Dietary supplementation of Platycodon grandiflorum polysaccharides mitigates weaning stress in piglets by modulating intestinal microbiota and improving gut health.

ABSTRACTAddressing weaning stress in modern swine production requires dietary interventions that improve gut health and reduce antibiotic use. It is crucial for sustainable animal husbandry, public health and environmental protection. The mannan‐rich fraction (MRF), a prebiotic, is known to modulate the intestinal microbiota. However, its effects on regulating intestinal flora and the potential mechanisms for alleviating diarrhea and weaning stress in piglets remain incompletely defined. Herein, we investigated the effects of dietary MRF supplementation on growth performance, intestinal morphology, gut microbiota composition, and inflammatory responses in Duroc × Large × White weaned piglets. MRF reduced the incidence of diarrhea to the same extent as antibiotics (p < 0.01). Compared with control group, MRF‐fed piglets exhibited increased ileal villus height (p < 0.01), higher abundance of Prevotella and Roseburia, and lower Campylobacter abundance (p < 0.01). MRF also elevated hindgut short‐chain fatty acids (SCFAs) concentrations, especially butyrate (p < 0.01). Mechanistically, molecular docking and transcriptomic analyses indicated that MRF can interact with Toll‐like receptor 5 (TLR5), thereby destabilizing the TLR5‐NF‐κB p65 complex. This interactions attenuated the phosphorylation of NF‐κB p65 (p < 0.01) and down‐regulated transcription of IL‐1β in the colon (p < 0.05). Collectively, our results demonstrate that MRF reshapes the gut microbiota, enhances barrier function, and potentially suppresses TLR5/NF‐κB‐mediated inflammation, offering a strategy to reduce antibiotic use in promoting piglet intestinal health.

Objective: To investigate the interindividual variation of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA expression level in the peripheral blood nucleated cells and its correlation with the severity of chemotherapy-induced myelosuppression. Methods: The expression level of Nrf2 mRNA in the peripheral blood nucleated cells from 30 cases of cancer was detected by semi-quantitative RT-PCR before chemotherapy, and the association between the Nrf2 mRNA expression and the chemotherapy-induced myelosuppression was analyzed. Results: The Nrf2 mRNA expression level in the peripheral blood mononuclear cells exhibited significant interindividual differences. The Nrf2 mRNA expression level was significantly higher in the patients with grades 0-1 leukopenia or neutropenia than that in the patients with grades 2-4 ( P <0.05), but it was not associated with the age, gender, chemotherapeutic regimen and the type of tumor. The association analysis showed that the Nrf2 mRNA expression level in the peripheral blood mononuclear cells was negatively associated with leukopenia and neutropenia ( r = –0.448, P = 0.013; r = –0.493, P = 0.006). Conclusion: The expression level of Nrf2 mRNA in the peripheral blood mononuclear cells is significantly correlated with chemotherapy-induced myelosuppression. It may be employed as an ideal marker to predict the severity of chemotherapy-induced myelosuppression and helpful for clinical decision-making and early prevention and treatment of myelosuppression.

