Discussion

Dietary fibre influences the intestinal microbiota composition and activity in pigs.^{(Reference Grześkowiak, Saliu and Martínez-Vallespín1)} Moreover, dietary fibre and its metabolites can have an impact on the intestinal physiology, immune system and may influence the BA homeostasis and host health.^{(Reference Grześkowiak, Saliu and Martínez-Vallespín1,Reference Kastl, Zong and Gershuni8)} The current study assessed whether fibre-enriched diets (either sugar beet pulp or lignocellulose) fed to sows during gestation and lactation have any distinct impact on the BA composition in sow colostrum and in gallbladder bile, and jejunal and colonic digesta collected from their 4-6-day-old offspring.

Here, we were capable of quantifying 12 BA species in colostrum. A more detailed analysis revealed that specific primary and secondary BA showed differences in colostrum from the two dietary groups. Specifically, secondary BA such as lithocholic and oxolithocholic acids were significantly increased in colostrum from sows who consumed diets enriched in sugar beet pulp, while the opposite fibre effect was observed for the primary 3α,7α,12α-trihydroxycholestanoic and secondary glycohyodeoxycholic BA. Therefore, high-fermentable fibre diets promoted elevated concentrations of secondary BA in colostrum, which might have resulted from the increased microbial bile acid transformations in the gut of the sows. Sow colostrum is known to be rich in nutrients and immunoglobulins.^{(Reference Hurley and Farmer14)} The specific immunoglobulins present in colostrum play a crucial role in shielding piglets from environmental antigens and enteric infections.^{(Reference Grześkowiak, Pieper and Kröger21)} We have previously identified biogenic amines and anti-C. difficile-toxin-IgG-antibodies in sow colostrum.^{(Reference Grześkowiak, Saliu and Wessels13,Reference Grześkowiak, Pieper, Huynh, Cutting, Vahjen and Zentek25)} However, reports on the BA in colostrum are very scarce. Recently, the profiling of metabolites in sow colostrum from three breeds of pigs confirmed the existence of breed-specific bile acids signatures.^{(Reference Cheng, Azad and Ding16)} In addition, two BA were recently quantified in colostrum from sows of different parity, but no differences were observed.^{(Reference Vötterl, Schwartz-Zimmermann and Lerch15)} In our study, we did not observe any significant differences in bile acid composition between gilts and sows. To our knowledge, however, our study is the first to quantify a wide range of BA in sow colostrum, thereby providing a more detailed BA profile of porcine colostrum. Although present at two- to four-fold lower concentration than in the piglet’s gut, their contribution to the biological actions of the colostrum and potential implications in early life of the suckling offspring warrants further investigation. BA have been detected in colostrum of healthy women (0.7 ± 0.2 µM of total BA) while women with intrahepatic cholestasis of pregnancy exhibited elevated levels of total BA (23.3 ± 14.8 µM).^{(Reference Brites and Rodriguez26)} It is possible that the presence of BA in colostrum in a healthy state has relevant biological functions. In fact, BA that were altered by the feeding of highly-fermentable fibre in this study are known to regulate intestinal barrier function, cell proliferation and immune function.^{(Reference Keating and Keely3)} In light of their pleiotropic actions, therefore, BA in colostrum could certainly have implications for the colostrum nutritional quality and potential health benefits in new-born piglets, such as promotion of intestinal barrier function through FXR activation.^{(Reference Keating and Keely3)}

Another aspect that remains poorly understood is the origin of BA in colostrum. BA have been detected in healthy murine brain (ca. 25 ng/g), which may suggest their signalling role on the central nervous system.^{(Reference Koike, Miyaji and Sano27)} A possible source of colostral BA could be through the systemic blood. However, the presence of BA in systemic blood (15–87 µM) has been related to serious illnesses such as hepatic failure in humans.^{(Reference Horvatits, Drolz and Roedl28)} Nevertheless, it is possible that small amounts of BA, without a harming effect, enter the systemic blood circulation after absorption in the gut from where they reach the mammary glands. Whether the type of fibre in the diet, either high- or low-fermentable, would have any impact on the BA transport to colostrum in sows, needs further investigation.

