Chemical coding of piglets small intestine neurons after prenatal exposure to β-hydroxy-β-methylbutyrate

To achieve high pork production efficiency and generate significant economic profits, the global swine industry must confront numerous challenges. These include the need to optimise nutrition, implement effective disease control and prevention measures, maintain proper hygiene and safety, and address issues such as extreme temperature fluctuations and low survival rates among newborn piglets (8, 24). The latter issue is closely linked to the optimisation of nutrition, which, beyond its obvious benefits in pig production, can also help address piglets’ general health status (18). The optimal nutrition of pregnant sows with a diet that is properly balanced and rich in key nutrients significantly improves reproductive performance, birth litter weight, weaning weight and the survival rate of newborn piglets. The composition of colostrum and milk is regulated by the mother’s diet, which directly influences the maturation of the newborns’ immune systems, providing protection against infections and supporting the development of a healthy gut microbiome. Overall intestinal health is, in turn, extremely important in combating piglet weaning stress, which is often combined with diarrhoea (11). However, the shaping of the offspring’s health starts earlier, in prenatal life. The prenatal programming theory states that postnatal development is shaped not only by genetics but also by prenatal non-genetic factors, such as the nutrients present in the pregnant female’s diet (2). One supplement that has been successfully used in prenatal nutritional programming is β-hydroxy-β-methylbutyrate (HMB) (26). This metabolite of leucine is commonly used as a dietary supplement to support protein metabolism, insulin activity and skeletal muscle hypertrophy. It is particularly popular among athletes and physically active individuals. In animals, HMB supplementation has shown various benefits, particularly in livestock, where it primarily leads to increased weight gain and improved production yields (29). Studies on pigs have demonstrated that HMB supplementation in sows’ diets as part of nutritional programming leads to heavier newborn piglets and faster attainment of market weight, because more protein is synthesised in the piglets’ skeletal muscles. Positive effects on the development of the offspring’s skeletal and digestive systems have also been observed (2, 5, 27). Additionally, it was noted that HMB may improve the uniformity of piglet birth weights, potentially mitigating the effects of intrauterine growth restriction (3).

As noted earlier, gut health plays a vital role in piglet development. The enteric nervous system (ENS), a key part of the digestive tract, is crucial for the early development of digestive functions. Intestinal innervation starts prenatally and continues to mature after birth, which is essential for newborn piglets as they transition to solid food (13). It is already known that HMB supplementation in pregnant sows significantly affects the expression of leptin, vasoactive intestinal peptide (VIP), and other gut barrier proteins in the offspring during weaning, while having a lesser impact on the basic morphology of the small intestine (26). There is presently insufficient data on the effect of HMB on the prenatal development of small intestine innervation in newborn pigs. Consequently, it is not known whether the effects observed by Tomaszewska et al. (26) were present from birth. Based on the available research, we hypothesise that HMB supplementation in pregnant sows will affect the chemical coding of the ENS in their newborn offspring. To verify this hypothesis, this study analyses the VIP, cocaine-and-amphetamine-regulated transcript (CART), neuronal nitric oxide synthase (nNOS) and substance P (SP) peptides in the small intestine of neonatal piglets after prenatal HMB supplementation, and evaluates the surface area of nerve fibres reacting with these proteins in comparison to piglets without prenatal supplementation.

The present study investigated the effects of prenatal HMB supplementation on the chemical coding of the ENS in the small intestine of neonatal piglets, focusing on the immunoexpression of VIP, CART, nNOS and SP peptides. There is limited information regarding how HMB influences the prenatal development of intestinal innervation, and whether the changes observed in previous studies manifest from birth. The results of the conducted study confirmed the initial assumption that prenatal HMB supplementation would affect the chemical coding of the ENS.

One of the most consistent findings across intestinal regions and layers was the significant decrease in VIP- and SP-reactive fibres in piglets prenatally supplemented with HMB, especially in the muscular layers and mucosa. These neuropeptides play critical roles in the regulation of intestinal motility, secretion and inflammation (15). Vasoactive intestinal peptide acts neuroprotectively and to counter inflammation, reducing the expression of pro-inflammatory cytokines and enlarging the production of anti-inflammatory cytokines (21). It also participates in the modulation of intestinal motility and blood flow (19), which affects digestion and protects the intestines from inflammatory conditions (6). Substance P, another neuropeptide, is in the tachykinin family and acts as a neurotransmitter and neuromodulator in the gastrointestinal tract (12). It modulates immune, vascular and motor responses and serves as a pro-inflammatory mediator by activating immune cells like mast cells, macrophages and T cells via the NK1 receptor (17). This activation leads to the release of cytokines and chemokines triggering inflammation and diarrhoea and stimulating intestinal motility. Production of SP is stimulated by IL-12 and inhibited by IL-10 and transforming growth factor β. Levels of SP rise with inflammation in inflammatory bowel disease, being higher in ulcerative colitis compared to Crohn’s disease and indicating a link between elevated SP levels and greater inflammation (17). The reduction in VIP- and SP-IR fibres may suggest that prenatal HMB supplementation possibly inhibits the activity of these pathways, potentially affecting gastrointestinal motility and immune responses in newborns. However, it is important to consider that the ENS in newborn pigs is still in a developmental phase, which means that changes in VIP and SP fibre density may not necessarily indicate impairment of the ENS due to HMB supplementation (8). Given that the ENS is undergoing maturation during this period (13), a decrease in VIP and SP may suggest a shift in the regulatory mechanisms that control intestinal function (28), possibly facilitating adaptation to postnatal life. Therefore, while the observed decrease in VIP and SP fibres raises questions about the influence of HMB on gut physiology, it does not automatically imply that HMB supplementation negatively impacts the development or functionality of the ENS in newborn pigs. Unknown mechanisms specific to newborns may be in play here, which require further investigation.

