Introduction

Intrauterine growth retardation (IUGR) impairs growth and development of the mammalian embryo/foetus or its organs during gestation [1]. IUGR in neonates is characterized by delayed postnatal growth, permanent mal-development and increased susceptibility to infection, resulting in high morbidity and mortality during the early life period [2]. Several studies have demonstrated that IUGR is associated with impaired intestinal development and poses a high risk of intestinal diseases in neonates [3–5]. Also, our previous studies found that both excessive and restricted nutrient intake impaired intestinal development and immune function in neonates with IUGR [6, 7]. The intestine is important for digestion and absorption of nutrients, and the gut-associated lymphoid tissue is the largest immune organ in the body [8]. Thus, new strategies to promote growth and intestinal function in neonates with IUGR are urgently needed.

Establishment of intestinal microbiota after birth plays an important role in the development of the gastrointestinal and immune systems [9]. Previous studies have reported that gut colonization by bacteria and the fermentation activity of the resulting intestinal microbiota are altered in neonates with IUGR, as compared with normal neonates, because of the effect of IUGR on the small intestine [10, 11]. Many recent studies have highlighted the beneficial role of probiotics in intestinal motor function. In humans, probiotics supplementation was found to reduce both the incidence and severity of necrotizing enterocolitis in newborns with IUGR [12]. In animals, moreover, probiotics seem to be a good alternative to the use of antibiotics to promote growth [13]. As we know, probiotics as microbial supplementation convey beneficial effects when administered in adequate quantities [14]. However, little is known about the effects of probiotics supplementation on growth, intestinal development and immune function in neonates with IUGR.

Bacillus subtilis is a facultative anaerobe that plays the vital roles in intestinal microecological balance by consumption of intestinal oxygen, which creates an anaerobic environment [15, 16]. Moreover, B. subtilis is preferred due to the high resistance of its spores to harsh environments and capacity for long-term storage at ambient temperatures [17]. Previous studies have reported that some Bacillus species improved porcine intestinal health by regulation of immune function to protect against pathogenic challenge [18, 19]. Moreover, some species can be used as potent producers of extracellular degrading enzymes to promote nutrient digestion and utilization [16, 18]. Pigs are multifoetal animals and exhibit serious IUGR occurrence, which has been recognized as an ideal model for the study of clinical nutrition [20]. In the present study, therefore, we investigated the effects of IUGR and B. subtilis PB6 supplementation on the growth performance, intestinal development and immune function of piglets during the suckling period.

Materials and methods

The animal experiment followed the actual law of animal protection and was approved by the Animal Care and Use Committee of the Sichuan Agricultural University and was performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Animal and treatment

Piglets with a birth weight near the mean litter birth weight (SD 0.5) were identified as normal birth weight (NBW), whereas those with at least 1.5 SD lower birth weight were defined as IUGR according to our previous study [6, 21]. A total of 14 pairs newborn boars (Pig Improvement Company 327 × 1050) of NBW with body weight (BW) at 1.49 (SD 0.06) kg and IUGR with BW at 0.92 (SD 0.03) kg were selected from 14 healthy sows (10 piglets/litter). All piglets were weaned at 7 days of age and moved to be individually fed with formula milk by bottle feeding every 3 h between 06:00 and 24:00 hours in nursing cages (0.8 m × 0.7 m × 0.4 m). In each litter, one of the IUGR and NBW piglets received the formula milk (FORM) or formula milk supplemented with B. subtilis PB6 (FORM-BsPB6), respectively. In total, four groups (BW-FORM) of piglets were created and studied: NBW with FORM; IUGR with FORM; NBW with FORM-BsPB6; IUGR with FORM-BsPB6 (n = 7 per group). The FORM was formulated according to previous study [6]. FORM-BsPB6 was prepared by supplementing the spores of B. subtilis PB6 (Kemin Industries, Inc., Des Moines, IA) at 60 g per 100 kg FORM powder, containing 2 × 109 cfu/kg. The liquid formula milk was prepared by mixing 1 kg of formula powder (DM 87.5 %) with 4 L of water, in which nutrients composition and levels were similar as sow milk [7]. All piglets had free access to drinking water. Room temperature was maintained at approximately 30 °C, and the humidity was controlled between 50 and 60 %. The BW and milk intake of piglets were recorded daily. The average daily DM intake (ADMI) was calculated via multiplying the average daily intake of milk by its DM content (%), while milk intake was calculated as the difference between the offered amounts and the refusals.