We have observed a few significant differences in the concentrations of primary and secondary BA from the gallbladder bile from both experimental groups of piglets. It is known that the fermentation of dietary fibres produces SCFA, which can affect BA synthesis in the liver. Particularly butyrate, has been shown to influence the expression of genes involved in BA metabolism.^{(Reference Sheng, Jena and Hu29)} Therefore, a link could exist between fibre-induced changes and the efficiency of fat digestion through the increased detergent properties of conjugated BA in the gastrointestinal tract. It is known that fat digestion begins in the duodenum by the secretion of bile and continues in the jejunum and ileum, where emulsified fats can be absorbed by the intestinal epithelium. Indeed, conjugated BA, such as the primary taurochenodeoxycholic acid and the secondary taurodeoxycholic acid, which showed group differences, have increased detergent properties and are therefore far more efficient at fat emulsification and digestion than unconjugated BA.^{(Reference Di Ciaula, Garruti and Baccetto30)} Interestingly, high concentrations of secondary bile acids in piglet bile may result from early microbial biotransformation in the intestine combined with enterohepatic recirculation, through which secondary metabolites are reabsorbed, conjugated in the liver, and secreted back into the bile.^{(Reference Hofmann31)}

We have demonstrated that piglets from the sugar beet pulp group had either significantly or numerically higher concentration of major secondary BA such as lithocholic acid and ursodeoxycholic acid, in the ileal digesta, as compared to piglets from the lignocellulose group. Pigs are recognised for their tendency towards coprophagy, and nursing piglets maintain constant exposure to the sow’s faeces after birth. This close contact may potentially impact the gut microecology of the offspring, influenced by that of the sow.^{(Reference Whatson and Bertram32)} Sugar beet pulp leads to a higher extent of microbial fermentation due to the presence of fermentable pectin and other carbohydrates. A modified metabolic activity of the piglet small intestinal microbiota, especially towards the end of the small intestine, might have contributed to BA deconjugation and higher lithocholic and ursodeoxycholic acids in this group of piglets. It is known that especially lactobacilli from the small intestine contain BA deconjugating enzymes,^{(Reference Khare and Gaur33)} which may in part explain the higher concentration of these BA in the piglets from the sugar beet pulp group. Additionally, BA are reabsorbed in the ileum, and their concentration can be influenced by the types of fibres present in the diet. Dietary fibre, especially insoluble fibre such as lignocellulose, can influence gut motility.^{(Reference Oliviero, Kokkonen, Heinonen, Sankari and Peltoniemi34)} Changes in the gut motility can affect the residence time of BA and bacteria in the small intestine, influencing their absorption and deconjugation, which could in part explain the observed differences in BA from the two dietary fibre groups. In addition, the interaction between BA and dietary fibres can affect the solubility and absorption of BA in the small intestine.^{(Reference Kahlon and Woodruff35)}

A small portion of microbially-produced (secondary) BA escapes reabsorption in the ileum and is transformed by colonic microbiota.^{(Reference Ridlon, Kang and Hylemon2)} Some of these secondary deconjugated and dexydroxylated BA can effectively influence microbial populations by either inhibiting or stimulating bacterial growth and activity. For instance, taurocholic, glycocholic and cholic acids are produced by the liver and act as potential germinant of C. difficile spores, whereas chenodeoxycholic acid inhibits germination and the secondary species deoxycholic and lithocholic acids inhibit growth and toxin synthesis by vegetative cells of C. difficile.^{(Reference Sorg and Sonenshein36)} Reports also show that, deoxycholic and chenodeoxycholic acids display toxicity against Bifidobacterium breve, Blautia coccoides and Bacteroides thetaiotaomicron.^{(Reference Watanabe, Fukiya and Yokota37)}

