Effects of dietary zinc/copper ratios on the metabolism of zinc and copper in weaned pigs.

This study compared the effects of different dietary zinc/copper ratios on zinc (Zn) and copper (Cu) metabolism in weaned pigs. One hundred and sixty piglets (7.81 ±â€…0.25 kg; 21 d of age) were used in a completely randomized 2 × 2 factorial design composed with high (H) and low (L) levels of added dietary Zn (100 and 3,000 mg/kg) and dietary Cu (6 and 130 mg/kg). Piglets were slaughtered at 21, 28, 35, and 42 d of age for blood and tissues collection. Serum, jejunum mucosa, liver, and kidney concentrations of Zn and Cu were analyzed as well as tissues mRNA abundance of genes related to their metabolism. Serum and liver Zn concentrations increased at days 28, 35, and 42 in HZn groups compared to pre-treatment levels (day 21; P ≤ 0.01) but for LZn animals, values decreased at days 28, 35, and 42 in liver (P ≤ 0.01) but remained stable vs. day 21 levels in serum (P ≥ 0.37). Serum, jejunum mucosa, liver, and kidney Zn concentrations were greater in HZn groups from day 28 (P ≤ 0.01). In jejunum mucosa, the mRNA expression of ZIP4 was lower in HZn piglets at day 28 (P ≤ 0.01) and at day 42 whereas HCu supplementation increased ZIP4 expression in LZn but not in HZn diets (P = 0.05). For ZNT1, MT3, and MT1, values of relative mRNA expression were greater for HZn animals in jejunum mucosa, liver, and kidney (P ≤ 0.01) from day 28. In kidney (P < 0.01) at day 42, HZn supplementation increased MTs expression in both LCu or HCu groups. Serum and liver Cu concentrations decreased at days 35 and 42 in all treatments compared to day 21 (P ≤ 0.04), except LZnHCu in liver that was not different from day 21 (P ≥ 0.17). Serum Cu concentrations were lower in HZn and greater in HCu groups at days 35 and 42 (P ≤ 0.01) whereas hepatic Cu was reduced by HZn diets in both LCu and HCu groups at days 35 and 42 (P ≤ 0.01). Jejunum Cu concentrations were increased by HCu diets in HZn but not in LZn groups at days 28 and 42 (P ≤ 0.04). Renal Cu concentrations were greater in HZn groups at day 28 (P < 0.01) whereas at day 42 HZn diets increased Cu values in both LCu and HCu groups (P ≤ 0.01). The expression of ATP7A in kidney at day 42 was greater for HZn groups (P = 0.02). In conclusion, high dietary Zn levels were not efficiently regulated by homeostatic mechanisms and significantly impaired Cu homeostasis. Low dietary Zn/Cu ratios allow a more efficient regulation of the metabolism of these trace minerals in post-weaning piglets. The current official recommendations for Zn and Cu to post-weaning piglets apparently do not fulfill their requirements.

Zinc oxide and copper sulfate are commonly used as growth promoters and alternatives to antibiotics to prevent diarrhea in weaned piglets but their use in post-weaning pigs diets has been challenged due to environmental issues and concerns related to bacterial resistance to antibiotics and heavy metals. Recently, it was reported that high dietary zinc levels interfere with copper status and may be detrimental to post-weaning piglets' health. In fact, the optimal dietary zinc/copper ratios need to be determined. Therefore, this experiment was conducted to evaluate the effects of different dietary zinc/copper ratios (3,000/130, 3,000/6, 100/130, and 100/6 mg/kg) on zinc and copper metabolism in weaned piglets. This study demonstrated that high dietary zinc/copper ratios impaired zinc and copper homeostasis but also that 100 mg/kg of dietary zinc and 6 mg/kg of dietary copper are apparently not sufficient to fulfill the piglets' requirements during the first weeks post-weaning.

Effects of dietary zinc oxide levels on the metabolism of zinc and copper in weaned pigs.