Blood sampling and analyses

Blood samples were collected by venepuncture in the morning (08:00) of day 21 after an overnight fast and were injected into two vacuum tubes containing sodium heparin. The vacuum tubes were immediately placed on ice until the examination of leucocytes and flow cytometry analysis, respectively (within 2 h). The differential leucocyte count was obtained using an ADVIA 2120 Hematology System (Bayer HealthCare, Tarrytown, NY). Total peripheral blood lymphocytes were separated from heparinized peripheral blood by separation medium and then were stained with mouse anti-porcine CD3e-SPRD (PE-Cy5) (catalogue no. 4510-13), CD4a-FITC (catalogue no. 4515-02) and CD8a-PE (catalogue no. 4520-09), which were purchased from Southern Biotechnology Associates (Birmingham, AL). PBS (1×, Gibco, Carlsbad, CA) and 1.0 % BSA (ICN Biomedicals, Aurora, OH) were used as diluent and washing buffer. Flow cytometry analysis was performed on a FACSCalibur flow cytometer (Becton–Dickinson, San Jose, CA) and repeated for the same sample.

Tissue sample collection

After blood sampling, all piglets were anaesthetized with an intravenous injection of pentobarbital sodium (50 mg/kg BW) and slaughtered. Piglets were weighed, and crown-rump length (CRL) was taken (the supine length of the piglet from the crown of its head to the base of its tail). Body mass index (BMI; BW/CRL2) was calculated for each piglet. The liver, spleen, kidney, heart and pancreas of each piglet were weighed immediately after slaughter. The length and weight of small intestine were measured after the removal of luminal contents. Duodenal, jejunal and ileal samples of approximately 2 cm in length were stored in 4 % paraformaldehyde solution for histological analyses. The other pieces of the jejunum and ileum (approximately 2 cm) were snap-frozen and then stored in fridge with −80 °C until further analysis. Finally, colonic digesta were collected immediately and frozen at −80 °C.

Small-intestinal morphology and goblet cell countings

The duodenal, jejunal and ileal samples were preserved in 4 % paraformaldehyde solution and then embedded in paraffin. Each of the samples (duodenum, jejunum and ileum) was used to prepare five slides, and each slide had three sections (5 µm thickness), which were stained with eosin and haematoxylin for intestinal morphology measurement by 20 well-oriented villi and crypts each section (Optimus software version 6.5; Media Cybergenetics), and villi–crypt ratio (VCR) was calculated. The goblets cells number per villi was measured (NIS-Elements BR 2.3; Nikon France SAS), and the values obtained from 10 villi by each small-intestinal segment were averaged.

Measurement of plasma immunoglobulin subset and cytokines

Commercially available enzyme immunoassays were performed according to the instructions from the manufacturer for the following markers: IgA (Bethyl Lab. Inc., Montgomery, USA), IL-1β (R&D Systems, Oxford, UK), TNF-α (R&D Systems, Oxford, UK), IL-10 (Bio Source/Med Probe, Camarillo, CA). Absorbance (450 nm) was determined using a Bio-Tek synergy HT microplate reader (Bio-Tek Instruments, VT). The detection limits were 12.5 ng/mL for IgA, 7.0 pg/mL for TNF-α, 30.0 pg/mL for IL-1β and 8.0 pg/mL for IL-10, respectively; the inter- and intra-assay coefficients of variation were less than 10 %.

