Early Weaning Stress Research Articles

Chloroquine Downregulation of Intestinal Autophagy Changed Intestinal Microbial Community Compositions and Metabolite Profiles in Piglets.

Our previous study demonstrated that moderate inhibition of intestinal autophagy was beneficial to alleviate early weaning stress in piglets, but the detailed mechanism behind this was unclear. Microbiota-mediated enterocyte autophagy helps maintain intestinal homeostasis. This study investigated the effects of inhibition or activation of autophagy in intestinal microbial community compositions and metabolite profiles in piglets. Eighteen 24-day-old weaned piglets were divided into three groups (each treatment of six piglets) and treated daily with rapamycin (RAPA), chloroquine (CQ) or a control volume of normal saline (CON group). Before the formal trial, the piglets were allowed to acclimatize for 3 days, and then the trial period was 14 days. Collected samples from the ileum and colon underwent 16S rRNA gene sequencing and metabolite analysis. Significant differences in microbial composition were observed in both the ileum and colon of the RAPA and CQ groups compared to the CON group (p < 0.05). In addition, the relative levels of abundance of Peptostreptococcus, Fusobacterium, Dialister, Selenomonas and Oceanobacillus in the ileum and Porphyromonas, Bacteroides, unidentified_Lachnospiraceae, Akkermansia, Sharpea, Peptococcus, Pseudoalteromonas, Peptoclostridium and unidentified_Acidobacteria in the colon were improved in piglets fed the RAPA diet, whereas the relative levels of abundance of Turicibacter, Rickettsiella and Sarcina in the ileum and Roseburia and Kroppenstedtia in the colon were enhanced in the CQ group (p < 0.05). Meanwhile, metabolomic analysis showed that there were significant differences in metabolites among all groups (p < 0.05), and KEGG enrichment analysis revealed that differential metabolites were mainly enriched in the ABC transporters and biosynthesis of amino acids pathways. Furthermore, these metabolites were closely related to differential microorganisms (p < 0.05). Overall, autophagy inhibition regulates the composition of intestinal microorganisms and their metabolites, and these differential metabolites are significantly correlated with differential intestinal microorganisms, which may in turn affect the production performance of weaned piglets.

Effects of dietary Chitosan oligosaccharides supplementation on Th17/Treg balance and gut microbiota of early weaned pigeon squabs

Our previous study found that early weaning is associated with decreased growth performance, intestinal barrier impairment, and an imbalance in Th17/Treg in pigeon squabs. Chitosan oligosaccharides (COS) has been substantiated to regulate gut microbiota and restore Th17/Treg equilibrium in mammals, thereby ameliorating growth performance. However, the potential effects of COS in altricial birds remain unclear. Three hundred healthy 7-day-old American king pigeon squabs were selected with similar body weights and randomly divided into 5 groups. The 5 treatment groups were as follows: the control group (CON), fed with artificial pigeon milk; 4 supplementation groups, fed with artificial pigeon milk +100 (COS1), 150 (COS2), 200 (COS3), and 250 (COS4) mg/kg COS, respectively. Results showed that dietary supplementation of COS significantly enhanced the growth performance of weaned squabs. Compared to the CON group, the COS groups exhibited increased villus length and villus area in the jejunum and ileum, accompanied by improvements in morphological structure and mucosal permeability. COS was found to reduce the levels of Th17-associated cytokines and increase the levels of Treg-associated cytokines. COS downregulated the expression of retinoic acid receptor-related orphan receptor C (RORC), a key transcription factor of Th17 cells, while upregulated the expression of Forkhead box protein P3 (FOXP3), a key transcription factor of Treg cells. Dietary COS supplementation increased gut bacterial diversity, altered the relative abundance of several bacteria taxa and enhanced the concentration of short-chain fatty acids (SCFA). Correlation analysis demonstrated a close association between gut microbiota, SCFAs, and indicators related to the Th17/Treg balance. Moreover, we found that SCFAs correlated more strongly with Th17/Treg-related indexes than gut microbiota. These results demonstrated that COS could relieve early weaning stress in pigeon squabs and the optimal dosage of dietary COS supplementation was suggested to be 200 mg/kg. In addition, COS had a protective effect on maintaining intestinal immune balance by modulating microbiota and Th17/Treg related signaling pathways, in which SCFAs might play a crucial role as messengers.

