Weaning stress and intestinal health of piglets: A review

1As the main site of nutrients digestion and absorption and an important defense line against the invasion of bacteria and endotoxins into the intestinal lumen, the intestinal tract is an important organ in response to stress in piglets. Therefore, it is important to maintain intestinal health in animal production. However, in actual production, piglets may suffer from many stresses, such as birth stress, weaning stress, heat stress, transport stress, etc., which usually affect the intestinal health of piglets, eventually, lead to economic losses.

2In modern intensive farming systems, early weaning techniques are often used to improve the productivity of sows, which can increase the annual litter size of sows, improve the utilization rate of breeding equipment, and bring more economic benefits for breeding enterprises. However, due to sudden separation from the sow, and the rapid changes in the diets, physical environments, and social environments, piglets may suffer from weaning stress, which jointly results in perturbation of intestinal microbiota, host physiological and biochemical functions, intestinal digestion and absorption capacity, and mucosal immune function, which eventually leads to decrease of feed intake, occurrence of post-weaning diarrhea (PWD), and growth restriction. That’s because, the intestinal development of the piglets is not mature, and the digestive system and immune system are not perfect at this stage, which leads to a poor ability of weaning piglets to adapt to the complex environment. Simultaneously, due to the insufficient secretion of digestive enzymes in the gastrointestinal tract, early-weaned piglets cannot digest solid food well, leading to the destruction of the intestinal physical barrier, including the destruction of the tight junctions (TJ), the reduction of mucins secretion, the increase of intestinal permeability and the un-balanced gut microbiota. When piglets suffered weaning stress, the intestinal environment is susceptible to invasion by pathogenic microorganisms such as Escherichia coli (E. coli), which stimulates the intestinal mucosa to secrete inflammatory factors and damage the function of the intestinal mucosal barrier. Weaning stress is not only closely related to the immune system and intestinal barrier function but also can damage the oxidation-antioxidant system and induce oxidative stress. Furthermore, weaning stress causes the dysbiosis of gut microbiota, and further increases the risk of gastrointestinal diseases in piglets. A correct understanding of the effects of weaning stress on intestinal health has an important guiding significance for nutritional regulation of intestinal injury caused by weaning stress. Therefore, here we reviewed the effects of weaning stress on the intestinal health of piglets, from the aspects of intestinal development, and intestinal barrier function, thereby providing a theoretical basis for nutritional strategies to alleviate weaning stress in mammals in future studies.

3The intestines display various functions including providing a main site for nutrient digestion and absorption as well as acting as a selective barrier to prevent the entry of exogenous harmful substances into the circulation system while allowing the selective absorption of nutrients including electrolytes and water. The integrity of the intestinal structure is the guarantee of nutrient digestion and absorption of piglets. The intestinal morphology, including villus height (VH), crypt depth (CD), and the ratio of villus height and crypt depth (VCR) reflect the health and absorption status of the intestinal function. The reduction in VH and VCR means that intestinal mucosal function was impaired, and intestinal digestion and absorption capacity was reduced.In contrast, a higher VH and VCR, and a lower CD of the intestines indicated better intestinal function. As is well-known that alterations in the villus−crypt structure are universal in weaned animals, such as intestinal villus shedding, crypt hyperplasia, and intestinal mucosa atrophy, which further destroys intestinal mucosal barrier function and digestive and absorptive capacity. For example, a study by Bomba et al. showed that, 5 days after weaning (33 days of age), the VH and VCR in the ileum of piglets were significantly lower than that before weaning (28 days of age). Similarly, Hu et al. verified the deterioration of intestinal morphology induced by weaning, which showed that VH and VCR on day 3 and day 7 postweaning were decreased compared with the preweaning stage, and VH and CD did not return to preweaning levels until day 14 postweaning. Furthermore, Boudry et al. reported that weaning induced long-lasting structural changes in the small intestine of piglets, which showed that the VH of the jejunum was still significantly lower on day 15 postweaning than preweaning. In addition, weaning stress has been shown to cause a decrease in the relative weight of the small intestine, with the total weight of the intestine at 15 days after weaning being only 50% of that before weaning. Taken together, early weaning can lead to intestinal morphological damage of piglets, including deeper CD, lower VH, reduced VCR, and lower intestinal relative weight. Therefore, to maximize pig production, it is necessary to reduce physiological changes in the small intestine caused by weaning stress.