There is a similar study that seemingly contradicts our results. Tomaszewska et al. (26) demonstrated an increased level of VIP-IR fibres in the submucosal plexus of weaned piglets prenatally supplemented with HMB. The course of the study was the same as ours; however, the difference was the age of the animals, where weaned piglets were enrolled by Tomaszewska et al. (26) and newborns by us. Such a result is most likely a reflection of the differing level of development in the ENS of the newborns in our study compared to the older animals. These contrasting outcomes reinforce the concept that the ENS undergoes dynamic, stage-specific shifts in neurochemical coding, which shape how prenatal HMB supplementation impacts gut physiology over time.

In comparison to SP- and VIP-reactive fibres, CART-reactive fibres extended over significantly more of the duodenal and ileal muscular layer and ileal submucosa areas. Cocaine-and-amphetamine-regulated transcript, known to be involved in regulating appetite and energy homeostasis, has been less studied in the context of the ENS (30). The proliferation of CART-reactive fibres suggests that HMB might influence the energy regulatory mechanisms of the gut (16), possibly improving nutrient absorption or promoting energy efficiency in neonatal piglets. Fibres reactive with nNOS also showed interesting patterns, particularly in the mucosa, where there was a consistent increase across all intestinal regions. Neuronal NOS is a marker for nitrergic neurons, which are essential for smooth muscle relaxation and regulation of intestinal motility via the production of NO (14). The observed increase in nNOS-reactive fibres in the mucosa, coupled with a lack of significant changes in the muscular layers (except in the jejunum), suggests that prenatal HMB supplementation might enhance nitrergic signalling in the mucosa, possibly improving mucosal blood flow and barrier function (14). Nitric oxide has well-known vasodilatory effects, which could enhance nutrient absorption and immune defence mechanisms in the intestinal mucosa (14).

Studies describing the impact of the toxin fumonisin and the toxic substance bisphenol A on the expression of the neuropeptides we investigated showed results opposite to ours, namely an increase in VIP- and SP-positive fibres and a decrease in CART- and nNOS- positive fibres (10, 23). In contrast, HMB as a supplement appears to regulate the expression of these neurotransmitters, which may indicate its protective effects. The decrease in SP and VIP levels following HMB administration could suggest a reduction in inflammation and an improvement in intestinal function, contrasting with the effect of toxins and toxic substances, which potentiates the activity of these peptides in response to the resultant damage (10, 22). The response to HMB supplementation varied across different regions of the small intestine. In the duodenum, a significant accrual of CART-reactive fibres in the muscular layer and a significant reduction in VIP-reactive fibres in both the submucosa and mucosa indicate a strong modulatory effect of HMB on the upper intestinal segments. In the ileum, however, HMB supplementation resulted in a marked increase in both CART-reactive fibres and nNOS-reactive fibres in the mucosa, suggesting that the effects of HMB may intensify towards the distal small intestine.

These regional differences could reflect the varying functional roles of the small intestine. The duodenum is primarily responsible for the initial stages of digestion and absorption, while the ileum plays a key role in the absorption of bile acids, vitamin B12 and other nutrients (9). The increased presence of CART and nNOS in the ileum might suggest a role for HMB in enhancing nutrient absorption and modulating motility towards the distal regions of the gut, where nutrient content is lower and energy absorption may be more critical. These findings provide new insights into how prenatal HMB supplementation may shape the development of the ENS and, by extension, gastrointestinal function in neonatal piglets. The reduction in SP- and VIP-reactive fibres, alongside the accrual of CART- and nNOS-reactive fibres, suggests that HMB exerts a selective influence on neurochemical pathways involved in gut motility, energy regulation and possibly immune function.

The beneficial effects of HMB on muscle preservation, immune modulation and anti-inflammatory responses in other tissues are known (1). The observed changes may reflect similar protective effects, potentially safeguarding the developing gut from inflammatory or oxidative damage during critical stages of growth. This could be particularly important for neonatal animals, as early-life gastrointestinal development has long-term consequences for health and productivity (31). However, the mechanisms by which HMB influences ENS development remain speculative. Further research is needed to elucidate the molecular pathways involved, potentially including interactions with growth factors, inflammatory mediators and oxidative stress responses, which are known to play a role in gut development and health.