Protein restriction and succedent realimentation affecting ileal morphology,ileal microbial composition and metabolites in weaned piglets

Dietary protein restriction is one of the effective ways to reduce post-weaning diarrhoea and intestinal fermentation in piglets, but it may also reduce growth performance. The compensatory growth induced by subsequent protein realimentation may solve the issue. However, little research has been done on the impact of protein realimentation on the gut. In this study, the effects of protein restriction and realimentation on ileal morphology, ileal microbial composition and metabolites in weaned piglets were investigated. Thirty-six 28-day-old weaned piglets with an average body weight of 6.47 ± 0.04 kg were randomly divided into a control group and a treatment group. The CP level in the diet of the control group was 18.83% for the entire experimental period. The piglets in the treatment group were fed 13.05% CP between days 0 and 14 and restored to a diet of 18.83% CP for days 14 to 28. On day 14 and 28, six pigs from each group were sacrificed and sampled. It was found that the abundance of Lactobacillus and Salmonella in the ileal digesta was significantly lower in the treatment group than the control group on day 14, whereas the abundance of Clostridium sensu stricto 1, Streptococcus, Halomonas and Pseudomonas significantly increased in the ileal digesta of the treatment group on day 14 compared with the control group. In addition, reduced concentrations of lactic acid, total short-chain fatty acids (total SCFAs), total branched chain fatty acids, ammonia and impaired ileal morphology and mucosal barrier were observed in the treatment group on day 14. However, diarrhoea levels decreased in the treatment group throughout the experiment. During the succedent protein realimentation stage, the treatment group demonstrated compensatory growth. Compared with the control group, the treatment group showed increased abundance of Lactobacillus and reduced abundance of Salmonella, Halomonas and Pseudomonas in the ileum on day 28. The concentrations of lactic acid and total SCFAs increased significantly, whereas the concentration of ammonia remained at a lower level in the treatment group on day 28 compared with the control group. Overall, protein realimentation could improve ileal morphology and barrier functions and promote ileal digestive and absorptive functions. In conclusion, ileal microbial composition and metabolites could change according to dietary protein restriction and realimentation and eventually influence ileal morphology and barrier functions.     

Denaturing Gradient Gel Electrophoresis Analysis of 16S Ribosomal DNA Amplicons To Monitor Changes in Fecal Bacterial Populations of Weaning Pigs after Introduction of Lactobacillus reuteri Strain MM53

The diversity and stability of the fecal bacterial microbiota in weaning pigs was studied after introduction of an exogenous Lactobacillus reuteri strain, MM53, using a combination of cultivation and techniques based on genes encoding 16S rRNA (16S rDNA). Piglets (n = 9) were assigned to three treatment groups (control, daily dosed, and 4th-day dosed), and fresh fecal samples were collected daily. Dosed animals received 2.5 × 10¹⁰ CFU of antibiotic-resistant L. reuteri MM53 daily or every 4th day. Mean Lactobacillus counts for the three groups ranged from 1 × 10⁹ to 4 × 10⁹ CFU/g of feces. Enumeration of strain L. reuteri MM53 on MRS agar (Difco) plates containing streptomycin and rifampin showed that the introduced strain fluctuated between 8 × 10³ and 5 × 10⁶ CFU/g of feces in the two dosed groups. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments, with primers specific for variable regions 1 and 3 (V1 and V3), was used to profile complexity of fecal bacterial populations. Analysis of DGGE banding profiles indicated that each individual maintained a unique fecal bacterial population that was stable over time, suggesting a strong host influence. In addition, individual DGGE patterns could be separated into distinct time-dependent clusters. Primers designed specifically to restrict DGGE analysis to a select group of lactobacilli allowed examination of interspecies relationships and abundance. Based on relative band migration distance and sequence determination, L. reuteri was distinguishable within the V1 region 16S rDNA gene patterns. Daily fluctuations in specific bands within these profiles were observed, which revealed an antagonistic relationship between L. reuteri MM53 (band V1-3) and another indigenous Lactobacillus assemblage (band V1-6).     