Plant extracts are considered to be an effective alternative to antibiotics in response to weaning stress in piglets. This study evaluated the effect of Illicium verum extracts (IVE) or Eucommia ulmoides leaf extracts (ELE) on growth performance, serum and liver antioxidant ability of nursery piglets, as well as the difference of IVE and ELE on Duroc×Landrace×Yorkshire (DLY) and Chinese native Licha-black (LCB) piglets. A total of 96 nursery piglets (48 DLY and 48 LCB piglets) with an average body weight of 11.22±0.32kg were randomly divided into four treatments in a 2×4 factorial design. Each treatment had four replicates with 3 DLY and 3 LCB piglets per replicate respectively. Treatments included: basal diet, basal diet+500mg/kg IVE, basal diet+250mg/kg ELE and basal diet+50mg/kg chlortetracycline (CHL). All piglets were housed individually for the 42days trial period after 7days adaptation. Results showed that there were significant interactions (p<.05) between piglets species and dietary treatments in average daily gain (ADG) and feed efficiency, serum and hepatic glutathione peroxidase (GSH-Px) and malondialdehyde (MDA), hepatic integral optical density (IOD) of α-tumour necrosis factor (TNF-α), hepatic relative mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2)/TNF-α and protein expression of TNF-α. Regardless of piglets species, supplementation with IVE and ELE increased (p<.05) ADG and feed efficiency, T-SOD and GSH-Px in serum and liver, hepatic IOD of Nrf2, hepatic mRNA and protein expression of Nrf2/TNF-α. However, CHL treatment resulted in lower (p<.05) serum GSH-Px and hepatic mRNA and protein expression of Nrf2/TNF-α, and higher hepatic MDA and IOD of TNF-α. Compared to LCB, DLY piglets had higher (p<.05) ADG and feed efficiency, serum and hepatic MDA, and protein expression of TNF-α, but lower (p<.05) ADFI, liver index, serum and hepatic GSH-Px, hepatic IOD of TNF-α, mRNA expressions of Nrf2/TNF-α were observed. In conclusion, Illicium verum (500mg/kg) and Eucommia ulmoides leaf (250mg/kg) extracts can increase the growth performance and antioxidant ability of DLY and LCB piglets, while chlortetracycline produces undesirable side-effects on the antioxidant ability of DLY and LCB piglets. Illicium verum and Eucommia ulmoides leaf extracts produced different antioxidant effects in DLY and LCB piglets with the Chinese native Licha-black pig responding better than Duroc×Landrace×Yorkshire.

This study was designed to determine the effect of early intervention with fecal microbiota transplantation combined C. butyricum and S. boulardii on anti-weaning stress capacity of piglets. 22 pregnant sows were selected in this study, 10 sows were divided into control group (CON), and 12 sows were divided into treatment group (FMT combined C. butyricum and S. boulardii, FMT+C+S). Piglets in CON were gavaged with placebo and piglets in treatment group were gavaged with bacterial solution once daily for the first 3 days after birth. Piglets were weaned at age of 28-day. To explore the effects of early intervention on weaning stress in piglets, after weaning, each group selected 65 healthy piglets to continue feeding for 4 weeks. The growth performance was measured by weekly individual weighing. ADFI during post-weaning period were measured daily. The blood biochemical markers for immunity function, intestinal barrier, and inflammation levels were determined by ELISA kits, and the indices of antioxidant ability were examined using the commercial assay kit. Isolated DNA from fecal were used for 16s rRNA amplicon sequencing to determine microbiota composition. FMT+C+S improved growth rate and decreased diarrhea rate both in sucking and post-weaning period in piglets. FMT+C+S significantly increased immunity function, intestinal barrier function, antioxidant capacity, and reduced inflammation levels in weaned piglets. FMT+C+S significantly increased the abundance of Firmicutes in feces before and after weaning (P &amp;lt; 0.01). In addition, at the genus level, several beneficial bacteria, such as Phascolarctobacterium, Oscillospira, Faecalibacterium, etc. were significantly enriched (P &amp;lt; 0.05) before weaning, and Lactobacillus, Lachnospira, Bacillus, Lysinibacillus, etc. were enriched significantly (P &amp;lt; 0.05) after weaning. These results suggested that early intervention with fecal microbiota transplantation combined C. butyricum and S. boulardii may be a effectively method to promote piglets growth and protect piglets from weaning stress, and even disease, through regulating gut microbiota composition.