4The intestinal tract is a dynamically self-renewing tissue, and its structural and functional integrity depends on the homeostatic maintenance of the dynamic balance between proliferation and apoptosis of intestinal mucosal epithelial cells. The renewal of intestinal epithelial cells is known to be primarily involved in the proliferation of crypt stem cells, the differentiation and shedding of villus cells. Crypt stem cells undergo symmetric differentiation to generate stem cells to maintain self-renewal or asymmetric differentiation to generate rapidly proliferating cells (progenitor cells). The progenitor cells then continuously migrate along the crypt–villus axis and finally differentiate into cells with specific functions, mainly including absorbing epithelial cells (enteroendocrine cells), Goblet cells, Endocrine cells, and Paneth cells. Apoptosis, also known as programmed cell death, is a physiological process of cell suicide that plays an important role in the growth and development of the body. However, due to physiological, environmental, and social challenges, piglets are easily prone to weaning stress, which disrupts the balance between cell proliferation and apoptosis of intestinal mucosal epithelial cells, and disorder of apoptosis and proliferation would increase intestinal mucosal permeability and affect intestinal barrier function. Increasingly, data are available showing that the expression of genes related to proliferation and differentiation was decreased, while the expression of genes related to apoptosis was increased in jejunal cells of weaned piglets. Montagne et al. suggested that weaning disrupts the balance between intestinal cell proliferation and apoptosis in piglets, resulting in intestinal villus atrophy and crypt hyperplasia, which further leads to intestinal morphological injury. Yang et al. reported that weaning-induced malnutrition in piglets would affect energy metabolism, macromolecular composition and localization, and protein metabolism, thus affecting the proliferation of intestinal crypt cells. Therefore, the reduction in intestinal cell proliferation and the increase of apoptosis caused by weaning stress may be the main mechanism responsible for intestinal mucosal injury during the postweaning period.

5The gastrointestinal digestive enzymes are involved in the regulation of growth and development of animals, because digestive enzymes can improve the feed efficiency by digestion and in turn to modulate the process of nutrient metabolism. Therefore, the activities and secretion of digestive enzymes in the small intestine are important indicators to evaluate intestinal development and digestive capacity in weaned pigs. Absorptive intestinal epithelial cells are the main cell type in the crypt-villus axis, which has the function of secreting a variety of digestive enzymes such as disaccharidase, peptidases, and phosphatase. In general, during the first 3 weeks after birth, the digestive system of piglets develops rapidly due to adequate nutrition intake from sows, and the activities of digestive enzymes such as intestinal lactase, protease, and lipase are significantly increased. However, due to the change in diet, the activities of enzymes on the brush border of the intestinal mucosa, such as disaccharidases, protease, and lipase are dramatically changed after weaning. It has been confirmed that intestinal morphology is associated with changes in intestinal digestive enzyme activities. Small intestinal disaccharidases (lactase, maltase, and sucrase) are the key enzymes of carbohydrate digestion and absorption in piglets. However, the activity and digestion ability of disaccharidases decreased significantly after weaning, which is considered to be an important cause of diarrhea in weaned piglets. In addition, the alkaline phosphatase (AKP) in the small intestinal villus epithelium is a landmark key enzyme associated with intestinal digestion and absorption function, which helps to increase the uptake and transport rate of nutrients, and also converts adenosine diphosphate (ADP) into adenosine triphosphate (ATP). Previous studies had demonstrated that early weaning significantly decreased small intestinal AKP activity in piglets, which indicated that weaning stress has adverse effects on intestinal digestion and absorption function. In conclusion, one possible explanation for the decrease of intestinal digestive enzymes activity in piglets caused by weaning may be due to the negative effect of weaning stress on intestinal morphology, which in turn inhibits the secretion of endogenous enzymes.