Lactobacillus reuteri 6475 Increases Bone Density in Intact Females Only under an Inflammatory Setting

MethodsA mild inflammatory state was induced in female mice by dorsal surgical incision (DSI). Following DSI animals were orally supplemented with L. reuteri or vehicle control for a period of 8 weeks. Gene expression was measured in the intestine and bone marrow by qPCR. Distal femoral bone density and architecture was analyzed by micro-CT.ResultsWe report that 8 weeks after DSI there is a significant increase in the weight of spleen, thymus and visceral (retroperitoneal) fat pads. Expression of intestinal cytokines and tight junction proteins are also altered 8 weeks post-DSI. Interestingly, L. reuteri treatment was found to display both intestinal region- and inflammation-dependent effects. Unexpectedly we identified that 1) L. reuteri treatment increased bone density in females but only in those that underwent DSI and 2) DSI benefited cortical bone parameters. In the bone marrow, dorsal surgery induced CD4⁺ T cell numbers, a response that was unaffected by L. reuteri treatment, whereas expression of RANKL, OPG and IL-10 were significantly affected by L. reuteri treatment.ConclusionOur data reveals a previously unappreciated effect of a mild surgical procedure causing a long-lasting effect on inflammatory gene expression in the gut and the bone. Additionally, we demonstrate that in intact female mice, the beneficial effect of L. reuteri on bone requires an elevated inflammatory status.     

Endogenous ileal losses of nitrogen and amino acids in pigs and piglets fed graded levels of casein

Abstract

In order to determine ileal losses of nitrogen (N) and amino acids (AA) and the coefficients of apparent and true ileal digestibility (AID, TID) of N and AA from casein in piglets and pigs, two experiments were conducted. In Experiment 1, 24 piglets were used. The piglets were weaned at 17 days of age, weighing 6.4 kg and cannulated at terminal ileum. Ileal digesta was collected at 28 – 29 and 35 – 36 days of age in period 1 and 2, respectively. Feed intake was 150 and 300 g · d^?1 during the first and second period. In Experiment 2, 16 castrates weighing 52.5 kg and cannulated at terminal ileum were used. The intake level of digestible energy was 2.5 times their maintenance requirement. The experiment lasted 7 days and ileal digesta was collected on day 6 – 7. Treatments consisted of four levels of N from casein: 8, 16, 24 and 32 g N · kg^?1 feed, respectively. Results showed that N level did not increase N or AA ileal losses. In piglets, N and AA ileal losses were similar between periods, except for period 2, where losses per kg DMI were about 47 and 64% higher for glycine and proline, respectively (p < 0.05). When ileal losses from pigs and piglets were compared, piglets had higher (p < 0.05) ileal losses of N and AA (excepted glutamic acid and alanine). A lower (p < 0.05) AID was observed in piglets in period 2 for N, methionine, glutamic acid, glycine and proline. With exception of glycine in pigs, all values for TID of N and AA of casein were superior to 0.90. Piglets had higher TID of N, leucine, isoleucine, valine and phenylalanine. These results showed that piglets have higher ileal losses than pigs.     

Lactobacillus reuteri KT260178 Supplementation Reduced Morbidity of Piglets Through Its Targeted Colonization,Improvement of Cecal Microbiota Profile,and Immune Functions

Supplementing suckling piglets with Lactobacillus reuteri isolated from a homologous source improves L. reuteri colonization number in the gastrointestinal tract, which can have health benefits. This study investigated dietary L. reuteri supplementation on the growth and health—including immune status—of piglets, as well as its colonization. A total of 60 sows with similar parity and body weight were allocated into one of three groups after secretion (n = 20 each, with 10 neonatal piglets of each): untreated control, L. reuteri supplementation, and antibiotic treatment. The experimental duration was 28 days, from birth of piglets to their group transferred. For the first 7 days after birth, all neonatal piglets were fed by sows. Piglets in the L. reuteri supplementation group were administered with 1.0 ml L. reuteri fermentation broth containing 5.0 × 10⁷ CFU. From 7 to 28 days, piglets were given basal feed (control), basal feed supplemented with L. reuteri (1.0 × 10⁷ CFU/g), or aureomycin (150 mg/kg). L. reuteri colonization in the distal jejunum and ileum was increased in piglets in the L. reuteri-supplemented as compared to the control group after 28 days, as determined by fluorescence in situ hybridization and real-time PCR analysis. Total Lactobacillus and Bifidobacterium counts in the cecum were higher whereas total aerobic bacteria (Escherichia coli and Staphylococcus) counts were lower in the L. reuteri as compared to the control group. L. reuteri supplementation also improved body antioxidant status and immune function relative to control animals. Strain-specific L. reuteri administered to piglets colonizes the intestinal mucosa and improves cecal microbiota profile and whole-body antioxidant and immune status, leading to better growth and lower morbidity and mortality rates.

     

Oral Microbiota Shift after 12-Week Supplementation with Lactobacillus reuteri DSM 17938 and PTA 5289; A Randomized Control Trial

Background

Lactobacillus spp. potentially contribute to health by modulating bacterial biofilm formation, but their effects on the overall oral microbiota remain unclear.

