Illicium verum and Eucommia ulmoides leaf extracts promote nutrient availability and antioxidant capacity in piglets by upregulating duodenal and jejunal Nrf2/TNF-α.

The effect of Illicium verum extracts (IVE) or Eucommia ulmoides leaf extracts (ELE) on nutrient availability, duodenal and jejunal antioxidant ability of Duroc ×Landrace × Yorkshire (DLY) and Chinese native Licha-black (LCB) piglets was investigated. Ninety-six piglets (48 DLY and 48 LCB respectively) without significant difference in body weight (11.22 ± 0.32 kg) were used in a 2 × 4 factorial design. Animals were randomly allocated to four treatments, and each had four replicates with three DLY and three LCB piglets. Treatments were basal diet (CON) and basal diet with 500 mg/kg IVE, 250 mg/kg ELE and 50 mg/kg chlortetracycline (CHL) respectively. Animals were placed individually for 7-days adaptation following 42-days test. Results showed the significant interaction (p < 0.05) between dietary treatments and pig species in activity of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), content of malondialdehyde (MDA), and α-tumour necrosis factor (TNF-α), nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2/TNF-α at mRNA and protein level in duodenum and jejunum of DLY and LCB piglets. The IVE and ELE increased (p < 0.05) activity of GSH-Px and T-SOD, and the Nrf2/TNF-α at mRNA and protein level, however, the decreased (p < 0.05) MDA content, and TNF-α at mRNA and protein level in duodenum and jejunum were observed. The CHL decreased (p < 0.05) activity of GSH-Px and T-SOD, TNF-α and Nrf2 at mRNA and protein level in duodenum, but increased (p < 0.05) MDA content and Nrf2/TNF-α in jejunum. DLY piglets had higher (p < 0.05) nutrient digestibility (organic matter, crude protein and gross energy), availability (biological value and net protein utilization), MDA content, and TNF-α at mRNA and protein level in jejunum, and had lower (p < 0.05) activity of GSH-Px and T-SOD, and Nrf2 and Nrf2/TNF-α at mRNA and protein level in duodenum and jejunum than LCB. In conclusion, the 500 mg/kg IVE and 250 mg/kg ELE improved the nutrient availability, and the improvement of antioxidant capacity is realized by activating the Nrf2/TNF-α of duodenum and jejunum. The CHL had adverse effects on antioxidant ability of DLY and LCB piglets. The results showed that the nutrient digestion and absorption capacity of DLY were stronger than that of LCB piglets, but the antioxidant capacity was lower than that of LCB piglets. Therefore, the IVE and ELE are recommended as a new potential alternative to antibiotics in piglets' diets.

Astaxanthin supplementation enriches productive performance, physiological and immunological responses in laying hens.

OBJECTIVE: Astaxanthin is a natural super antioxidant. The present study was carried out to investigate the effect of astaxanthin rich Phaffia rhodozyma (PR) supplementation in diets on laying production performance, egg quality, antioxidant defenses and immune defenses in laying hens. METHODS: A total of five hundred and twelve 60-week-old Lohmann Brown laying hens (2,243±12 g) were randomly assigned to four groups, each including 4 replicates with 32 birds per replicate. Astaxanthin rich PR was added to corn-soybean meal diets to produce experimental diets containing 0 (Control), 800 mg/kg, 1,200 mg/kg, and 1,600 mg/kg PR, respectively. The astaxanthin content in the diet was 0.96 mg/kg, 1.44 mg/kg and 1.92 mg/kg respectively. RESULTS: Results showed that dietary PR supplementation tended to increase daily feed intake (p = 0.0512). There was no effect of astaxanthin rich PR on Haugh units, albumen height, egg shape index, eggshell strength, and eggshell thickness at weeks 6 (p>0.05). However, egg yolk color was significantly improved (p<0.05). In addition, astaxanthin rich PR supplementation significantly increased serum glutathione peroxidase and superoxide dismutase activity (p<0.05), increased serum immunoglobulin G content (p<0.05), and reduced malondialdehyde content (p<0.05) in laying hens. CONCLUSION: In conclusion, astaxanthin rich PR can improve the color of egg yolk, enhance the antioxidant defenses, and regulate the immune function.