6The negative effects of weaning on the development of intestinal function can be explained as follows: first, weaning will destroy intestinal morphology and then destroy the balance between intestinal cell proliferation and apoptosis, resulting in reduced secretion of digestive enzymes; second, increased intestinal cell apoptosis further aggravated intestinal morphological damage and the decrease of digestive enzyme activity; finally, decreased digestive enzyme activity also affects intestinal morphology and apoptosis ( Figure 1 ). Therefore, the decrease in digestion and absorption related enzyme activities is an important reason for the growth retardation of weaned piglets.

7The intestinal tract is constantly in contact with foreign substances, selectively absorbs effective nutrients, and resists and eliminates the invasion of toxins and enteric pathogens, and the normal operation of this mechanism depends on the integrity of intestinal barrier function. The intestinal barriers are mainly composed of intestinal epithelial cells, intestinal mucus layer, immune cells, and normal microorganisms and their metabolites, and are often artificially divided into the mechanical (physical) barrier, chemical barrier, immune barrier, and microbial barrier, which cooperate in structure and function to effectively maintain intestinal homeostasis. Separation of piglets from sows at weaning is known to induce immediate and long-term deleterious effects on gut defense mechanisms, including intestinal barrier dysfunction, intestinal inflammation, and increased intestinal permeability. Intestinal barrier function was impaired at the beginning of weaning and recovered after 2 weeks of weaning. However, studies have shown that the earlier weaning occurs, the longer barrier impairment persists.

8The intestinal epithelial barrier is mainly formed by a continuous monolayer of proliferating and differentiating intestinal epithelial cells tightly linked by the apical junctional complex (AJC). AJC is mainly composed of TJs, adherens junctions (AJ), and desmosomes ( Figure 2 ), which establishes the cellular polarity and reduces the space between adjacent cells, therefore selectively allowing the absorption of nutrients and limiting the access of pathogens, toxins, and xenobiotics from the intestinal lumen to the mucosal tissues. Among these structures, TJs constitute the main determinant of the intestinal physical barrier. TJs are formed by a multiple-protein complexes located in the apical portion of the lateral membrane of intestinal epithelial cells, mainly composed of transmembrane proteins, such as Claudin, Occludin, and junctional adhesion protein molecule-A (JAM-A), Myosin, F-actin, Myosin light chain kinase (MLCK), as well as cytoplasmic proteins such as zonula occludens (ZO)-1, ZO-2 and ZO-3 ( Figure 2 ), which play an important role in maintaining the intestinal mechanical barrier and regulating intestinal permeability.

9Previous studies have shown that weaning stress could lead to impaired physical barrier function in piglets, characterized by the disruption of intestinal epithelial TJs and increased intestinal permeability. Increased intestinal permeability makes it easier for intestinal pathogenic microorganisms, endotoxins, and other antigenic substances to break the intestinal mucosal barrier and enter other tissues, organs, and the blood circulation system, which can eventually cause enteroborne infections. In general, intestinal permeability can be reflected by the measure of transepithelial electrical resistance (TER) of the intestinal mucosa using the Ussing chamber system. A decreased TER reflects an altered intestinal barrier. For instance, Cao et al., Hu et al., Boudry et al., Smith et al., and Wijtten et al. used Ussing chamber system to detect the intestinal TER, and the results all showed that early weaning resulted in a decreased TER in piglets, indicated that weaning stress had a destructive effect on intestinal barrier function. The detection of intestinal TJ protein expression also can be used as an important index to evaluate intestinal mechanical barrier function. Weaning stress has been established to disrupt multiple TJ proteins, including claudin-1, occluding, ZO-1 ZO-2, and ZO-3, possibly by activating mitogen-activated protein kinase (MAPK) and transforming growth factor-β1 (TGF-β1) signaling pathways, although further studies are needed to confirm this. The effects of weaning stress on the intestinal mechanical barrier function of piglets are summarized in Table 1. These studies suggested that weaning can damage intestinal TJ structures and increase intestinal permeability.