Methods and Findings

Oral microbiota was characterized via 454-pyrosequencing of the 16S rDNA hypervariable region V3-V4 after 12 weeks of daily Lactobacillus reuteri DSM 17938 and PTA 5289 consumption. Forty-four adults were assigned to a test group (n = 22) that received lactobacilli lozenges (10⁸ CFU of each strain/lozenge) or a control group that received placebo (n = 22). Presence of L. reuteri was confirmed by cultivation and species specific PCR. Tooth biofilm samples from 16 adults before, during, and after exposure were analyzed by pyrosequencing. A total of 1,310,292 sequences were quality filtered. After removing single reads, 257 species or phylotypes were identified at 98.5% identity in the Human Oral Microbiome Database. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria, and Actinobacteria were the most abundant phyla. Streptococcus was the most common genus and the S. oralis/S. mitis/S. mitis bv2/S. infantis group comprised the dominant species. The number of observed species was unaffected by L. reuteri exposure. However, subjects who had consumed L. reuteri were clustered in a principal coordinates analysis relative to scattering at baseline, and multivariate modeling of pyrosequencing microbiota, and culture and PCR detected L. reuteri separated baseline from 12-week samples in test subjects. L. reuteri intake correlated with increased S. oralis/S. mitis/S. mitis bv2/S. infantis group and Campylobacter concisus, Granulicatella adiacens, Bergeyella sp. HOT322, Neisseria subflava, and SR1 [G-1] sp. HOT874 detection and reduced S. mutans, S. anginosus, N. mucosa, Fusobacterium periodicum, F. nucleatum ss vincentii, and Prevotella maculosa detection. This effect had disappeared 1 month after exposure was terminated.

Conclusions

L. reuteri consumption did not affect species richness but induced a shift in the oral microbiota composition. The biological relevance of this remains to be elucidated.

Trial Registration

ClinicalTrials.gov NCT02311218     

Influence of <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> I5007 on the intestinal and systemic immune responses of healthy and <Emphasis Type="Italic">E. coli</Emphasis> challenged piglets

The effect of feeding Lactobacillus fermentum I5007 on the immune system of weaned pigs with or without E. coli challenge was determined. Twenty-four weaned barrows (6.07 ± 0.63 kg BW) were randomly assigned to one of four treatments (N = 6) in a factorial design experiment. The first two treatments consisted of healthy piglets with half of the pigs receiving no treatment while the other half was orally administered with L. fermentum I5007 (10⁸ CFU/ml) at a daily dose of 20 ml. Pigs in the second two treatments were challenged on the first day with 20 ml of E. coli K88ac (10⁸ CFU/ml). Half of these pigs were not treated while the remaining pigs were treated with 20 ml of L. fermentum I5007 (10⁸ CFU/ml). Peripheral blood lymphocytes subsets were determined using flow cytometry. The intestinal mucosal immunity of the pigs was monitored by real time polymerase chain reaction. The cytokine content of the pig’s serum was also analyzed. Oral administration of L. fermentum I5007 increased blood CD4⁺ lymphocyte subset percentage as well as tumor necrosis factor-α and interferon-γ expression in the ileum. Pigs challenged with E. coli had elevated jejunal tumor necrosis factor-α while interferon-γ expression was increased throughout the small intestine. There was no difference in the concentration of the cytokines interleukin-2, interleukin-6, tumor necrosis factor-α and interferon-γ in the serum. CD8⁺ and CD4⁺/CD8⁺ in peripheral blood were not affected by treatment. In conclusion, L. fermentum I5007 can enhance T cell differentiation and induce ileum cytokine expression suggesting that this probiotic strain could modulate immune function in piglets.     

Colonization and Immunomodulation by Lactobacillus reuteri ATCC 55730 in the Human Gastrointestinal Tract

