Early feeding leads to molecular maturation of the gut mucosal immune system in suckling piglets

IntroductionDiet-microbiota-host interactions are increasingly studied to comprehend their implications in host metabolism and overall health. Keeping in mind the importance of early life programming in shaping intestinal mucosal development, the pre-weaning period can be utilised to understand these interactions in suckling piglets. The objective of this study was to investigate the consequences of early life feeding on the time-resolved mucosal transcriptional program as well as mucosal morphology.MethodsA customised fibrous feed was provided to piglets (early-fed or EF group; 7 litters) from five days of age until weaning (29 days of age) in addition to sow’s milk, whereas control piglets (CON; 6 litters) suckled mother’s milk only. Rectal swabs, intestinal content, and mucosal tissues (jejunum, colon) were obtained pre- and post-weaning for microbiota analysis (16S amplicon sequencing) and host transcriptome analysis (RNA sequencing).ResultsEarly feeding accelerated both microbiota colonisation as well as host transcriptome, towards a more “mature state”, with a more pronounced response in colon compared to jejunum. Early feeding elicited the largest impact on the colon transcriptome just before weaning (compared to post-weaning time-points), exemplified by the modulation of genes involved in cholesterol and energy metabolism and immune response. The transcriptional impact of early feeding persisted during the first days post-weaning and was highlighted by a stronger mucosal response to the weaning stress, via pronounced activation of barrier repair reactions, which is a combination of immune activation, epithelial migration and “wound-repair” like processes, compared to the CON piglets.DiscussionOur study demonstrates the potential of early life nutrition in neonatal piglets as a means to support their intestinal development during the suckling period, and to improve adaptation during the weaning transition

Dietary diversity affects feeding behaviour of suckling piglets

Stimulating solid feed intake in suckling piglets is important to facilitate the weaning transition, exemplified by the positive correlation between pre- and post-weaning feed intake. The present study compared the effect of dietary diversity (i.e. offering two feeds simultaneously) and flavour novelty (i.e. regularly changing the flavour of one feed) on the feeding behaviour and performance of suckling piglets until weaning at day 22. It was hypothesized that presentation of the feed in a more diverse form, by varying multiple sensory properties of the feed, stimulates pre-weaning feed intake. Piglets received ad libitum feed from 2 days of age in two feeders per pen (choice feeding set-up). One group of piglets (dietary diversity (DD), n = 10 litters) were given feed A and feed B which differed in production method, size, flavour, ingredient composition and nutrient profile, smell, texture and colour. The other group of piglets (flavour novelty (FN), n = 9 litters) received feed A plus feed A to which one of 4 flavours were added from day 6 in a daily sequential order. Feeding behaviour was studied by weighing feed remains (d6, 12, 16, 22) and by live observations (4-min scan sampling, 6 h/d; d9, 14, 21; n = 6 litters per treatment). Observations were also used to discriminate ‘eaters’ from ‘non-eaters’. All piglets were weighed at d2, 6 and 22. Piglets did not prefer feed A (d2–22: 1.4 ± 0.16 kg/litter) over B (1.6 ± 0.18) within DD nor had a preference for feed A with (d6–22: 1.1 ± 0.06 kg/litter) or without additional flavours (0.9 ± 0.07) within FN. Nevertheless, DD-litters (d2–22: 3.0 ± 0.32 kg) ate significantly more than FN-litters (2.0 ± 0.12 kg; P = 0.02) and explored the feed 2.6 times more at d14 (P = 0.001). Furthermore feed A, the common feed provided in DD and FN, was more consumed in DD (d2–22: 1.4 ± 0.16 kg) compared to FN (1.0 ± 0.07 kg; P = 0.04). The percentage of eaters within a litter did not differ over time between DD (d9: 26%, d14: 78%, d21: 94%) and FN (20%, 71% and 97%) and no effect was found on pre-weaning weight gain. In conclusion, this study showed that provision of dietary diversity to suckling piglets stimulated their feed exploration and intake more than dietary flavour novelty only, but did not enhance the percentage of piglets within a litter that consume the feed or their growth performance. These data suggest that dietary diversity could be an innovative feeding strategy to stimulate solid feed intake in suckling piglets.</p

Early-life feeding in piglets: the impact on intestinal microbiota and mucosal development

Early-life bacterial colonisation can be of particular importance to the overall growth and health of an animal, especially influencing intestinal and immune system development with long-term implications. This is especially relevant in pig production where post-weaning enteric infection is one of the major concerns related to the gut health of pigs, and is associated with economic losses and welfare problems. Commercial pig production systems involves early and abrupt weaning, which contrasts with the gradual transition from mother’s milk to solid feed in nature. Due to such abrupt weaning, a piglet is challenged with multiple stressors (including environmental-, nutritional- and psychological-) which is usually associated with changes in gut microbiota and a high incidence of diarrhoea. Modulating intestinal microbiota to reduce weaning-associated problems in pigs, is getting increasing scientific and commercial interest, as microbial dysbiosis (or imbalance) has been identified as a leading cause of post-weaning intestinal infections. The overall aim of this thesis was to understand the molecular effects of early-life feeding (pre-weaning provision of fibrous feed), associated with the pattern of intestinal microbiota colonisation and gut maturation. We hypothesised that early feeding of a fibrous diet containing both soluble and insoluble fermentable fibres will affect the microbiota and physiological development of neonatal or suckling piglets, facilitating a better preparation for the weaning transition.First, we focussed on assessing an optimal sampling method to study microbiota development in neonatal piglets. We showed that rectal swabs are a suitable alternative sample type to study the porcine microbiome development in early-life, when faecal sampling is challenging. Although rectal swab samples bring a certain degree of variability with the presence of mucosa-adhered population, they also provide the opportunity to assess the impact of an intervention on the mucosa-adhered populations. The second aim of this thesis was to investigate the age-related gut microbiota colonisation and the impact of fibrous feed on the gut microbiota colonisation as well as intestinal physiology in neonatal piglets. We performed a longitudinal study (using rectal swabs) to evaluate the impact of early feeding (fibrous feed) on the microbiota colonisation at pre- and post-weaning time-points. The results revealed that the early-fed (EF) piglets had an accelerated maturation of the microbiota, compared to the control (CON) piglets that consumed milk exclusively. Accelerated maturation at pre-weaning time-points were characterised by the simultaneous emergence of typical post-weaning-associated microbial groups (such as Prevotella, Roseburia, Faecalibacterium, Ruminococcus, Megasphaera, Subdoligranulum) and a more rapid decline of typical early-life/pre-weaning microbial genera (e.g., Fusobacterium, Finegoldia, Bacteroides, Eschechichia-Shigella). Moreover, we found a quantitative association between eating behaviour of EF piglets (video scores) and their microbiota signature, indicating that the piglets who spent more time at the feeding trough had a higher abundance of ‘accelerated’ microbial groups. Furthermore, early feeding altered the colonic microbiota composition, increased microbial fermentation products (SCFA) in the colon and modulated intestinal development i.e., increased weights and lengths of several intestinal tract segments, as well as a decreased villus-crypt ratio in jejunal mucosa and an increased abundance of proliferative cells in colon mucosa, just before weaning. The third aim of this thesis was to evaluate the host mucosa transcriptome response due to pre-weaning fibrous feed. Maximum impact of early feeding was detected at weaning (day29; compared to other time-points), which was followed by convergence of the transcriptome three weeks post-weaning (day+21) in EF and CON piglets. We showed that early feeding not only accelerates the gut microbiota, but also the host transcriptome maturation at weaning. The results revealed that in the EF group, oxidative phosphorylation, cholesterol biosynthesis and oxidative stress-related pathways were significantly enriched, whereas sirtuin signalling and immune response pathways were downregulated in the colon mucosa at day29. Remarkably, three days after weaning, the EF piglets displayed a stronger mucosal responsiveness compared to the CON piglets, reflected in the increased expression of genes (transcriptomics) related to immune activation, epithelial migration and “wound-repair" like processes necessary to maintain gut barrier integrity (during weaning transition) as well as jejunal morphometry. In the last chapter, we explored fundamental aspects of host-microbe interactions, by evaluating the associations between the intestinal microbiota and behaviour of suckling piglets. We aimed to tentatively reveal associations between the intestinal microbiota composition and piglet behaviour in a test for coping style, (i.e. a personality trait), as well as anxiety- and exploration-related behaviour in a novel environment test. A number of microbial groups such as Coprococcus, CAG-873, Atopobium and Prevotella were identified to be associated with anxiety- and/or exploration-related behaviour, although these results are premature and need further validation for their biological relevance. Overall, the findings of this thesis indicate that EF piglets show an “alerted system” with an enhanced responsiveness to external stimuli of feed and microbiome development, compared to the CON group, thus exemplifying the potential of early-life programming to modulate intestinal development in piglets. Furthermore, the results of this thesis indicate that early feeding of fibrous feed has considerable potential to better prepare young piglets for the weaning transition

Assessing the Impact of Diet on the Mucosa-Adhered Microbiome in Piglets Using Comparative Analysis of Rectal Swabs and Colon Content

Previously, we demonstrated that rectal swabs provide a legitimate alternative to faecal sampling for the assessment of the intestinal microbiota in young piglets. However, we also reported that mucosa-adhered microbial populations were more represented in rectal swabs compared to faecal samples, albeit to a degree that varied per swab-sample. Here, we explored the possibility to exploit this variable enrichment of adhered populations in the rectal swabs to assess the impact of diet on mucosa-adhered microbiota in pre-weaning piglets. Paired samples of rectal swabs and colon luminal contents were collected from piglets just before weaning during two independent but similarly designed animal experiments [n = 28 piglets (experiment 1); n = 16 piglets (experiment 2)], with an early feeding treatment (EF) group that had access to customised fibrous feed in addition to sow’s milk and a control (CON) group exclusively reared on sow’s milk. The intestinal microbiome composition in rectal swabs and colon samples collected at 29 days of age were subjected to metataxonomic analysis. The results identified the genera Escherichia-Shigella, Anaerococcus, Peptostreptococcus, Enterococcus, Trueperella, Actinomyces, and Peptoniphilus as discriminative taxa enriched in rectal swabs compared to colon. Apart from Escherichia-Shigella (10–11% average relative abundance), most of these mucosa-adhered microbial genera display relatively low abundance. Rectal swab microbiota was found to be more variable, which is likely due to variable enrichment of mucosa-adhered microbes. Although almost exclusively driven by one of the experiments, the post-weaning diarrhoea-associated taxa Escherichia-Shigella, was enriched in CON compared to the EF group, suggesting that early life feeding may suppress post-weaning-diarrhoea-related problems in piglets. Our findings demonstrate that rectal swabs allow the investigation of the mucosa-adhered microbial populations as a function of dietary treatment in piglets. This offers opportunities to further study dietary approaches that suppress the abundance of the post-weaning diarrhoea associated adherent microbes like Escherichia-Shigella. Furthermore, we demonstrate that the paired swab-colon microbiota information (obtained from a subset of animals) can predict the mucosa-adhered populations or “mucosity factor” in rectal swab samples, facilitating the analysis of the adhered microbiota in large animal cohort studies using readily obtainable rectal swabs

Ultrafast differential flexibility of Cro-protein binding domains of two operator DNAs with different sequences

The nature of the interface of specific protein–DNA complexes has attracted immense interest in contemporary molecular biology. Although extensive studies on the role of flexibility of DNA in the specific interaction in the genetic regulatory activity of lambda Cro (Cro-protein) have been performed, the exploration of quantitative features remains deficient. In this study, we have mutated (site directed mutagenesis: SDM) Cro-protein at the 37th position with a cysteine residue (G37C) retaining the functional integrity of the protein and labelled the cysteine residue, which is close to the interface, with a fluorescent probe (AEDANS), for the investigation of its interface with operator DNAs (OR3 and OR2). We have employed picosecond resolved polarization gated fluorescence spectroscopy and the well known strategy of solvation dynamics for the exploration of physical motions of the fluorescent probes and associated environments, respectively. Even though this particular probe on the protein (AEDANS) shows marginal changes in its structural flexibility upon interaction with the DNAs, a non-covalent DNA bound probe (DAPI), which binds to the minor groove, shows a major differential alteration in the dynamical flexibility in the OR3–Cro complex when compared to that of the OR2 complex with the Cro-protein. We attempt to correlate the observed significant structural fluctuation of the Cro-protein binding domain of OR3 for the specificity of the protein to the operator DN

Effects of Creep Feed Provision on Behavior and Performance of Piglets Around Weaning

Creep feed provision may ease weaning, hence we determined the impact of providing fibrous creep feed before weaning and adding this feed to the post-weaning diet on piglet behavior and performance. Pre-weaning, litters with on average 12 piglets were given creep feed (CF, n = 12 litters) or not (NF, n = 10 litters). Post-weaning, piglets (n = 8 pens with 4 piglets/treatment) were given a weaner diet (CON) or weaner diet supplemented with creep feed (CS). Behaviors were scored in the home pen at d11, 16, 22 and 27 after birth and at wk 1 and 2 post-weaning. Feed intake, growth and fecal consistency were measured up to d14 post-weaning. A blood sample was taken at d2, 15 and 29 after birth and d2, 5 and 14 post-weaning. CF-piglets consumed on average 397 ± 71 g creep feed before weaning. CF-piglets grew faster in the last week before weaning than NF-piglets (249 ± 7 vs. 236 ± 11 g/d, F(1, 18) = 5.81, P = 0.03). However, CF- and NF-piglets did not differ in weaning weight, within-litter coefficient of variation in weaning weight, behaviors in the farrowing and weaner pen, and haptoglobin concentrations. Creep feed supplementation enhanced feed exploration at wk 2 post-weaning (0.29 ± 0.08 vs. 0.11 ± 0.03%, F(1, 27) = 5.27, P = 0.03), but did not affect other post-weaning behaviors. Pre-weaning creep feed provision and post-weaning creep feed supplementation did not affect overall feed intake, growth, feed efficiency and fecal consistency for the first 14 days post-weaning, neither body weight at d14 post-weaning. Nevertheless, CF-piglets had a lower within-pen coefficient of variation in body weight at d14 post-weaning than NF-piglets (13.6 ± 1.9 vs. 15.1 ± 1.5%, F(1, 26) = 6.89, P = 0.01). In conclusion, pre-weaning creep feed provision and post-weaning creep feed supplementation had no clear effects on piglet behavior and performance.</p

Transcriptome profiling of porcine jejunum tissue, its derived organoids over long-term culture, and transformed cell line IPECJ2 as reference model

Purpose: Organoids are gaining more traction as physiologically relevant models for host physiology, but their transcriptional stability has not been identified over long term culture. Comparing this to its host-derived tissue and a transformed cell line of this intestinal location could provide insight into the stability and maintenance of cellular programs in vitro. Methods: Jejunum organoids were derived from 2 5-week old piglets, and grown separately in triplicate for 3 and 12 weeks. Tissue homogenate, organoids from both time points (divided in triplicate at start of experiment to monitor culture-dependent variability) and three IPEC-J2 passage rates were isolated for RNA using an RNEasy mini kit following manufacturers protocols. Results: RNA was enriched for PolyA and sequenced on an illumina sequencer. Reads were checked for quality, trimmed, and mapped following the tuxedo suite pipeline (modified to fit our parameters) and CLC Genomics Workbench to identify unknown gene locations. Conclusions: Organoids cultured for 12 weeks showed strong inter- and intra variability, congruence between independent cultures, and strong epithelial resemblance to host tissues. It further exemplified a more extensive transcriptome than IPEC-J2, showing more physiologically relevant genes

Specific DNA sequences allosterically enhance protein–protein interaction in a transcription factor through modulation of protein dynamics: implications for specificity of gene regulation

Most genes are regulated by multiple transcription factors, often assembling into multi-protein complexes in the gene regulatory region. Understanding of the molecular origin of specificity of gene regulatory complex formation in the context of the whole genome is currently inadequate. A phage transcription factor λ-CI forms repressive multi-protein complexes by binding to multiple binding sites in the genome to regulate the lifecycle of the phage. The protein–protein interaction between two DNA-bound λ-CI molecules is stronger when they are bound to the correct pair of binding sites, suggesting allosteric transmission of recognition of correct DNA sequences to the protein–protein interaction interface. Exploration of conformation and dynamics by time-resolved fluorescence anisotropy decay and molecular dynamics suggests a change in protein dynamics to be a crucial factor in mediating allostery. A lattice-based model suggests that DNA-sequence induced allosteric effects could be crucial underlying factors in differentially stabilizing the correct site-specific gene regulatory complexes. We conclude that transcription factors have evolved multiple mechanisms to augment the specificity of DNA–protein interactions in order to achieve an extraordinarily high degree of spatial and temporal specificities of gene regulatory complexes, and DNA-sequence induced allostery plays an important role in the formation of sequence-specific gene regulatory complexes