56 Early Weaning Stress Relocates Epithelial Enteroendocrine Cells and Alters Intestinal Nutrient Sensing and Transport Functions in Pigs

Abstract Intestinal nutrient sensing and enteroendocrine functions are critical in controlling feed intake and nutrient transport, yet have been overlooked in agricultural animals. Enteroendocrine cells (EECs) residing on intestinal epithelium monitor luminal chemical environment and orchestrate body neuronal and hormonal responses. After nutrient stimulation, the EECs release hormones, such as glucagon-like peptide 2 (GLP2), which dynamically regulates intestinal nutrient transport and epithelial barrier homeostasis. In pigs, early weaning stress has negatively impacted intestinal development and functions. The objective of the present study is to evaluate effects of early weaning on intestinal nutrient sensing functions, EEC-derived GLP2 production, and epithelial nutrient transport in pigs. Commercial pig littermates (females, n = 9) were split-weaned at the age of d15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were all raised under the same conditions and euthanized at 35 days of age. Intestinal tissues were collected and preserved for later immunohistochemistry staining and gene expression analyses. Immediately post euthanasia, the mucosal layers from jejunum and ileum were mounted on the Ussing chambers to investigate physiological functions. Nutrients transport activities were measured by short circuit current changes (ΔIsc) in response to luminal nutrients (glucose, alanine, lysine, and di-peptide) applications. Nutrient sensing function was measured by GLP2 release rate following glucose stimulation in Ussing chambers. Serum and intestinal tissue concentrations of GLP2 were also measured by ELISA. Data were analyzed by the student’s t-test. Our results showed altered electrogenic nutrients and ion transport activities between EW and LW pigs. The ileal basal Isc was significantly less in EW pigs, indicating a reduction in ions transport. The EW pigs significantly downregulated electrogenic lysine transport, while upregulated active transport of dipeptide. The EECs number in ileum was reduced in EW pigs, indicated by less ChgA (EEC cell marker) mRNA and significantly less ChgA positive-stained cells. In addition, EW pigs exhibited a pattern of EECs relocation, in which the ChgA-positive cells enriched towards the lower half of villi. Nutrient sensing activities of the EECs were suppressed in both jejunum and ileum in EW pigs, indicated by a significant reduction in the glucose-stimulated GLP2 release on Ussing chambers. Moreover, in contrast with the significantly greater GLP2 concentration in ileum, the serum GLP2 concentration was significantly less in the EW pigs, which reflected a decreased GLP2 release rate. In summary, our results suggested that early weaning stress altered EECs number and locations on epithelium, reduced EECs nutrient sensing activities and endogenous GLP2 release, and changed nutrient transport activities. Understanding the mechanism by which early weaning stress regulates intestinal nutrient sensing and transport functions will lead to novel feeding strategies that improve intestinal health and animal production.

PSV-8 Early Life Adversity Impacts Intestinal Epithelial Development and Stem Cells Activities in Pigs

Abstract Early life adversity significantly impacts organ development and predisposes animals towards greater risk of diseases later in life. In the previous research in pigs, long lasting intestinal barrier dysfunctions have been found to be associated with early weaning stress. However, the underlying mechanism is not fully elucidated. Intestinal epithelial functions are associated with the number and maturation of multicellular components, including enterocytes, goblet cells, Paneth cells, and enteroendocrine cells (EEC). Intestinal epithelial stem cells (IESC) give rise to the whole epithelium within 5 to 7 days. Such rapid turnover rate allows intestinal adaptation to stress conditions. We hypothesized that the early life stress impacted stem cells activities which would contribute to the long-lasting post-stress functional changes in intestinal epithelium. The objective of the present study is to evaluate the epithelium component cells and IESC proliferation and differentiation activities in an early weaning stress model in pigs. Piglets (n = 18) were obtained from commercial litters and were pair-weaned at the age of d 15 (early weaning, EW) and d 26 (late weaning, LW). Piglets were co-housed and raised under the same conditions. At the age of d 35, ileal tissues were isolated and fixed for morphological measurements. Intestinal crypts were isolated and cultured in the Matrigel matrix to form enteroids. The proliferation activities of IESCs were measured by enteroids morphology; differentiation capacity was measured by quantification of epithelial cell markers with and without chemicals-induced enteroids differentiation. Gene expressions and (or) staining of specific markers for IESCs (Olfm4, BMI1), enterocytes (ALP1), goblet cells (Muc2), Paneth cells (LYZ), and EECs (ChgA) in ileal tissue and ileal enteroids were also quantified. Data were analyzed by the student’s t-test. Our results showed that EW significantly (P &amp;lt; 0.05) reduced ileal villus width and crypt fission rate, while no changes were found on villus height and crypt depth compared with LW control. The alterations of ileal epithelium components in EW pigs were indicated by significantly fewer number of EECs, greater number of the goblet cells, and reduced ALP1 marker expression. The enteroids formation and budding rates were both significantly suppressed in EW pigs, which indicated a reduced IESC proliferating activity. In the fully developed enteroids, gene expressions of LYZ, ALP1, BMI1, Olfm4, and Hes1 were least in EW, but Muc2 expression was greater compared with control. In the differentiation culture media, the enteroids from EW pigs exhibited significantly enhanced potential for goblet cell differentiation but reduced capacity for enterocyte differentiation compared with LW pigs. Overall, the results indicate that early weaning stress affects the programming and activity of IESCs, which leads to an altered epithelium cell population and intestinal functions. Further understanding of mechanisms regulating IESCs activity will shed light on novel strategies mitigating stress-associated intestinal dysfunctions.