Lactobacillus reuteri ATCC 55730 is a probiotic (health-promoting) bacterium widely used as a dietary supplement. This study was designed to examine local colonization of the human gastrointestinal mucosa after dietary supplementation with L. reuteri ATCC 55730 and to determine subsequent immune responses at the colonized sites. In this open clinical investigation, 10 healthy volunteers and 9 volunteers with ileostomy underwent gastroscopy or ileoscopy and biopsy samples were taken from the stomach, duodenum, or ileum before and after supplementation with 4 × 10⁸ CFU of live L. reuteri ATCC 55730 lactobacilli per day for 28 days. Biopsy specimen colonization was analyzed using fluorescence in situ hybridization with a molecular beacon probe, and immune cell populations were determined by immunostaining. Endogenous L. reuteri was detected in the stomach of 1 subject and the duodenum of 3 subjects (out of 10 subjects). After L. reuteri ATCC 55730 supplementation, the stomachs of 8 and the duodenums of all 10 subjects were colonized. Three ileostomy subjects (of six tested) had endogenous L. reuteri at baseline, while all six displayed colonization after L. reuteri supplementation. Gastric mucosal histiocyte numbers were reduced and duodenal B-lymphocyte numbers were increased by L. reuteri ATCC 55730 administration. Furthermore, L. reuteri administration induced a significantly higher amount of CD4-positive T-lymphocytes in the ileal epithelium. Dietary supplementation with the probiotic L. reuteri ATCC 55730 induces significant colonization of the stomach, duodenum, and ileum of healthy humans, and this is associated with significant alterations of the immune response in the gastrointestinal mucosa. These responses may be key components of a mechanism by which L. reuteri ATCC 55730 exerts its well-documented probiotic effects in humans.     

Feed Fermentation with Reuteran- and Levan-Producing Lactobacillus reuteri Reduces Colonization of Weanling Pigs by Enterotoxigenic Escherichia coli

This study determined the effect of feed fermentation with Lactobacillus reuteri on growth performance and the abundance of enterotoxigenic Escherichia coli (ETEC) in weanling piglets. L. reuteri strains produce reuteran or levan, exopolysaccharides that inhibit ETEC adhesion to the mucosa, and feed fermentation was conducted under conditions supporting exopolysaccharide formation and under conditions not supporting exopolysaccharide formation. Diets were chosen to assess the impact of organic acids and the impact of viable L. reuteri bacteria. Fecal samples were taken throughout 3 weeks of feeding; at the end of the 21-day feeding period, animals were euthanized to sample the gut digesta. The feed intake was reduced in pigs fed diets containing exopolysaccharides; however, feed efficiencies did not differ among the diets. Quantification of L. reuteri by quantitative PCR (qPCR) detected the two strains used for feed fermentation throughout the intestinal tract. Quantification of E. coli and ETEC virulence factors by qPCR demonstrated that fermented diets containing reuteran significantly (P < 0.05) reduced the copy numbers of genes for E. coli and the heat-stable enterotoxin in feces compared to those achieved with the control diet. Any fermented feed significantly (P < 0.05) reduced the abundance of E. coli and the heat-stable enterotoxin in colonic digesta at 21 days; reuteran-containing diets reduced the copy numbers of the genes for E. coli and the heat-stable enterotoxin below the detection limit in samples from the ileum, the cecum, and the colon. In conclusion, feed fermentation with L. reuteri reduced the level of colonization of weaning piglets with ETEC, and feed fermentation supplied concentrations of reuteran that may specifically contribute to the effect on ETEC.     

Dietary supplementation of weaned piglets with a yeast-derived mannan-rich fraction modulates cecal microbial profiles,jejunal morphology and gene expression

The development of nutritional strategies to improve microbial homeostasis and gut health of piglets post-weaning is required to mitigate the high prevalence of post-weaning diarrhea and subsequent growth checks typically observed during the weaning transition. Therefore the objective of this study was to determine the effect of supplementing piglet creep and nursery feed with a yeast-derived mannan-rich fraction (MRF) on piglet growth performance, cecal microbial profiles, and jejunal morphology and gene expression. Ten litters of piglets (n=106) were selected on postnatal day (PND) 7 and assigned to diets with or without MRF (800 mg/kg) until weaning (n=5 litters/treatment; initial weight 3.0±0.1 kg). On PND 21, 4 piglets per litter (n=40) were selected and weaned into the nursery where they remained on their respective diets until PND 42. A two-phase feeding program was used to meet nutrient requirements, and pigs were switched from phase 1 to phase 2 on PND 28. Feed intake and piglet weights were recorded on PND 7, 14, 21, 28, 35 and 42. On PND 28 and 42, ten piglets per treatment were euthanized to collect intestinal tissue and digesta. Piglets supplemented with MRF had 21.5% greater (P<0.05) average daily feed intake between PND 14-21. However, MRF supplementation did not affect piglet growth performance compared to control. On PND 28, jejunal villus height was 16.8% greater (P<0.05) in piglets consuming MRF supplemented diets. Overall microbial community structure in cecal digesta on PND 28 tended to differ in pigs supplemented with MRF (P=0.076; analysis of similarities (ANOSIM)) with increased (P<0.05) relative abundance of Paraprevotellaceae genera YRC22 and CF231, and reduced (P<0.05) relative abundance of Sutterella and Prevotella. Campylobacter also tended to reduce (P<0.10) in MRF supplemented piglets. On PND 28 differential gene expression in jejunal tissue signified an overall effect of supplementing MRF to piglets. Downstream analysis of gene expression data revealed piglets supplemented with MRF had enriched biological pathways involved in intestinal development, function and immunity, supporting the observed improvement in jejunal villus architecture on PND 28. On PND 42 there was no effect of MRF supplementation on jejunal morphology or overall cecal microbial community structure. In conclusion, supplementing Actigen™, a MRF, to piglets altered cecal microbial community structure and improved jejunal morphology early post-weaning on PND 28, which is supported by enrichment of intestinal development pathways.     

