Journal of Animal Science and Biotechnology
Research

Grass hay mixed-in creep feed or separately-fed differentially affects digestive development in pre- and post-weaning piglets

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Abstract

Background

Based on observations in feral pigs, the role of dietary fibre and structure may be underestimated in suckling piglet nutrition. This study investigated the effect of grass hay offered to suckling piglets either separately or included in their creep feed, combined with nursery diets with or without grass pellet inclusion on growth performance and gastrointestinal development.

Methods

Thirty-six litters (14–15 piglets per litter) were divided into three equal groups of 12 litters per treatment during the suckling phase: control group (CON) received regular creep feed; GH group received chopped grass hay as-is in separate feeders alongside regular creep feed; PGH group received regular creep feed but barley and wheat were replaced by 28% grass pellets. After weaning (d 23), each litter was split into two dietary treatments in a split-plot design (pre-wean treatment as main plot). Two of the pre-wean diets were also offered until d 14 post-weaning, i.e., CON (CON nursery diet, CON-C, GH-C, PGH-C) and PGH (GH nursery diet, CON-GH, GH-GH, PGH-GH). Thereafter, transitioning to a diet containing 13% wheat/barley or grass pellets, respectively, until d 39 post-weaning. Gastrointestinal morphology, gene expression of intestinal nutrient transporters and barrier proteins, metabolite profile and microbiota were assessed on the day before weaning, d 10 and d 38 post-weaning. A total of 24 piglets were sacrificed at each dissection point.

Results

At weaning, GH group had consumed 7 g/piglet grass hay, and PGH group had consumed 46 g/piglet creep feed. One day before weaning, GH piglets showed heavier emptied small intestine (P= 0.044) and colon (P= 0.065), higher SCFA production in proximal segments and lower SCFA production in colon (P< 0.05). Higher abundance ofPrevotellaceae NK3b31 groupwas observed in caecal and colonic content of PGH compared to GH group (P< 0.05), and PGH group showed a lower energy conversion ratio (net energy intake/gain,P= 0.035). Following weaning, GH nursery group had a reduced average daily gain (226 vs. 183 g,P< 0.001) during d 0–14, while this group showed compensatory growth afterwards (P= 0.056). Main plot effects on increased expressions ofCLDN3andFFAR2were observed in GH and PGH by d 38 post-weaning (P< 0.05). An interaction effect showed greater luminal abundance of thePrevotellaceae NK3b31 groupin GH-GH and PGH-GH groups compared to CON-GH on d 38. The GH nursery diet showed a better energy conversion ratio (P= 0.006) with no influence on body weight and their SCFA production shifted towards proximal segments.

Conclusion

In conclusion, feeding a structured and fibre-rich diet to suckling piglets enhance their digestive tract development and adapt their microbiome to fibre digestion in later life. Maintaining a fibre-rich diet from suckling to nursery is recommended, though this come with a transient reduction in weight gain caused by lower feed intake that, however, can be recovered afterwards accompanied with an optimized energy conversion ratio.

Introduction

At the end of the suckling phase, farmed piglets need to abruptly transition from a sow milk-based diet to a solid diet. This requires a resilient gastrointestinal tract (GIT) but developing this continues to pose an issue [1]. In addition to the dietary transition, piglets face complex challenges, including separation from the sow, new housing conditions and stress from newly established social hierarchies. These stressors, combined with the immature physiological conditions in these piglets, result in anorexia, diarrhoea, growth stasis and potentially, mortality [2,3,4,5]. Creep feeding—applying a supplemental diet to piglets during the suckling period—is widely applied to accelerate GIT development and familiarize piglets with solid feed prior to weaning. Typically, creep feeds are formulated to be nutrient-dense, highly digestible and palatable. However, creep feed intake remains variable within and among litters, potentially resulting in health and performance problems in later life phases [6, 7]. Meanwhile, excessive fermentation of poorly digested creep feed residues in the distal digestive tract promotes microbial dysbiosis, eventually exacerbating the weaning stress [8].

In contrast to the farm setting, piglets in the wild start ingesting solid, fibre-rich items from the first week of life onward, which is associated with faster development of their stomachs [9]. Studies across diverse wild animal species have reported a drive for particular dietary profiles that are assumed to match their nutritional requirements [10,11,12]. The observation in wild piglets, therefore, presents promising possibilities for incorporating fibrous and structure-rich items into the diet of suckling piglets. For suckling piglets in farm settings, the precise nutritional support and proper development of digestive organs should be prioritized to support resilience in later phases, rather than solely aiming for maximal growth rate, as is done for the fattening period. Therefore, the previous study reported that replacing starch with insoluble fibre, such as cellulose and oat hulls, in creep feed increased the weight and length of the GIT and subtly altered the microbiome composition in the colon [13, 14]. To further simulate the natural conditions, our previous study demonstrated similar effects in suckling piglets offered chopped grass hay in a separate feeder alongside common creep feed [15]. Grass hay fostered the growth of the GIT and was well-accepted by suckling piglets. The variable physical and chemical properties of different sources of dietary fibre, however, may result in distinct modes of action [16]. Meanwhile, whether the more robust GIT development observed at weaning in the previous study can be maximally utilized or enhanced by continuing grass hay inclusion in the post-weaning diet, remains to be clarified [15]. Thus, further research is warranted to optimize the provision of dietary fibre and structure both in the pre- and post-weaning phases.

Given that finely milled and pelleted diets are commonly applied in practice and postulating that some outcomes in our previous study may be attributed to limited sample size, this current study not only replicated the chopped grass hay treatment on a larger-scale, but also used a pelleted grass-containing diet for suckling piglets. Additionally, this study aimed to explore the interactive effect of feeding grass hay during the suckling and nursery phase on growth performance, gastrointestinal development, gene expression of nutrient transporters and intestinal barrier proteins, gastrointestinal metabolites, and microbiota. We hypothesized that a long-term, fibre-enriched diet initiated at a young age would further benefit the overall development of piglets.