Higher concentrations of some secondary BA such as deoxycholic acid, lithocholic acid, taurodeoxycholic acid, or the primary BA such as chenodeoxycholic acid, may alter epithelial barrier function, cell signalling and immune response.^{(Reference Keating and Keely3,Reference Mühlbauer, Allard and Bosserhoff38)} Here, we showed that the total secondary BA including their species such as lithocholic and ursodeoxycholic acids were increased in the piglets whose dams were fed sugar beet pulp, as compared to lignocellulose. Higher concentrations of secondary BA may reflect increased microbial activity due to high-fermentable fibres, compared to low-fermentable fibres. Interestingly, we have previously reported that the expression of zonula occludens 1 (ZO-1) and transforming growth factor β (TGF-β) genes in the colonic tissue were upregulated in the piglets from the sows fed sugar beet pulp as compared to lignocellulose.^{(Reference Grześkowiak, Saliu and Wessels13)} Indeed, micromolar concentrations of cholic, deoxycholic and chenodeoxycholic acids but not ursodeoxycholic acid decreased transepithelial electrical resistance and increased paracellular permeability in a Caco-2 cell model.^{(Reference Raimondi, Santoro and Barone39)} In addition, elevated cholic acid in the colon has been associated with inflammation or disease processes in the gut,^{(Reference Kajiura, Ohkusa and Okayasu40)} which may enhance the expression and activation of TGF-β, contributing to tissue damage and remodelling.^{(Reference Raimondi, Santoro and Barone39)}

BA, depending on their concentration and origin (primary or secondary), have been reported to either stimulate or inhibit the intestinal epithelial cell proliferation. Specifically, higher concentrations of certain secondary BA have inhibitory or cytotoxic activity while low concentrations can be beneficial to the epithelial cells.^{(Reference Rolo, Palmeira and Wallace41)} Dietary fibre influences colonic secondary lithocholic BA levels, as seen in previous studies. For instance, the inclusion of soluble flaxseed meal in feed, compared to insoluble oat hulls, has been shown to increase faecal lithocholic acid concentration in growing pigs (ca. 25 kg body weight) (3.97 vs. 2.26 mg/g).^{(Reference Ndou, Kiarie, Thandapilly, Walsh, Ames and Nyachoti42)} Despite this, knowledge regarding the effect of lithocholic acid on porcine intestinal cells remains limited. Nevertheless, in cancer research, the impact of lithocholic acid on the inhibition of colonic cell proliferation is widely studied.^{(Reference Nordling, Glinghammar, Karlsson, De Kok and Rafter4)} In our previous observations, significant increases in colonic proliferating cell nuclear antigen gene expression (PCNA) were noted in piglets from the sugar beet pulp group, which coincided with higher lithocholic acid concentrations in this group.^{(Reference Grześkowiak, Saliu and Wessels13)}

The body weight of piglets or sow feed intake were unlikely to affect BA levels in colostrum and intestinal digesta, respectively, as these characteristics were controlled and remained similar across both feeding groups. Besides, reports on the impact of dietary fibre on animal performance and relation to BA levels in colostrum or intestinal digesta are scarce.^{(Reference Fang, Zhang and Meng6)} This feeding experiment would benefit from the microbiota composition and metabolomics analysis in intestinal digesta of the study sows and piglets. Such data could offer valuable insights into the microbial activity’s potential to metabolise BA. Additionally, in future BA analysis of sow milk should be performed, to allow potential associations with BA levels in piglet digesta. However, such analysis goes beyond the scope of the current study aims.

Insights gained from the current study may also contribute to understanding how maternal diet influences BA profiles in colostrum and infant gut. These findings could support dietary strategies to enhance maternal-infant health in human medicine. The research may also provide valuable implications for preventing BA-related diseases and optimising nutrient absorption through targeted dietary interventions. The role of BA in colostrum in healthy mothers should be further investigated and pigs can be considered as valuable model species.

In summary, incorporating specific dietary fibres into the diet of sows could modify the composition of certain BA in their colostrum. BA in colostrum may affect the BA balance in piglets. Additional studies are needed to fully understand how fibre-rich diets influence the presence of BA in colostrum, the digestive system of new-born piglets, and the broader aspect of piglet health.