Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
DAIRY SCIENCE
Non Technical Summary
Periparturient disorders are not only disastrous to the farmer but they are responsible for substantial decreases in overall herd health. Periparturient paresis results in a 14% decrease in milk production in the subsequent lactation, with a cost of $334/animal per incidence (Erb et al., 1984; Oetzel, 1988). Additionally, subclinical hypocalcemia affects 25-50% of cows and costs $125/animal/incidence. Prevention of hypocalcemia in dairy cattle could save the dairy industry approximately $140 million each year. This project focuses on delineating a novel pathway for the regulation of calcium mobilization of bone during the transition period. A cow that survives the transition period free of disease can be expected to have a successful lactation period, with high hopes for efficient reproductive performance. The ability to produce an adequate amount of milk, as well as to reproduce is absolutely critical to the dairy farmer maintaining economic viability of the farm operations, as well as to satisfy human food needs. Hypocalcemia has been demonstrated to be responsible for blunted immune cell response to stimuli, reduction of smooth muscle contraction, and increased risk for reproductive disorders placing animals at a higher risk of mastitis, retained fetal membranes, displaced abomasum, dystocia, and ketosis, all of which lead to a decreased production life (Erb et al., 1984; Kimura et al., 2006; Goff, 2008; Reinhardt et al., 2010). The average reduction in productive life of an animal overcome with milk fever is 3.4 yr (Horst et al., 1997). Reduced animal productivity is a major economic detriment to the dairy farmer, particularly when the primary source of income is derived from the amount of milk each animal successfully produces in a respective lactation cycle. Therefore, this project is important because it will be critical to demonstrating a novel mechanism for the regulation of hypocalcemia in transiton cattle.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
To determine the role of serotonin in regulating calcium mobilzation during the periparturient period in dairy cattle.
Project Methods
Experiment 1. Determine circulating and milk concentrations of 5-HT, PTHrP and calcium in Holstein and Jersey cattle throughout their pregnancy/lactation cycle. This goal of this experiment is to determine the patterns of 5-HT, PTHrP and calcium in the circulation and milk throughout the pregnancy/lactation cycle (d -30 partum through an entire 305 day lactation cycle) of multiparous Holstein and Jersey cattle from local commercial dairy farms in Wisconsin. Whole blood will be collected to analyze 5-HT (ELISA, Enzo Life Sciences) and calcium ( Additionally, we seek to determine the bone turnover of jersey cattle throughout the pregnancy/lactation cycle (d -30 partum through an entire 305 day lactation cycle) through the measurement of a bone turnover marker, C-telopeptide (CTx) fragments of collagen type I in serum (Serum CrossLaps Elisa; Immunodiagnostics systems; Holtenius and Ekelund, 2005). Samples will be collected on d -20, and d -5 through d +5, d +30, d +60, d +90, d +150, and d +300 relative to parturition. Milk samples will be collected daily for the first 10 d post-partum to measure PTHrP and calcium. We will collect samples from 25 Jersey and 25 Holstein cows, for a total of 50 animals, as this will convey a power of 0.9786 when performing R-square tests for multiple correlations between variables. Additionally, in order to compare variables between breeds, 25 animals per breed will provide a power of 0.7915, with a detectable contrast of 0.80 when conducting a balanced ANOVA. Additionally, we will obtain herd records for each animal to determine the incidence of various transition related disorders, as well as to determine daily milk yield in each of these animals. Experiment 2a. Determine the ability of selective 5-HT receptor agonists to stimulate PTHrP, and the ability of selective 5-HT receptor antagonists to inhibit PTHrP in lactogenic cultures of bovine mammary epithelial cells. Primary bovine mammary epithelial cells (pBMEC) possess five 5-HT receptor subtypes (Hernandez et al., 2009). Studies suggest that the 5-HT2B receptor subtype is responsible for 5-HT induction of PTHrP in