Materials and methods

The housing, rearing and any other procedures on animals were in compliance with the European Union Directive 2010/63/EU, and assessed by the Dutch Central Committee on Animal Experimentation (CCD), under application number AVD20400202316684.

Animal housing and management

Thirty-six Hypor Libra sows (Hendrix Genetics, Boxmeer, The Netherlands; average parity 3.16, ranging from 1 to 6,) were inseminated (Hypor Maxter) at the Swine Research Centre (Trouw Nutrition R&D, Sint Anthonis, The Netherlands). They were moved to individual farrowing pens (200 cm × 260 cm) one week prior to the expected farrowing date. Ten farrowing pens were located in each climate-controlled room, where the lights were on between 6:00 and 22:00. Each pen was equipped with separate drinking nipples for the sow and the piglets, and an elevated feed trough for the sow designed to prevent piglets from consuming sow feed. Sows had ad libitum access to drinking water and were fed daily rations according to a step-up scheme after farrowing. The day when most sows started farrowing was set as d 0 of this trial, and litter size was equalized over treatments to 14–15 piglets per sow within 2 d after farrowing. Ear tagging, tail docking and iron injection were performed within 3 d after birth of piglets. All piglets were vaccinated against Escherichia coli serotype F18 (Ecoporc Shiga, IDT Biologika GmbH, Dessau-Rosslau, Germany) on d 3 of age, and a triple-vaccine against Mycoplasma hyopneumoniae, porcine circovirus type 2 (PCV2) and porcine reproductive and respiratory syndrome (PRRS) virus (Ingelvac Mycoflex, Circoflex and PRRSflex, respectively, Boehringer Ingelheim GmbH, Ingelheim, Germany) was administered 1 week prior to weaning. Weaning occurred on a fixed day, with piglets averaging 23 d of age (SD = 0.8 d) and 6.6 kg body weight (SD = 0.5 kg).

Experimental design and dietary treatments

This study was designed as split-plot with 3 dietary treatments pre-weaning as main plot (experimental unit was litter) and 2 dietary treatment post-weaning as split plot (experimental unit was pen; Fig. 1). Pre-weaning, litters were randomly allocated to a treatment based on parity and farrowing date of the sow. From d 2 of age until weaning, the piglets in the control group (CON, n = 12 litters) only received the control creep feed which consisted of a basal diet containing highly digestible, finely ground ingredients and 28% of a 50:50 mix of wheat and barley in a round feeder (Table 1). In addition to the control creep feed, piglets in the GH group (n = 12 litters) were given chopped grass hay (analysed nutrients: 8.7% crude protein, 1.5% crude fat, 59.35% neutral detergent fibre and 32.4% acid detergent fibre) in a separate feeder alongside the creep feed feeder. The positions of the two feeders were alternated daily to avoid location preference bias in pens of the GH group. The third group (PGH, n = 12 litters) received the control diet in which the 28% wheat and barley were fully replaced by a grass pellet (mean particle size: 0.36 mm). The diets were fed as crumble pre-wean and for the first 14 d post-wean and their composition can be found in Table 1. The inclusion level of the grass pellet was selected based on the result of voluntary feed intake observed in a previous study [15]. The feeders in the pen were positioned identically across all litters and near the head of the sow. Creep feed, grass hay and water were available ad libitum for piglets.

Fig. 1
figure 1

The schematic diagram from the start of the treatments (d 2 of age) to the end of the experiment (d 39 post-weaning)

Table 1 Composition (ingredients, nutrients) of the experimental creep feeds fed from d 2 to weaning
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Feed leftovers were collected and completely replaced by fresh feed at 7:00 each day. Concurrently, feeders were replenished with fresh feed when empty which was checked twice daily during the first two days, and subsequently four times daily. Three days prior to weaning, piglets were identified as creep feed eaters by adding a blue colorant (0.5% indigo carmine from Sigma-Aldrich) to the crumbles for faecal colour assessment from d 8 to 4 prior to weaning. Two days prior to weaning, 8 litters per treatment (out of the 12 in total) were selected based on number of eaters with 4 litters per treatment having at least 1 eater and 4 other litters per treatment having at least 5 eaters.

Following weaning, piglets were moved to a nursery unit. The 12 piglets closest to median weight of each litter was split into two equal groups of 6 piglets and housed in a pen (dimensions 200 cm × 150 cm), ensuring a balanced body weight distribution between the two pens per litter. The piglets selected for post-wean sampling were evenly distributed in two nursery pens. One pen from each litter received the control feed (CON nursery group: CON-C, GH-C PGH-C), while the other pen received the diet with 28% grass pellet (GH nursery group: CON-GH, GH-GH, PGH-GH) during the first 14 d post-weaning. These diets were the same as used pre-weaning. On d 14 post-weaning, both groups transitioned to a link diet which consisted of a basal link diet to which either 13% of a 50:50 mix of wheat and barley was added (CON nursery group) or 13% grass pellet (GH nursery group, Table 2). Diets were fed as 4 mm pellets until the end of the study (d 39 post-weaning).

Table 2 Composition (ingredients and nutrients) of the experimental nursery feed fed from d 14 to 39 post-weaning
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Sampling and measurement

The individual body weight of piglets was measured at birth, 24 h of age (to calculate colostrum intake), d 10 during lactation (with d 0 being the start of the treatments), the day prior to weaning, d 14 post-weaning, and the last day of the study (d 39 post-weaning). Average daily gain (ADG, g/pig/d) was calculated at litter or pen level per period (d 0 to weaning, weaning to d 14 post-weaning and d 14 to 39 post-weaning). Feed intake during suckling was measured daily and total feed intake of creep feed (g/piglet) and GH intake was calculated (g/litter). During the nursery phase, feed intake was measured on d 14 and 39 and average daily feed intake (ADFI, g/pig/d) was calculated. Feed conversion ratio and energy (FCR) conversion ratio (ECR) were also calculated. Upon discovering a deceased piglet, the animal was weighed, and feed was weighed back to adjust feed intake records accordingly.