Based on the theory of "Gongsun (SP4) connects the Thoroughfare Vessel" of the "eight confluence acupoints", the effect of electroacupuncture (EA) at SP4 on Kelch-like epichlorohydrin associated protein factor 1 (Keap1)/ nuclear factor-erythroid 2 related factor 2 (Nrf2)/ heme oxygenase-1 (HO-1) signaling pathway was observed in rats with premature ovarian insufficiency (POI) for exploring its underlying mechanisms in treating POI. Thirty-two female SD rats were randomly divided into 4 groups:blank, model, EA and medication, with 8 rats in each group. The POI model was established by intraperitoneal injection of cyclophosphamide. EA (2 Hz, a strength inducing slight limb tremor) was applied to bilateral SP4 for 20 min, once a day for a total of 14 d. Rats of the medication group were treated by gavage of solution of estradiol valerate tablets (0.09 mg/kg) once a day for 14 d. During the experiment, the changes of body mass in each group were recorded and the general conditions were observed. After the interventions, the contents of follicle-stimulating hormone (FSH), estradiol (E2), anti-mullerian hormone (AMH), malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) were detected by ELISA. The morphological changes of ovarian tissue were observed under light microscope after H.E. staining. The protein and mRNA expression levels of Keap1, Nrf2 and HO-1 in the ovarian were detected by immunohistochemistry, Western blot, and PCR, respectively. After modeling, the model rats had poor mental state, reduced activity, irritability, emaciated body, dry and dull hair, decreased food intake, and occasionally yellow soft stools, black stools, and bloody secretions around the eyes and the tip of the nose. After the interventions, the mental state of rats in the EA and medication groups was recovered, and had brown oval feces, with no bloody secretion. Compared with the blank group, the body mass, levels of serum E2, AMH, SOD, GSH-Px, immunoactivity of Nrf2 and HO-1, and expression levels of Nrf2 and HO-1 proteins and mRNAs in the ovarian tissue were significantly decreased (P<0.01), and the contents of serum FSH and MDA, immunoactivities and expressions of Keap1 protein and mRNA were obviously increased (P<0.01) in the model group. Following the interventions, both the decrease in the levels of E2, AMH, SOD, GSH-Px, and the protein and mRNA expression levels of Nrf2 and HO-1 and the increase of contents of serum FSH and MDA, immunoactivities and expressions of Keap1 protein and mRNA were reversed in both EA and medication groups (P<0.01, P<0.05), without significant differences between the two groups in all the indexes mentioned above. H.E. staining showed a decrease in the primordial and growing follicles in the ovarian cortex and an increase in the atretic follicles and disordered and loose arrangement of the granular layer in the model group, and an increase in the primordial and growing follicles in the ovarian cortex, reduction in the atretics follicles, thickened granular layer and closer arrangement in the EA and medication groups. EA of SP4 can promote the restoration of the body mass, regulate the level of serum sex hormones, improve the body's antioxidant capacity, reduce the damage of oxidative stress of ovaries and promote the normal development of follicles in POI rats, which may be related to its function in regulating Keap1/Nrf2/HO-1 signaling pathway.

To observe the effect of electroacupuncture (EA) on nuclear transcription factor E2 related factor 2 (Nrf2)/NOD-like receptor family pyrin domain-containing protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1) pathway in the substantia nigra (SN) of mice with Parkinson's disease (PD), so as to explore the neuroprotective mechanism of EA. Forty C57BL/6 male mice were randomly divided into 4 groups, namely, control, PD model, EA and sham-EA groups, with 10 mice in each group. The PD mouse model was established by gavage of rotenone for 4 weeks. Mice in the EA group were given EA stimulation (1 mA, 2 Hz) at "Fengfu" (GV36), bilateral "Taichong" (LR3) and "Zusanli" (ST36) for 30 min, once daily for 2 consecutive weeks. And mice in the sham-EA group were given acupuncture at the subcutaneous areas of the same acupoints without EA stimulation. The open-field test was used for assessment of mouse behavior. The levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in the serum were detected by enzyme-linked immunosorbent assay . The positive expression of tyrosine hydroxylase (TH) in SN was determined by immunohistochemistry. The mRNA expression levels of Nrf2, NLRP3, Caspase-1, gasdermin D(GSDMD), IL-1β, IL-18 and the protein expression levels of Nrf2, NLRP3, Caspase-1 and GSDMD in the SN were detected by quantitative real-time PCR and Western blot, separately. After modeling, compared with the control group, the behavioral score was increased (P<0.01), the total exercise time, the total distance and the average speed were decreased (P<0.01), and the positive expression of TH and the mRNA and protein expression levels of Nrf2 in the SN were decreased (P<0.01), while the contents of IL-1β and IL-18 in serum, the mRNA and protein expression levels of NLRP3, Caspase-1 and GSDMD and the mRNA expression levels of IL-1β and IL-18 in the SN were up-regulated (P<0.01) in the PD model group. Following EA intervention, the behavioral score was decreased(P<0.01), the total exercise time, total distance and average speed were increased (P<0.01), the positive expression of TH and the mRNA and protein expressions of Nrf2 in SN were up-regulated (P<0.01, P<0.05), while the contents of IL-1β and IL-18 in serum, the mRNA and protein expression levels of NLRP3, Caspase-1 and GSDMD as well as the mRNA expression levels of IL-1β and IL-18 in the SN were down-regulated (P<0.01, P<0.05) in the EA group relative to the PD model and sham-EA groups. There were no significant differences in the above indicators between the PD model and sham-EA groups. EA stimulation of GV36, LR3 and ST36 can improve motor deficits, reduce the loss of dopamine neurons in the SN, and inhibit neuroinflammatory responses in mice with PD, which may be related to its effects in regulating the Nrf2/NLRP3/Caspase-1 pathway mediated pyroptosis.