Effects of oral supplementation with Spirulina and Chlorella on growth and digestive health in piglets around weaning

Weaning of piglets is associated with important changes in gut structure and function resulting from stressful events such as separation from the sow, moving to a new facility and dietary transition from a liquid to a solid feed. This may result in post-weaning diarrhoea and a decrease in feed intake and growth. In humans, the cyanobacterium Spirulina platensis (SP) and the freshwater microalga Chlorella vulgaris (CV) are known for their beneficial health effects. This study aimed to determine the effects of early oral administration of Spirulina and Chlorella in piglets on mucosal architecture and cytokine expression in the intestine around weaning, and consequences on growth performance and diarrhoea incidence. The experiment was conducted on 108 suckling piglets of 14 days of age (initial BW=4.9±0.7 kg) and weaned at 28 days of age (day 0). Animals received orally 385 mg/kg BW per day of SP or CV, or water (negative control (NC)) during 4 weeks from day −14 to day 14 and their growth performance was measured daily. After weaning, growth, feed intake and diarrhoea incidence were measured daily. Intestinal morphology and functionality were assessed at day −1, day 2, and day 14. During the suckling period, average daily gain (ADG) in SP piglets was higher, resulting in a higher weaning BW compared to NC and CV piglets (P<0.05). No significant difference between treatments was observed for ADG, average daily feed intake, and gain to feed (G : F) ratio after weaning, but the extent of growth retardation after weaning was the lowest in piglets supplemented with Chlorella (P<0.01). Supplementation with Spirulina reduced diarrhoea incidence by 50% from day 0 to day 14 (P<0.05). Mucosal architecture at the jejunum was unaffected by Spirulina or Chlorella administration (P>0.10). Shorter ileal villi were measured in SP and CV piglets than in NC piglets (P<0.05). Cytokine expression did not differ between treatments in response to weaning. At day 14, IL-8 expression in the ileum was higher in SP piglets, while IL-1β expression in the jejunum was higher in CV piglets (P<0.05). This study shows that Spirulina administration around weaning alleviates diarrhoea in weaned piglets, without marked modulation of local inflammation.     

In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence

Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1×10¹⁰ CFU and luminescence signals at doses ranging from 1×10⁵ to 1×10¹⁰ CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.     

Antiobesity effect of Lactobacillus reuteri 263 associated with energy metabolism remodeling of white adipose tissue in high-energy-diet-fed rats

Obesity is a serious and costly issue to the medical welfare worldwide. Probiotics have been suggested as one of the candidates to resolve the obesity-associated problems, but how they combat obesity is not fully understood. Herein, we investigated the effects of Lactobacillus reuteri 263 (L. reuteri 263) on antiobesity using four groups of Sprague–Dawley rats (n=10/group), namely, C (normal diet with vehicle treatment), HE [high-energy diet (HED) with vehicle treatment], 1X (HED with 2.1×10⁹ CFU/kg/day of L. reuteri 263) and 5X (HED with 1.05×10¹⁰ CFU/kg/day of L. reuteri 263), for 8 weeks. L. reuteri 263 improved the phenomenon of obesity, serum levels of proinflammatory factors and antioxidant enzymes. More importantly, L. reuteri 263 increased oxygen consumption in white adipose tissue (WAT). The mRNA expressions of thermogenesis genes uncoupling protein-1, uncoupling protein-3, carnitine palmitoyltransferase-1 and cell death-inducing DFFA-like effector-a were up-regulated in WAT of the 5X group. Moreover, L. reuteri 263 might induce browning of WAT due to the higher mRNA levels of browning-related genes peroxisome proliferator-activated receptor-γ, PR domain containing-16, Pparγ coactivator-1α, bone morphogenetic protein-7 and fibroblast growth factor-21 in the 1X and 5X groups compared to the HE group. Finally, L. reuteri 263 altered the expressions of genes involved in glucose and lipid metabolisms in WAT, including increasing the levels of glucose transporter type 4 and carbohydrate-responsive element-binding protein and decreasing the expression of Acetyl-CoA carboxylase-1. The results suggest that L. reuteri 263 may treat obesity through energy metabolism remodeling of WAT in the high-energy-diet-induced obese rats.     