This study compared different dietary zinc oxide (ZnO) levels on zinc (Zn) and copper (Cu) metabolism in weaned pigs. One hundred twenty weaned piglets (7.96 ± 1.17 kg; 21 d of age) were used in a completely randomized 3 × 4 factorial design composed with three levels of dietary ZnO at 100 (100Zn), 1,000 (1,000Zn), or 3,000 mg/kg (3,000Zn) and four ages at slaughter at 21 (day 21), 23 (day 23), 35 (day 35), and 42 d (day 42). Dietary Cu levels were constant at 130 mg/kg. Serum, jejunum, liver, and kidney levels of Zn and Cu as well as mRNA abundance of genes related to Zn and Cu metabolism were analyzed. Zinc levels were greatest in 3,000Zn piglets from day 35 in all tissues (P ≤ 0.01). In 3,000Zn piglets, mRNA expression of ZIP4 was reduced in jejunum whereas ZnT1 and MT3 were stimulated in jejunum and liver and MT1 in kidney (P ≤ 0.04) from day 35. Copper levels were greatest in jejunum (P = 0.06) and kidney (P ≤ 0.01; days 35 and 42 only) and lowest in liver and serum (P ≤ 0.01) of 3,000Zn piglets. In conclusion, the treatment containing 3,000 mg ZnO/kg triggered Zn homeostatic mechanisms in weaned pigs and impaired Cu metabolism through high enterocyte and kidney Cu sequestration.

Zinc oxide (ZnO) is commonly used in post-weaning pig diets as growth promoter alternative to antibiotics to prevent diarrhea. The use of supranutritional levels of ZnO in post-weaning pigs diets has been challenged due to environmental issues and concerns related to bacterial resistance to antibiotics and heavy metals. However, the limited knowledge of the consequences of high levels of dietary ZnO on the metabolism of trace minerals has hampered advances to replace this nutritional strategy without compromising piglets health. Therefore, this experiment was conducted to evaluate the effects of increasing levels of dietary ZnO (i.e., 100, 1,000, and 3,000 mg/kg) on Zn and Cu metabolism in weaned piglets. In this experiment, it was demonstrated that systemic Zn levels were not effectively regulated with supplementation levels at 3,000 mg of ZnO/kg of diet. In addition, this level of dietary ZnO increased the intestinal intracellular sequestration of Cu and impaired its renal reabsorption, negatively impacting hepatic, and systemic serum Cu concentrations. These results emphasize the potential risk of Cu deficiency under long-term supranutritional supplementation of dietary ZnO during the post-weaning period, with potentially detrimental impacts on piglets growth.

Weaning differentially affects mitochondrial function, oxidative stress, inflammation and apoptosis in normal and low birth weight piglets.

Weaning is associated with increased occurrence of infections and diseases in piglets. Recent findings indicate that weaning induces mitochondrial dysfunction and oxidative stress conditions that more severely impact smaller piglets. The objective of this study was to characterize the molecular mechanisms underlying these physiological consequences and the relation with systemic inflammatory status in both normal and low birth weight (NBW and LBW) piglets throughout the peri-weaning period. To conduct the study, 30 sows were inseminated, and specific piglets from their litters were assigned to one of two experimental groups: NBW (n = 60, 1.73 ± 0.01 kg,) and LBW piglets weighing less than 1.2 kg (n = 60, 1.01 ± 0.01 kg). Then, 10 piglets from each group were selected at 14, 21 (weaning), 23, 25, 29 and 35 days of age to collect organ and plasma samples. Specific porcine RT2 Profiler™ PCR Arrays related to mitochondrial function, oxidative stress, inflammation and apoptosis processes were first used to target genes that are modulated after weaning in NBW piglets (d 23 and d 35 vs. d 14). Expression of selected genes was evaluated by quantitative PCR. These analyses revealed that expression of inflammatory genes CXCL10 and CCL19 increased after weaning in intestinal mucosa, while expression of genes encoding subunits of the mitochondrial respiratory chain was downregulated in liver and kidney of both groups. Interestingly, major modulators of mitophagy (BNIP3), cell survival (BCL2A1) and antioxidant defense system (TXNRD2, GPx3, HMOX1) were found to be highly expressed in NBW piglets. The systemic levels of TNF-α and IL1-ß significantly increased following weaning and were higher in NBW piglets. These results provide novel information about the molecular origin of mitochondrial dysfunction and oxidative stress observed in weaned piglets and suggest that clearance of dysfunctional mitochondria, antioxidant defenses and inflammatory response are compromised in LBW piglets.