Fecal microbiota transplantation (FMT) could shape the structure of intestinal microbiota in animals. This study was conducted to explore the changes that happen in the structure and function of microbiota caused by weaning stress, and whether early-life FMT could alleviate weaning stress through modifying intestinal microbiota in weaned piglets. Diarrheal (D) and healthy (H) weaned piglets were observed, and in the same farm, a total of nine litters newborn piglets were randomly allocated to three groups: sucking normally (S), weaned at 21 d (W), and early-life FMT + weaned at 21 d (FW). The results demonstrated that differences of fecal microbiota existed in group D and H. Early-life FMT significantly decreased diarrhea incidence of weaned piglets. Intestinal morphology and integrity were improved in the FW group. Both ZO-1 and occludin (tight junction proteins) of jejunum were greatly enhanced, while the zonulin expression was significantly down-regulated through early-life FMT. The expression of IL-6 and TNF-α (intestinal mucosal inflammatory cytokines) were down-regulated, while IL-10 (anti-inflammatory cytokines) was up-regulated by early-life FMT. In addition, early-life FMT increased the variety of the intestinal microbial population and the relative amounts of some beneficial bacteria such as Spirochaetes, Akkermansia, and Alistipes. Functional alteration of the intestinal microbiota revealed that lipid biosynthesis and aminoacyl-tRNA biosynthesis were enriched in the FW group. These findings suggested that alteration of the microbiota network caused by weaning stress induced diarrhea, and early-life FMT alleviated weaning stress in piglets, which was characterized by decreased diarrhea incidence, improved intestinal morphology, reduced intestinal inflammation, and modified intestinal bacterial composition and function.

