PMCA1 in GMEC.

Material and Methods

Ethics statement

All experimental procedures were carried out in accordance with the Institutional Animal Care and Use Committee in the College of Animal Science and Technology, Northwest A&F University, Yangling, Xianyang, Shaanxi, China (protocol number 15-516).

Animals and experimental design

A total of 30 multiparous Guanzhong dairy goats were selected from the Hongxing dairy goat breeding farm in Xi’an. These goats were randomly assigned to 2 experimental groups of 15 goats each (5-HTP injection group and saline injection group). Goats were enrolled in the experiment 2 wk before the initiation of sample collection to allow for acclimation. 5-HTP (5 mg/mL, 4350-09-8, Sigma-Aldrich, St. Louis, MO) was dissolved in sterile normal saline. Intramuscular injection of saline or 1 mg/kg body weight of 5-HTP was performed at 0800 hours from 10 d before the predicted parturition date until the day of parturition. Six goats (three goats in each group) were eliminated because they had not given birth after 10 d which may be due to individual differences among goats or the efficiency of MOET of the farm. Each of the remaining animals (12 goats in each group) received at least 7 d and at most 10 d of injections. The composition and nutrient levels of diet are shown in Table 1.

Table 1.

Composition and nutrient levels of basal diet (air-dry basis)

Items Content, %
Ingredient
 Corn silage 49.00
 Corn grain 16.50
 Soybean meal 7.50
 Wheat bran 3.60
 Rapeseed meal 0.90
 CaHPO4 0.45
 NaCL 0.45
 Premix1 0.60
 Alfalfa hay 21.00
 Total 100.00
Nutrient level
 Dry matter 86.54
 Crude protein 16.67
 ADF 7.54
 NDF 22.86
 Calcium 1.06
 Phosphorus 0.74
 NE, MJ/kg 6.78
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The premix provided the following per kg of diets: VA 100,000 IU, VD3 250,000 IU, VE 350 IU, Mn 950 mg, Zn 1,400 mg, Fe 1,320 mg, Cu 300 mg, Co 8 mg, I 30.4 mg, Se 8 mg, Ca 650 mg, P 142.5 mg.

Sample collection

During the period from 7 d before delivery to 5 d after delivery, The blood samples without anticoagulant of the goats were harvested via the jugular vein at 0700 hours daily. The blood samples were centrifuged at 4,000 × g for 20 min at 4 °C to harvest serum. The serum samples were stored at −20 °C until analysis. Approximately 50 mL colostrum (days 0 and 1) or milk samples (days 2 to 5) were collected by hand milking at 0700 and 1600 hours during the first 6 d of lactation. Two colostrum or milk samples of the same goat on the same day were evenly mixed at 1:1. The determination of milk contents via milk component analyzer (Foss FT120, Denmark) was completed on the day of sampling and the remaining samples were stored at −80 °C until analysis.

Serum and milk laboratory analysis

The concentrations of serum total calcium were determined by using a quantitative colorimetric calcium assay kit (C004-2-1, Nanjing Jiancheng Bioengineering Institute, China) as described by Zang et al. (2018). Serum samples were analyzed for serum component analysis (alkaline phosphatase [ALP] levels, total serum protein levels, and serum albumin levels) by the Yangling Demonstration Zone Hospital. The ALP levels in serum were analyzed by ALP assay kit (SFBC rate method, Hu‘nan YongHeYangGuang Biotechnology Co. Ltd., China) according to the manufacturer’s instructions, and the intra-assay coefficient of variance (CV) was <5.0% and the inter-assay CV was <5.0%. The total serum protein levels in serum were analyzed by total protein assay kit (Doumas method, Hu‘nan YongHeYangGuang Biotechnology Co. Ltd.) according to the manufacturer’s instructions, and the intra-assay Cv was <2.0% and the inter-assay CV was <5.0%. The serum albumin levels in serum were analyzed by albumin assay kit (Bromocresol Green method, Hu‘nan YongHeYangGuang Biotechnology Co. Ltd.) according to the manufacturer’s instructions, and the intra-assay Cv was <2.0% and the inter-assay CV was <5.0%.

