Proteomic analysis reveals altered expression of proteins related to glutathione metabolism and apoptosis in the small intestine of zinc oxide-supplemented piglets

Zinc is an important dietary factor that regulates intestinal amino acid and protein metabolism in animals. Recent work with the piglet, an established animal model for studying human infant nutrition, has shown that supplementing high levels of zinc oxide (ZnO) to the diet ameliorates weaning-associated intestinal injury and growth retardation. However, the underlying mechanisms are largely unknown. This study tested the hypothesis that zinc supplementation affects expression of proteins related to glutathione metabolism and oxidative stress in the gut. Using two-dimensional gel electrophoresis and mass spectrometry, we identified 22 up-regulated and 19 down-regulated protein spots in the jejunum of weanling piglets supplemented with ZnO (3,000 mg/kg Zn) compared with the control pigs (100 mg/kg Zn). These proteins are related to energy metabolism (increased level for succinyl-CoA transferase and decreased level for creatine kinase M-type); oxidative stress (decreased levels for 78 kDa glucose-regulated protein and glutathione-S-transferase-ω); and cell proliferation and apoptosis (increased levels for A-Raf-1 and calregulin). Consistent with the changes in protein expression, the ratio of reduced glutathione to oxidized glutathione was increased, whereas glutathione-S-transferase and glutathione peroxidase activities as well as the protein level of active caspase-3 were reduced in ZnO-supplemented piglets. Collectively, these results indicate that ZnO supplementation improves the redox state and prevents apoptosis in the jejunum of weaning piglets, thereby alleviating weaning-associated intestinal dysfunction and malabsorption of nutrients (including amino acids).     

Effect of excessive dietary fluoride on nutrient digestibility and retention of iron, copper, zinc, and manganese in growing pigs

Ninety-six crossbred growing pigs were used to evaluate the effects of fluoride levels on growth performance, nutrient digestibility, and the retention of minerals in tissues. Four dietary treatments were formulated by supplementing fluorine (as NaF) to a corn-soybean basal diet (39.75 mg/kg F) to provide the following added fluorine levels: 0, 50 100, and 150 mg/kg fluorine. The results showed pigs of the 100 and 150 mg/kg fluorine-added groups had decreased average daily gain (ADG) and increased feed gain ratio (F/G) compared to the control (p<0.05). Apparent digestibility of protein and calcium in 100 and 150 mg/kg fluorinetreated groups was significantly lower than that of the control (p<0.05). On the other hand, iron, copper, zinc, and manganese levels in most tissues of the 100 and 150 mg/kg fluorine groups were markedly changed compared to the control (p<0.05). However, growth performance, nutrient digestibility, and mineral concentrations in all tissues of pigs were not significantly affected by the addition of 50 mg/kg fluorine (p>0.05). Thus, this study suggested that excess fluoride levels could decrease growth performance and change the retention of iron, copper, zinc, and manganese in pigs.     

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.     

Zinc attenuates forskolin-stimulated electrolyte secretion without involvement of the enteric nervous system in small intestinal epithelium from weaned piglets

In a previous study, we found that secretagogue-stimulated electrolyte secretion was attenuated by dietary and serosal zinc in piglet small intestinal epithelium in Ussing chambers. Several studies show that the enteric nervous system (ENS) is involved in regulation of electrolyte and/or fluid transport in intestinal epithelium from many species. The aim of the present study is to examine the mechanisms behind the attenuating effect of zinc on electrolyte secretion and to study whether the ENS is involved in this effect of zinc in vitro. Twenty-four piglets (six litters of four piglets) were allocated randomly to one of two dietary treatments consisting of a basic diet supplemented with 100 mg zinc/kg (Zn(100)) or 2500 mg zinc/kg (Zn(2500)), as ZnO. All the piglets were killed at 5-6 days after weaning and in vitro experiments with small intestinal epithelium in Ussing chambers were carried out. Furthermore, zinc, copper, alkaline phosphatase (AP) and metallothionein (MT) in mucosa, liver, and plasma were measured. These measurements showed that zinc status was increased in the Zn(2500) compared to the Zn(100) fed piglets. The in vitro studies did not confirm previous findings of attenuating effects of dietary zinc and zinc in vitro on the 5-HT induced secretion. But it showed that the addition of zinc at the serosal side attenuated the forskolin (FSK) (cAMP-dependent) induced ion secretion in epithelium from piglets fed with Zn(100) diet. Blocking the ENS with lidocaine or hexamethonium apparently slightly reduced this effect of zinc in vitro, but did not remove the effect of zinc. Consequently, it is suggested that zinc attenuates the cAMP dependent ion secretion mainly due to an effect on epithelial cells rather than affecting the mucosal neuronal pathway.     