(1) Background: Weaning is a challenging and stressful event in the pig's life, which disrupts physiological balance and induces oxidative stress. Microbiota play a significant role during the weaning process in piglets. Therefore, this study aimed to investigate key gut microbiota and metabolites associated with weaning stress in piglets. (2) Methods: A total of ten newborn piglet littermates were randomly assigned to two groups: S (suckling normally) and W (weaned at 21 d; all euthanized at 23 d). Specimens of the cecum were dehydrated with ethanol, cleared with xylene, embedded in paraffin, and cut into 4 mm thick serial sections. After deparaffinization, the sections were stained with hematoxylin and eosin (H&E) for morphometric analysis. Cecal metagenomic and liver LC-MS-based metabolomics were employed in this study. Statistical comparisons were performed by a two-tailed Student's t-test, and p < 0.05 indicated statistical significance. (3) Results: The results showed that weaning led to intestinal morphological damage in piglets. The intestinal villi of suckling piglets were intact, closely arranged in an orderly manner, and finger-shaped, with clear contours of columnar epithelial cells. In contrast, the intestines of weaned piglets showed villous atrophy and shedding, as well as mucosal bleeding. Metagenomics and metabolomics analyses showed significant differences in composition and function between suckling and weaned piglets. The W piglets showed a decrease and increase in the relative abundance of Bacteroidetes and Proteobacteria (p < 0.05), respectively. The core cecal flora in W piglets were Campylobacter and Clostridium, while those in S piglets were Prevotella and Lactobacillus. At the phylum level, the relative abundance of Bacteroidetes significantly decreased (p < 0.05) in weaned piglets, while Proteobacteria significantly increased (p < 0.05). Significant inter-group differences were observed in pathways and glycoside hydrolases in databases, such as the KEGG and CAZymes, including fructose and mannose metabolism, salmonella infection, antifolate resistance, GH135, GH16, GH32, and GH84. We identified 757 differential metabolites between the groups through metabolomic analyses-350 upregulated and 407 downregulated (screened in positive ion mode). In negative ion mode, 541 differential metabolites were identified, with 270 upregulated and 271 downregulated. Major differential metabolites included glycerophospholipids, histidine, nitrogen metabolism, glycine, serine, threonine, β-alanine, and primary bile acid biosynthesis. The significant differences in glycine, serine, and threonine metabolites may be potentially related to dysbiosis caused by weaning stress. Taken together, the identification of microbiome and metabolome signatures of suckling and weaned piglets has paved the way for developing health-promoting nutritional strategies, focusing on enhancing bacterial metabolite production in early life stages.

Lycopene Affects Intestinal Barrier Function and the Gut Microbiota in Weaned Piglets via Antioxidant Signaling Regulation

During weaning, piglets are susceptible to intestinal inflammation and impairment in barrier function. Dietary fiber (DF) plays an active role in alleviating weaning stress in piglets. However, the effects of different sources of dietary fiber on the performance of weaned piglets are inconsistent, and the mechanisms through which they affect intestinal health need to be explored. Therefore, in this study, sixty weaned piglets were randomly divided into three treatment groups: basal diet (control, CON), beet pulp (BP), and alfalfa meal (AM) according to the feed formulation for a 28-day trial. The results showed that both AM and BP groups significantly reduced diarrhea rate and serum inflammatory factors (IL-1β and TNF-α) and increased antioxidant markers (T-AOC and SOD), in addition to decreasing serum MDA and ROS concentrations in the AM group. At the same time, piglets in the AM group showed a significant reduction in serum intestinal permeability indices (LPS and DAO) and a substantial increase in serum immunoglobulin levels (IgA, IgG, and IgM) and expression of intestinal barrier-associated genes (Claudin1, Occludin, ZO-1, and MUC1), which resulted in an improved growth performance. Interestingly, the effect of DF on intestinal inflammation and barrier function can be attributed to its modulation of gut microbes. Fiber-degrading bacteria enriched in the AM group (Christensenellaceae_R-7_group, Pediococcus and Weissella) inhibited the production of TLR4- through the promotion of SCFAs (especially butyrate). MyD88-NF-κB signaling pathway activation reduces intestinal inflammation and repairs intestinal barrier function. In conclusion, it may provide some theoretical support and rationale for AM to alleviate weaning stress and improve early intestinal dysfunction, which may have implications for human infants.