Three dissection points were chosen: the day before weaning to evaluate the effect of treatments during lactation and d 10 and d 38 post-weaning. Piglets selected for euthanasia and sampling had similar colostrum intake and a median body weight on d 10 prior to weaning. Thus, on 2 d prior to weaning, all piglets subjected to sampling were selected. Eight piglets per treatment were sacrificed on the day prior to weaning, and 4 piglets per pre-wean * post-wean treatment combination were sacrificed on d 10 and d 38 (n = 24 in total per dissection point).

Before dissection, piglets were sedated using a mixture of Zoletil (250 mg zolazepam and 250 mg tiletamine; VIRBAC, Carros, France) and 20 mL Sedanum (20 mg xylazine/mL; Dechra Pharmaceuticals, Northwich, UK) at 1 mL per 10 kg BW and subsequently killed via intra-cardiac injection with 40% barbiturate pentobarbital (390 mg pentobarbital sodium and 50 mg phenytoin sodium per mL, Euthasol, Virbac, Carros, France). A midline laparotomy was performed to expose internal organs. Different sections of the GIT were identified, and clamps were used to avoid mixing of luminal content. The full weights of stomach, small intestine, cecum and colon were measured, their content was sampled for further analysis by gently squeezing, and subsequently their empty weights were recorded. The length of the small intestine and colon was also measured. Additionally, approximately 0.5 cm2 tissue samples were collected at consistent positions from the middle of the ileum and colon and preserved in RNAlater solution (R0901, Sigma-Aldrich, USA) at 4 °C for subsequent gene expression analysis. Representative and homogenized digesta from stomach, ileum, cecum and mid-colon were collected. A subsample of the cecum and mid-colon content was collected in a cryo tube and stored at −80 °C for analyses of microbiota. The remaining content samples were stored at −20 °C until further analysis.

Diet composition analysis

The crude protein, fat, fibre in the feed were analysed by proximate analysis [17]. The combustion method (method 990.03; LECO FP 528 MI, USA) was used to determine the level of nitrogen with protein calculated as N × 6.25. Fat was extracted from the feed using diethyl ether acid hydrolysis. A feed sample was combusted at a temperature of 550 °C after which the residue was weighed to determine ash content [18]. The analysis of cellulose, hemicellulose and lignin was based on the previous methods described in the literature [19].

Luminal contents’ metabolic profile

The short-chain fatty acids (SCFA) in digesta were analysed using a gas-chromatographic method which is described by Gadeyne et al. [20]. In short, a homogenous 1 g sample was diluted in 5 mL distilled water with internal standard (1 mg of 2-ethyl butanoic acid). After 15 min of centrifugation (22,000 × g at 4 °C), the supernatant was filtered, and an aliquot was transferred into a 1.5-mL glass vial for analysis. The SCFA concentrations were measured by gas chromatography (HP 7890 A, Agilent Technologies, Diegem, Belgium), equipped with a flame ionization detector and a Supelco Nukol capillary column (30 m × 0.25 mm × 0.25 μm, Sigma-Aldrich, Diegem, Belgium).

Ammonia-N (nitrogen as NH4+ and NH3) was analysed using colorimetry using the Berthelot reaction as described by Chaney and Marbach [21].

RNA extraction and quantitative real-time PCR (qRT-PCR)

The gene expressions of SGLT1, B0AT1 and PepT1 in the ileum, and ZO-1, CLDN3, FFAR2 and MCT1 in the colon were determined by qRT-PCR. The messenger RNA (mRNA) of samples were entirely extracted using TRIzol reagent (Sigma-Aldrich, Overijse, Belgium) in accordance with the manufacturer’s protocol. After examination of the concentration and quality of RNA, the complementary DNA (cDNA) was synthesized from 200 ng of total RNA using the PrimeScript™ RT Reagent Kit (RR037 A, Takara; Saint-Germain-en-Laye, France). mRNA expression was performed in the Lightcycler 480 II detection system (Roche) with Fast SYBR Green Master Mix (Takara). All samples were run in triplicate. The genes hydroxymethylbilane synthase (HMBS), succinate dehydrogenase complex subunit A (SDHA), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), topoisomerase II beta (TOP2B), beta-actin (β-actin) and TATA-box binding protein (TBP) were all used as endogenous control. The overview of primers is listed in Additional file 1: Table S1. Subsequently, the 2−ΔΔCt method was used to analyse the relative fold changes.

Microbiota analysis in cecum and mid-colon based on 16S rRNA

The microbial DNA in samples were extracted using QIAamp DNA Fecal Mini Kit (Qiagen, Hilden, Germany). DNA samples with concentrations of 1 ng/μL were sent out to Novogene (Beijing, China), where the 16S rRNA gene V3–V4 hypervariable region was amplified. With 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs), the react conditions were carried out for 0.2 µmol/L of forward and reverse primers, and about 10 ng template DNA. Thermal cycling consisted of initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, and elongation at 72 °C for 30 s and 72 °C for 5 min. The Illumina libraries were pooled and size-selected by preparative gel electrophoresis. Sequencing was performed on the Illumina NovaSeq platform (Beijing, China). Raw sequences from all samples underwent filtering, denoising, merging, and chimera removal to form operational taxonomic units (OTU) by the DADA2 plugin in Qiime2 software.