Illicium verum extracts and probiotics with added glucose oxidase promote antioxidant capacity through upregulating hepatic and jejunal Nrf2/Keap1 of weaned piglets.

Accumulating evidences indicate that plant extracts and probiotics are effective antioxidant substitutes which play important roles in animal production. However, the comparative study of the mechanism underlying the antioxidant property of Illicium verum extracts (IVE) and probiotics with added glucose oxidase (PGO) on piglets remains to be explored. This study evaluated the difference and the interaction effect of IVE and PGO on serum, liver, and jejunum antioxidant capacity of weaned piglets. A total of 32 weaned piglets (Duroc × Landrace × Yorkshire) at the age of 28 d with an average body weight of 14.96 ± 0.32 kg were randomly divided into four treatments with eight replicates per treatment in a 2 × 2 factorial arrangement. Treatments included basal diet (IVE-PGO-), basal diet + 1,000 mg/kg PGO (IVE-PGO+), basal diet + 500 mg/kg IVE (IVE+PGO-), and basal diet + 500 mg/kg IVE + 1,000 mg/kg PGO (IVE+PGO+). All the piglets were housed individually for the 42-d trial period after 7-d adaptation. The piglets were euthanized at the end of the experiment and the liver and jejunum samples were taken and subjected to immunohistochemistry, Western blotting, as well as antioxidant and qRT-PCR analysis. Significant interactions were observed between IVE and PGO for total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) in serum (42 d), liver, and jejunum; malondialdehyde (MDA) in serum (21 d); and mRNA and protein expression of kelch sample related protein-1 (Keap1) and nuclear factor erythroid-2 related factor (Nrf2)/Keap1 in the liver and jejunum (P < 0.05). Both IVE and PGO improved (P < 0.05) T-SOD and GSH-Px in the serum (42 d), liver, and jejunum, and the mRNA and protein expression of Nrf2 and Nrf2/Keap1 in the liver and jejunum, but decreased (P < 0.05) MDA in the serum (21 d) and the mRNA and protein expression of Keap1 in the liver and jejunum. Immunohistochemical results confirmed that IVE and PGO enhanced the positive reactions of Nrf2 but weakened Keap1 in both the liver and jejunum. In conclusion, the results confirmed that IVE (500 mg/kg) and PGO (1,000 mg/kg) can improve the antioxidant capacity of weaned piglets and that the interaction effect between IVE and PGO is significant. At the same time, the fact that IVE and PGO activate the Nrf2/Keap1 in the liver and jejunum signaling pathway suggests that they play an important role in the ameliorative antioxidant capacity of weaned piglets. Therefore, the combination of IVE and PGO could be recommended as a new potential alternative to antibiotics in piglets' diets.

Effects of zearalenone-induced oxidative stress and Keap1-Nrf2 signaling pathway-related gene expression in the ileum and mesenteric lymph nodes of post-weaning gilts.