Digestive enzyme activities

After thawing, the frozen jejunal tissue was weighed and homogenized (5 min) in the 9 times volume of 50 mM Tris–HCl buffer, pH 7.0, centrifuged (3000×g, 10 min). The supernatant was collected and stored at −20 °C for enzyme assay. Total proteins were extracted, and their concentration was determined according to the procedure of bicinchoninic acid (Solarbio, Inc.), with bovine serum albumin as standard. Disaccharidases (including maltase, sucrase and lactase) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer’s instructions. The absorbance was determined with spectrophotometer (Beckman Coulter DU-800; Beckman Coulter, Inc.). The activities of disaccharidases were expressed as U/mg protein. One unit (U) was defined as 1 nmol maltase, sucrase and lactase as substrate for the enzymatic reaction.

Microbial population determination

Bacterial DNA was extracted from colonic digesta using the Stool DNA Kit (Omega Bio-Tek) according to the manufacturer’s instructions. Primers and probes (Table 1) were designed with Primer Premier 5.0 (Premier Biosoft International, Palo Alto, CA) and followed 16S rRNA sequences of maximum species of each genus homology downloaded from GenBank database, European Molecular Biology Laboratory and DNA Data Bank of Japan to obtain specific amplification, and the sequences of all the genera taken from the database were submitted to DNAStar (MegAlign) program (DNASTAR, Inc., Madison, WI), as described by Zhang et al. [22]. Next, these sequences were submitted to alignment, in which the maximum number of species belonging to one genus was gathered and the regions showing conservations were picked up as genus-specific primers and probes. All the primers and probes used in this experiment were commercially synthesized by Invitrogen (Shanghai, China).

Table 1 Oligonucleotide primers and probes used for bacteriological analysis
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Quantitative real-time PCR was conducted with CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA) with optical-grade 96-well plates. For the quantification of total bacteria, the reaction mixture (25 μL) contained 1 μL forward and 1 μL reverse primers (100 nM), 12.5 μL SYBR Premix EX Taq (Takara, Dalian, China), 1 μL template DNA and 9.5 μL nuclease-free water. The thermal cycling conditions were an initial predenaturation step at 95 °C for 10 s, 40 cycles of denaturation at 95 °C for 5 s, annealing at 64.5 °C for 25 s and extension at 72 °C for 60 s. For the quantification of Lactobacillus, Escherichia coli, Bifidobacterium and Bacillus, real-time PCR was conducted in a reaction volume of 20 μL with 1 μL probe enhancer solution, 0.3 μL probe (100 nM), 1 μL forward and 1 μL reverse primers (100 nM), 8 μL RealMasterMix (Tiangen, Beijing, China), 1 μL template DNA and 7.7 μL nuclease-free water. The PCR conditions involved 10 s at 95 °C and 50 cycles for 5 s at 95 °C, 25 s at annealing temperature (Table 1) and 60 s at 72 °C. The threshold cycle (CT) values and baseline settings were determined by automatic analysis settings, and the copy numbers of the target group for each reaction were calculated from the standard curves.

For the quantification of bacteria in the test samples, specific standard curves were generated by constructing standard plasmids, as presented by Han et al. [15]. Deoxyribonucleic acid concentrations of standard plasmids were detected using a spectrophotometer (Beckman Coulter DU 800; Beckman Coulter, Fullerton, CA). A series of tenfold dilution (1 × 109 to 1 × 101 copies/μL) of plasmid DNA were used to construct their respective standard curves. Each standard curve was generated by a linear regression of the plotted points with the logarithm of template copy numbers as the abscissa and the CT values as the ordinate. The gene copy numbers were calculated by the following formula: (6.0233 × 1023 copies/mol × DNA concentration (μg/μL))/(660 × 106 × DNA size (bp)).