The purpose of the present study was to investigate the effects of drinking water alkaline mineral complex (AMC) supplementation on growth performance, intestinal morphology, inflammatory response, immunity, antioxidant defense system, and barrier functions in weaned piglets. In a 15-d trial, 240 weaned piglets (9.35 ± 0.86 kg) at 28 d of age (large white × landrace × Duroc) were randomly divided into two groups: the control (Con) group and the AMC group. Drinking water AMC supplementation improved (P < 0.01) final body weight (BW) and average daily gain (ADG) in weaned piglets compared to the Con group. Importantly, AMC reduced (P < 0.01) the feed-to-gain (F:G) ratio. AMC water improved the physical health conditions of piglets under weaning stress, as reflected by the decreased (P < 0.05) hair score and conjunctival score. Moreover, there was no significant (P > 0.05) difference in relatively small intestinal length, organ (liver, spleen, and kidney) indices, or gastrointestinal pH value in weaned piglets between the two groups. Of note, AMC significantly promoted the microvilli numbers in the small intestine and effectively ameliorated the gut morphology damage induced by weaning stress, as evidenced by the increased (P < 0.05) villous height (VH) and ratio of VH to crypt depth. Additionally, AMC lessened the levels of lipopolysaccharide (LPS, P < 0.01) and the contents of IL1β (P<0.05), and TNF-α (P<0.05) in the weaned piglet small intestine. Conversely, the gut immune barrier marker, secretory immunoglobulin A (sIgA) levels in serum and small intestine mucosa were elevated after AMC water treatment (P < 0.01). Furthermore, AMC elevated the antioxidant mRNA levels of (P < 0.05) SOD 1-2, (P < 0.01) CAT, and (P < 0.01) GPX 1-2 in the small intestine. Likewise, the mRNA levels of the small intestine tight junction factors Occludin (P < 0.01), ZO-1 (P < 0.05), Claudin 2 (P < 0.01), and Claudin 5 (P<0.01) in the AMC treatment group were notably higher than those in the Con group. In conclusion, drinking water AMC supplementation has an accelerative effect on growth performance by elevating gut health by improving intestinal morphology, the inflammatory response, the antioxidant defense system, and barrier function in weaned piglets.

The present study evaluated the mechanism underlying the protective effect of sulforaphane (SFN) on cadmium (Cd)-induced Sertoli cell (TM4 cells) injury in mice. The apoptosis rate of cells in each group was detected by flow cytometry. It was determined the effect of SFN on the expression of downstream molecular targets of Nrf2/ARE axis and on the lipid peroxide content. The related genes involved in the nuclear factor E2-related factor 2(Nrf2)/antioxidant response element (ARE) signaling pathway were evaluated by RT-PCR; for example, the mRNA expression levels of Nrf2, heme oxygenase-1 (HO-1), glutathione peroxidase (GSH-Px), quinone oxidoreductase 1 (NQO1), and γ-glutamylcysteine synthetase (γ-GCS), while the protein expression levels were assessed by Western blot. Our results showed that the mRNA and protein expression levels of Nrf2, HO-1, NQO1, GSH-Px, and γ-GCS were increased in various degree when the Sertoli cells were to added different concentrations of SFN. Our results also showed that SFN reduced the apoptosis rate, increased the activity of T-SOD, inhibited the increase of the MDA content caused by Cd. Meanwhile, SFN could increase the mRNA and protein expression levels of Nrf2, HO-1 and NQO1 and reduced the mRNA and protein expression levels of GSH-Px and γ-GCS caused by Cd in Sertoli cells (p < 0.01). Taken together, SFN could improve the antioxidant capacity of Sertoli cells, and exert a protective effect on the oxidative damage and apoptosis of Cd-induced Sertoli cells through the activation of Nrf2/ARE signal transduction pathway.

Weaning stress in piglets can lead to poor health outcomes and reduced production. We investigated the effects of probiotics, one potential antibiotic alternative, on the growth performance, serum biochemical parameters, intestinal morphology, mucosal immunity, hypothalamic neurotransmitters, and colonic microflora in weaned piglets. Thirty-six weaned piglets were fed a basal diet, a diet supplemented with colistin sulphate antibiotic, or a diet supplemented with probiotics including Clostridium butyricum, Bacillus subtilis, and B. licheniformis. Probiotics significantly increased the feed : gain ratio, improved the average day gain from day 1 to day 28, and decreased the diarrhoea index. Probiotics also lowered the serum concentrations of AST, ALT, and ALP on day 14 and lowered the serum concentration of ALT on day 28 compared with the control. Probiotic supplementation caused fewer ileal apoptotic cells. The serum and ileal concentrations of TNF-α and IL-1β on day 28 were significantly lowered, and the serum concentrations of IL-6 were significantly lowered on days 14 and 28. Probiotic-fed piglets exhibited higher contents of hypothalamic serotonin and dopamine as well as serum γ-aminobutyric acid along with higher colonic concentrations of butyrate and valerate on day 28. High-throughput sequencing showed 972 core operational taxonomic units among all groups, of which 48 were unique to the probiotic-treated group. The relative abundance of genus Bacillus and species Bacillus velezensis was enriched in probiotic piglets; the phylogenetic investigation of communities by the reconstruction of unobserved states indicated that amino acid metabolism, DNA repair, replication and recombination proteins, and secretion systems were enriched with probiotics. In conclusion, the Clostridium butyricum-based probiotics improved growth performance, enhanced intestinal morphology, changed hypothalamic neurotransmitters and modulated colonic microflora in weaned piglets.