Dietary supplementation with zinc oxide decreases expression of the stem cell factor in the small intestine of weanling pigs

Dietary supplementation with a high level of zinc oxide (ZnO) has been shown to reduce the incidence of diarrhea in weanling pigs, but the underlying mechanisms remain largely unknown. Intestinal–mucosal mast cells, whose maturation and proliferation is under the control of the stem cell factor (SCF), play an important role in the etiology of diarrhea by releasing histamine. The present study was conducted to test the novel hypothesis that supplementing ZnO to the diet for weanling piglets may inhibit SCF expression in the small intestine, thereby reducing the number of mast cells, histamine release, and diarrhea. In Experiment 1, 32 piglets (28 days of age) were weaned and fed diets containing 100 or 3000 mg zinc/kg (as ZnO) for 10 days (16 piglets per group). In Experiment 2, two groups of 28-day-old piglets (8 piglets per group) were fed the 100- or 3000-mg zinc/kg diet as in Experiment 1, except that they were pair-fed the same amounts of feed. Supplementation with a high level of ZnO reduced the incidence of diarrhea in weanling piglets. Dietary Zn supplementation reduced expression of the SCF gene at both mRNA and protein levels, the number of mast cells in the mucosa and submucosa of the small intestine and histamine release from mucosal mast cells. Collectively, our results indicate that dietary supplementation with ZnO inhibits SCF expression in the small intestine, leading to reductions in the number of mast cells and histamine release. These findings may have important implications for the prevention of weaning-associated diarrhea in piglets.     

Effects of dietary supplementation with grape seed procyanidins on nutrient utilisation and gut function in weaned piglets

Grape seed procyanidins (GSPs), widely known for their beneficial health properties, fail to bring about the expected improvement in piglets’ growth performance. The effects of dietary supplementation with GSPs on nutrient utilisation may be a critical influencing factor. Hence, the purpose of this study was to investigate the effects of dietary supplementation with GSPs on nutrient utilisation and gut function in weaned piglets. One hundred and twenty crossbred piglets were allocated randomly to four treatment groups, with three replicate pens per treatment and 10 piglets per pen. Each group was given one of the four dietary treatments: the basal diet (control group) or the basal diet with the addition of 50-, 100- or 150-mg/kg GSPs. The trial lasted 28 days. Faeces were collected from d 12 to 14 and from d 26 to 28 for measuring the coefficient of total tract apparent digestibility (CTTAD) of the nutrients. Blood samples were collected on d 14 and 28 for detecting the blood biochemical parameters. Two piglets per pen were slaughtered to collect the pancreas and intestinal digesta for evaluating the digestive enzyme activity and the coefficient of ileal apparent digestibility (CIAD) of the nutrients. On d 14 and 28, supplementation with 150-mg/kg GSPs significantly decreased the CTTAD of DM and CP in piglets. On d 14, GSPs supplementation at a concentration of 150 mg/kg led to a remarkable decrease in the CIAD of CP and gross energy (GE). On d 28, GSPs supplementation at a dose of 150 mg/kg generated a marked decline in the CIAD of DM, GE, CP and ether extract. Grape seed procyanidins supplementation at concentrations of 100 or 150 mg/kg inhibited the activities of lipase and amylase. In contrast, the jejunum mucosa maltase and sucrase activities increased due to the inclusion of GSPs at a concentration of 100 mg/kg in the piglet diet. Compared with the levels of the control group, the serum glucose and total protein levels were enhanced significantly by supplementation with GSPs at 100 mg/kg and reduced dramatically at 150 mg/kg. The serum diamine oxidase activity and endotoxin levels were decreased by GSPs supplementation in piglet diets. In conclusion, higher concentrations of GSPs in weaned piglet diets attenuated nutrient digestion and inhibited digestive enzyme activity; however, suitable concentrations of GSPs could promote brush-border enzyme activity, enhance serum glucose and total protein concentrations and decrease epithelial permeability.     