Statistical analysis

A linear mixed model in SPSS version 29.0 software (IBM SPSS Inc., USA) was used to determine the statistical difference, using litter in the suckling phase, pen during the post-weaning phase, and piglet for post-mortem measurements as the experimental unit. For measurements during the suckling period including post-mortem measurements, sow parity and litter were included as random factors and suckling treatment as fixed factor. Average birth weight after cross-fostering, exact weaning age for performance data during the suckling phase, and body weight at dissection for GIT macroscopy measurements were included as covariates. For measurements during the nursery period, suckling treatment, post-weaning treatment, and their interaction were included as fixed effects and suckling treatment nested in litter was included as random factor. Feed conversion ratio was calculated as ADFI divided by ADG and energy conversion ratio was calculated as (net energy level of the diet × ADFI) divided by ADG. The data of metabolic profiles that was below the detection limit were considered missing values. The Tukey–Kramer correction was applied for multiple comparisons of estimated marginal means. The abundance results at the genus level of microbiota were compared by Kruskal–Wallis H test with Welch’s post-hoc test. A principal coordinates analysis (PCoA) of OTUs based on Unweighted UniFrac distances combined with ANOSIM test. P < 0.05 was considered significant and 0.05 ≤ P < 0.10 was interpreted as a trend.

Results

Performance metrics

Birth weight was 10% lower in the GH group than in the two other pre-weaning groups (P = 0.039; Table 3). Before weaning, no statistical difference was observed in creep feed intake among groups (Table 3). Litters from the GH group consumed 7.3 g/piglet chopped grass hay during the complete lactation period (21 d). Even though ADG (P = 0.389) was not influenced by suckling treatments, the ECR was significantly improved in the PGH group compared to CON (P = 0.035).

Table 3 Growth performance of suckling piglets fed control creep feed (CON), control creep feed and chopped grass hay (GH), or creep feed containing 28% grass pellets (PGH)
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After weaning, five piglets died and subsequently removed (1 in CON-C, 1 in GH-C, 1 in GH-GH and 2 in PGH-C). A tendency for an interactive effect was observed for body weight at d 14, with only PGH-GH showing a tendency of higher weight compared to the other suckling treatments within GH nursery group (P = 0.084; Table 4). The GH nursery group showed significant reduction in growth performance, with lower ADFI (223 vs. 261 g, P = 0.001) and ADG (181 vs. 226 g, P < 0.001) compared to those in the CON nursery group (Table 4). However, the ECR was lower in the GH nursery group (P = 0.001). When the grass pellet inclusion was switched from 28% to 13% at d14 post-weaning, the ADFI and ADG in the GH nursery group increased and exceeded those in the CON nursery group (P = 0.002 and P = 0.056, respectively), with the ECR remaining lower and increasing FCR compared to the CON nursery group (P = 0.006 and P = 0.001; Table 4).

Table 4 Growth performance of piglets fed with a control diet (CON nursery treatment) or a diet containing 28% (phase 1; d 0–14) or 13% (phase 2; d 14–39) grass pellets (GH nursery treatment) during the post-weaning period after being fed a control creep feed (CON), control creep feed and chopped grass hay (GH), or creep feed containing 28% grass pellets (PGH) during the suckling phase
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Gastrointestinal morphometrics

On the day prior to weaning, the inclusion of chopped GH increased the empty small intestine weight to 222 g and length to 851 cm (P = 0.044 and P = 0.012, respectively) compared to CON (214 g and 792 cm, respectively) and PGH (202 g and 758 cm, respectively). Meanwhile, a trend towards a heavier empty colon in GH piglets was observed (P = 0.065, Table 5).

Table 5 Gastrointestinal tract morphometrics of piglets euthanized on the day prior to weaning1
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At d 10 post-weaning, heavier full and empty colon weights were observed in the GH nursery group (P = 0.015 and P = 0.048, respectively). Greater colon length was also found in the GH nursery group, averaging 170.7 cm compared to 155.3 cm in the CON nursery group (P = 0.043; Table 6). At d 38, the empty weights of stomach (P = 0.088), small intestine (P = 0.099), and colon (P = 0.010) were 17%, 8% and 24% greater, respectively, in the GH nursery group compared to the CON nursery group. However, the SI was shorter in the GH suckling group compared to other two suckling treatments on d 38 post-weaning (P = 0.026; Table 6).

Table 6 Gastrointestinal tract morphometrics of piglets euthanized at post-weaning d 10 or d 381
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The mRNA expression of intestinal nutrient transporters and barrier function in ileum and mid-colon

In the ileum at the day before weaning, no statistical differences were observed in expression levels of SGLT1, B0AT1 and PepT1 (Fig. 2A). By d 10 post-weaning, piglets from the PGH suckling group showed higher expression of all evaluated genes compared to piglets from other suckling treatments within each nursery treatment (P < 0.05; Fig. 2B). Additionally, the SGLT1 in the CON nursery group showed higher expression level than in the GH nursery group (P = 0.041). At d 38 post-weaning, no significant differences were based on suckling treatments, but all three transporter genes exhibited higher expression levels in the GH nursery group compared to the CON nursery group (P < 0.05).