Weaning stress in piglets poses a significant challenge to the swine industry, often leading to compromised intestinal health, reduced feed intake, diarrhea, and increased mortality. Although antibiotics and pharmacological doses of zinc oxide have traditionally been used to alleviate these issues, rising concerns over antimicrobial resistance and environmental sustainability have driven the search for effective alternatives. The purpose of this study was to evaluate the effects of plant essential oils (PEO) as dietary supplements in a low-zinc, antibiotic-free diet on the growth performance, blood parameters, and intestinal health of weaned piglets. A total of 100 weaned piglets (7.90 ± 0.2 kg) were randomly assigned to five dietary treatments: ZnO, 0PEO, 30PEO, 60PEO, and 120PEO. Each treatment included four replicates of five piglets. The ZnO group received a basal diet (65 mg/kg Zn) supplemented with 1600 mg/kg zinc oxide from d 1 to 14 post-weaning. The 0PEO, 30PEO, 60PEO, and 120PEO groups received the basal diet with 0, 30, 60, and 120 mg/kg PEO, respectively. On d 28, one piglet per replicate with a body weight (BW) closest to the treatment average was selected and euthanized for sampling of duodenum, jejunum, and ileum tissue and colonic digesta (n = 4). Parameters included growth, serum indices, intestinal morphology, colonic volatile fatty acid (VFA), and colonic microbiota. Data were analyzed using SAS 9.4 (Cary, NC, USA). Polynomial contrasts were used to test the linear and quadratic effects of increasing PEO levels. A pre-planned contrast was used to compare the ZnO and 0PEO groups. Results showed a quadratic effect (P &amp;lt; 0.05) of PEO supplementation on hemoglobin concentration, with the predicted peak at 53 mg/kg. Villus height and the villus-to-crypt ratio in the duodenum, jejunum, and ileum increased linearly (P &amp;lt; 0.05) with PEO levels. Colonic VFA concentrations, including total VFAs, acetic, propionic, butyric, and valeric acids, were significantly affected by PEO in both linear and quadratic (P &amp;lt; 0.05) manners, with 52 mg/kg achieving total VFAs levels equivalent to the ZnO group. Additionally, colonic microbiota alpha diversity responded quadratically (P &amp;lt; 0.05) to PEO concentrations. In terms of growth performance, piglets in the 0PEO group had lower (P &amp;lt; 0.05) BW than those in the ZnO group by d 28. PEO supplementation showed a quadratic tendency (P = 0.055) on BW, with the highest estimated value at 69 mg/kg, comparable to that of the ZnO group. Although regression analyses identified slightly varying optimal PEO concentrations for different parameters, the 60 mg/kg level consistently improved multiple indicators. In conclusion, dietary supplementation with 60 mg/kg of PEO improved intestinal morphology, microbial fermentation, and growth performance, suggesting its potential as a natural alternative to pharmacological ZnO in antibiotic-free diets for weaned piglets.