Total RNA extraction and real-time RT-PCR

Total RNA was extracted from frozen ileal samples using TRIzol reagent (catalogue no. 15596-026; Invitrogen) according to the manufacturer’s instructions, and the quality and purity of RNA samples were assessed by electrophoresis on 1.0 % agarose gels (Egel; Invitrogen, Carlsbad, CA, USA) and nucleic acid analyser (A260/A280, Beckman DU-800; Beckman Coulter, Inc.), respectively. Subsequently, the RNA was performed at 37 °C for 15 min, followed by RT inactivation at 85 °C for 5 s using PrimeScript™ RT reagent kit (catalogue no. RR047A, Takara). A portion of the RT products (1μL) was used directly for real-time PCR. Real-time PCR assays were performed on complementary DNA samples in 384-well optical plates on a 7900HT ABI Prism Sequence Detection System (Applied Biosystems, Foster City, CA, USA) using the SYBR green system (catalogue no. RR820A, Takara). Primers for individual genes were designed using Primer Express 3.0 (Applied Biosystems) and are given in Table 2. The reaction mixture (10 μL) contained 5 μL of freshly SYBR® Premix Ex Taq II (Tli RNaseH Plus) and 0.2 μL ROX Reference Dye II (50×), 0.8 μL of the primers, 1 μL of RT products and 3 μL diethylpyrocarbonate-treated water. The PCR protocol was used as following: 1 cycle (95 °C 30 s), 40 cycles (95 °C 5 s, 60 °C 31 s) and 1 cycle (95 °C 15 s, 60 °C 1 min and 95 °C 15 s). The standard curve of each gene was run in duplicate and three times for obtaining reliable amplification efficiency values as described previously [23]. The correlation coefficients (r) of all the standard curves were more than 0.99, and the amplification efficiency values were between 90 and 110 %. At the end of amplification, dissociation analyses of the PCR product were performed to confirm the specificity of PCR products. The relative mRNA abundance of analysed genes was calculated using the method of 2−ΔΔCt, as described previously [24]. The most stable housekeeping genes (β-actin and GADPH) were chosen for normalization. Finally, the mRNA level of each target gene for IUGR-FORM group was set to 1.0.

Table 2 Primer sequences of the target and reference genes
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Western blotting

Protein extracts were obtained by homogenizing ileal tissues with a total protein extraction kit (Beyotime Biotechnology, Jiangsu, China), according to the manufacturer’s guide. The protein content was measured using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). The antibody was used in our experiment: goat polyclonal anti-ZO-1 (sc-8146, Santa Cruz Biotechnology, Santa Cruz, CA, USA), goat polyclonal anti-claudin-1 (sc-17658, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-β-actin (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Western blot analysis was performed as previously described [25]. Chemiluminescence detection was performed using the ECL Plus™ Western Blotting Detection System (Amersham, Arlington Heights, IL, USA), according to the manufacturer’s instructions. The relative expression of target protein was normalized using β-actin as the internal protein, and then, the normalized values were used for the comparison of the expression of target protein in groups.

Statistical analysis

The data were analysed by Duncan’s multiple comparisons for the 2 × 2 factorial experimental design using the general linear model (GLM) procedure of SPSS statistical software (Ver. 20.0 for Windows, SPSS, Chicago, IL, USA) in the following model: y ijk  = μ + a i  + b j  + (ab) ij  + e ijk (i = 1, 2, j = 1, 2, k = 1, 2, …, n ij ), where y ijk represents the dependent variable, μ is the mean, a i is the effect of BW (IUGR, NBW), b j is the effect of diet (FORM, FORM-BsPB6), (ab) ij is the interaction between BW and diet, and e ijk is the error term. Results are presented as means with their standard errors (SEM). Differences were considered as significant when P < 0.05, and a tendency was recognized when P < 0.10.

Results

Growth performance

Regardless of diet, both ADG (−28 %, P < 0.001) and ADMI (−31 %, P < 0.001) were significantly decreased in IUGR piglets compared with NBW piglets, while the FCR had no significant difference, and accordingly, the final BW and net BW gain of IUGR piglets were lower (−28 to 31 %, P < 0.001) than those of NBW piglets (Table 3). Regardless of BW, FORM-BsPB6 had no significant effects on the ADG, ADMI, final BW and net BW gain, but the FCR was markedly decreased (−10 %, P = 0.043) in piglets fed FORM-BsPB6. During the whole experimental period, furthermore, IUGR piglets fed FORM-BsPB6 had significantly lower FCR compared with piglets fed FORM (−17 %, P < 0.050).