Piglet weight gain during the first two weeks of lactation influences the immune system development.

The aim of this study was to investigate the effect of the piglet growth during the first week of life on ileal expression of genes and on development of the immune system. Eight litters adjusted to 12 piglets were used. Within each litter, the piglet that showed the lowest weight gain (LWG; n = 8) and the one that showed the highest weight gain (HWG; n = 8) in their first week of life were enrolled. Peripheral blood mononuclear cells (PBMC) were isolated on days 8 and 16 to characterize cellular population profiles and to assess ex-vivo secretion of interleukin-10 (IL-10), IL-6 and tumor necrosis factor-α (TNF-α). On day 16, piglets were euthanized and ileum samples were collected to extract RNA for microarray analysis and gene expression by qPCR. As expected, growth performance of LWG piglet was impaired compared to HWG piglets (P < 0.05). From day 8 to 16, the percentage of CD21+ B cells significantly increased in blood of heavier HWG piglets while the percentage remained constant in smaller LWG piglets (P weight x day = 0.01). For the CD4+CD8α- Th cells, a marked increase was observed in LWG piglets from 8 to 16 days of age (P = 0.002) whereas no significant change occurred in HWG piglets. Percentages of CD14+ monocytes and other MHC-II+ cells were respectively higher and lower on day 8 compared to day 16 for both groups of piglets (P < 0.01). On day 8, LPS-activated PBMC from LWG piglets produced less IL-6 compared to HWG piglets (P < 0.05). Microarray analysis of gene expression in piglets' ileum tissue indicated that several genes involed in defense response and response to oxidative stress were modulated differently in LWG compared to HWG. Gene analysis by Q-PCR confirmed microarray results and revealed that IL-10, SOD1, NOS2, NOD2, TLR4, TLR9, CD40 and CD74 expressions were significantly decreased (P < 0.05) in LWG in comparison to HWG piglets, while MYD88 and NFkBiA showed a tendency to decrease (0.05 ≤ P < 0.07). These results suggest that birth weight and milk intake affect the growth performances and the development of immunity by modulating the expression of genes associated with immunity and oxidative stress in piglets' intestinal tissue, and by affecting the leukocyte populations involved in innate and cell-mediated immunity in nursing piglets. Therefore, impaired development of immune system in LWG piglets might have an impact on their resistance to infections later in life.

Impact of deoxynivalenol (DON) contaminated feed on intestinal integrity and immune response in swine.

This study was performed to characterize the influence of consuming DON naturally contaminated feeds on pig's intestinal immune defenses, antibody response and cellular immunity. Sixteen 4-week-old piglets were randomly allocated to two dietary treatments: control diet or diet contaminated with 3.5 mg DON/kg. At days 7 and 21, animals were immunized with ovalbumin (OVA). On day 42, intestinal samples were collected for measurement of gene expression involved in immune response, oxidative status and barrier function. Primary IgG antibody response to OVA was increased in pigs fed DON diet compared to control animals. In the ileum of pigs fed DON diet, claudin, occludin, and vimentin genes involved in integrity and barrier function were down-regulated compared to controls. Results also revealed that expression of two chemokines (IL-8, CXCL10), interferon-γ, and major antioxidant glutathione peroxidase 2 (GPX-2) were up-regulated whereas expression of genes encoding enzymatic antioxidants including GPX-3, GPX-4 and superoxide dismutase 3 (SOD-3) were down-regulated in pigs fed DON-contaminated diet. These results strongly suggest that ingestion of DON naturally contaminated feed impaired intestinal barrier and immunological functions by modulating expression of genes coding for proteins involved in tight junctions, tissue remodelling, inflammatory reaction, oxidative stress reaction and immune response.

Hypoxia regulates the expression of extracellular matrix associated proteins in equine dermal fibroblasts via HIF1.