Zearalenone (ZEA) contamination of feed affects animal husbandry and the human health. Currently, the molecular mechanism underlying small intestine-related diseases caused by ZEA-induced oxidative stress is not well understood. In this study, we aimed to identify the mechanisms involved in ZEA (0.5-1.5 mg/kg)-induced oxidative stress in the ileum and mesenteric lymph nodes (MLNs) and the role of the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in post-weaning gilts. Forty post-weaning gilts (Landrace × Yorkshire × Duroc) with an average body weight of 14.01 ± 0.86 kg were randomly allocated to four groups and fed a corn-soybean meal basal diet supplemented with < 0.1, 0.5, 1.0, or 1.5 mg/kg ZEA. The results showed that the activity of total superoxide dismutase and glutathione peroxidase decreased (p < 0.05) linearly and quadratically and that the content of malondialdehyde increased (p < 0.05) quadratically in the ileum and MLNs with increasing ZEA in the diet. Immunohistochemical analysis showed that the expression of Nrf2 and glutathione peroxidase 1 (Gpx1) immunoreactive proteins in the ileum and MLNs were significantly enhanced with increasing ZEA. The relative mRNA and protein expression of Nrf2, Gpx1, quinone oxidoreductase 1 (Nqo1), hemeoxygenase 1 (Ho1), modifier subunit of glutamate-cysteine ligase (Gclm), and catalytic subunit of glutamate-cysteine ligase (Gclc) increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Keap1 decreased (p < 0.05) linearly and quadratically in the ileum with increasing ZEA concentrations in the diet. Further, the relative mRNA and protein expression of Nrf2 and Gpx1 increased (p < 0.05) linearly and quadratically, and the relative mRNA and protein expression of Nqo1, Ho1, and Gclm decreased (p < 0.05) quadratically in the MLNs as ZEA concentrations increased in the diet. Our results provide valuable genetic information on ZEA-induced oxidative stress in the ileum and MLNs of post-weaning gilts and have elucidated the key regulatory genes involved in the Keap1-Nrf2 signaling pathway. Results indicated that the Keap1-Nrf2 signaling pathway might be a key target to further prevent and treat ZEA-induced injury to the ileum in post-weaning gilts.

Effect of Illicium verum or Eucommia ulmoides leaf extracts on the anti-stress ability, and mRNA and protein expression of Nrf2 and TNF-α in Duroc × Landrace × Yorkshire and Chinese native Licha-black nursery piglets.

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 ± 0.32 kg 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 + 500 mg/kg IVE, basal diet + 250 mg/kg ELE and basal diet + 50 mg/kg chlortetracycline (CHL). All piglets were housed individually for the 42 days trial period after 7 days 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 (500 mg/kg) and Eucommia ulmoides leaf (250 mg/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.

Comparative study of stress response, growth and development of uteri in post-weaning gilts challenged with zearalenone and estradiol benzoate.

The objective of this study was to evaluate the effects of zearalenone (ZEA) and estradiol benzoate (EB) on stress injury and uterine development in post-weaning gilts. Thirty healthy post-weaning female gilts (Duroc × Landrace × Large White) aged 28-32 days were randomly allocated to three treatments as follows: (a) basal diet (Control), (b) basal diet plus 1.0 mg/kg purified ZEA (ZEA) and (c) basal diet plus 0.75 ml (1.5 mg) EB per pig at 3-days intervals by intramuscular injection (EB). The serum estradiol (E2 ), the final and the increased vulvar area, uterine index, thickness of the myometrium and endometrium, and protein expression of heat shock protein 70 (HSP70) in ZEA group were higher than those in the control group (p < .05), but lower than those in the EB group (p < .05). The serum luteinizing hormone in ZEA group was lower than that of the control group (p < .05), but higher than that in the EB group (p < .05). Higher serum follicle-stimulating hormone and progesterone were observed in the ZEA and control groups than those in the EB group (p < .05). The serum glutathione peroxidase activity in the ZEA group was lower than that in the control and EB groups (p < .001), and the malondialdehyde in the ZEA group was higher than that in the control and EB groups (p < .001). Moreover, the relative mRNA and protein expression of growth hormone receptor (GHR) and relative mRNA expression of HSP70 in the ZEA and EB groups were higher than those in the control group (p < .05). In conclusion, both ZEA (1.0 mg/kg) and EB (1.5 mg at 3 days intervals by intramuscular injection) stimulated vulvar swelling and uterine hypertrophy by disordering serum hormones and up-regulating GHR expression, and induced stress by different mechanisms in this study. Furthermore, the observed up-regulating HSP70 expression challenged by ZEA or EB may be part of the mechanism to resist stress injury.

Zearalenone induced oxidative stress in the jejunum in postweaning gilts through modulation of the Keap1-Nrf2 signaling pathway and relevant genes1.