Group lactation from 7 or 14 days of age reduces piglet aggression at weaning compared to farrowing crate housing

Early life experiences can affect social behaviour in later life, but opportunities for socio-behavioural development are often overlooked in current husbandry practices. This experiment investigated the effects of rearing piglets in two-stage group lactation (GL) system from 7 or 14 days of age on piglet aggression at weaning. Three lactation housing treatments were applied to a total of 198 piglets from 30 litters of multiparous sows. All dams farrowed in standard farrowing crates (FCs). Group lactation litters were transferred with their dam at 7 (GL₇) or 14 days (GL₁₄) postpartum to GL pens (one pen of five sows at 8.4 m²/sow and one pen of seven sows at 8.1 m²/sow, per GL treatment). Farrowing crate litters remained with their dam in a single litter until weaning. At weaning, 10 to 14 piglets from two unfamiliar litters from the same housing treatment were mixed into pens (n=5 pens/treatment) and their behaviour was continuously recorded for 3.5 h. For each pen, the frequency of aggressive bouts (reciprocal and non-reciprocal aggression lasting <5 s), the frequency and duration of fights (reciprocal aggression lasting ⩾5 s) and bullying events (non-reciprocal aggression lasting ⩾5 s) were recorded, along with whether interactions involved familiar or unfamiliar piglets. Aggressive bouts delivered by FC piglets were approximately 1.5 and 3.0 times more frequent than that delivered by GL₇ and GL₁₄ piglets, respectively (40.5, 16.7 and 9.9 bouts/pig, respectively; P<0.05). Fighting was more frequent (1.6, 0.3 and 0.4 fights/pig, respectively; P<0.001) and fights were longer (83, 15 and 32 s fight/pig, respectively; P<0.001) between FC piglets than between GL₇ or GL₁₄ piglets. Bullying did not differ between housing treatments (P>0.05). GL₇ and GL₁₄ piglets engaged in a similar number of fights with unfamiliar as familiar piglets, but FC piglets had almost three times as many fights with unfamiliar than with familiar piglets (P<0.05). This experiment confirms the benefits of GL housing for pig social development. Further investigation is required to determine whether mixing before 14 days postpartum has implications for other indicators of animal welfare and productivity in a two-stage GL housing system.     

两种消浪树种幼苗光合特性对模拟潮汐水淹的响应

滨海滩涂消浪林是沿海防护林体系的重要组成部分,在消浪护岸、促淤保滩、防御来自海洋自然灾害等方面具有不可代替的作用。本研究以中亚热带两种木本消浪植物海滨木槿（Hibiscus hamabo）和蜡杨梅（Myrica cerifera）为研究对象,模拟海水水淹,系统研究了不同海水水淹程度下两木本植物光合特性的响应规律。结果发现,随着水淹胁迫的增加,海滨木槿最大净光合速率逐渐下降、光补偿点逐渐增加,表观量子效率在7、21天时先降低后增加,后期呈现逐渐降低的趋势,说明胁迫初期通过增加光合利用率来适应水淹胁迫,暗呼吸速率在7、21天逐渐增加,而在42、70天时W8（水淹8 h,水深45 cm）处理显著小于其它处理,说明W8处理已显著影响了该树种的光合特征;蜡杨梅最大净光合速率、表观量子效率表现为逐渐降低的趋势,而光补偿点和暗呼吸速率则逐渐增加,42天后W6（水淹6 h,水深35 cm）、W8处理各项指标显著小于其它处理。经多元方差分析发现,水淹21天内海滨木槿的光合指标要显著好于蜡杨梅,而在42天后则基本相同;海滨木槿在水淹胁迫6 h内可以生长,而蜡杨梅则只能在4 h内,这可为消浪林构建提供理论依据。     

Endogenous ileal losses of nitrogen and amino acids in pigs and piglets fed graded levels of casein