To investigate the protective effect of paraoxonase 1 (PON1) gene against liver oxidative stress injury in mice due to dichlorvos poisoning. Experiment 1: 12 male Balb/c mice were randomly divided into three groups, with 4 mice in each group: control group, green fluorescent protein lentivirus control group (Lv-GFP group), and recombinant PON1 lentivirus group (Lv-PON1 group). 2 × 10⁷ TU of Lv-GFP or Lv-PON1 was transfected via tail vein, while normal saline was given to those in control group. Blood was collected on 0, 1, 3, 5, 7, 9 days via fundus venous plexus for the assay of serum PON1 activity. PON1 mRNA and protein expression levels were respectively determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot on the 3rd post-lentivirus transfection day. Experiment 2: according to the random number table method, another 96 male Balb/c mice were divided into four groups of 24 mice in each control group, dichlorvos group, Lv-GFP intervention group, and Lv-PON1 intervention group. Lv-GFP or Lv-PON1 was transfected via tail vein followed by intraperitoneal injection of dichlorvos 9 mg/kg, while those in control group were given normal saline. Six mice in each group were sacrificed respectively at 6, 12, 24, 48 hours, and liver tissue was collected. PON1 mRNA and nuclear factor E2-related factor 2 (Nrf2) mRNA expression levels were determined by RT-PCR, and PON1 protein level was determined by Western Blot. The content of malondialdehyde (MDA) and glutathione (GSH) in the liver tissue were determined by chemical colorimetry. The activity of superoxide dismutase (SOD) and catalase (CAT) were measured by double antibody sandwich enzyme linked immunosorbent assay (ELISA). Experiment 1: after Lv-PON1 was transfected to normal mice, PON1 activity in serum gradually increased and maintained a high level on 3rd day, while that of the control group and Lv-GFP group showed a normal low level. On the 3rd post-lentivirus transfection day, PON1 mRNA and PON1 protein expressions in liver were significantly higher than those of control group and Lv-GFP group. Experiment 2: compared with control group, the mice in dichlorvos group showed significant decreases in PON1 mRNA, PON1 protein, Nrf2 mRNA as well as GSH, SOD, CAT levels at 6 hours [PON1 mRNA (gray value): 0.237 ± 0.075 vs. 0.674 ± 0.011, PON1 protein (gray value): 0.602 ± 0.086 vs. 0.998 ± 0.124, Nrf2 mRNA (gray value): 0.089 ± 0.012 vs. 0.126 ± 0.010, GSH (mg/g): 3.84 ± 0.33 vs. 5.52 ± 0.40, SOD (μg/g): 0.383 ± 0.040 vs. 0.564 ± 0.052, CAT (ng/g): 7.32 ± 1.28 vs. 12.46 ± 1.54, all P < 0.05 ], and remarkable increase in MDA content (nmol/g: 7.78 ± 0.41 vs. 2.34 ± 0.25, P < 0.05). With the extension of time, PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels gradually increased, MDA content gradually decreased, Nrf2 mRNA expression level had risen to the level of control group at 24 hours (0.133 ± 0.019 vs. 0.126 ± 0.009, P > 0.05), and it was higher than that of the control group at 48 hours ( 0.206 ± 0.028 vs. 0.124 ± 0.010, P < 0.05). Compared with that of the dichlorvos group, Lv-PON1 intervention group showed a significant increase in PON1 mRNA, PON1 protein, Nrf2 mRNA and GSH, SOD, CAT levels [PON1 mRNA (gray value): 0.726 ± 0.021 vs. 0.237 ± 0.075, PON1 protein (gray value): 0.739 ± 0.050 vs. 0.602 ± 0.086, Nrf2 mRNA (gray value): 0.158 ± 0.007 vs. 0.089 ± 0.012, GSH (mg/g): 4.30 ± 0.22 vs. 3.84 ± 0.33, SOD ( μg/g): 0.454 ± 0.062 vs. 0.383 ± 0.040, CAT (ng/g): 8.98 ± 1.02 vs. 7.32 ± 1.28, all P < 0.05 ], and a decrease in MDA content (nmol/g: 6.56 ± 0.44 vs. 7.78 ± 0.41, P < 0.05). Regulation of PON1 gene can reduce MDA content, enhance SOD and CAT activities, increase GSH content, and it may also up-regulate Nrf2 mRNA expression to play a protective effect against oxidative stress of liver injury induced by dichlorvos poisoning.