Table 3 Effects of B. subtilis PB6 supplementation on the growth performance of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Organ indices

As shown in Table 4, regardless of diet, IUGR significantly decreased (−11 to 35 %, P < 0.010) the weights of internal organs such as heart, liver, spleen, kidney, pancreas, intestine and the BMI on day 28; moreover, the intestinal length and 28 CRL of IUGR piglets were shorter (−13 to 18 %, P < 0.010) than those of NBW piglets. However, the relative intestinal length to intestinal weight and the relative intestinal length to BW in IUGR piglets were higher (+18 to 22 %, P < 0.050) than NBW piglets. FORM-BsPB6 had no significant influence on the organ indices of piglets.

Table 4 Effects of B. subtilis PB6 supplementation on the organ indices of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Plasma immunoglobulin and cytokines

As shown in Table 5, irrespective of diet, IUGR markedly decreased the plasma concentrations of IgA (P < 0.001) and IL-1β (P = 0.006) and the ratio of IL-1β to IL-10 (P < 0.001). However, FORM-BsPB6 had no significant influence on plasma immunoglobulin subset and cytokines. Furthermore, IL-1β concentration in IUGR piglets fed FORM-BsPB6 had no significant difference with NBW piglets.

Table 5 Effects of B. subtilis PB6 supplementation on the concentrations of IgA, TNF-α, IL-1β and IL-10 of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Composition of peripheral leucocytes and lymphocyte percentages

IUGR significantly decreased the count (P = 0.021) and percentage (P = 0.025) of lymphocytes but markedly increased the percentage of neutrophils (P = 0.023) and CD8+ (P = 0.009); moreover, the counts of leucocyte (P = 0.057) and monocytes (P = 0.063) and the percentage of monocytes (P = 0.097) had a tendency to decrease in IUGR piglets (Tables 6, 7). However, FORM-BsPB6 had no significant influence on composition of peripheral leucocytes and lymphocyte percentages in piglets. Furthermore, the counts of monocytes, the percentages of neutrophils, lymphocytes and monocytes in IUGR piglets fed FORM-BsPB6 had no significant difference with NBW piglets.

Table 6 Effects of B. subtilis PB6 supplementation on the count and percentage of blood leucocytes, neutrophils, lymphocytes and monocytes of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Table 7 Effects of B. subtilis PB6 supplementation on percentage of CD3+, CD4+, CD8+ T lymphocytes and the ratio of CD4+/CD8+ in intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Intestinal morphology and goblet cell density

As shown in Table 8, regardless of diet, IUGR significantly decreased (P = 0.039) villous height in the duodenum of piglets while increased goblet cell number per villous in the duodenum (P < 0.001) and jejunum (P = 0.019). Regardless of BW, FORM-BsPB6 significantly increased villous height (P = 0.023) and the VCR (P < 0.001) while decreased the crypt depth (P = 0.012) in the ileum.

Table 8 Effects of B. subtilis PB6 supplementation on the intestinal morphology of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Digestive enzyme activities

In the jejunum, IUGR significantly decreased the activity of maltase (P = 0.011) while increased the activity of lactase (P = 0.030). Regardless of BW, piglets fed FORM-BsPB6 had a tendency to increase the activities of maltase (P = 0.082) and sucrase (P = 0.095) compared to piglet fed FORM. Furthermore, the activity of maltase in IUGR piglets fed FORM-BsPB6 had no significant difference with NBW piglets (Table 9).

Table 9 Effects of B. subtilis PB6 supplementation on enzyme activities in the jejunum of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Gene expression in the ileum

As shown in Table 10, IUGR markedly decreased the mRNA abundance of TLR-9 (P = 0.020) and TOLLIP (P = 0.001); moreover, the mRNA abundance of MyD88 (P = 0.098) and TLR2 (P = 0.065) had a tendency to decrease, while the mRNA abundance of SIGIRR (P = 0.081) had a tendency to increase in the ileum of piglets with IUGR. FORM-BsPB6 markedly increased the mRNA abundance of ZO-1 (P = 0.001) in the ileum of piglets. In addition, the significant interaction between BW and diet was observed for the mRNA abundance of TOLLIP (P = 0.046) in the ileum.