Fig. 2
figure 2

The mRNA expression of nutrient transporters, tight-junction proteins and receptors in ileum and mid-colon of piglets fed a control diet (CONnur) or a diet containing 28% (phase 1; d 0–14) or 13% (phase 2; d 14–39) grass pellets (GHnur) during the post-weaning period after being fed a control creep feed (CON), control creep feed and chopped grass hay (GH), or creep feed containing 28% grass pellets (PGH) during the suckling phase. A and B The mRNA expression of SGLT1, BOAT1 and PepT1 in the ileum at weaning and post weaning. C and D The mRNA expression of ZO-1, CLDN3, FFAR2 and MCT1 in the mid-colon at weaning and post weaning. Bars in the graph represent means ± SD (with each bar based on n = 8 at weaning and n = 4 post weaning). SGLT1 = sodium/glucose cotransporter 1; B0AT1 = Sodium-dependent neutral amino acid transporter B(0)AT1; PepT1 = Peptide transporter 1; ZO-1 = Tight junction protein ZO-1; CLDN3 = Claudin 3; FFAR2 = Free fatty acid receptor 2; MCT1 = Monocarboxylate transporter 1. a,bBars with different superscripts within nursery treatments differ significantly based on suckling treatments at P < 0.05; *Indicates a significant difference within a protein based on nursery treatments at P < 0.05

The CLDN3 expression in the colon was higher in piglets from the GH suckling group than the other two suckling treatments and this effect lasted throughout the study (Fig. 2C and D, P < 0.05). Relative to the baseline set as CON, MCT1 in the colon of piglets from both the GH and PGH suckling groups showed a lower expression on the day prior to weaning (P = 0.024; Fig. 2C). Greater expression of FFAR2 was observed in piglets from the PGH suckling group irrespective of nursery treatment at d 10 post-weaning. For nursery treatments, ZO-1 and FFAR2 expression levels at d 10 post-weaning, and FFAR2 and MCT1 at d 38 post-weaning showed greater expression in the CON nursery group, while CLDN3 expression was higher in the GH nursery group at d 38 post-weaning (P < 0.05; Fig. 2D).

Metabolic profiles

At the day prior to weaning, the GH suckling group had higher concentrations of acetate (P = 0.021) and propionate (P = 0.047) in cecum compared to CON, while ammonia concentration (P = 0.009) was lower than CON with PGH being in between and not different. The colonic acetate was higher in CON compared to the other two groups (175 vs. 140 vs. 141 µmol/g DM, P = 0.018; Table 7).

Table 7 Metabolic profiles in gastrointestinal sections of piglets euthanized on the day prior to weaning1
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At d 10 post-weaning, the caecal acetate and propionate of the GH suckling group still exhibited higher concentrations than those from the other two (P < 0.05), but this effect was only observed within the CON nursery group (interaction P = 0.037). An interactive effect was observed, with higher colonic acetate levels in piglets from PGH-GH compared to GH-GH. However, both GH-C and PGH-C showed higher acetate levels than CON-C (suckling P < 0.05; interaction P = 0.062). Nursery treatment effects were evident at d 10 post-weaning, with higher concentrations of caecal propionate, butyrate and ammonia, and colonic concentrations of propionate, butyrate and BCFA in the CON nursery group compared to the GH nursery group (P < 0.05; Table 8). Furthermore, within the CON nursery group, the PGH suckling group had the highest colonic acetate and propionate concentrations (P < 0.05).

Table 8 Metabolic profiles in gastrointestinal sections of piglets euthanized at post-weaning d 10 and d 381
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At d 38 post-weaning, caecal acetate concentration was higher in the GH nursery group than in the CON nursery group (932 vs. 888 µmol/g DM, P < 0.05), while colonic propionate and BCFA concentrations were higher in the CON nursery group (213 vs. 184, 12 vs. 10 µmol/g DM, respectively, P < 0.05; Table 8). Notably, PGH as suckling treatment led to a long-term effect, with lower levels of colonic BCFA compared to the other two suckling treatments (P < 0.05; (Tables 8 and 9).

Table 9 Alpha-diversity of caecal and colonic microbiota at weaning1
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Microbiota composition in cecum and mid-colon

The microbiota community in both cecum and colon showed no difference in α-diversity at the day prior to weaning as assessed by the Shannon, Simpson and Chao1 indices (P > 0.05). Post-weaning, the Chao1 index of the caecal microbiota was higher (P < 0.05) in the GH nursery group compared to the CON nursery group on d 10. The Shannon index was higher (P < 0.05) in the GH nursery group compared to the CON nursery group on d 38 (Table 10). In the colon, the Chao1 was higher (P < 0.05) in the GH nursery group compared to the CON nursery group on d 38. In the β-diversity analysis, significantly distinct microbiota communities among all groups were observed only on d 10 post-weaning, both in cecum (P = 0.003, R = 0.1951) and colon (P = 0.027, R = 0.1653, Fig. 3).

Table 10 Alpha-diversity of caecal and colonic microbiota on d 10 and d 38 post-weaning1
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Fig. 3
figure 3

Beta-diversity of microbiota in cecum and mid-colon. Principal coordinates analysis (PCoA) of OTUs based on Unweighted UniFrac distances with ANOSIM test. A–C PCoA plots of cecal microbiota at weaning, day 10 and day 38 post-weaning. D–F PCoA plots of colonic microbiota at weaning, d 10 and d 38 post-weaning. CON = control group; GH = chopped grass hay group; PGH = pellet grass hay group. CON-C, GH-C and PGH-C represents piglets from CON, GH or PGH groups before weaning and followed by control nursery feed. CON-GH, GH-GH and PGH-GH represents piglets from CON, GH or PGH groups before weaning and followed by grass hay nursery feed

The microbiota structures were further analysed in cecum and mid-colon. Firmicutes (70.5%), Bacteroidetes (24.1%) and Proteobacteria (1.2%) were the three most abundant phyla in the cecum, while Firmicutes (63.3%), Bacteroidetes (30.6%) and Synergistota (1.8%) were predominant in the mid-colon at weaning (Fig. 4A and D). At genus level, Prevotellaceae_NK3B31_group and Megasphaera showed significantly higher abundance in the PGH suckling group compared to the GH suckling group within the top 20 abundant genera of cecum at weaning (P < 0.05, Fig. 4B). Prevotellaceae_NK3B31_group and Ruminococcus in the mid-colon were also more abundant in the PGH suckling group compared to other two groups (P < 0.05; Fig. 4E). LEfSe analysis revealed that the caecal microbiota in the CON suckling group compared to PGH and GH were rich in Oscillospira, Escherichia-Shigella abundance at the genus level and Enterobaceriaceae at the family level. The GH suckling group exhibited higher abundance in genera Lactobacillus reuteri, Limosilactobacillus and Rikenellaceae family in cecum, while the genera Lactobacillus johnsonii and Prevotellaceae_NK3B31_group were prominent in the PGH suckling group (Fig. 4C). In the colon, Limosilactobacillus and Lactobacillus reuteri remained notable in the GH suckling group, and Ruminococcaceae and Prevotellaceae families showed higher abundance in the PGH suckling group compared to the other two groups, as indicated by LEfSe analysis (Fig. 4F).