Yeast Peptides Improve the Intestinal Barrier Function and Alleviate Weaning Stress by Changing the Intestinal Microflora Structure of Weaned Lambs.

Early weaning stress in lambs leads to decreased feed intake, damage to intestinal morphology, changes in the microbial flora structure, and subsequent complications. Yeast peptides are antimicrobial peptides with anti-inflammatory, antioxidant, and bacteriostasis effects. To study the effects of yeast peptides on relieving weaning stress in lambs, 54 lambs were randomly divided into three groups: ewe-reared (ER), yeast-peptide-treated (AP), and early-weaned (EW) lambs. The body weight and dry matter intake did not significantly differ among all groups. After weaning, the daily gain and feed conversion rate decreased significantly (p < 0.01), but AP showed an upward trend. In the EW group, immunoglobulin (Ig) levels changed significantly post-weaning (IgG decreased; IgA and IgM increased); the villi shortened, the crypt depth increased, and the villi height/crypt depth decreased (p < 0.001). The abundance and diversity of microflora among all groups were not significantly different. A column coordinate analysis showed significant differences in the intestinal microbial structure between the AP and EW groups. Lactobacillus, Aeriscardovia, Ruminosaceae_UCG-014, and Catenisphaera may play key roles in alleviating weaning stress in lambs. Our study provides new clues for alleviating weaning stress in lambs by describing the influence of yeast peptides on the intestinal microflora during weaning.

Expression Profile of miR-199a and Its Role in the Regulation of Intestinal Inflammation.

Early weaning stress impairs intestinal health in piglets. miRNAs are crucial for maintaining host homeostasis, while their implication for animal health remains unclear. To identify weaning-associated miRNAs, piglets were sampled at day 0, 1, 3, 7 and 14 after weaning. The data indicated that the highest levels of miR-199a-5p in jejunal villus upper cells were observed on day 14 after weaning, while the lowest levels in crypt cells were noted on day 7 and 14. In contrast, miR-199a-3p was down-regulated in both of these two cells on day 7 after weaning compared with day 0. Both miR-199a-5p and -3p were differently expressed along the villus-crypt axis. To further clarify the function of miR-199a, mice deficient in miR-199a were exposed to dextran sulfate sodium (DSS) to induce colitis. Results revealed that silencing of miR-199a enhanced sensitivity to DSS-induced colitis. Moreover, the increased morbidity and mortality were correlated with enhanced inflammatory cell infiltration, elevated pro-inflammatory cytokine expression, impaired barrier function, and a concomitant increase in permeability-related parameters. Bioinformatic analysis further demonstrated that lipid metabolism-related pathways were significantly enriched and Ndrg1 was verified as a target of miR-199a-3p. These findings indicate that miR-199a may be important for animal health management.

Asinine milk mitigates stress-mediated immune, cortisol and behavioral responses of piglets to weaning: A study to foster future interventions in humans.

The present study assessed whether asinine milk supplementation improved the immune and behavioral responses of piglets during an early life weaning stress event as a model for its future use in humans. For this, 48 piglets from 4 different litters were used. At 20 days of age, piglets were weighed and allocated with their litter and dam into group pens until 28 days of age. Four piglets from each litter were then randomly assigned to either (1) asinine milk supplementation (n = 16) (2), skimmed cow milk supplementation (n = 16) or (3) no supplementation (n = 16; control group). The supplementations were voluntarily administered for 3 days preweaning and 3 days postweaning using a baby bottle. The effects on the weaning stress response were assessed through salivary cortisol measurements; behavioral tests such as the open field, novel object end elevated plus maze tests; and gene expression of HSD11B1, NR3C1 and IL1B in PBMCs, which was determined by RT-qPCR and normalized to GAPDH and UBB. To test the effect of the supplementations on weight, milk intake, gene expression, and behavior, a randomized block design was used with repeated measurements over time by the PROC MIXED procedure. The effects on salivary cortisol were determined using the ratio between the morning and afternoon concentrations, considering the time before and after the weaning event. Principal component analysis (PCA) and Fisher's test were performed to evaluate the behavior test data. When comparing salivary cortisol concentrations between the pre- and postweaning periods, there was a difference (p < 0.05) between the supplementation groups in the afternoon period, suggesting that piglets fed asinine milk had lower afternoon cortisol concentrations postweaning than their counterparts. For the behavioral tests, the supplementations had no measurable effects. No difference was between groups pre- and postweaning for the expression of HSD11B2, which codes for an enzyme that breaks down cortisol. However, the expression of NR3C1, which encodes the glucocorticoid receptor, was significantly upregulated in piglets supplemented with cow milk (mean 1.245; p < 0.05). Asinine milk downregulated 1L1B gene expression, which codes for an inflammatory cytokine. In conclusion, these results suggest that supplementation with asinine milk may represent a strategy to diminish the damage associated with an early life event by modulating IL1B expression and reducing salivary cortisol levels in piglets undergoing weaning stress. Further transcriptomic and metabolomic studies may improve our understanding of the molecular pathways that mediate this systemic immune-mediated response.