Table 10 Effects of B. subtilis PB6 supplementation on the mRNA abundance of innate immune-related genes in the ileum of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Gut microbial population

Regardless of diet, there were no significant differences in microbial populations in colonic digesta between IUGR and NBW piglets (Table 11). FORM-BsPB6 significantly increased the copy number of Bacillus (P = 0.010) in colonic digesta of piglets.

Table 11 Effects of B. subtilis PB6 supplementation on the microbial populations (log10 copies/g of wet digesta) in colonic digesta of intrauterine growth retarded (IUGR) and normal birth weight (NBW) neonates
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Protein abundances of ZO-1 and claudin-1

Regardless of diet, IUGR had no significant influences on the protein abundances of ZO-1 and claudin-1 in the ileum of piglets. However, FORM-BsPB6 markedly increased the protein abundances of ZO-1 (Fig. 1a) and claudin-1 (Fig. 1b) in the ileum of piglets (P < 0.050).

Fig. 1
figure 1

Relative protein expression levels of ZO-1 (a) and claudin-1 (b) in the ileum of IUGR and NBW piglets fed FORM or FORM-BsPB6. Values are means, with standard errors represented by vertical bars, n = 7. The value of protein expression = densitometry units of selected protein/densitometry units of β-actin detected by Western blotting. IUGR intrauterine growth retardation, NBW normal birth weight, FORM formula milk, FORM-BsPB6 formula milk supplemented with B. subtilis PB6, ZO-1 zonula occludens 1. a,bMean values with unlike letters were significantly different (P < 0.05). *P < 0.05 for the respective sources of variation (the diet)

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Discussion

IUGR is associated with increased neonatal mortality and morbidity in both animals and humans [26, 27]. Numerous studies using porcine models have shown that IUGR delayed postnatal growth and gut development [4, 7, 11]. Meanwhile, increased bacterial adhesion in the intestine due to IUGR is a predisposing factor to intestinal pathologies [10, 11]. B. subtilis, as a well-tolerated facultative anaerobe, may facilitate growth performance and intestinal homoeostasis in mammals [28]. In this study, we investigated the effects of B. subtilis PB6 supplementation in formula milk on growth performance, intestinal development and immune function of IUGR piglets during the early postnatal period.

First, the ADMI and ADG were reduced in IUGR piglets, as compared to NBW piglets. As a result, the net weight gain and final BW were much lower in IUGR than in NBW piglets. These findings indicate that IUGR negatively affects birth weight and postnatal growth of neonates, consistent with the results of previous studies [29–31]. Dietary supplementation with B. subtilis PB6 had no significant influences on the ADMI and ADG, but significantly improved feed efficiency, as indicated by the lower FCR value. Similarly, previous studies indicated that B. subtilis improves nutrient digestibility and utilization in both weaning and finishing pigs via modulation of microbial populations [13, 32, 33]. The results of the current study also showed that dietary supplementation of B. subtilis PB6 markedly increased Bacillus populations in colonic digesta, indicating the crucial role of B. subtilis PB6 on microbial composition.

It was reported that maternal nutrition can be selectively allocated for the growth of internal organs [34]. In this study, the relative weights of internal organs of IUGR piglets were similar to those of NBW piglets, although the absolute weights of the internal organs were lower. In accordance with the findings of a previous study [35], CRL and BMI were lower in piglets with IUGR than NBW piglets. BMI could be used as an indicator of survival, with a higher BMI value associated with greater survival [36]. In this study, the lower BMI in IUGR piglets may partially explain the greater mortality in IUGR neonates [37].