Large doses of zinc oxide increases the activity of hydrolases in rats

The effect of pharmacological doses of zinc oxide (1000; 2500; 5000 mg per kg diet) and two levels of dietary protein on pancreatic and intestinal hydrolase activity in rats were studied. It was hypothesized that ZnO would increase intestinal and pancreatic hydrolase enzyme activity. Male Wistar rats, averaging 64 g body weight, were randomly allocated to dietary treatments (chow diets- meeting all NRC requirements) containing 10% or 15% protein supplemented with additional ZnO (above 100 mg/kg ZnSO(4)) as follows: 0.0; 0.1; 0.25; 0.5% w/w. Water and food were provided ad libitum. Animals were fed the diets for 10 days and body weights were recorded; after decapitation blood and organ samples were collected. Amylase, lipase, trypsin, and total protease activity of pancreatic homogenates and small intestinal contents were determined. ZnO supplementation dose dependently increased the plasma Zn concentration and significantly increased amylase, lipase, trypsin and total protease activity in pancreatic homogenates and small intestinal contents. The statistical analysis showed significant protein and ZnO interaction on the activity of amylase in the pancreas, and amylase, trypsin and total-protease in the small intestinal content. Therefore ZnO at high dietary concentration may influence the digestion of nutrients via increased hydrolase activity.     

Dietary Zinc Glycine Chelate on Growth Performance,Tissue Mineral Concentrations,and Serum Enzyme Activity in Weanling Piglets

One hundred twenty crossbred piglets (Duroc × Landrace × Yorkshire) were used to determine the effects of dietary zinc glycine chelate on growth performance, tissue mineral concentrations, and serum enzyme activity. All pigs were allotted to four treatments and fed with basal diets supplemented with 0, 50, and 100 mg/kg Zn as zinc glycine chelate or 3,000 mg/kg Zn as zinc oxide (ZnO). After the 35-day feeding trial, results of the study showed that, compared to the control, average daily gain was improved (P < 0.05) for pigs fed 100 mg/kg Zn from zinc glycine chelate or 3,000 mg/kg Zn from ZnO and Zn concentrations in serum and M. longissimus dorsi were significantly enhanced by 100 mg/kg dietary zinc glycine chelate and 3,000 mg/kg ZnO. In addition, supplementation of 100 mg/kg zinc glycine chelate decreased (P < 0.05) the liver Fe level, liver Zn level, spleen Cu level, and kidney Cu level compared to that of the 3,000-mg/kg ZnO group. For feces mineral excretion, 3,000 mg/kg Zn from ZnO greatly increased the concentration of fecal Zn (P < 0.01) and Mn (P < 0.05) compared to that of the control or the 100-mg/kg zinc glycine chelate group. Moreover, alkaline phosphatase and Cu/Zn superoxide dismutase activities of pigs in 100 mg/kg zinc glycine chelate and ZnO treatments were greatly higher than that of the control. The results of present study showed that supplementation with zinc glycine chelate could improve growth and serum enzyme activities and could also decrease zinc excretion in feces in weanling pig compared to high dietary ZnO.     

Evaluation of the effects of pharmacological zinc oxide and phosphorus source on weaned piglet growth performance,plasma minerals and mineral digestibility

