Performing Department
(N/A)
Non Technical Summary
Pigs get most of their nutrients from food, but stress messes with their guts and hinders their growth. Our research focuses on how stress disrupts digestion, aiming to find ways to keep them healthy and maximize nutrient absorption even when stressed. We suspect that hidden players in the gut, like nerves and hormone-producing cells, get thrown off-balance by stress, messing up how well pigs absorb nutrients. The experiment will involve stressing pigs by weaning them at different ages. We'll then compare how well they absorb nutrients from their food and see if the nerves and hormone functions in their guts play a role. We expect early weaning to damage both the gut's nerve and hormone system, leading to poorer nutrient absorption. This research is expected to reveal key insights into how stress affects nutrient transporters, paving the way for new dietary approaches. These approaches could either provide optimal nutrition for stressed pigs or develop strategies to improve their lifelong ability to utilize nutrients.
Animal Health Component
(N/A)
Research Effort Categories
Basic
90%
Applied
(N/A)
Developmental
10%
Goals / Objectives
The long-term goal of our research program is to elucidate the basic physiological mechanisms by which production stressors in pig negatively impact gastrointestinal functions, in order to design strategies to optimize gut health and efficiency in animals. By the following aims, we will test our central hypothesis that production stressors alter the functional efficiency of gut epithelial AA transport which is involved with ENS and enteroendocrine modulations.Aim 1: Define how does weaning and weaning age influence intestinal AAs transporter expression and function in pigs.Aim 2: Identify stress-induced alterations in epithelial EECs activities and their modulation on intestinal AA transporter expression.Aim 3: Identify enteric nerve contribution on AAs transporter function.
Project Methods
Experimental design. Total 36 female piglets from 6 litters will be used in this study. Piglets will be randomly assigned to one of three weaning age groups: weaning at 15d (early weaning stress, EW), 20d (industrial weaning age, IW), and 25d of age (late-wean pig, LW). At respective weaning ages, piglets will be weaned to an on-site nursery (2 pigs/pen), while unweaned piglets assigned to other weaning groups will remain with their sows. All weaned pigs will be offered ad libitum access to water and the standard commercial nursery diet. After more than 2 weeks of post weaning recovery, the long-term effects of weaning stress on intestine development and AA transporter functions will be evaluated at d40 of age for all EW, IW, and LW pigs.Sample collection. Intestinal samples of jejunum and ileum will be collected. The mucosal scraping of each section will be snap frozen in LN2 and stored at -80C for later isolations of BBMV, RNA, and protein samples. Immediately following euthanasia, a 10 cm segment of each section will be collected for the Ussing chamber analyses. Intestinal samples will also be fixed in 10% NBF and 4% PFA until later histological analysis and immunohistochemistry staining.Intestinal electrogenic AA transport function. The ileal mucosa will be used on the Ussing chamber. We will record basal transepithelial electrical resistance (TER; ?•cm2), spontaneous PD (mV/cm2) and short-circuit current (Isc; µA/cm2). We will measure the Isc changes (ΔIsc) in response to serial additions of 3 selected AAs: glutamine (neutral transporter B0AT1), glutamate (anionic transporter EAAT3)10, and lysine (cationic transporter b0,+AT) to represent 3 distinct AA transport functions in the apical membrane of epithelial cells.Transporter expression. BBM of intestinal epithelium is the functional site of all nutrient transporters. BBM will be isolated from intestinal mucosal scraping to quantify the protein level of major AA transporters (B0AT1, rBAT/b0,+AT , EAAT3) by Western blot. We will quantify the expression of AA transporters by RT-PCR to identify the transcriptional changes.Growth performance and blood indicators. The body weight and feed intake of each pen will be measured. Growth performance and feed efficiency will be calculated. Blood samples will be collected from pigs prior to euthanasia. All blood samples will be processed for the subsequent measurement, such as serum endocrine indicators and stress hormones.Statistical Analysis. Data will be analyzed by one-way ANOVA and post hoc multiple comparisons among weaning ages. Pen will be experimental unit (n = 6) for growth performance. Individual pigs will be used as experimental unit (n = 12) for all the other measurement analyses.Serum EECs hormones. Blood level of EECs products (CCK, GLP2, PYY) will be quantified by ELISA kits, to evaluate enteroendocrine functional changes by early weaning stress.Ex vivo EECs secretion. Ussing chamber technique will be used to evaluate EECs function in nutrient sensing. We will mount jejunum and ileum mucosa on the Ussing chamber (described in Aim 1). We will apply nutrients on mucosal chamber of the tissue, collect supernatant from serosal side of chamber before and after nutrients stimulation. The difference of EECs contents (GLP2, PYY) released in chamber supernatants will indicate EECs secretory function.In vitro Enteroids culture. We will isolate intestinal crypt cells from ileum and develop enteroids culture on Matrigel matrix with L-WRN conditioned medium. We will monitor the proliferation of enteroids by measuring the size, number, budding/expansion rate by bright field microscopy. We will also examine the differentiation of enteroids, by quantifying the epithelial cell population by using specific staining, and biomarkers for gene expression.EECs nutrient sensing function. We will introduce EECs specific differentiation media in enteroids culture to increase EECs number. Then we will determine AA sensing signals of EECs by stimulating enteroids with AAs in the culture medium and assessing the release of signaling products (5-HT, GLP2, and GIP) by ELISA using commercially available assay kits. We will quantify the mRNA expression of AA sensors T1R1/T1R3 and GPCR6A by qRT-PCR.Statistical Analysis. Data will be analyzed by student t-test between EW and LW pigs. Intestinal tissues (n = 12) and enteroids (n = 8) from individual pigs will be used as experimental units.Ex vivo neural input of AA transporter functions. We will block the overall basal nerve function by TTX (tetrodotoxin) pretreatment, and (or) specific neural receptors antagonists Propranolol (beta-adrenoceptor blocker), Hexamethonium (nicotinic receptor blocker), and Atropine (muscarinic receptor blocker). We will compare the difference of AA electrogenic transport (ΔIsc in response to AA application on chamber) with and without neural blockade treatment on the chambers. We will also isolate BBMV from the Ussing chamber drug-treated tissues to quantify the protein of AA transporters after blocking ENS activity, to elucidate the basal contribution of neural input on AA transporter location and function.In vitro enteroids culture. We will culture enteroids isolated from the ileum of EW and LW pigs. We will apply ENS specific neurotransmitters (vasoactive intestinal polypeptide, acetylcholine, substance P) in the enteroids culture to mimic the altered neural microenvironment in pigs. We will use RT-PCR to measure the AA transporter gene expression change after neurotransmitter treatments, in order to evaluate input of ENS on transcription.Statistical Analysis. Data will be analyzed by student t-test between EW and LW pigs. Data from each neural blockade treatment on Ussing chambers or neurotransmitters in enteroids culture will be normalized to non-treated control tissue. Intestinal tissues (n = 12) and isolated enteroids (n = 8) from individual pigs will be used as experimental units for all the analysis.