BACKGROUND: Exuberant granulation tissue (EGT), a fibrotic healing disorder resembling the human keloid, occurs almost exclusively in limb wounds of horses and may be caused in part by a relative state of hypoxia within the wound. OBJECTIVE: The objectives of this study were therefore to (1) assess the effects of hypoxia on equine dermal fibroblast (EDF) proliferation and apoptosis, (2) study the effects of hypoxia on the expression of key extracellular matrix (ECM) associated proteins and determine if such effects are dependent on hypoxia-inducible factor (HIF), and (3) determine if EDFs from the body or limb respond differently to hypoxia. METHODS: EDFs were isolated and cultured from skin from body or limb under normoxic or hypoxic conditions for up to 7days. RESULTS: Hypoxia significantly stimulated EDF proliferation, but had no effect on cell survival. The hypoxia-mimetic agent CoCl(2) up-regulated COL1A1 expression and down-regulated MMP2 expression, suggesting an increase in ECM synthesis and a decrease in turnover. Both regulatory effects were inhibited by the addition of echinomycin, indicating that they are mediated by the transcriptional regulatory activity of HIF. No differences were observed between EDFs originating from body or limb for any effect of hypoxia or CoCl(2), suggesting that EGT development does not depend on intrinsic properties of limb fibroblasts. CONCLUSIONS: We conclude that hypoxia regulates ECM remodeling via HIF1 in EDFs, and that this may be an important determinant in the pathogenesis of equine EGT.

Constitutive expression of hypoxia-inducible factor-1 α in keratinocytes during the repair of skin wounds in horses.

As a transient hypoxic state exists within skin wounds in horses and may be important for the healing process, this study sought to identify a molecular hypoxia response occurring in horse limb and body wounds healing by second intention. Hypoxia-inducible factor 1α (HIF1α) protein expression was studied throughout repair by Western blotting and immunofluorescence. Paradoxically, HIF1α was strongly expressed in intact skin and its expression decreased dramatically following wounding (p<0.01), despite the expected hypoxic state within the wounded tissue. HIF1α levels reincreased in parallel with the epithelialization process, and more rapidly in body wounds than in limb wounds (p<0.01). HIF1α localized predominantly to the keratinocyte layer, in which it was constitutively expressed throughout healing. The HIF1α target gene cyclin-dependent kinase inhibitor 1A (CDKN1A) showed a pattern of expression similar to HIF1α throughout the healing process and also localized to the keratinocyte layer, suggesting that HIF1α may regulate its constitutive expression. The HIF1α target genes vascular endothelial growth factor A (VEGFA) and solute carrier family 2 (facilitated glucose transporter) member 1 (SLC2A1) however did not have a pattern of expression similar to HIF1α, at the mRNA level. We conclude that HIF1α is expressed in a continuous and hypoxia-independent manner in equine keratinocytes in both intact and wounded skin, and may regulate the expression of CDKN1A in this cell type.

Regional differences in wound oxygenation during normal healing in an equine model of cutaneous fibroproliferative disorder.

Wound repair in horse limbs is often complicated by the development of exuberant granulation tissue (EGT) and excessive scarring while body wounds tend to repair uneventfully. EGT resembles the human keloid. While the events leading to keloid formation are not fully elucidated, tissue hypoxia has been proposed as a major contributing factor. The objective of this study was to investigate tissue oxygen saturation in healing full-thickness wounds created on the horse limb and body, using near-infrared spectroscopy. Spectroscopic reflectance data were collected from both anatomic sites at specific times following wounding. The oxygen saturation values of limb wounds were significantly inferior to those of body wounds during the early period of healing, indicating a temporary, relative state of hypoxia in the former during the inflammatory phase of repair. Horses present a weak, persistent inflammatory response to wounding, especially at the limb level. The relative hypoxia present acutely in limb wounds of horses may promote a feeble yet prolonged inflammatory response, which could interfere with and retard the subsequent phases of healing. Ongoing low-grade inflammation in horse wounds is accompanied by up-regulation of various inflammatory and profibrotic mediators, which might ultimately promote the development of fibroproliferative disorders such as EGT.