10The intestinal chemical barrier is formed by the mucus layer, which is composed of mucins (MUCs) secreted by goblet cells and antimicrobial proteins secreted by epithelial cells. The intestinal mucus layer is the first line of defense to protect intestinal epithelial cells from pathogenic microorganisms, which can effectively prevent the colonization of pathogenic microorganisms and plays an important role in maintaining the homeostasis of the intestinal environment. Mucins are glycosylated proteins with a high molecular weight characterized by an important element, the ‘mucin domain’, which comprises the main components of intestinal mucus. Mucins can be classified into gel-forming secretory mucins (eg, MUC2, MUC5, and MUC6) and membrane-bound mucins (e.g., MUC1, MUC3, MUC4, MUC13, and MUC17) according to their structure and localization. Among all mucins, MUC2 forms the bulk of intestinal mucus, which participates in intestinal lubrication, pathogenic bacteria antagonism, and intercellular signal transduction.

11Mucins are synthesized mainly by goblet cells in the gut. Therefore, any factors that affect the differentiation of goblet cells would affect the secretion of intestinal mucins. Weaning stress has been reported to injures secretory cells (mucus-producing goblet cells) differentiation and thus resulted in decreased mucins secretion. For instance, Hedemann and Jensen indicated that early weaning would not only lead to a decrease in intestinal mucin secretion but also change the glycosylation pattern of mucin, thereby weakening the intestinal chemical barrier function and increasing the probability of intestinal infection. Similarly, Yang et al. reported that the MUC2 gene was negatively regulated in weaned piglets, suggesting that weaning destructed the chemical barrier in the intestinal tract. Normal mucins secretion and expression is very important for maintaining intestinal barrier function ( Figure 3 ). Firstly, when the secretion of intestinal mucins decreased, the intestinal mucosa mucus layer became thinner, and pathogenic microorganisms could easily pass through the mucus layer to destroy the function of the intestinal chemical barrier; secondly, invasive pathogenic microorganisms compete with the normal microbiota on the surface of the intestinal mucosa for adhesion sites, destroying the normal microbial barrier; thirdly, invasive pathogenic microorganisms such as salmonella and shigella could destroy the intestinal mucosal mechanical barrier by inducing apoptosis of intestinal epithelial cells as well as by disrupting the distribution of TJ proteins between intestinal mucosal cells; finally, changed in mucins secretion and expression would cause inflammation and damage the intestinal mucosal immune barrier.

12The intestinal immune barrier is a well-developed and complex local immune system, which mainly composed of immune organs, immune cells (intraepithelial lymphocytes, lamina propria lymphocytes, neutrophils, and macrophages), and immune molecules (antibacterial peptides, immunoglobulins, and cytokines). The intestinal immune barrier is important for recognizing exogenous antigenic stimuli while ensuring that the animal body is not over-sensitive to harmless antigens. Generally, intestinal epithelial cells recognize pathogenic molecules and beneficial substances through pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and nucleotide binding oligomerization domain (NOD) like receptors (TLRs). It promotes the immune response and inhibits inflammation by regulating the signaling pathways of the nuclear factor kappa B (NF-κB), MAPK, and peroxisome proliferator-activated receptor γ (PPAR-γ).