Lycopene Affects Intestinal Barrier Function and the Gut Microbiota in Weaned Piglets via Antioxidant Signaling Regulation

In pig production, early and abrupt weaning frequently causes weaning stress, which manifests as oxidative damage, barrier disruption, and digestion and absorption capacity declines. Lycopene exhibits beneficial antioxidant capacity in both humans and other animal models. The present study aimed to investigate the effects of lycopene supplementation on early weaning stress in piglets and the underlying mechanisms by examining the oxidative stress state, gut intestinal barrier function, and the gut microbiota. Twenty-four 21-day-old weaned piglets [Duroc × (Landrace × Yorkshire); castrated males; 5.48 ± 0.10 kg initial body weight] were randomly assigned to 2 treatments. The piglets were fed a basal diet (control treatment) or a basal diet supplemented with 50 mg/kg lycopene (lycopene treatment) for 28 days. The serum lipid levels, serum and jejunum enzyme activities, jejunum morphology, mRNA and protein expression, and gut microbiota were determined. Compared with the control treatment, lycopene supplementation increased the serum catalase activity (P = 0.042; 62.0%); serum total cholesterol concentration (P = 0.020; 14.1%); and jejunum superoxide dismutase activity (P = 0.032; 21.4%), whereas it decreased serum (P = 0.039, 23.0%) and jejunum (P = 0.047; 20.9%) hydrogen peroxide concentrations. Additionally, lycopene increased the mRNA and protein expression of NFE2-like bZIP transcription factor 2 (214.0% and 102.4%, respectively) and CD36 (100.8% and 145.2%, respectively) in the jejunum, whereas it decreased the mRNA and protein expression of Kelch-like ECH-associated protein 1 (55.6% and 39.8%, respectively ). Lycopene also improved jejunal morphology, increasing the villus height (P = 0.018; 27.5%) and villus:crypt ratio (P < 0.001; 57.9%). Furthermore, it increased the abundances of potentially beneficial bacterial groups, including Phascolarctobacterium and Parasutterella, and decreased those of potentially pathogenic bacterial groups, including Treponema_2 and Prevotellaceae_unclassified. Lycopene supplementation strengthens the intestinal barrier function and improves the gut microbiota in weaned piglets by regulating intestinal antioxidant signaling.

PSIX-20 Nrf2 Pathway Mediates Resistance to Weaning Stress-Induced Intestinal Injury in Piglets of Different Breeds

Abstract The intestine is the central organ of early weaning stress in piglets; however, the response of intestinal stem cells (ISCs) that drive epithelial renewal between the different breeds of piglets is not yet known. Therefore, this investigation was to explore the sensitivity of ISCs to early weaning stress. Taoyuan Black and Duroc piglets were slaughtered at 21 days of age (on the day of weaning) and 24 days of age (3 days after weaning) with 10 pigs, respectively. And the results showed that Taoyuan Black pigs are more tolerant to early weaning stress, while the mucosal mass of jejunum was significantly decreased in 24-day-old Duroc piglets compared with the 21-day-old Duroc piglets (P &amp;lt; 0.05). The weaning stress damaged the jejunal structure, decreased the expression of tight junction related proteins ZO-1, Occludin, and Claudin-1, and increased intestinal permeability in Duroc piglets (P &amp;lt; 0.05). The ex vivo intestinal organoids (IOs) derived from the jejunal crypt of Duroc piglets after weaning exhibited lower expansion efficiency (P &amp;lt; 0.05). Furthermore, the levels of proliferating cell marker PCNA, terminally differentiated cell marker KRT20, absorptive cell marker Villin, goblet cell marker MUC2, and Paneth cell marker lysozyme were significantly reduced in the jejunum, crypt, and jejunal organoids of 24-day-old Duroc (P &amp;lt; 0.05). ROS levels in the jejunum of Duroc piglets were increased along with the inhibition of the Nrf2 pathway compared with 21-day-old Duroc piglets (P &amp;lt; 0.05). Collectively, the Taoyuan Black pigs had stronger tolerance to weaning stress; however early weaning suppressed the Nrf2 pathway, decreased proliferation and differentiation activity of ISCs, and disrupted intestinal homeostasis in Duroc piglets.

Early Weaning Stress Induces Intestinal Microbiota Disturbance, Mucosal Barrier Dysfunction and Inflammation Response Activation in Pigeon Squabs.