Crossbred pigs (n=720; average age=28±3 days and weight=9.5±0.3 kg) were used in a 20-day trial in order to determine the influence of phosphorus (P) source and various doses of pharmacological zinc (Zn) on growth performance, plasma minerals and mineral digestibility. Pigs (five intact males and five females per pen) were randomly allotted to treatments in a 3×3 factorial arrangement with three sources of dietary P (4.5 g/kg digestible P, 4.5 g/kg digestible P plus 2500 phytase units (FTU)/kg, or 5.5 g/kg digestible P) and three dietary levels of supplemental Zn (0, 1750 or 3500 mg/kg) from ZnO (82% Zn) with eight pens per treatment. Diets were formulated to exceed all nutrient requirements, including calcium (Ca), P and Zn from day 0 to 20. Zn supplementation increased (quadratic P<0.05) average daily feed intake. There was a significant Zn level×P source interaction on average daily gain and feed conversion ratio (FCR). Pigs fed 4.5 g/kg digestible P without or with 2500 FTU/kg phytase gained more per day (quadratic P<0.05) and had better FCR (quadratic P<0.05) when they were fed 1750 mg/kg supplemental Zn. However, pigs fed 5.5 g/kg digestible P gained more per day (linear P<0.05) and were more efficient (linear P<0.05) when they were fed 3500 mg/kg supplemental Zn. Plasma Zn and Zn digestibility increased (linear P<0.05) as pharmacological Zn supplementation increased from 0 to 3500 mg/kg, irrespective of P source. However, Ca, P, sodium (Na), potassium (K) and copper (Cu) digestibility were reduced (P<0.05) as pharmacological Zn supplementation increased, and this was mitigated or exacerbated by the supplementation of 5.5 g/kg digestible P or phytase. In conclusion, increasing the dietary inclusion of pharmacological Zn may impact growth performance in young pigs through the interaction with minerals such as Ca, P, Na and K. Pharmacological Zn may reduce Na or K digestibility and indirectly reduce water secretion into the lumen, resulting in an increase in faecal dry matter as pharmacological Zn supplementation in the diet increased.     

Effect of avilamycin and tylosin on the metabolizable energy in growing and finishing pigs

In two metabolism trials with growing and finishing pigs the influence of the antibiotic feed additives Avilamycin and Tylosin on the metabolizable energy was investigated at different levels of dietary protein content. In the first experiment (growing pigs) the antibiotics were supplied at levels of 0 mg/kg, 40 mg/kg Avilamycin and 40 mg/kg Tylosin to diets containing 18.5%, 17.5%, 16.5% and 14.0% of crude protein. In the second experiment (finishing pigs) 0 mg/kg antibiotics, 20 mg/kg Avilamycin and 20 mg/kg Tylosin were used in diets containing 16.5% and 14.0% of crude protein. The body weight of the animals averaged 46 kg (growing pigs) and 68 kg (finishing pigs).

In growing pigs the supplementation of Tylosin increased the digestibility of dry matter and energy at 1 percentage unit each, while in finishing pigs no effects were observed. Since the urinary energy excretion was not affected by antibiotics, there was only in Tylosin treated growing pigs a slight rise in dietary contents of metabolizable energy by 1.6%. The reduction of the dietary protein content resulted in increased digestibility of dry matter and energy, reduced urinary energy excretions and increased dietary contents of metabolizable energy.     

Effect of dietary bovine lactoferrin on performance and antioxidant status of piglets

The experiment investigated the effect of dietary bovine lactoferrin (bLF) on growth performance and antioxidant systems of piglets. Duroc–Landrace–Yorkshire crossbred female piglets (n = 120, 35 days of age, live-weight 9.70 ± 0.71 kg) were fed a diet containing 0, 1250 or 2500 mg/kg bLF for 30 days. After completion of the feeding experiment, eight animals from each treatment were randomly selected to determine malondialdehyde (MDA) and total antioxidant capacity (TAOC), copper–zinc–superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx), catalase (CAT) activities in serum and longissimus muscle, and CuZnSOD, GPx and CAT mRNA levels in longissimus muscle. Results showed that supplementation with bLF improved average daily gain (ADG), average daily feed intake (ADFI) and reduced diarrhea ratios of piglets with the linear and quadratic polynomial contrasts being significant (P<0.01). The highest weight gain and the best feed conversion occurred at 2500 mg/kg bLF. Serum and longissimus muscle CuZnSOD and GPx, and serum TAOC linearly and quadraticly increased (P<0.01), serum CAT and longissimus muscle CAT and TAOC linearly increased (P<0.01), whereas MDA concentrations in serum and longissimus muscle linearly decreased (P<0.01) as dietary bLF supplementation increased. Increasing dietary bLF levels improved (P<0.01) mRNA expression of CuZnSOD and CAT in longissimus muscle of piglets. The results indicated that exogenous antioxidant bLF was effective in improving growth performance and enhancing antioxidant enzymes activity and mRNA levels of piglets.     