the mammary gland (Hernandez et al., 2012). However the contribution of the other four receptor subtypes have yet to be investigated in bovine mammary epithelial cells in relation to stimulation of PTHrP. pBMEC will be cultured in a 3-D lactogenic model as previously described (Stiening et al., 2008; Hernandez et al., 2008; Hernandez et al., 2009). After 8 d in culture with serum-free proliferation media, pBMEC will be switched to a lactogenic media in combination with gel release (Stiening et al., 2008; Hernandez et al., 2008; Hernandez et al., 2009). Concentration response curves (1/2 log) will be performed on pBMEC cultured in a 3-D lactogenic model and treated with the following selective agonists/antagonists, respectively, (5-HT1B: CP-94253 hydrochloride/SB-216641 hydrochloride; 5-HT2A: TCB-2/R-96544 hydrochloride; 5-HT2B: BW-732C86/SB-204741; 5-HT4: zacopride hydrochloride/GR-113808; 5-HT7: LP-44/SB-258719) for 72 hr. pBMEC will then be collected for RNA isolation using the TriReagent Protocol (Molecular Research Products) and subsequently will be reverse transcribed into cDNA (iScript Reverse Transcription Kit; BioRad). Quantitative real-time PCR (qPCR) will then be performed for PTHrP using ssoFast Eva Green (BioRad). qPCR data will be analyzed using the 2-ΔΔCt method (Livak and Schmittgen, 2001; Schimttgen and Livak, 2008). Results will be analyzed using a one-way analysis of variance (ANOVA) with a Tukey's post-test to make pair-wise comparisons. Experiment 2b. Delineation of the molecular signaling pathway responsible for 5-HT induction of PTHrP in the bovine mammary gland through siRNA-mediated knockdown of the 5-HT2B receptor. This experiment is designed as a follow-up to the pharmacological experiments performed demonstrating the role of the 5-HT2B receptor in calcium regulation in the mammary gland through 5-HT induction of PTHrP (preliminary experiments). We will utilize RNAi technology in pBMEC isolated from both Jersey and Holstein cattle to demonstrate the contribution of the 5-HT2B receptor to the regulation of PTHrP expression in the bovine mammary gland. We will be using either transient or stable expression of ON-TARGET plus siRNA dual strand chemical modification for receptor of interest in our experiments. This method has been validated by Dharmacon (Thermo Scientific) to reduce miRNA-induced off targets by preventing sense strand from being blocked by RISC uptake to favor the antisense strand loading and reducing sense strand-induce off targets. This protocol is also modified within seed region to destabilize miRNA-like activity and improve specificity to the desired targets for the most potent knockdown. Target sequences will be designed according to published and proprietary rules. Sequences will be BLAST aligned to the bovine genome sequences. At least four siRNA sequences for each target mRNA (5-HT2B) will be synthesized and tested for effectiveness at silencing the gene. Transfection will be performed using a lipid-based tranfection protocol, INTERFERin in vitro siRNA protocol (Polyplus) in pBMEC in monolayer cultures. A double transfection will be performed for the respective siRNAs to ensure effective silencing (of at least 70%) of the gene of interest. Additionally, a control gene siRNA (GAPDH) and non-targeting siRNA will be used as controls to determine transfection efficiency. Knockdown efficiency will be measured through qPCR for respective genes to demonstrate the percent inhibition of respective genes. The RNAi sequence for the 5-HT2B target that provides the most effective knockdown will be utilized for subsequent experiments. The variables that will be subsequently assayed upon silencing the 5-HT2B receptor include PTHrP mRNA expression and protein levels, and phospholipase C-β (PLC-β) activity levels. Additionally, we will examine PKC activity, as this molecule is downstream of PLC-β. We will also determine activity levels of ERK1/ERK2 MAP kinases, as this pathway has been shown to be regulated by the 5-HT2B receptor (Raymond et al., 2001). mRNA levels will be quantified using qPCR techniques previously described. Activity levels for PLC-β, PKC, and ERK1/2 MAP kinases will be determined using activity assays per manufacturer's instructions from Worthington Biochemical Corporation, Enzo Life Sciences, and Chemicon International, respectively.