Generally, intestinal morphology is an important factor reflecting intestinal development [38]. Previous findings showed that IUGR impaired cell proliferation, as well as absorptive and digestive function in the small intestine [8, 39]. Similarly, our results indicated that IUGR significantly decreased villous height in the duodenum of piglets. However, dietary supplementation with B. subtilis PB6 markedly increased villous height and decreased crypt depth in the ileum, resulting in a significant increase in the VCR. Lee et al. [33] reported that increased villus height and VCR in weaning piglets fed a diet supplemented with B. subtilis LS 1-2. The VCR is an important parameter to evaluate nutrient digestion and absorption capacity [40]. Therefore, an increase in villus height and VCR of piglets receiving B. subtilis PB6 suggests better intestinal development.

Several studies have reported that IUGR decreased the activity of digestive enzymes in the intestine of neonates [10, 21]. In the present study, IUGR was found to inhibit maltase activity, but enhanced lactase activity in the jejunum of piglets. The relatively higher lactase activity in IUGR piglets may be a compensatory response to enteral nutrition, which is consistent with the findings of our previous study [7]. However, dietary B. subtilis PB6 supplementation tended to increase the activities of maltase and sucrase in the jejunum of piglets. In particular, maltase activity in IUGR piglets receiving B. subtilis PB6 was normalized to similar levels as NBW piglets. The increasing activities of disaccharidases by B. subtilis PB6 indicate improved digestive capability of piglets.

IUGR neonates suffer from persistent immunological impairment throughout childhood [41]. The immunotype of blood is an important tool in the diagnosis of immunological disorders. In the present study, concentrations of IgA and IL-1β, numbers or percentages of lymphocytes and monocytes and the IL-1β: IL-10 ratio in the peripheral blood were lower in IUGR than in NBW piglets. Previous studies have demonstrated that IUGR alters cytokine profiles in the placenta and foetus [42, 43]. Meanwhile, decreased proliferation of lymphocytes in the thymus and cytokine levels in the peripheral blood were observed in rat and sheep models of IUGR [44, 45]. In this study, the increase in the number of CD8+ T cells indicates impaired T cell development in IUGR neonates. It has been shown that dietary probiotics supplementation could enhance cellular and humoral immune function in weaned piglets [46]. In this study, the levels of peripheral IL-1β, neutrophils, lymphocytes and monocytes in IUGR piglets receiving B. subtilis PB6 were consistently similar to those of NBW piglets, suggesting that the impairment of cellular immune function by IUGR can be alleviated by supplementation with probiotics.

Generally, intestinal epithelial cells provide immunological regulation to microbial invasion through both innate and adaptive immune responses [47]. It has been demonstrated that Toll-like receptors (TLRs) are typical pattern recognition receptors that mediate the innate host defence to maintain mucosal and commensal homoeostasis [48]. In this study, immune function was impaired in the intestine of IUGR piglets, as indicated by the decreased expression levels of TLR-9 and TOLLIP in the ileum of IUGR compared to NBW piglets. However, these gene expressions had not been markedly affected by dietary supplementation with B. subtilis PB6. The doses and strains of B. subtilis may affect the modulating effects of the animal immune function, as reported previously [49]. In addition, the gastrointestinal tract plays a central role as a physiological barrier between the outer environment and the body [50]. The paracellular barrier function of intestinal epithelia is thought to be regulated by tight junction proteins [51, 52]. The results of the current study indicate that dietary supplementation with B. subtilis PB6 increased expressions of ZO-1 and claudin-1 in the ileum of piglets. Both ZO-1 and claudin-1 are essential structural and functional components of tight junctions [25]. Hence, increased expression of tight junction proteins suggests improved intestinal barrier function in response to dietary supplementation of B. subtilis PB6.

This study provides new insights into the effects of dietary probiotics supplementation on growth performance, intestinal development and immune function of neonates. The results showed that IUGR delayed postnatal growth, intestinal development and impaired immune function in piglets. However, dietary B. subtilis PB6 supplementation improves growth rate, intestinal development and immune function, as indicated by the better digestive capability, barrier function and blood immunotype of piglets. These findings suggest that probiotics treatment could play an important role in the intestinal development and immune function of neonates during the early postnatal period.