Source: UNIVERSITY OF DELAWARE submitted to NRP
UNDERSTANDING THE INTESTINAL AMINO ACIDS TRANSPORT ADAPTATIONS AND ENTERIC NEUROENDOCRINE CONTRIBUTION DURING WEANING STRESS IN PIG
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032172
Grant No.
2024-67016-42328
Cumulative Award Amt.
$300,000.00
Proposal No.
2023-07735
Multistate No.
(N/A)
Project Start Date
Aug 1, 2024
Project End Date
Jul 31, 2026
Grant Year
2024
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
UNIVERSITY OF DELAWARE
(N/A)
NEWARK,DE 19717
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%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053510102050%
3063510102050%
Knowledge Area
305 - Animal Physiological Processes; 306 - Environmental Stress in Animals;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1020 - Physiology;
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.

Progress 08/01/24 to 07/31/25

Outputs
Target Audience:This work will benefit animal scientists, biologists, and physiologists by advancing the understanding of how early-life stress influences intestinal development and its long-term impact on health. The early weaning model and enteroid culture methods developed in this study will be valuable tools for researchers investigating intestinal physiology and developmental programming. Additionally, the applied component of this research has direct relevance to the swine industry, offering insights that can inform optimal weaning strategies to support animal health and production efficiency. Changes/Problems:Due to the graduation of a previous student and personnel changes, we experienced a gap in skilled personnel capable of efficiently conducting the enteroid culture assays. Current limitations in international student recruitment have further complicated efforts to fill this gap. However, we have been actively seeking and training new motivated graduate students. Two new students have recently joined the group, which will strengthen our research capacity moving forward, though it may result in some delays in project completion. To accommodate this, we are in the process of submitting a request for a one-year no-cost extension. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?One manuscript is under preparation. We plan to spread out our work by manuscript and poster publications. What do you plan to do during the next reporting period to accomplish the goals?In addition to the animal trials that we have done and described above, one animal trial involving three weaning ages is currently underway. We have harvested nine pigs and are in the process of analyzing the collected samples. Another pig trial is scheduled for late September 2025, which will include 27 pigs. This trial will build upon the methodologies established in our previous studies. Intestinal enteroids will be isolated to investigate EEC cell function and their role in regulating amino acid transporter expression. Additionally, Ussing chamber electrophysiological measurements will be employed to assess the contribution of enteric nervous system activity to transporter function in the upcoming trials.

Impacts
What was accomplished under these goals? Project Progress Report We have completed one preliminary pig trial comparing early weaning (EW) and late weaning (LW) conditions to capture the maximal differences between these two extremes. This trial successfully established a weaning stress model and enabled the quantification of intestinal brush-border enzyme activities, nutrient transport efficiency, and intestinal enteroid culture development. Major progress toward each specific aim is summarized below. Aim 1: Determine how weaning age influences intestinal amino acid transporter expression and function We observed that aminopeptidase activity was significantly higher in EW pigs, suggesting increased digestive enzyme activity. Nutrient transport responses were not uniformly altered. Specifically: Glucose and dipeptide absorption were increased in the jejunum of EW pigs. Alanine absorption remained unchanged. Lysine absorption was significantly decreased in EW pigs. These findings suggest that early-weaned pigs may adapt to weaning stress by shifting from free amino acid to dipeptide-based transport mechanisms. However, this adaptive response may predispose pigs to lysine deficiency, which could impact long-term growth and health. Aim 2: Identify stress-induced changes in enteroendocrine cell (EEC) activity and their role in modulating amino acid transport We successfully isolated intestinal crypts and established enteroid cultures from both EW and LW pigs. Enteroids from EW pigs exhibited reduced formation and budding efficiency, indicating impaired intestinal epithelial stem cell function following weaning stress. Gene expression analysis revealed a deficiency in functional EEC cell development in EW pigs. We hypothesize that EECs in these pigs may shift their function from nutrient sensing toward supporting damaged stem cells. This functional shift is further supported by reduced circulating levels of EEC-derived hormones. The specific role of EECs in regulating amino acid transporters remains unclear and will be further investigated in upcoming trials. Aim 3: Examine the contribution of enteric nervous system activity to amino acid transport We observed significantly increased enteric nervous system activity in EW pigs. However, its direct influence on amino acid transporter expression and function has not yet been fully characterized. This will be a focus in the next phase of the study. Summary and Next Steps This preliminary trial has allowed us to: Establish a robust early weaning stress model. Quantify key functional outcomes in intestinal physiology. Successfully culture intestinal enteroids for mechanistic studies. Our findings support the hypothesis that early weaning induces distinct adaptive responses in nutrient absorption and intestinal epithelial function. Moving forward, we will expand our study to include refined weaning age groups: early, industry-standard, and late weaning. This will enable a more detailed, quantitative analysis of how weaning age affects intestinal adaptation and function.

Publications