Early weaning stress has been reported to impair intestinal health in mammals. Like mammals, weaning of the pigeon squab, an altricial bird, is associated with social, environmental and dietary stress. However, understanding of weaning stress on intestinal functions is very limited in altricial birds, especially in squabs. This study was aimed to evaluate the effects of early weaning stress on intestinal microbiota diversity, architecture, permeability, the first line defense mechanisms, mucosal barrier functions, and immune cell responses. A total of 192 newly hatched squabs were randomly allocated into two groups, one weaned on day 7 and the other remained with the parent pigeons. Mucosal tissue and digesta in ileum, as well as blood samples, were collected from squabs (n = 8) on days 1, 4, 7, 10, and 14 postweaning. Our results showed that weaning stress induced immediate and long-term deleterious effects on both growth performance and intestinal barrier functions of squabs. Early weaning significantly increased ileal bacterial diversity and alters the relative abundance of several bacteria taxa. Weaning stress can also cause morphological and functional changes in ileum, including an atrophy in villi, an increase in permeability, and a variation in the mRNA expression of genes encoding mucins, immunoglobulins, tight junction proteins, toll-like receptors, and cytokines, as well as the concentration of secretory IgA. We concluded that the impaired intestinal barrier functions accompanied with early weaning stress seems to be the main reason for the poor growth rate after weaning in squabs. In addition, the disturbance of intestinal microbiota of early weaning stress in squabs coincided with dysfunction of intestinal mucosal barrier and activation of inflammation cell responses that were possibly mediated via the activation of toll-like receptors.

Gut microbiota-derived metabolites contribute negatively to hindgut barrier function development at the early weaning goat model.

Early weaning induces intestinal injury, leading to a series of long-term symptoms such as inflammation, malabsorption and diarrhea. In this study, we hypothesized that microbes and their metabolites modulate the host's inflammatory response to early weaning stress in a goat model. A total of 18 female Tibetan goat kids (n = 9) were weaned from their mothers at 28 d (D28) and 60 d (D60) postpartum. D60 and D28 groups were fed the same solid diet ad libitum from weaning to 75 d of age. The colonic epithelium was subject to RNA-sequencing, the caecal digesta metabolomics were assessed by liquid chromatography–tandem mass spectrometry (LC-MS/MS), and the caecal microbiota composition was analysed by 16S ribosomal RNA gene sequencing. We found that early weaning substantially increased the colonic pro-apoptotic gene expression of B-cell lymphoma associated X (Bax), caspase-9, and caspase-3, and decreased the expression of zonula occludens-1 (ZO-1) and claudin-1 (P < 0.01). In addition, a significant Bacteroides acidifaciens enrichment was observed in the hindgut of early-weaned goats (P < 0.01), which negatively correlated with lysophosphatidylcholine products. Similarly, the chemokine signaling, IL-17 signaling, and peroxisome proliferators-activated receptor (PPAR) signaling pathways were upregulated in the colonic mucosa of the early-weaned goats. By applying caecal microbiota transplantation from goats to defaunated C57/6J mice, we confirmed that caecal microbiota of D28 goat kids increased the relative abundance of B. acidifaciens and significantly up-regulated the genes of Bax, G protein–coupled receptor (GPR) 109A, GPR 43, fatty acid binding protein 6, nuclear receptor subfamily 1 group H member 3, angiotensin converting enzyme 2, and IL-6 expression (P < 0.05), and decreased ZO-1, and claudin-1 protein expression in the mice jejunum and colon (P < 0.001). These results proposed that the hindgut microbiota and metabolites mediate the barrier function weakening during early weaning, and the relative abundance of B. acidifaciens was negatively correlated with the hindgut barrier gene expression. This study demonstrates how weaning stress can affect key host–microbe interaction regulators in the hindgut, in a lysophosphatidylcholine dependent and independent manner. Furthermore, based on our mice data, these results are transferable to other mammal species.

Multi-omics unravel the compromised mucosal barrier function linked to aberrant mucin O-glycans in a pig model

Early weaning stress (EWS) in piglets is associated with intestinal dysfunction. Here, utilizing a pig EWS model to mimic early-life stress (ELS) in humans, we investigated the mechanism of ELS-induced intestinal diseases through integrated multi-omics analyses of proteome, glycome, and microbiome. Our results demonstrated that EWS resulted in disrupted the ileal barrier integrity by reducing tight junction-related gene expression and interfering with cell-cell adhesion paralleled the increased proportion of pathogens such as Escherichia_Shigella and Helicobacter. Furthermore, Proteome data revealed that the accumulation of unfolded proteins and insufficient unfolded protein response (UPR) process caused by EWS led to ER stress. Data from proteome and glycome found that EWS induced aberrant mucin O-glycans, including truncated glycans, reduction in acidic glycans, and increased in fucosylated glycans. In addition, correlation test by taking fucose and inflammatory response into account suggested that enhancement of fucose expression might be a compensatory host response. Taken together, these results extend the comprehensive knowledge of the detrimental impacts and pathogenesis of EWS and help to provide intervention targets for ELS-induced intestinal diseases in the future.