The concentrations of 5-HT and PTHrP in serum and colostrum and milk were analyzed by ELISA kit (ml061860 and ml061858, Shanghai Enzyme-linked Biotechnology Co. Ltd.) according to the manufacturer’s instructions. The intra-assay Cv was <9.7% and the inter-assay CV was <13%. Before the concentrations of PTHrP in colostrum and milk were analyzed, samples were diluted 1:1000 in order to fall within the range of the standard curve of the assay.

Cell culture and treatments

GMEC were isolated from the mammary gland of Guanzhong dairy goats as described by Li et al. (2016). Briefly, the GMEC were obtained from mammary gland biopsies of three goats at peak of lactation. Under sterile conditions, mammary gland tissue sections were dissected and washed with D-Hank’s solution. The granular acinar tissue was cut into pieces and then cultured with a complete medium until cells separated from the tissue. The culture medium composition is as follows: Basal DMEM/F12 medium (D6570, Solarbio, Beijing, China) supplemented with 5 μg/mL insulin (11070-73-8, Sigma-Aldrich), 100 U/mL penicillin and 100 mg/mL streptomycin, 10 ng/mL epidermal growth factor (PHG0311, Sigma-Aldrich), 1 μg/mL hydrocortisone (H0888, Sigma-Aldrich), and 10% fetal bovine serum (Gibco, Gaithersburg, MD). The GMEC were cultured in a humidified atmosphere with 5% CO2 at 37 °C. The cells were cultured in the lactogenic medium with prolactin (L6520, 2 μg/mL, Sigma-Aldrich) for 48 h before performing the following experiments.

Serotonin HCL (153-98-0, Selleck, China) and 4-chloro-dl-phenylalanine (PCPA, 7424-00-2, Selleck) were dissolved in dimethyl sulfoxide, and further dilutions were made in a complete medium. The GMEC were seeded in 12-well plates at a density of 2 × 105 cells per well and treated with 100 μM 5-HT (Zang et al., 2018) or 30 μM PCPA (Zhang et al., 2021), respectively, when the confluence reached about 70% to 80%. Cells were collected for RNA isolation after 24 h incubation and were collected for protein extraction after 48 h incubation.

Measurement of intracellular calcium

As described by Zhang et al. (2021), the intracellular calcium trafficking in GMEC was detected using Fluo-3, AM (4 μM, F8841, Solarbio). Briefly, Fluo-3, AM was dissolved with anhydrous DMSO to prepare 2 mM storage solution. An equal volume of 20% pluronic F127 solution was added to the storage solution. About 4 μm Fluo-3, AM working solution was prepared by diluting with Hanks’ balanced salt solution (HBSS, Solarbio). The working solution was then added to the cells. After culturing at 37 °C for 20 min, five times the volume of HBSS containing 1% fetal bovine serum was added to GMEC for 40 min. The cells were washed three times and then resuscitated with HEPES buffer saline (Solarbio) to make 1 × 105 cells per mL solution. Cells were cultured for 10 min, and intracellular calcium trafficking was detected by laser scanning confocal microscopy. The intensity of fluorescence was measured by ImageJ software.

The calcium assay kit (C004-2-1, Nanjing Jiancheng Bioengineering Institute) was also used to determine the concentrations of total intracellular calcium. Briefly, when the cells grow 90% to 100% in a six-well plate, 100 μL sample lysate buffer was added to each well for 1 min. Then, the cell lysate is centrifuged at 10,000 to 14,000 × g, 4 °C for 5 min, and the supernatant was collected for calcium concentration detection according to the instruction. Besides, the protein concentration of the supernatant was determined by the Bradford method.

Quantitative real-time PCR

Total RNA was extracted by using Trizol Reagent (15596026, Invitrogen, Shanghai) according to the manufacturer’s instructions. The PrimeScript RT kit (RR047A, Takara Bio Inc., Japan) was used to synthesize the first-strand complementary DNA. Sequences of quantitative real-time PCR (qPCR) primers are shown in Supplementary Table S1. The amplification efficiency of the primers was confirmed to be in the range of 95% and 105%, and the primer specificity was evaluated by the presence of a single temperature dissociation peak. The qPCR was performed in triplicate in a Bio-Rad master cycler using the SYBR Green PCR Master Mix (RR420L, Takara Bio Inc.) according to the manufacturer’s protocol. A negative RT sample and water control were run on all plates. Ubiquitously expressed transcript, ribosomal protein S9, and mitochondrial ribosomal protein L39 were used as reference genes (Bionaz and Loor, 2007). The internal reference CT value of each sample was calculated by the weighted average CT value of the three internal reference genes. The 2−ΔΔCt method was used for analyzing the qPCR data with the control-treated GMEC serving the basis of comparison.

Western blotting

As described by Zang et al. (2018), cells were collected from different treatment groups and lysed in radioimmunoprecipitation assay buffer. The protein concentration was determined by the Bradford method. Proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes and subsequently blocked with milk powder solution for 4 h at room temperature and incubation with the primary antibody for 8 h at 4 °C. Anti-PTHrP (1:1,000), anti-PMCA1 (1:1,000), and anti-β-actin (1:3,000) were purchased from Abcam (ab239527, ab190355, and ab8226, Abcam, Cambridge, MA). The membranes were then washed with PBS-tween and incubated for 1 h with secondary antibodies. Protein bands were detected after treatment of SuperSignal West Femto agent of Thermo (34094, Thermo Scientific, Karlsruhe, Germany).

Statistical analysis

Statistical analyses were assessed by SPSS 20.0 (SPSS, Chicago, IL). Data were tested for normality using the Shapiro–Wilk test. An individual animal was considered the experimental until in all analyses. The general linear model was used for statistical analysis. The model used was Y = μ + T + D + T*D + e, where μ was the overall average of the dependent variable, T (treatment) was the fixed effect of treatment with 5-HTP or saline, D was the fixed effect of the days relative to parturition (DRTP), T*D was the interaction effect of DRTP and T, and e was the random error effect. 5-HTP treatment was used as between-subject effect and time was used as within-subject effect. Data for milk composition content (fat, protein, lactose, nonfat solid, total fat solid, and urea nitrogen), blood biochemical index (ALP, total protein, and albumin), concentrations of calcium, concentrations of 5-HT, and PTHrP were analyzed using repeated measures ANOVA. When the tested dependent variable does not obey Mauchly’s Test of Sphericity (P < 0.05), the multivariate (multivariate analysis of variance) of GLM was chosen for further analysis. In the multivariate method, the data of each day were considered as dependent variables, DRTP and treatment were chosen as fixed factors. The data are presented as mean ± standard error of the means (SEM) for at least three independent experiments. P-values < 0.05 were considered statistically 
significant (*P < 0.05).

Results

Goat milk component concentrations are not affected by 5-HTP infusion

Goat milk component concentrations, including fat, protein, lactose, nonfat solid, and total fat solid, were evaluated as a measurement of goat lactation status. There were no differences in all component indicators between the goats that received injection of 5-HTP and goats that received injection of the corresponding amount of saline (P > 0.05; Figure 1A–F).

Figure 1.

Figure 1.

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Goat milk contents of multiparous dairy goat injected with saline or 1.0 mg/kg bodyweight of 5-HTP reconstituted in saline (12 goats per group). Effect of 5-HTP injection on milk fat concentration (A). Effect of 5-HTP injection on milk protein concentration (B). Effect of 5-HTP injection of lactose concentration (C). Effect of 5-HTP injection on nonfat solid concentration (D). Effect of 5-HTP injection on total fat solid concentration (E). Effect of 5-HTP treatment injection on milk urea nitrogen (F). Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

Circulating 5-HT concentrations are increased by 5-HTP infusion

The mean serum 5-HT concentrations of the whole injection 
period were increased by 5-HTP injection treatment (2.526 ± 0.072 ng/mL vs. 2.969 ± 0.072 ng/mL, P < 0.05, Figure 2A). 5-HTP injection treatment and DRTP had an effect (P < 0.001) on circulating 5-HT concentrations. There was also an effect of treatment by time interaction (P = 0.009) with 5-HTP-injected goats having higher serum 5-HT concentrations than saline-injected goats on day −5 (P = 0.003), day −4 (P = 0.005), day −2 (P = 0.002), and day 5 (P < 0.001; Figure 2B). There was a treatment by time interaction for colostrum 5-HT concentration, because the colostrum 5-HT concentration on day 1 was higher in 5-HTP injection group than that in saline injection group (5.227 ± 0.362 ng/mL vs. 4.475 ± 0.356 ng/mL, P < 0.05, Figure 2C).

Figure 2.

Figure 2.

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Circulating 5-HT concentrations of multiparous dairy goats injected with saline or 1.0 mg/kg bodyweight of 5-HTP reconstituted in saline (12 goats per group). Effect of 5-HTP injection on average serum 5-HT concentration during the entire injection period (A). Effect of 5-HTP injection on serum 5-HT concentrations on each day (B). Effect of 5-HTP injection on milk 5-HT concentrations on each day (C). Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

Contents of serum calcium and ALP are increased by 5-HTP infusion

There was a significant effect of 5-HTP injection treatment on serum calcium concentration (P = 0.006, Figure 3A). 5-HTP injection treatment by time interaction had an effect on serum calcium concentrations because the serum calcium concentration of the 5-HTP injection group was higher than that of the saline injection group on day 4 after parturition (P < 0.05, Figure 3A). There was no treatment effect or treatment by time interaction effect on the colostrum or milk calcium concentration (P > 0.05, Figure 3B). The 5-HTP treatment increased serum contents of ALP on days −4 and 0 (P < 0.05, Figure 3C). No differences in serum albumin and globulin were observed (Supplementary Figure S1).

Figure 3.

Figure 3.

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Circulating calcium and alkaline phosphatase concentrations of multiparous dairy goats injected with saline or 1.0 mg/kg bodyweight of 5-HTP reconstituted in saline (12 goats per group). Effect of 5-HTP injection on serum calcium concentrations on each day (A). Effect of 5-HTP injection on milk calcium concentrations on each day (B). Effect of 5-HTP injection on serum alkaline phosphatase concentrations before or after parturition (C). Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

Circulating PTHrP concentrations are increased by 5-HTP infusion

There was a treatment by time interaction for serum PTHrP concentrations because the serum PTHrP concentrations of the 5-HTP-treated group were increased on day −5 (P < 0.001), day −4 (P = 0.031), day −1 (P = 0.006), day 3 (P = 0.002), day 4 (P = 0.001), and day 5 (P < 0.001) compared with the saline injection group (P < 0.05), while no differences were found on days −2, 0, 1, and 2 (P > 0.05, Figure 4A). 
There was also a treatment by time interaction for milk PTHrP concentrations because the milk PTHrP concentration on day 5 was higher in the 5-HTP injection group compared with the saline injection group (P = 0.002, Figure 4B).

Figure 4.

Figure 4.

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Circulating PTHrP concentrations of multiparous dairy goats injected with saline or 1.0 mg/kg bodyweight of 5-HTP reconstituted in saline (12 goats per group). Effect of 5-HTP injection on serum PTHrP concentrations on each day (A). Effect of 5-HTP injection on milk PTHrP concentrations on each day (B). Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

5-HT increases intracellular calcium in GMEC

To investigate whether 5-HT could regulate the calcium homeostasis, GMEC were treated with 100 μM 5-HT or 30 μM PCAP (inhibitor of 5-HT synthesis), respectively. Calcium staining results showed that intracellular calcium contents in GMEC were increased by the treatment of 5-HT and decreased by the treatment of PCPA (P < 0.05, Figure 5A and B). 
Calcium level measurement showed that 5-HT treatment increased the intracellular calcium by 20% and PCPA decreased the intracellular calcium by 50% (1.658 ± 0.032 mM vs. 2.107 ± 0.038 mM vs. 0.792 ± 0.041 mM, P < 0.05, n = 6, Figure 5C).

Figure 5.

Figure 5.

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Effect of 5-HT on the concentrations of intracellular calcium in goat mammary epithelial cells. Cells were treated with 5-HT (100 μM), inhibitor of THP1 (PCPA, 30 μM), or DMOS (negative control). Intracellular calcium ion was stained by Fluo-3, AM and imaged by Laser scanning Confocal Microscope (A, B). Fluorescence intensity was measured by ImageJ software (B). Intracellular calcium level was measured by the quantitative colorimetric calcium assay kit (C). All experiments were conducted in triplicate. Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

5-HT increased expression of PTHrP and altered genes related to calcium transportation in GMEC

Changes in the expression of calcium homeostasis-related genes caused by 5-HT or PCPA treatment were further evaluated. Protein abundance of PHTrP in GMEC was increased with 5-HT treatment but decreased with PCPA treatment (P < 0.05, Figure 6A). 5-HT treatment decreased the protein abundance of PMCA1, which is responsible for maintaining intracellular calcium homeostasis of mammary epithelial cells, and PCPA treatment increased the protein abundance of PMCA1 (P < 0.05, Figure 6A). Besides, the 5-HT treatment decreased the mRNA expression of PMCA1 and SPCA1 and increased the mRNA expression of PTHrP, PMCA2, and SPCA2 in GMEC (P < 0.05, Figure 6B–F). On the contrary, mRNA expression of PMCA1 and SPCA1 were increased and mRNA expression of PTHrP, PMCA2, and SPCA2 were decreased when GMEC were treated with PCPA compared with the control group (P < 0.05, Figure 6B–F).

Figure 6.

Figure 6.

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Effect of 5-HT treatment on the expression of PTHrP and genes related to calcium transportation in GMEC. Cells were treated with 5-HT (100 μM), inhibitor of THP1 (PCPA, 30 μM), or DMOS (negative control). Western blotting for PTHrP and PMCA1 protein in each group (A). Band densitometry was estimated by ImageJ. Relative mRNA expression of genes related to calcium transportation of cells (B–F). All experiments were conducted in triplicate. Asterisks indicate statistical difference between group means (P < 0.05). All values are reported as mean ± SEM.

Discussion

From pregnancy to lactation, mammals have to face physiological calcium challenges from the delivery process and colostrum synthesis (Kovacs and Kronenberg, 1997; Horseman and Collier, 2014; Hernandez-Castellano et al., 2018). This period is the primary reason why dairy goats or cows are prone to calcium-related disorders, specifically clinical and subclinical hypocalcemia. 5-HT regulates various aspects of mammary gland homeostasis, such as lactoprotein biogenesis, calcium transportation, and glucose metabolism (Matsuda et al., 1998; Laporta et al., 2015a; Hernandez-Castellano et al., 2017). Studies by Laporta and colleagues have demonstrated the role of 5-HT in modulating calcium homeostasis in late lactation cows (Laporta et al., 2015a; Weaver et al., 2016a). In addition, the regulatory effects of 5-HT on calcium homeostasis are unique and dependent on the breeds of cattle (Weaver et al., 2016b). To date, up to now, no research has been reported on the effect of 5-HT on dairy goats. The results of the current in vivo experiment suggested that treatment of dairy goats with 5-HTP had positive effects on calcium status during the transition period. In addition, the results of the in vitro experiment show that changes in 5-HT concentrations affect goat mammary tissue by increasing intracellular calcium levels in GMEC.

The composition of colostrum is very different from that of normal milk (Zhang et al., 2020). To evaluate the effects of 5-HTP injection, the composition of colostrum, milk fat, milk protein, lactose, total fat solids, nonfat solids, and milk urea nitrogen were monitored from days 0 to 5 relative to the delivery day. The colostrum composition changed greatly, which indicated that the imbalance of energy metabolism in the body was caused by parturition. In comparison, the concentrations of milk urea nitrogen on day 1 were higher than that of the saline injection group. Milk urea nitrogen could be used as an indicator of protein metabolism and energy intake of lactating ewes (Cannas et al., 1998). As reported by Laporta et al. (2015a), the energy metabolism of dairy cows was also altered by increasing 5-HT concentrations. Therefore, it is still worth studying whether 5-HT can be used as a regulator of energy-protein metabolism balance in perinatal dairy goats.

The concentrations of 5-HT synthesized in the mammary glands are the highest during lactation (Collier et al., 2012; Hernandez et al., 2012; Laporta et al., 2013c). Exogenous 5-HTP has been shown to increase concentrations of circulating 5-HT and PTHrP and milk calcium concentrations after parturition in mice and rats (Hernandez et al., 2012; Laporta et al., 2013a). In the current study, 5-HTP injection successfully increased blood 5-HT level at days −5, −4, and −2 before parturition. Colostrum 5-HT levels of the 5-HTP injection group were also higher than that in the saline injection group. Hernandez-Castellano et al. (2018) found that supplementation of colostrum and milk with 5-hydroxy-l-tryptophan increased the adaptive immune system but had no effect on growth performance in calves (Hernandez-Castellano et al., 2018). In a study by Miao et al. (2019), dietary supplementation of tryptophan to the lactating sows significantly increased milk yield, milk 5-HT concentration, milk calcium level, the feed intake of sows, and the average daily gain of piglets, meanwhile, treatment of tryptophan promoted the fatty acid synthesis, lactose synthase, and β-casein production in PMEC. Those studies showed that increased 5-HT concentrations may be beneficial to the health of developing offspring either by enhancing immunity or by increasing milk nutritional pathways (Moore et al., 2015; Weaver et al., 2016b). It is necessary to evaluate the role of 5-HT on milk yield, the colostrum quantity, and lamb growth performance in our further studies.

Subclinical or clinical hypocalcemia (milk fever) is usually due to the inability to extract sufficient calcium from the bone in multiparous dairy goats (Oetzel, 1988; Liesegang et al., 2006; Kovacs, 2015). This study analyzed the effects of 5-HT on calcium homeostasis in perinatal dairy goats from different perspectives. In the saline injection group, the average postpartum serum calcium level was lower than that of the prepartum level, indicating that the dairy goats were challenged with calcium homeostasis under normal conditions (Possamai et al., 2015). No significant difference was observed between the prepartum or postpartum serum calcium levels in the 5-HTP injection group. Serum ALP levels, the indicator of cell osteogenic differentiation (Ying et al., 2013), were increased in the 5-HTP injection group. These results suggested that 5-HTP treatment was helping to maintain higher circulating calcium concentrations around parturition.

PTHrP is reported to be a regulator of perinatal calcium homeostasis (Ardeshirpour et al., 2006) and can play a feedback role in regulating both high and low serum calcium levels (Eller-Vainicher et al., 2012). Previous studies had shown that there was a correlation between serum PTHrP and serum calcium in cows or goats on the day of parturition (Laporta et al., 2013a; Zang et al., 2018). In this study, both prepartum and postpartum serum PTHrP levels in the 5-HTP injection group were higher than that of the saline injection group. It was also observed that 5-HTP injection can only improve the serum 5-HT levels of prepartum, but the higher PTHrP levels can be maintained to the fifth day of postpartum. This is probably due to the promoting effect of 5-HT on PHTrP being sustainable. During the transitional period, the main source of calcium in the circulation system was bone calcium reabsorption, and the pathways of calcium loss included milk calcium secretion, urine calcium excretion, the requirements of fetal development, and so on. In dairy cows, milk production was not affected by 5-HTP infusion while the calcium concentrations in the milk were increased during the transitional period (Laporta et al., 2015a; Weaver et al., 2016b). In this study, 5-HTP treatment had no effect on the colostrum or milk calcium concentrations. Due to the limited conditions, the milk yield of goats was not recorded and there was no way to calculate the total amount of colostrum or milk calcium in the present study. Therefore, full-scale monitoring of calcium homeostasis is still necessary for our future studies.

5-HT promoted the expression of PTHrP in goat or cow mammary epithelial cells (Hernandez et al., 2012; Zang et al., 2018). PTHrP, secreted into the circulation system, stimulates calcium resorption from bone (Dedic et al., 2018). To further study the mechanism of 5-HT regulating calcium homeostasis in breast tissue, we treated GMEC with exogenous 5-HT and PCPA (5-HT synthesis inhibitor), and measured the calcium levels in GMEC. 5-HT treatment significantly increased calcium levels in GMEC. Meanwhile, changes in PTHrP expression in cells were consistent with changes in calcium levels in cells. In the mammary gland, PMCA1 was present with a basolateral localization and PMCA2 localization appeared more pronounced toward the apical membrane. Ist is envisaged that direct transport of calcium across the apical membrane by PMCA2 accounts for the majority of calcium in milk (60% to 70%). In contrast to PMCA2, PMCA1 had a more basolateral distribution, consistent with a possible role in the regulation of cytosolic free calcium in cells of the mammary gland (Faddy et al., 2008). The downregulation of PMCA1 and SPCA1 and upregulation of PMCA2 and SPCA2 caused by 5-HT treatment suggested that more calcium would be stored in the cells, and the GMEC tend to transport calcium into the milk. 5-HT synthesis deficiency caused by tryptophan hydroxylase 1 (TPH1) knockout in mouse module (Hernandez et al., 2012) or si-TPH1 in GMEC (Zang et al., 2018) led to a decrease in expression of PTHrP. In the present study, PCPA, the specific inhibitor of TPH1, decreased calcium levels and the expression of PTHrP significantly. It also supports the conclusion that serum PTHrP concentration was correlated with serum calcium concentration (Zang et al., 2018). However, it seems that there is more than one way for 5-HT to regulate calcium metabolism in cells. Our recent study suggested that 5-HT can increase intracellular calcium of mammary epithelial cells via the miRNA-99a-3p/PMCA1 axis (Chen et al., 2020), which was consistent with the decreased PMCA1 expression levels induced by 5-HT treatment.

5-HT is mostly known as a neurotransmitter in the central nervous system (De Deurwaerdere and Di Giovanni, 2021), however, 98% of total 5-HT is produced in the body(Berger et al., 2009). There is increasing evidence supporting the importance of peripheral 5-HT in physiological functions, immune response, and other metabolic processes (Ahern, 2011; Itsumi et al., 2020; Suleimanova et al., 2020). The establishment of new roles and functions for 5-HT on the goat during the transition period helps to improve lactation performance and efficiency. 5-HT has been reported to regulate various functions in the mammary glands such as cell turnover, lactoprotein gene expression, calcium signaling, and tight junction formation (Pai et al., 2015; Kessler et al., 2019). 5-HT can also regulate glucose metabolism, lipid genesis, energy metabolism, and immune processes in different tissue and organs (Fu et al., 2018; Valente et al., 2021). The lack of peripheral 5-HT in mammary glands significantly impacted insulin signal and glucose metabolism (Laporta et al., 2015b). The current study suggests that 5-HT has comprehensive and simultaneous effects on physiological metabolism across and within tissues, which agrees with previous research.

Conclusions

This study presents evidence that prepartum injection of 5-HTP can increase the concentrations of serum 5-HT on days −5, −4, −2, and 5, relative to parturition and further increase the concentrations of serum PTHrP on days −5, −4, −1, 3, 4, and 5 relative to parturition. 5-HT could increase intracellular calcium level via increasing PTHrP and decreasing PMCA1 in GMEC. Understanding the regulatory role of 5-HT on calcium homeostasis may contribute to the prevention or decrease the occurrence prevent or relieve the occurrence of hypocalcemia in periparturient dairy goats.

Supplementary Material

skac065_suppl_Supplementary_Material
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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No.31572368) and the Science and Technology Transformation Project of Shaanxi Provincial Department of Agriculture (NYKJ-2020-YL-05).

Glossary

Abbreviations

5-HT

5-hydroxytryphtamine, serotonin

5-HTP

5-hydroxy-l-tryptophan

CasR

calcium-sensitive receptor

ELISA

enzyme-linked immunosorbent assay

GMEC

goat mammary epithelial cells

MRPL39

mitochondrial ribosomal protein L39

PCPA

4-chloro-dl-phenylalanine

PMCA1

plasma membrane Ca2+-ATPases1

PMCA2

plasma membrane Ca2+-ATPases2

PTHrP

parathyroid hormone-related protein

RPS9

ribosomal protein S9

SPCA1

secretory-pathway Ca2+-ATPases1

SPCA2

secretory-pathway Ca2+-ATPases2

SRECA2

sarcoplasmic reticulum Ca2+-ATPase2

TPH1

tryptophan hydroxylase 1

UXT

ubiquitously expressed transcript

Conflict of Interest Statement

The authors declare no conflict of interest.

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