Researches have shown that dietary zearalenone (ZEA) caused oxidative stress in the liver and reproductive organs of postweaning gilts. However, information on the effects of ZEA on oxidative stress of the small intestine in the piglets is limited. The objective of this study was to determine the effects of ZEA exposure on oxidative stress, the Kelch-like erythroid cell-derived protein with CNC homology (ECH)-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and on immunohistochemistry of the jejunum in postweaning gilts. A 35-d feeding experiment using 40 postweaning gilts (Landrace × Yorkshire × Duroc) with an average BW of 14.01 ± 0.86 kg in 4 groups fed corn-soybean meal-based diets containing 0, 0.5, 1.0, and 1.5 mg ZEA/kg was conducted. The jejunum was obtained at the end of the experiment and used for analyses. The results showed that the activities of total superoxide dismutase and glutathione peroxidase and the relative expressions of Keap1 mRNA and protein in the jejunum linearly and quadratically decreased (P < 0.05) with increasing concentrations of ZEA in the diets. The malondialdehyde content, the integrated optical density of Nrf2 and glutathione peroxidase 1 (GPX1), and the relative expressions of Nrf2, GPX1, quinone oxidoreductase 1 (NQO1), and modifier subunit of glutamate-cysteine ligase (GCLM) mRNA and proteins linearly and quadratically increased (P < 0.05) with increasing levels of ZEA. Immunohistochemical analysis showed that Nrf2 and GPX1 immunoreactivity was enhanced by the ZEA treatments, and block localization of yellow and brown immunoreactive substances in the jejunum was observed with increasing levels of ZEA. The results suggest that ingested ZEA induced oxidative stress in the jejunum in postweaning gilts through upregulation of the Keap1-Nrf2 signaling pathway and downstream target genes NQO1, HO1, and GCLM, indicating the important role of the Keap1-Nrf2 signaling pathway in oxidative stress induced by ZEA in the jejunum of the postweaning piglets.

Zearalenone-Promoted Follicle Growth through Modulation of Wnt-1/ß-Catenin Signaling Pathway and Expression of Estrogen Receptor Genes in Ovaries of Postweaning Piglets.

Feedstuffs are severely contaminated by zearalenone (ZEA) worldwide. A specific dietary level of ZEA could cause malformations of the reproductive organs of sows, false estrus, decreased litter size, and abortion. However, the underlying mechanisms are still not clear. The objectives of the present study were to assess the effects of ZEA on morphology, distribution, and expression of estrogen receptors (ERα and ERß) in the ovaries of postweaning piglets. Furthermore, the relationship between ERs/glycogen synthase kinase (GSK)-3ß-dependent pathways mediated by ZEA and the Wnt-1/ß-catenin signaling pathway was examined. Forty healthy weaning piglets were allocated to the following four treatment groups: piglets fed with basal diet only (control), and ZEA0.5, ZEA1.0, and ZEA1.5, which were fed basal diets supplemented with ZEA at 0.5, 1.0, and 1.5 mg·kg-1, respectively. Then, the expression of GSK-3ß, ERα, ERß, and Wnt-1/ß-catenin were examined histomorphologically and immunohistochemically. Results showed that the proportion of primordial follicles (PrF's) decreased ( p < 0.001) but that of atretic primordial follicles (APFs) increased ( p < 0.001) with increasing dietary ZEA levels. More interestingly, the immunopositivity of ERß in the ovaries was stronger than that of ERα with the same treatment. The relative mRNA and protein expression levels of ERα, ERß, Wnt-1, ß-catenin, and GSK-3ß in the ovaries of postweaning gilts increased linearly ( p < 0.05) as dietary ZEA concentrations increased. Moreover, the accumulation of Wnt-1 and ß-catenin in the ovaries indicated that ZEA activated the Wnt-1/ß-catenin pathway, mediated by ERs/GSK-3ß. Our results strongly suggested that ovarian follicles in the ZEA (0.5-1.5 mg·kg-1)-treated groups were highly proliferative state, indicating that ZEA promoted ovarian development. The results also suggested that ZEA activates the ERs/GSK-3ß-dependent Wnt-1/ß-catenin signaling pathway, indicating its important role in accelerating development of the ovaries.