In order to determine ileal losses of nitrogen (N) and amino acids (AA) and the coefficients of apparent and true ileal digestibility (AID, TID) of N and AA from casein in piglets and pigs, two experiments were conducted. In Experiment 1, 24 piglets were used. The piglets were weaned at 17 days of age, weighing 6.4 kg and cannulated at terminal ileum. Ileal digesta was collected at 28-29 and 35-36 days of age in period 1 and 2, respectively. Feed intake was 150 and 300 g x d(-1) during the first and second period. In Experiment 2, 16 castrates weighing 52.5 kg and cannulated at terminal ileum were used. The intake level of digestible energy was 2.5 times their maintenance requirement. The experiment lasted 7 days and ileal digesta was collected on day 6-7. Treatments consisted of four levels of N from casein: 8, 16, 24 and 32 g N x kg(-1) feed, respectively. Results showed that N level did not increase N or AA ileal losses. In piglets, N and AA ileal losses were similar between periods, except for period 2, where losses per kg DMI were about 47 and 64% higher for glycine and proline, respectively (p < 0.05). When ileal losses from pigs and piglets were compared, piglets had higher (p < 0.05) ileal losses of N and AA (excepted glutamic acid and alanine). A lower (p < 0.05) AID was observed in piglets in period 2 for N, methionine, glutamic acid, glycine and proline. With exception of glycine in pigs, all values for TID of N and AA of casein were superior to 0.90. Piglets had higher TID of N, leucine, isoleucine, valine and phenylalanine. These results showed that piglets have higher ileal losses than pigs.     

Administration of a novel plant extract product via drinking water to post-weaning piglets: effects on performance and gut health

The present study evaluated the effects of a novel plant extract (PE) product (Grazix^TM) on the performance and gut health of weaned piglets challenged with Escherichia coli. The PE was a standardised mixture of green tea leaves (Camellia sinensis) and pomegranate fruit (Punica granatum) obtained by using the LiveXtract^TM process. A total of 144 piglets were weaned at 24 days and allocated to 8 for a 35-day experiment with a 2×2×2 factorial design comparing different treatments (water without product (CT) or 8 μl/kg per day PE in drinking water (PE)), feeding regimens (ad libitum (AD) or restricted (RE)) and oral E. coli challenges on day 9 (sham (−) or infected (+)). There were six pens per group with three piglets per pen. On day 35, 24 of the RE feeding piglets were slaughtered. It was found that PE supplementation increased the average daily gain (ADG) from day 28 to day 35 (P=0.03) and increased the gain to feed ratio (G : F) from day 7 to day 14 (P=0.02). RE feeding led to lower feed intake in piglets during the 1^st week (P<0.01), 2^nd week (P=0.06), 3^rd week (P=0.05), and throughout the course of the overall study period (P=0.05). E. coli challenge decreased the ADG and G : F ratio from day 7 to day 14 (P=0.08 and <0.01, respectively) and increased the faecal score (higher values indicate more severe diarrhoea) on days 14, 21, 28 and 35 (P<0.01). PE supplementation decreased the faecal score in the challenged piglets during the 1^st week post-challenge (P<0.01). E. coli challenge increased the faecal E. coli level on day 14 (P=0.03) and increased the Enterobacteriaceae level on day 35 (P<0.01). Reduced faecal E. coli was observed on days 14 and 35 (P=0.05 and 0.02, respectively), and reduced Enterobacteriaceae (P<0.01) was found on day 35 in the PE animals. RE feeding increased the faecal Lactobacillus, Enterobacteriaceae and E. coli levels on day 35 (P=0.02, <0.01 and <0.01, respectively). These results suggest that PE supplementation may improve the gut health status of post-weaning piglets and counteract some of the negative effects that occur when piglets are challenged with E. coli.     

Specific Response of a Novel and Abundant Lactobacillus amylovorus-Like Phylotype to Dietary Prebiotics in the Guts of Weaning Piglets

Using 16S rRNA gene-based approaches, we analyzed the responses of ileal and colonic bacterial communities of weaning piglets to dietary addition of four fermentable carbohydrates (inulin, lactulose, wheat starch, and sugar beet pulp). An enriched diet and a control diet lacking these fermentable carbohydrates were fed to piglets for 4 days (n = 48), and 10 days (n = 48), and the lumen-associated microbiota were compared using denaturing gradient gel electrophoresis (DGGE) analysis of amplified 16S rRNA genes. Bacterial diversities in the ileal and colonic samples were measured by assessing the number of DGGE bands and the Shannon index of diversity. A higher number of DGGE bands in the colon (24.2 ± 5.5) than in the ileum (9.7 ± 4.2) was observed in all samples. In addition, significantly higher diversity, as measured by DGGE fingerprint analysis, was detected in the colonic microbial community of weaning piglets fed the fermentable-carbohydrate-enriched diet for 10 days than in the control. Selected samples from the ileal and colonic lumens were also investigated using fluorescent in situ hybridization (FISH) and cloning and sequencing of the 16S rRNA gene. This revealed a prevalence of Lactobacillus reuteri in the ileum and Lactobacillus amylovorus-like populations in the ileum and the colon in the piglets fed with fermentable carbohydrates. Newly developed oligonucleotide probes targeting these phylotypes allowed their rapid detection and quantification in the ileum and colon by FISH. The results indicate that addition of fermentable carbohydrates supports the growth of specific lactobacilli in the ilea and colons of weaning piglets.     