Fig. 4
figure 4

The microbiota composition and differential analysis in cecum and mid-colon across treatments and development stages. A Relative abundance of cecal microbiota on genus level at weaning. B Clustered heatmap of top 20 abundant genus in cecum based on log2-transformed data and Z-score normalization at weaning. C Histogram generated from linear discriminant analysis (LDA) effect size (LEfSe) analysis results for cecal microbiota at weaning. D Relative abundance of colonic microbiota on genus level at weaning. E Clustered heatmap of top 20 abundant genus in colon based on log2-processed and Z-score normalization. F Histogram generated from LEfSe results for colonic microbiota at weaning. Highlighting significant difference, taxa with difference at an LDA score greater than 2.5 were displayed in LEfSe histogram. G and H Clustered heatmap of top 20 abundant genus in cecum on day 10 and day 38 post-weaning. I and J Clustered heatmap of top 20 abundant genus in colon on d 10 and d 38 post-weaning. CON-C, GH-C and PGH-C represents piglets from CON, GH or PGH groups before weaning and followed by control nursery feed. CON-GH, GH-GH and PGH-GH represents piglets from CON, GH or PGH groups before weaning and followed by grass hay nursery feed. Kruskal–Wallis H test with Welch’s post-hoc test were applied in heatmap. a,bBox with different superscripts differ significantly based on suckling treatments at P < 0.05; *Indicates a significant difference within a genus based on nursery treatments at P < 0.05

The top 20 abundant genera were clustered in a heatmap to illustrate differences post-weaning, and LEfSe analysis results for each group were also listed (Additional file 1: Fig. S1). At d 10 post-weaning and compared to the GH nursery group, [Eubacterium] coprostanoligenes group, Prevotella 7, Escherichia shigella, Megasphaera, Clostridium sensu stricto 1 were more abundant in caecal microbiota of the CON nursery group (P < 0.05; Fig. 4G). By d 38, only Lactobacillus exhibited higher abundance in the CON nursery group (P < 0.05), whereas the Prevotellaceae NK3B31 group was more abundant in the GH nursery group in caecal microbiota (P < 0.05; Fig. 4H). Within the GH nursery group, both GH-GH and PGH-GH suckling treatment showed a higher abundance of Prevotellaceae NK3B31 group than the CON-GH suckling treatment (P < 0.05) both in cecum and colon on d 38 post-weaning (Fig. 4H and J). In the mid-colon, the CON nursery group showed higher abundances of Prevotella 7, Prevotella 9, Faecalibacterium, Clostridium sensu stricto 1 and Phascolarctobacterium on d 10 post-weaning with Clostridium sensu stricto 1 remaining elevated on d 38 (P < 0.05; Fig. 4I and J). In contrast, the GH nursery group showed a higher abundance of Lactobacillus on d 10 post-weaning, and Prevotellaceae NK3B31 group and Prevotella on d 38 post-weaning (P < 0.05; Fig. 4I and J).

Discussion

We here demonstrate that introducing a fibrous diet item such as grass hay in the suckling phase, either separately or mixed in the creep feed, can influence the development of the GIT. It also can modify the fermentation and microbiota profile, with persisting effects into later life. Fibrous diets have been long recognized as beneficial for growing pigs and sows [22,23,24]. However, the variability in feed intake and unpredictable challenges associated with neonatal piglets have led to hesitation in exploring the fibre application for this age group.

Unlike our previous study, where the feed intake of chopped grass hay reached 54 g per piglet at weaning, the intake in the present study was limited to approximately 7 g per piglet (assuming equal intake among piglets in a litter) [15]. The shorter suckling duration (23 vs. 28 d) and the potential breed variation (Topigs Norsvin TN70 × Belgian Pietrain vs. Hypor Libra × Hypor Maxter) likely contributed to the difference in grass hay intake, as seen in other studies [25, 26]. In the previous study, a considerable increase in solid feed consumption was observed during the last week of lactation. Moreover, another study reported that piglets consumed only 5 g/piglet solid feed until d 20 of age, but this amount increased to 63 g/piglet in the following week [26]. This suggests that extending the suckling phase by one more week has a substantial impact on piglets’ overall appetite, highlighting the crucial role of suckling duration on grass hay consumption and its effects on GIT development. Particularly in light of the EU animal welfare legislation, the requirement for a longer suckling duration in practice may enhance the functional benefits of grass hay [27].