Mast cell histamine mediates intestinal immune response to early life stress in piglets via histamine 1 receptor (H1R)

Abstract Mast cells are important immune modulators of stress-related gastrointestinal (GI) disorders. Our previous studies investigating early life stress in piglets showed that histamine, a major mast cell granule mediator is released within minutes of early weaning stress and is followed by increased intestinal mucosal expression of histamine receptor subtypes. The precise contribution of histamine to stress-induced GI immune responses is unknown. The objective of this study was to test the hypothesis that stress-induced intestinal immune response is mediated through the histamine 1 receptor (H1R). Fifteen-day-old female Yorkshire piglets were administered either saline vehicle or the H1R antagonist, desloratadine (2 mg/kg; intramuscular), 30 minutes prior to early weaning stress. At weaning, piglets were weaned from their dams and housed in nursery pens with ad libitum access to water. At 24 hours post-weaning, mid-jejunum and mesenteric lymph nodes (MLN) were collected for subsequent qPCR gene expression as well as IHC localization and protein expression analyses. Unweaned control piglets remained with the sow and were collected immediately at weaning. Gene expression for H1R was not influenced by weaning; however, immunohistochemical analysis of H1R revealed increased expression and localization in lamina propria cells and myofibroblasts in jejunal villi and expression in the longitudinal and circular muscle layers in early weaned piglets. In the MLN, weaning increased the expression of TNFα, TGFβ, and β-Integrin. This response was reduced in the group treated with the H1R antagonist. Together, these data demonstrate that histamine via H1R plays an important role in early weaning stress-induced intestinal immune responses.

Interaction between biological sex and early life adversity on gastrointestinal (GI) immune development

Abstract Early postnatal life represents a critical window of GI immune development when lifelong immune function is shaped. Yet, little is known on how early life events impact long-term immune development in animals and people. Based on our previous research, we investigated how biological sex and early weaning (EW) stress interact to shape long-term GI immune development in the pig. Female (F), male intact (MI) and male castrated (MC) piglets (Yorkshire-cross) were weaned at 17 d (EW) or at 26 d of age (Later weaning; LW). At 77 d of age, tissue samples were harvested for RNA-seq, qPCR, and immunoassays. Serum cytokine and ileal mucosal IgG responses were measured following oral and intramuscular vaccination for Lawsonia intracellularis (LI) and PCV2, respectively. Compared with LW pigs, EW MC and MI pigs had reduced body weight gain (P&amp;lt;0.05). The impact of biological sex and EW were observed in vaccine responses. Serum from F showed higher PCV2-specific IgG titers and LI-antibody levels compared with MC and MI pigs. EW F had higher IgG titers in response to vaccination compared with LW F. In contrast, EW MC had suppressed antibody titers compared with LW MC. Pathway analysis of RNA-seq data from ileal mucosa revealed enrichment of genes associated with increased in immune-related pathways (e.g. inflammation) in EW F. In contrast, EW MC exhibited enrichment for genes associated with a down-regulation of immune response (e.g. immune cell migration). Together, these studies showed that biological sex and EW have a major impact on GI immune development in the pig. An understanding of the role and underlying sex-specific mechanisms driving altered immune development in EW pigs could reveal new targets for enhancing early and long-term immune development.

Chloroquine Downregulation of Intestinal Autophagy to Alleviate Biological Stress in Early-Weaned Piglets.

Simple SummaryWeaning is one of the biggest challenges in a pig’s life. Autophagy is a catabolic process aimed at recycling cellular components and damaged organelles in response to diverse stress conditions. There are two autophagy-modifying agents, rapamycin (RAPA) and chloroquine (CQ), that are often used in vitro and in vivo to regulate this process. We speculated that the regulation of autophagy may have some effect on weaning pressure. In this study, we try to understand the role of autophagy in intestinal barrier function and inflammation during the first week after weaning. We examined the effects of modulation of autophagy via RAPA and CQ on growth performance, immunity, inflammation profile, and the intestinal barrier to find potential value for CQ as a feed additive agent for ameliorating weaning stress.Early weaning stress impairs the development of gastrointestinal barrier function, causing immune system dysfunctions, reduction in feed intake, and growth retardation. Autophagy was hypothesized to be a key underlying cellular process in these dysfunctions. We conjectured that rapamycin (RAPA) and chloroquine (CQ), as two autophagy-modifying agents, regulate the autophagy process and may produce deleterious or beneficial effects on intestinal health and growth. To explore the effect of autophagy on early weaning stress in piglets, 18 early-weaned piglets were assigned to three treatments (each treatment of six piglets) and treated with an equal volume of RAPA, CQ, or saline. The degree of autophagy and serum concentrations of immunoglobulins and cytokines, as well as intestinal morphology and tight junction protein expression, were evaluated. Compared with the control treatment, RAPA-treated piglets exhibited activated autophagy and had decreased final body weight (BW) and average daily gain (ADG) (p < 0.05), impaired intestinal morphology and tight junction function, and higher inflammatory responses. The CQ-treated piglets showed higher final BW, ADG, jejuna and ileal villus height, and lower autophagy and inflammation, compared with control piglets (p < 0.05). Throughout the experiment, CQ treatment was beneficial to alleviate early weaning stress and intestinal and immune system dysfunction.

PSXII-1 Effects of early weaning on feed intake, growth performance, and diarrhea rate of Hu lambs

Abstract The objective of this study was to evaluate the effects of early weaning on growth performance, feed intake and diarrhea rate of Hu lambs, and establish the experimental model of early weaning stress in lambs. A total of 60 neonatal male Hu lambs were randomly allotted into 10 pens and 6 lambs in each pen with their ewes. Five pens of lambs were weaned at 21days of age as early weaning group (EW group), and the other 5 pens of lambs were remained with their ewes as control group (CON group). Body weight (BW) were recorded at birth (d0) and at d7, 14, 21, 22, 24, 26, 28, 35,42 and 49 after birth. Solid feed intake of lambs in each pen was recorded and diarrhea incidence was also monitored on daily basis. Data was analyzed using the MIXED model of SAS, and statistical significance was determined at P &amp;lt; 0.05. There was no difference in BW at birth and d21 between the two groups of lambs (P &amp;gt; 0.05). However, BW and average daily gain (ADG) of the lambs in EW group were significantly lower than those in CON group (P &amp;lt; 0.01) since weaning. Solid feed intake of the lambs in EW group were obviously higher than that in CON group (P &amp;lt; 0.01). In addition, the diarrhea rate was significantly higher than that in CON group from d21 to d35 (P &amp;lt; 0.01). The results indicated that lambs weaned at d21 showed strong stress response by significantly decreasing in BW and ADG, and had more feed intake but higher diarrhea rate. Future study is warranted to determine the changes in gut microbiota and barrier function to assess the mechanism of early weaning on health status and growth performance of lambs, and to investigate nutritional intervention methods to alleviate weaning stress.

Hippophae rhamnoides polysaccharides protect IPEC-J2 cells from LPS-induced inflammation, apoptosis and barrier dysfunction in vitro via inhibiting TLR4/NF-κB signaling pathway

Inflammatory response caused by early weaning stress in piglets is associated with various diseases. The Hippophae rhamnoides polysaccharide (HRP) exhibits anti-inflammatory activity and immunomodulatory properties. The mechanisms for the protective effects of HRP on barrier function, inflammatory damage and apoptosis in intestinal porcine epithelial cells (IPEC-J2) induced by the lipopolysaccharide (LPS) are unknown. In this study, we first demonstrated the cytotoxicity of HRP-induced IPEC-J2 cells by reducing cell viability. IPEC-J2 cells were treated with 0–800 μg/mL doses of HRP, and 0–600 μg/mL doses were used in further experiments. Upon exposure to LPS, the viability of IPEC-J2 cells, ROS production, immunoglobulin levels (immunoglobulin M (IgM), immunoglobulin A (IgA) and immunoglobulin G (IgG)) and tight junction protein level (zonula occludens-1 (ZO-1), occluding, claudin-1) decreased. Inflammatory factors (interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α)) and apoptosis (Bcl-2, Bax, caspase-3, caspase-8 and caspase-9) were increased. Cell morphology and internal structure were damaged in the LPS treatment. Pre-treating cells with HRP (0–600 μg/mL) reduced inflammatory factors levels, apoptosis rate, increased immunoglobulins, tight junction protein levels and relieved cell surface morphology damage. Pre-treatment with HRP also reduced the levels of the Toll-like receptor 4 (TLR4) and Myeloid differentiation factor 88 (MyD88) and inhibited the phosphorylated NF-κB factor-kappa B (NF-κB) in cells induced by LPS. These results show that pre-treatment with HRP protected against LPS-induced IPEC-J2 cell damage through its anti-inflammatory activity.

Effect of Early Weaning on the Intestinal Microbiota and Expression of Genes Related to Barrier Function in Lambs.

Weaning stress has been reported to impair intestinal health. The gut microbiota plays a vital role in the long-term health of the host. However, our understanding of weaning stress on gut microbiota and barrier function is very limited in livestock species, especially lambs. We investigated the effects of early weaning stress on intestinal bacterial communities and intestinal barrier function in lambs. A total of 24 neonatal male Hu lambs were randomly allocated into two groups, one weaned on day 28 and the other weaned on day 56. At 42 and 84 days, six lambs from each group were randomly selected and sacrificed. Ileal tissue and ileal digesta were collected to compare the differences in ileal microbiota and the mRNA levels of Toll-like receptors (TLRs) and tight junction proteins between the early weaning group and the control group at day 42 when the early weaning group have been weaned for 14 days, and at day 84 when the 28 and 56 days weaning groups had been weaned for 56 and 28 days, respectively. 16S rRNA gene sequencing of ileal samples revealed that the ileal microbiota was very different between the two groups, even at 84 days of age. Early weaning significantly increased alpha diversity and altered the relative abundance of several bacterial taxa. The expression of genes related to intestinal barrier function was affected by early weaning. Early weaning significantly increased ileal mRNA levels of TLR1 on days 42 and 84; TLR2, TLR4, and TLR5 on day 84; claudin1 and claudin4 on day 42; and occludin on day 84. We demonstrate that early weaning not only altered the ileal microbiota on day 42 (compared with lambs that were not weaned), but also had lasting effects on the ileal microbiota at day 84; furthermore, early weaning impacts expression levels of genes related to intestinal barrier function.