Regulation of Zinc Transporter 1 Expression in Dorsal Horn of Spinal Cord After Acute Spinal Cord Injury of Rats by Dietary Zinc

Zinc concentrations in the dorsal horn of spinal cord are important for wound healing, neurological function, and reproduction. However, the response of the spinal cord to alterations in dietary zinc is unknown in rats after spinal cord injury (SCI). The current study explored cellular zinc levels and zinc transporter 1 (ZnT1) expression in the dorsal horn of spinal cord with different dietary zinc after SCI. A hundred and forty-four male Wistar rats were randomly divided into four groups: sham-operated group (30?mg Zn/kg), zinc-high dietary SCI model group (ZH, 180?mg Zn/kg), zinc-adequate dietary SCI model group (30?mg Zn/kg), and marginal zinc-deficient dietary SCI model group (MZD, 5?mg Zn/kg). To test the hypothesis that dietary zinc may regulate role of ZnT1 expression in dorsal horn after acute SCI, we traced ZnT1 proteins and zinc ions with immunohistochemistry, western blot, and autometallography. Zinc and ZnT1 levels of the dorsal horn in ZH significantly increased after surgery (P?<?0.05), reached peak level (P?<?0.05) on the seventh day, and subsequently levels of their expression began to decrease. But zinc levels and ZnT1 expression of spinal cord in MZD dietary groups decreased (P?<?0.05) in SCI. There was a positive correlation between ZnT1 protein and zinc content in spinal cord (R?=?0.49880, P?=?0.0492). We found that both zinc and ZnT1 expressions in spinal cord are regulated by dietary zinc. These results indicate that dietary zinc may regulate the expression of ZnT1 in the dorsal horn of spinal cord after SCI. ZnT1 may, at the same time, play a significant role in the maintenance of zinc homeostasis in SCI.     

Concentration and chemical form of dietary zinc shape the porcine colon microbiome,its functional capacity and antibiotic resistance gene repertoire

Despite a well-documented effect of high dietary zinc oxide on the pig intestinal microbiota composition less is it yet known about changes in microbial functional properties or the effect of organic zinc sources. Forty weaning piglets in four groups were fed diets supplemented with 40 or 110 ppm zinc as zinc oxide, 110 ppm as Zn-Lysinate, or 2500 ppm as zinc oxide. Host zinc homeostasis, intestinal zinc fractions, and ileal nutrient digestibility were determined as main nutritional and physiological factors putatively driving colon microbial ecology. Metagenomic sequencing of colon microbiota revealed only clear differences at genus level for the group receiving 2500 ppm zinc oxide. However, a clear group differentiation according to dietary zinc concentration and source was observed at species level. Functional analysis revealed significant differences in genes related to stress response, mineral, and carbohydrate metabolism. Taxonomic and functional gene differences were accompanied with clear effects in microbial metabolite concentration. Finally, a selection of certain antibiotic resistance genes by dietary zinc was observed. This study sheds further light onto the consequences of concentration and chemical form of dietary zinc on microbial ecology measures and the resistome in the porcine colon.Subject terms: Microbiome, Applied microbiology     

The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs

The effect of a dietary supplementation of zinc oxide (ZnO) on the stability of the intestinal flora and on the composition of coliforms in weaned pigs was investigated. Faecal floras were characterized by their metabolic activities and fermentative capacity (FC) using the Phene Plate generalized microplate. Coliforms were characterized by conventional enumeration and by the Phene Plate-RS plates. The latter measured FC, phenotypic diversity, persistence of each coliform strain in piglets, and similarity among the coliform populations within groups. From weaning onwards, the control pigs (n = 5) were fed a basal diet ad libitum, while experimental pigs (n = 5) were given the same food supplemented with 2500 ppm ZnO. Metabolic fingerprinting of faecal floras indicated marked differences between the composition of floras of treated and control pigs during the first 2 weeks post-weaning. The FC of faecal flora in both groups decreased as pigs aged, but it was significantly (P 相似文献   

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy-two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn^high; 50 ppm, Zn^low). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn^high and E. faecium groups gained weight after infection while those in the control group (Zn^low) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn^high+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn^high and E. faecium groups at single time points after infection compared to the Zn^low control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology.