Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
DAIRY SCIENCE-GEN
Non Technical Summary
Subclinical hypocalcemia affects 50% of all dairy cows in the United States. Cows that succumb to subclinical hypocalcemia are more likely to develop other metabolic disorders and diseases that are common in early lactation dairy cows. The overall objective of the project aims to describe the contribution of serotonin to the regulation of calcium homeostasis in lactating dairy cows. We will use in vitro laboratory techniques combined with cow experiments to demonstrate that serotonin controls maternal calcium concentrations by regulating calcium transport and parathyroid hormone-related protein production in the mammary gland. Parathyroid hormone-related protein production is critical for calcium mobilization from bone. Serotonin has been shown to coordinate calcium transport and signaling in the mammary gland and is critical to regulating the communication between the bone and mammary gland during lactation. Therefore, dissecting the mechanisms by which serotonin regulates maternal calcium homeostasis will lead us to develop novel methods to prevent hypocalcemia. Current methods for the prevention of hypocalcemia in dairy cows are insufficient. Our project aims to improve the productivity and health of the dairy cow by reducing hypocalcemia during the periparturient period. The overall impact of our research aims to result in a novel method to improve maternal calcium homeostasis during the early lactation period through manipulation of the serotonin-calcium-parathyroid hormone related-protein axis.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Goals / Objectives
The overall objective of this research is to dissect the contribution of serotonin to the regulation of maternal calcium homeostasis during lactation in dairy cows. Our central hypothesis is that serotonin stimulates Ca transfer from blood into the mammary gland, resulting in a transient hypocalcemia. This is the critical step that causes mammary gland secretion of PTHrP. Our research will establish serotonin as a novel therapeutic intervention for prevention of SCH and CH. This will result in decreased metabolic disorders and culling, and increase animal productivity and health.The major goals of this project are:1) Directly evaluate the role of serotonin and the calcium sensing receptor in regulation of parathyroid hormone related-protein production and expression of calcium transporters in the bovine mammary gland epithelium;2) Determine the indirect effect of the serotonin-parathyroid hormone related-protein axis in response to subclinical hypocalcemia, and;3) Elucidate the contribution of the serotonin-parathyroid hormone related-protein axis to reduction of subclinical hypocalcemia and clinical hypocalcemia in dairy cows receiving a negative dietary cation-anion difference pre-calving.
Project Methods
Methods for the three objectives are as follows:Objective 1a: After 8 d in culture with serum-free proliferation media, pBMEC will be switched to a lactogenic medium in combination with gel release in order to maximize lactogenic capacity of the pBMEC. MAC-T cells will be grown to confluence in monolayers in a proliferation medium and will subsequently be treated with a lactogenic complex for 72 hr to induce a lactogenic response. Treatments will be as follows for pBMEC collagen cultures: lactogenic medium alone, lactogenic medium + 200 μM serotonin hydrochloride, lactogenic medium + 1.0 μM EGTA, lactogenic medium + 200 μM serotonin hydrochloride + 1.0 μM EGTA. Treatments will be as follows for MAC-T cells: lactogenic media alone (control), lactogenic media + 200 μM serotonin hydrochloride, lactogenic media + 0.6 μM EGTA, lactogenic media + 200 μM serotonin hydrochloride + 0.6 μM EGTA. Experiments will be performed in triplicate and repeated in four independent experiments (N=4). Total RNA will be isolated from BMEC and MAC-T using the TriReagent Protocol and subsequently reverse transcribed into cDNA. Quantitative real-time PCR (qPCR) will then be performed for PTHrP and the Ca pumps using ssoFast Eva Green. Additionally, PTHrP (IRMA) and serotonin (ELISA) will be measured in the media collected from the pBMEC and MAC-T in response to all treatments. Results will be analyzed using a mixed-model ANOVA with a Tukey's post-test for pair-wise comparisons.Objective 1b: We will test the necessity of CaSR for PTHrP production in pBMECs and MAC-T cells grown in lactogenic conditions as described in objective 1a. Graded concentrations of the CaSR agonist (strontium chloride, Tocris) or antagonist (NPS 2143 hydrochloride, Tocris), with and without 200 μM serotonin hydrochloride will be added and Ca pump and PTHrP mRNA abundance measured 72 hr later. Experiments will be performed in triplicate and repeated in 4 independent experiments (N=4). Media will be collected and analyzed for serotonin (ELISA) and PTHrP (IRMA). RNA will be isolated, reverse transcribed, quantified, and analyzed as described in Aim 1a. Results will be analyzed using a mixed model ANOVA with a Tukey's post-test for pair-wise comparisons.Objective 2: Ten late-lactation, non-pregnant multiparous lactating Holstein cows with similar milk production, and ten non-pregnant, non-lactating Holstein cows, for a total of 20 cows, will be enrolled on this experiment and housed at the UW-Madison Dairy Cattle Center. We are using both lactating and non-lactating cows in order to illustrate the importance of the mammary serotonin-PTHrP axis in the control of maternal hypocalcemia during lactation. We are also using non-pregnant cows to remove the influence of pregnancy hormones on mammary gland development and function. A power analysis based on the outcome of a 0.2 mM decrease in total Ca concentration with a standard deviation of 0.2, and a power of 0.8, results in n=5 per treatment group. Lactating and non-lactating cows will be randomly assigned to one of two treatments in a completely randomized design. Cows in Group 1 will receive a continuous 24 h IV infusion of sterile saline (control; n=5 lactating (CL), n=5 non-lactating (CNL)). Cows in Group 2 will receive a continuous 24 h IV infusion of 5% EGTA (EGTA; n=5 lactating (EGTA-L), n=5 non-lactating (EGTA-NL)), which is selective for binding Ca. A controlled infusion pump will be utilized, and the rate of infusion will be held constant between groups. The EGTA group will receive an infusion of 500 ml/h initially, until blood ionized Ca reaches 1.0 mM (equivalent to approximately 2.0 mM total Ca), which is a level considered to be SCH, after which the infusion rate will be modified hourly, based on ionized blood Ca concentrations. Concentrations of ionized blood Ca will be monitored with a handheld cow-side biochemical analyzer using CG8+ cartridges. Blood samples will be collected immediately prior to the infusion, as well as hourly during the infusion, to measure concentrations of serotonin (ELISA), total Ca (colorimetric assay), PTHrP (IRMA), PTH (ELISA; Alpco) and carboxyterminal cross-linked teleopeptide of collagen I (ICTP; ELISA), a bone resorption marker. Additional blood samples will be collected at 4, 8, 12, 24, 48, and 72 hr after the end of the infusion period. Urine samples will be collected immediately prior to infusion and every 4 h during the infusion, and at 12, 24, 48 and 72 hr after the infusion to measure deoxypyridinoline (DPD; ELISA), a bone resorption marker and total Ca concentrations. Feed samples from the total mixed ration will be collected the week of the experiment, dried at 60°C, and analyzed at Dairyland Laboratories for crude protein, neutral detergent fiber, lignin, fat, ash, and major minerals including Ca, phosphorous, potassium, sodium, chloride, magnesium, and sulfur using wet chemistry analysis. Mammary gland biopsies will be performed 1 d prior to the start of the infusions, immediately after the end of the infusions, and 72 hr after the infusions have been terminated. Samples will be analyzed for mRNA expression. Additionally, serotonin (ELISA), and PTHrP (IRMA) concentrations will be measured in the mammary gland samples collected, as previously described. Data will be analyzed using a mixed model ANOVA with time included as a repeated measure due to the multiple blood and urine samples taken over time, and cow within treatment considered a random effect. Means separation will be done using Tukey's post-hoc for pair-wise comparisons.Objective 3: A randomized complete block design with a 2x2 factorial arrangement of treatments in which there will be four treatment groups: 1) Positive DCAD diet (+130 meEq/kg) fed for 21 d pre-calving (PDCAD), 2) positive DCAD diet plus intravenous infusion of 1.0 mg/kg 5-HTP for 7 days pre-calving (PDCAD+5HTP), 3) negative DCAD diet (-130mEq/kg) fed for 21 days pre-calving (NDCAD), and 4) negative DCAD diet plus intravenous infusion of 1.0 mg/kg 5-HTP for 7 days pre-calving (NDCAD+5HTP). Cows not receiving infusion of 5-HTP will be infused with a similar amount of saline solution. 32 multiparous Holstein cows will be blocked by anticipated calving date then randomly allocated to one of the four treatments to yield 8 cows per treatment. An n=8 will be used for each treatment group for a total of 32 cows. This sample size was chosen based on a power analysis using total Ca concentrations as the response variable. We expect to see a 0.2 mM change in total Ca concentrations, with a standard deviation of 0.25, resulting in a power of 0.8. Blood and urine samples will be taken at -9, -7, -5, -3, -1, 0, 1, 2, 3, and every 3 d until d 30 of lactation. Milk samples will be collected on the same days as the post-calving blood samples. Mammary gland biopsies will be taken on the day prior to the initiation of 5-HTP treatment, 24 hr after calving and every 7 days until d 30 of lactation. Milk yield and feed intake will be recorded daily, and feed samples will be collected weekly for dry matter analysis and nutrient analysis as described in Experiment 2. Serum harvested from blood will be analyzed for total Ca, ionized Ca, serotonin and ICTP as described in experiment 2. Plasma harvested from blood will be analyzed for PTHrP. Urine samples will be analyzed for total Ca and DPD concentrations as described in experiment 2. Urine pH will also be monitored to ensure diet acidification. Milk samples will be analyzed for total Ca concentrations. Mammary gland samples will be analyzed for mRNA expression (qPCR) of TPH1, PTHrP, PMCA2, CaSR, ORAI1, and SPCA2 as described in objective 1 of the proposal. Data will be analyzed using a mixed model ANOVA with time included as a repeated measurement, block, cow within block, and treatment considered random effects, and means separation using Tukey's post-hoc test for pair-wise comparisons.