In the current study, despite the low grass hay intake, it is remarkable that substantial effects on intestinal development and microbial activity were found. The creep feed intake showed high variability between litters, with an approximate 70% coefficients of variation, as is commonly observed [6, 28, 29]. Growth performance during the suckling period is mainly determined by sow milk intake rather than creep feed intake, as shown in the current study [30]. Despite lower net energy consumption in the PGH fed piglets, this feed did not affect growth until weaning. Moreover, it may be due to the overestimation in the energy requirement of creep feed may in the modern farming practicing. In terms of dietary energy dilution, it seemed that the 28% inclusion rate of grass pellets (25.9% IDF) was more than the piglets could compensate for shortfall in energy intake. Interestingly their growth recovered and even surpassed the CON nursery groups when the grass pellet inclusion was reduced to 13% after d 14 post-weaning. Meanwhile, the optimized ECR was observed in PGH group throughout the study. In analogy, diets supplemented with 5% or 6% wheat bran enhanced growth performance in weaned piglets in contrast with a lack of effect of a higher inclusion rate of 14.9% in other studies [31,32,33]. Meanwhile, similar inclusion ratios of insoluble fibre, yet from different sources, yielded varying growth outcomes in weaned piglets [31]. Therefore, the optimal proportion of grass pellets in the nursery feed could be further optimized, considering the catch-up growth after an initial post-weaning growth retardation due to a high-fibre diet as shown in the present study. Considering the common challenge of highly variable creep feed intake, which was still observed in the current study, a further larger-scale experiment covering more piglet breeds across different regions may strengthen the findings.

In contrast to the grass pellet incorporated into the feed, the provision of chopped grass hay in a separate feeder increased the size of the GIT at weaning. This finding suggests that the physical form and the method of provision of chopped grass hay promoted this change. The large, coarse matter of insoluble fibre were reported to reduce digesta segregation (from mouth to ileocecal junction) in growing pigs [34], likely increasing bulk and retention time within specific sections of the GIT. A similar observation in finishing pigs, where those fed with coarse straw exhibited a longer median retention time throughout the intestinal tract compared to those fed fine straw [35]. This bulk, in turn, may enhance nutrient absorptive processes by activating mechanoreceptors (stretch activated neural cells), which promote digestive tract development [36]. This process also aligns with the observed long-term increase in tight junction protein expression in the colon as observed in this study. The lower post-weaning colon weight due to pre-weaning grass hay provision (independently of the nursery diet) may be the result of the sudden removal of grass hay as a physical stimulus, which is supported by the heavier colon when nursery piglets still received insoluble fibre through the pellet. Although both the ADFI and ADG were reduced in the GH nursery group compared to the CON nursery group, the FCR shows that the reduction in ADG is not fully explained by the reduction in ADFI. For instance, intestinal development requires a substantial amount of energy expenditure [37], which may explain the association between hay-induced intestine development and (temporary) overall growth reduction. This effect on colon development did not seem to be a measure of fermentative capacity, as colon weight effects often coincided with the inverse effect on SCFA production. Still, the LEfSe analysis revealed that taxa that were abundant in colon of piglets from PGH at weaning, such as Ruminococcus, Prevotella, Prevotella 9 and Prevotella sp. RS2, became more prevalent in the colon of piglets from GH-GH on d 10 post-weaning. The management of GH-GH piglets, which initially had access to two feeders with free-choice feeding before weaning, transitioning to a single unfamiliar diet after weaning may also contributed to this intestinal growth stasis. Moreover, the abrupt shift of microbiota mentioned above in GH-GH may have intensified the weaning challenge [38, 39]. However, a better adaptation (similar abundant microbes) was seen in piglets that remained on feed that included grass pellets from pre- to post-weaning. The observed trend of an interactive effect in body weight of PGH-GH group may further suggest this adaptation mechanism besides its effect on luminal microbiota. Eventually, a long-term exposure to nursery feed containing the grass pellets, i.e., high insoluble fibre, resulted in an enlargement of the stomach and small and large intestines in piglets by the end of study. This is consistent with findings from other studies involving high- (insoluble) fibre diets [40,41,42,43].

The elevated expression of nutrient transporters and intestinal barrier proteins due to grass hay reflected the increased exposure to substrates as well as nutrient absorption efficiency within the intestinal lumen. Piglets that received the PGH diet during suckling had upregulated nutrient transporters, including SGLT1, B0AT1 and PepT1 in ileum, up to d 10 post-weaning, and piglets that received the GH nursery diet showed the same activation of these genes compared to the CON nursery diet by d 38 post-weaning. The results of transporters in ileum, together with the observed lower ammonia concentration in ileum at d 10 post-weaning, suggest that proximal intestinal segments of piglets fed a diet containing grass pellets at a young age became more capable of digesting dietary protein or producing monosaccharides [44,45,46,47]. However, additional data of on ileal microbiota and digestive enzyme activity may strengthen this conclusion. In the colon, the lower non-fibre carbohydrate level in GH nursery diets accompanied by lower concentrations of substrate availability, unsurprisingly resulted in lower expression levels of FFAR2 and MCT1 on d 38 post-weaning, consistent with previous studies linking lower expression levels to substrate availability [48,49,50]. Conversely, the higher SCFA levels in the colon of piglets from the PGH suckling group compared to the other two suckling treatments across both nursery treatments likely contributed to stimulation of the expression of MCT1.

Similar to findings from studies on (insoluble) fibre, fermentation primarily occurred in the cecum and proximal colon in pigs, as observed through the slaughter method [51,52,53]. A higher SCFA production was noted in the cecum of piglets supplemented with chopped GH on the day prior to weaning, whereas no such increase was shown in piglets fed a diet containing grass pellets during suckling. According to the data of caecal and colonic microbiota, this outcome was likely not due to a lack of carbohydrate fermentation capacity but rather the limited availability of fermentable substrates in PGH diets, including starch. Compared to the other two suckling treatments, piglets in the PGH group displayed a higher abundance of Prevotellaceae NK3b31 group, Prevotella 7, Lachnospiraceae, and Lachnoclostridium in the cecum before weaning. These abundant genera play crucial roles in degrading various types of dietary fibre in the diet [54,55,56,57], indicating that the inclusion of grass pellets in a diet induced more fibre fermentation in the cecum than separate provision of chopped grass hay. Because the voluntary intake of the separate grass hay was lower than anticipated, the fibre intake was higher through the inclusion of the grass pellet in the PGH group, which is a plausible explanation for the stronger effect on fermentation. Long-term feeding of grass hay pellet showed that the carbohydrate sources from the diet, exposed to the well-established taxa mentioned above in caecum, may be priorly fermented in caecum and absorbed in the longer colon. The results of SCFA distribution along GIT of piglets in GH nursery diet also match this hypothesis, along with an optimized ECR across the GH nursery group. Additionally, the higher SCFA levels in the proximal sections with lower pH environment can also downregulate bacterial urease activity, as reported in other studies [58,59,60]. Consequently, the resulting decrease in ammonia levels of digesta reduced the burden on the distal sections, as elevated ammonia concentrations can irreversibly deteriorate the intestinal barrier function [61].