13Conventionally, the intestinal immune system of pigs does not reach the level of adult pigs until 7 weeks of age. However, in modern pig production, piglets are usually weaned at 3-4 weeks of age, when the intestinal immune barrier is not mature, which resulted in increased disease susceptibility. The intestine can be regarded as the largest immune organ in animals, where up to 70% of the immune cells are localized in the mucosa and submucosa of the intestine. Due to physiological and psychological factors, piglets would encounter many pathogenic and nonpathogenic challenges after weaning, which disrupt the immune barrier function. Firstly, weaning resulted in intestinal immune cells, such as T lymphocytes, and intestinal mast cell dysfunction. McCracken et al. showed that during the first 2 days postweaning, the number of intestinal inflammatory T cells and matrix metalloproteinase (i.e., stromelysin) were significantly increased; and Spreeuwenberg et al. showed that the CD4+/CD8+ T lymphocytes ratio decreased after weaning, which indicated that weaning induced transient gut inflammation in pigs. Mast cell hyperactivation is an important pathophysiological mechanism in inflammatory diseases, such as irritable bowel syndrome. Studies have shown that compared with late-weaned pigs (weaned at 28 days of age), the early-weaned pigs (weaned at 19 days of age) have higher numbers of intestinal mast cells at 24 h after weaning and sustained hyperplasia of intestinal mast cells at 7 weeks, 9 weeks, and 20 weeks after weaning. Secondly, weaning stress activates the intestinal immune system and produces a large number of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6, IL-8, and the overproduction of these cytokines can lead to intestinal damage and dysfunction. Thirdly, weaning leads to the destruction of intestinal integrity, microbial invasion stimulates the expression of secreted immunoglobulin A (sIgA) and defensin in the jejunum of piglets, which is beneficial to recovering intestinal barrier function after weaning. The effects of weaning stress on the intestinal immune function of piglets are summarized in Table 2. These studies suggested that weaning stress can induce transient intestinal inflammation and damage the intestinal immune barrier in piglets.

14Hundreds of millions of microbiota populations inhabit the mammalian gastrointestinal tract, which play an important role in the regulation of nutrients digestion, intestinal barrier, immune response, endocrine and other physiological processes. It has been basically clear that dysregulation and translocation of the intestinal microbiota can cause intestinal cell apoptosis, intestinal physical barrier damage, and intestinal immune dysfunction, which is not conducive to animal intestinal health. Previous studies have demonstrated that the development of the intestinal microbiota in pigs is age-dependent, with birth and weaning being the most critical periods of life. At birth, colonization of the gut microbiota (such as Escherichia coli and Streptococcus spp.) begins once the newborn is exposed to microbes from the mother and the surrounding environment and is influenced by the consumption of colostrum and milk in sows. Subsequently, aerobic bacteria, facultative anaerobes, and obligate anaerobes gradually colonized the intestinal tract of piglets, and about 2 days later, the intestinal tract was completely colonized by microorganisms, and Lactobacillus was the dominant bacteria.

15Weaning is one of the most stressful events in piglet life, this particular period also provides an important window to shape the gut microbiota. In early weaning, the diet of piglets is changed from good digestible breast milk to poor digestible solid feed, piglets cannot fully digest and utilize these nutrients due to the immature digestive system, which provides a good source of nutrients for some pathogenic bacteria to multiply, thereby altering the composition of the gut microbiota (e.g., increasing the ratio of Escherichia coli to Lactobacillus) and destroying the microbial barrier function of the intestinal tract. Diarrhea is a common symptom of weaning stress, which can also lead to changes in the intestinal microbiota of piglets. Yang et al. showed that the diarrheic piglets had lower relative abundances of Bacteroides, Ruminococcus, Bulleidia, and Treponema, the genera that play key roles in nutrient metabolism than healthy piglets after weaning. Similarly, Sun et al. showed that the diarrheic pigs had lower relative abundances of Bacteroidales than non-diarrheic pigs during weaning. While the disruption of gut microbiota, in turn, aggravates post-weaning diarrhea of piglets. Of note, some pathogens can also disrupt the microbiome composition of weaned piglets. For example, Arguello et al. showed that there was an increase in pathogenic bacteria (Citrobacter), and a decrease in the population of beneficial bacteria such as Bifidobacterium and Lactobacillus at the ileum mucosa of weaned piglets infected with Salmonella Typhimurium.