Lactobacillus frumenti Facilitates Intestinal Epithelial Barrier Function Maintenance in Early-Weaned Piglets.

Increased intestinal epithelial barrier function damages caused by early weaning stress have adverse effects on swine health and feed utilization efficiency. Probiotics have emerged as the promising antibiotic alternatives used for intestinal barrier function damage prevention. Our previous data showed that Lactobacillus frumenti was identified as a predominant Lactobacillus in the intestinal microbiota of weaned piglets. However, whether the intestinal epithelial barrier function in piglets was regulated by L. frumenti is still unclear. Here, piglets received a PBS vehicle or PBS suspension (2 ml, 108 CFU/ml) containing the L. frumenti by oral gavage once a day during the period of 6–20 days of age prior to early weaning. Our data demonstrated that oral administration of L. frumenti significantly improved the intestinal mucosal integrity and decreased the serum endotoxin and D-lactic acid levels in early-weaned piglets (26 days of age). The intestinal tight junction proteins (including ZO-1, Occludin, and Claudin-1) were significantly up-regulated by L. frumenti administration. The serum immunoglobulin G (IgG) levels, intestinal secretory immunoglobulin A (sIgA) levels, and interferon-γ (IFN-γ) levels were significantly increased by L. frumenti administration. Furthermore, our data revealed that oral administration of L. frumenti significantly increased the relative abundances of health-promoting microbes (including L. frumenti, Lactobacillus gasseri LA39, Parabacteroides distasonis, and Kazachstania telluris) and decreased the relative abundances of opportunistic pathogens (including Desulfovibrio desulfuricans and Candida humilis). Functional alteration of the intestinal bacterial community by L. frumenti administration was characterized by the significantly increased fatty acids and protein metabolism and decreased diseases-associated metabolic pathways. These findings suggest that L. frumenti facilitates intestinal epithelial barrier function maintenance in early-weaned piglets and may be a promising antibiotic alternative used for intestinal epithelial barrier function damage prevention in mammals.

Early weaning in pigs induces long‐term alterations in intestinal nutrient transporter function and expression

Early life adversity (ELA) is associated with increased disease risk throughout the lifespan but the developmental mechanisms underlying the association remain elusive. Altered intestinal nutrient transporter function has been implicated as a mechanism contributing to a number of diseases associated with ELA such as Type 2 diabetes and inflammatory and functional GI disorders in humans and impaired health and growth performance in agricultural animals. Using an early weaning stress model of ELA in pigs, we investigated the link between ELA and long‐term intestinal nutrient transporter function and expression. Yorkshire breed female piglets were weaned from their sows at 15 d (Early Weaning; EW) or 28 d of age (Late Weaning; LW). Early and LW piglets were co‐housed and received the same diets. At 70 d of age, mid‐jejunum and distal ileum were harvested from EW and LW pigs and mounted on Ussing chambers to evaluate Na+‐dependent electrogenic nutrient transporter function, determined as the change in short‐circuit current (ΔIsc) in response to select luminal nutrients. Villus morphology and epithelial gene expression of nutrient transporters were assessed. Compared with age‐matched LW pigs, EW pigs exhibited reduced jejunal and ileal Na+ glucose linked transporter (SGLT1) function (by ~30%; P &lt; 0.05) and amino transporter function for glutamate, glutamine, and alanine by (~30–35%; P &lt; 0.05). In contrast, jejunal and ileal lysine transporter function was upregulated (by 110% and 25%, respectively; P &lt; 0.05) in EW pigs. Gene expression for nutrient transporters (SGLT1, EAAT3, and B0AT1) were reduced (P &lt; 0.05) while the lysine transporter CAT1 was enhanced in the EW jejunum. There were no differences observed in villus morphology between EW and LW pigs. Enteric neuronal inhibitors tetrodotoxin, atropine and propranolol, enhanced ΔIsc responses to glucose, lysine and alanine transporter function in EW but not in LW pig ileum. Together, these data show that EW in pigs induces lasting and divergent alterations in small intestinal nutrient transporter function and expression which is mediated in part by the enteric nervous system. These findings could provide new insight into the mechanisms of increased disease risk linked with ELA and thus lead to novel dietary or therapeutic strategies.Support or Funding InformationNIH R01 HD072968USDA‐NIFA #2017‐67015‐26673This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.