It is, however, worth mentioning that the insoluble fibre content of grass hay is likely very unfermentable in young piglets especially when provided in coarse form. Therefore, its stimulating effect on fermentation is more likely due to passage rate kinetics rather than as substrate on its own. A similar observation was done in a piglet study comparing soluble with insoluble fibre where also the insoluble fibre had the largest effect on promoting fermentation [13]. The dominant caecal genera in piglets from the GH suckling group, such as Limosilactobacillus and Lactobacillus_reuteri, which are primarily utilizing glucose, lactose and maltose, coupled with higher SCFA levels, suggest that chopped grass hay effectively promoted fermentation of carbohydrate from the control creep feed or sow milk [62,63,64]. Additionally, the relatively lower caecal abundance of pathogens such as Escherichia coli and Shigella in the GH and PGH suckling groups compared to CON at weaning may also be attributed to the presence of SCFA substrates and acid-producing microbes [65,66,67]. Subsequently, a greater proportion of undigested sugars or starch from CON diets likely entered the colon, where they were fermented into higher concentrations of SCFA, facilitated by microbes such as Streptococcus and Hydrogenoanaerobacterium which were abundantly present [68, 69]. In contrast, in the PGH suckling group, the residual grass pellet content may have continued to support the growth of complex carbohydrate- or fibre-degrading genera in the colon, including Prevotellaceae NK3b31 group, Ruminococcus, and Prevotella 9. The similarity of abundant luminal microbes between the PGH suckling group and the GH nursery group might have facilitated the adaption to the diet transition around weaning, ultimately minimizing the negative impact on growth performance despite the 28% inclusion of grass pellets in the post-weaning diet and even showing improved performance switching to 13% inclusion of grass pellets.

Interestingly, after weaning, genera enriched in the caecum or colon of piglets fed the PGH diets during suckling, such as Prevotella 7 and Prevotella 9, decreased when piglets were transitioned to GH nursery diets compared to CON nursery diets. This pattern suggests that pre-weaning abundance of these two genera in the PGH group may primarily be attributed to the components of sow milk, such as porcine milk oligosaccharides. Concurrently, the higher proportions of digestible carbohydrates in the CON nursery diets likely contributed to the abundance of Clostridium sensu stricto 1 which has been reported as butyrate-forming pioneer in the early age of infants [70]. By the end of the study, with longer term of feeding GH nursery feed, the SCFA distribution in the proximal and distal segments exhibited a pattern similar with that observed in the GH and PGH suckling groups. Genera such as Rikenellaceae RC9 gut group, Prevotellaceae NK3B31 group and Treponema in the cecum, involved in fibre fermentation, may contribute to this pattern through prior exposure to other carbohydrates in the cecum during the digesta transit [54, 71, 72].

After all, a longer tracking on growth performance, such as following piglets until the slaughter phase, would provide a more comprehensive assessment of the results of larger GIT, enhanced intestinal barrier function, and altered SCFA distribution along with microbiota community shifts in large intestine induced by grass content.

Conclusion

Providing chopped grass hay in a separate feeder next to a control creep feed to suckling piglets stimulated GIT development, hindgut fibre-degrading microbiota and shifted fermentation to more proximal parts of the intestinal tract, despite low intakes level during 23-day lactation. The GIT growth promotion was not seen when including 28% grass pellets in the creep feed, but that diet performed best in terms of ECR during the suckling and nursery phases.

After weaning, a 28% grass pellet inclusion in the feed reduced feed intake and weight gain for the first 2 weeks, yet piglets were shown to compensate and even increase weight gain which was mediated by an increase in feed intake once the grass pellet inclusion rate was reduced to 13%. Our study demonstrated that it is worth stepping away from the common approach of low-fibre and finely ground diets for young piglets. A consistent provision of a high-fibre diet for piglets is recommended if introduced during the suckling phase, given the beneficial effects on ECR and GIT health. Additionally, the inclusion rate of grass pellets right after weaning should be reconsidered and kept below 28%.

Data Availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

  • ADF:: Acid detergent fibre
  • ADL:: Acid detergent lignin
  • ADG:: Average daily gain
  • ADFI:: Average daily feed intake
  • BW:: Body weight
  • DE:: Digestible energy
  • DM:: Dry matter
  • ECR:: Energy conversion ratio
  • FCR:: Feed conversion ratio
  • GIT:: Gastrointestinal tract
  • IDF:: Insoluble dietary fibre
  • NDF:: Neutral detergent fibre
  • NE:: Net energy
  • OTU:: Operational taxonomic unit
  • PCoA:: Principal coordinates analysis
  • SCFA:: Short-chain fatty acid
  • SID:: Standard ileal digestibility

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Acknowledgements

We sincerely acknowledge the staff of the Swine Research Centre of Trouw Nutrition for their support and expertise throughout the course of this study.

Funding

Not applicable.

Ethics Declaration

Ethics approval and consent to participate

The housing, rearing and any other procedures on animals were in compliance with the European Union Directive 2010/63/EU, and assessed by the Dutch Central Committee on Animal Experimentation (CCD), under application number AVD20400202316684.

Consent for publication

All authors have read and agreed to the published version of manuscript.

Competing interests

The authors declare that they have no competing interests.

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