16Studies have shown that Firmicutes, Proteobacteria, Bacteroidetes, Tenericutes, and Spirochaetes were the five dominant bacterial phyla in the intestinal tract of piglets, regardless of weaning or not. Although weaning stress generally did not change the species of the phyla, it did change the relative abundance of some phyla, especially the levels of families and genera in the corresponding phyla. For example, the abundance of Prevotella, a bacterium closely related to timely intake of solid feed, decreased significantly within 3 days after weaning due to weaning stress-induced feeding refusal. However, once newly weaned piglets started to consume food, the abundance of Prevotella increased rapidly. Furthermore, Li et al. showed that the proportion of Alloprevotella and Oscillospira decreased; and Zhong et al. showed that during the first week after weaning, the relative abundances of the dominant bacterial families Erysipelotrichaceae and Lachnospiraceae increased. Alloprevotella has been suggested to mainly produce succinate and acetate, and the Oscillibacter species can produce butyrate, which plays a role in improving the intestinal barrier and exhibits anti-inflammatory function. Butyrate production is negatively correlated with the relative abundances of Erysipelotrichaceae and Lachnospiraceae. Recent reports demonstrated that Erysipelotrichaceae has a potential role in host physiology and/or inflammation related diseases in the gastrointestinal tract and the increased abundance of Lachnospiraceae would lead to increased secretion of pro-inflammatory cytokines in the intestine. Taken together, changes in intestinal microbiota caused by weaning stress may be related to the short-chain fatty acids (SCFA) driven by microorganisms, which might be a key modulator in the maintenance of intestinal homeostasis after weaning ( Figure 4 ).

17In summary, weaning stress plays a vital role in host health, which is believed to be closely associated with the function of the intestinal barrier, including the physical barrier, chemical barrier, immune barrier, and microbial barrier. First, weaning stress disrupts the intestinal mucosal mechanical barrier by reducing the expression of tight junction proteins, which makes it easier for bacteria and toxins to break through the intestinal mucosal barrier, and cause the release of a variety of cellular inflammatory factors, thereby leading to the occurrence of intestinal inflammation. Second, weaning stress disrupts the intestinal chemical barrier by inhibiting goblet cell differentiation and mucins secretion, and decreased mucins secretion further disrupts the intestinal mechanical barrier, immune barrier, and microbial barrier. Third, weaning stress disrupts the intestinal chemical barrier by promoting the secretion of inflammatory factors, while pro-inflammatory cytokines including IL-1β, TNF-α, and IFN-γ, have important intestinal TJ barrier-modulating actions. Finally, weaning stress destroys the intestinal microbial barrier by inducing intestinal microbial disorder, which also adversely affects the function of the intestinal mechanical barrier, immune barrier, and chemical barrier.

18Early weaning is considered one of the most critical periods in pig production, during which the animal must face multiple stressors, such as separation from their mothers and littermates, sudden changes in feeding patterns, and environment, eventually leading to varying degrees of weaning stress symptoms. Weaning stress has a great influence on the intestinal function of piglets. At the early stage of weaning, due to the incomplete development of the intestinal function of piglets, intestinal morphology, digestive function, and the balance between apoptosis and proliferation of intestinal epithelial cells of piglets are damaged to a certain extent after sudden weaning. At the same time, weaning stress also leads to damage to the intestinal barrier function of piglets, manifested as the damage of intestinal epithelial tight junction structure; the proliferation of intestinal goblet cells and reduced secretion of intestinal mucins; increased release of intestinal pro-inflammatory factors; and increased colonization of the so called “harmful bacteria”. In conclusion, weaning stress is detrimental to gut health, but this disadvantage gradually disappears as piglets adapt to the new environment. At present, the use of nutritional interventions to alleviate stress from weaning is the mainstream research direction. Due to this, we need to correct the understanding of the effects of weaning stress on intestinal health, which is of important guiding significance for the nutritional regulation of weaning stress-induced intestinal injury.

19All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

20This research was supported by grants from the China Overseas Expertise Introduction Program for Discipline Innovation (D17016); the Key Science and Technology Program of Guizhou Provence (No. 5411 2017 Qiankehe Pingtai Rencai); Guizhou Normal University Academic New Seedling Fund project (Qianshi Xinmiao [2021] B16); the Natural Science Research Project of Education Department of Guizhou Province (Qianjiaohe KY Zi [2021] 294).

21We thank all the authors who contributed references to this article, including those listed in the references and those not included in the references due to our omissions.

22The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

23All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.