Dietary supplementation with a nucleotide-rich yeast extract modulates gut immune response and microflora in weaned pigs in response to a sanitary challenge

An experiment was carried out to evaluate the short-term effect of supplementing a nucleotide-rich yeast extract (NRYE) on growth performance, gut structure, immunity and microflora of piglets raised under sanitary and unsanitary conditions. A total of 84, 21-day old piglets were used in this study; 42 piglets were raised in a room designated as the clean room that was washed once per week, whereas the other 42 piglets were raised in a room designated as the unclean room in which 7 kg of manure from the sow herd was spread on each pen floor on day 1 and 7 and the room was not washed throughout the experiment. The pigs were fed a corn–soybean meal-based diet without or with 0.1% NRYE. Each treatment had 7 replicate pens in each room, and each pen housed 3 pigs. Feed disappearance and BW were recorded on day 1 and 14. On day 14, one pig per pen was euthanized to collect ileum, mesenteric lymph nodes and spleen tissues, and cecum and colon digesta. Overall, NRYE supplementation did not affect growth performance in both clean and unclean conditions, improved kidney weight in both clean (P=0.0002) and unclean room (P<0.0001) and tended to improve the villus height/crypt depth ratio in the clean room (P=0.073). Supplementing NRYE was associated with upregulation of Ileal programmed cell death gene-1 (P=0.0003), interleukin (IL)-1β (P<0.0001), IL-6 (P=0.0003), IL-10 (P<0.0001) and tumor necrosis factor-α (TNF-α) (P<0.0001) in pigs raised in the unclean room. Supplementing the NRYE in pigs raised in the clean room suppressed growth of cecal Enterobacteriacea (P<0.0001) members and colonic Enterococcus spp. (P<0.019), improved proliferation of cecal Lactobacillus spp. (P<0.002) and colonic Clostridium cluster IV (P<0.011) and XVIa members (P<0.0002). Supplementing the NRYE in the unclean room improved proliferation of cecal Clostridium cluster IV (P<0.026) and suppressed proliferation of colonic Enterococcus spp. (P<0.037). In conclusion, supplementing the NRYE to piglets under unsanitary conditions improved ileal immune response by upregulating inflammatory cytokines, and positively modulated proliferation of beneficial gut bacteria and suppression of harmful ones in both clean and unclean rooms.     

Changes in the diversity and composition of gut microbiota of weaned piglets after oral administration of <Emphasis Type="Italic">Lactobacillus</Emphasis> or an antibiotic

The gut microbiota plays important roles in the health and well-being of animals, and high-throughput sequencing facilitates exploration of microbial populations in the animal gut. However, previous studies have focused on fecal samples instead of the gastrointestinal tract. In this study, we compared the microbiota diversity and composition of intestinal contents of weaned piglets treated with Lactobacillus reuteri or chlortetracycline (aureomycin) using high-throughput sequencing. Nine weaned piglets were randomly divided into three groups and supplemented with L. reuteri, chlortetracycline, or saline for 10 days, and then the contents of three intestinal segments (jejunum, colon, and cecum) were obtained and used for sequencing of the V3–V4 hypervariable region of the 16S rRNA gene. The microbiota diversity and composition in the jejunum were different from those in the colon and cecum among the three treatments. In the jejunum, treatment with L. reuteri increased the species richness of the microbiota, as indicated by the ACE and Chao1 indexes, compared with the chlortetracycline group, in which several taxa were eliminated. In the colon and cecum, relative abundances of the phylum Firmicutes and the genus Prevotella were higher in the chlortetracycline group than in the other groups. Distances between clustered samples revealed that the L. reuteri group was closer to the chlortetracycline group than the control group for jejunum samples, while colon and cecum samples of the L. reuteri group were clustered with those of the control group. This study provides fundamental knowledge for future studies such as the development of alternatives to antibiotics.