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The Expression of Zinc Transporters Changed in the Intestine of Weaned Pigs Exposed to Zinc Chitosan Chelate

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The Expression of Zinc Transporters Changed in the Intestine of Weaned Pigs Exposed to Zinc Chitosan Chelate Biol Trace Elem Res DOI 10.1007/s12011-016-0732-1 The Expression of Zinc Transporters Changed in the Intestine of Weaned Pigs Exposed to Zinc Chitosan Chelate Mengyuan Lv1 & Xiongfeng Fu1 & Luansha Hu1 & Xiaojing Yue1 & Xinyan Han1 Received: 6 March 2016 /Accepted: 29 April 2016 # Springer Science+Business Media New York 2016 Abstract This study was conducted to investigate the effect of Introduction zinc chitosan chelate (CS-Zn) on zinc transporter expression and content of tissue zinc in weaned piglets. A total of 90 Zinc (Zn) is not only an essential component of many en- weaned pigs (Duroc × Landrace × Yorkshire) were randomly zymes but also influences diverse physiological processes in- allocated to treatment groups with supplementation of cluding cell division, DNA synthesis, immune functioning, 100 mg/kg zinc as ZnSO4, 100 mg/kg zinc as mixture of and other important biological systems [1, 2]. Zinc is added ZnSO4 and chitosan, or 100 mg/kg zinc as CS-Zn, respectively. into the diet of weaned pig, always as inorganic form (ZnO or After 30 days of trial, 18 piglets (six pigs per treatment) were ZnSO4), to improve performance and lessen diarrhea [3]. The killed and the samples of duodenal mucosa were taken for National Research Council (NRC) recommended usage of analysis of zinc transporter mRNA expressions and protein zinc in growing pig is 100 mg/kg. But in the actual production, abundance. The results show that CS-Zn more effectively in- the usage of zinc is up to 3000 mg/kg during the post-weaning creases (p < 0.05) the average daily gain (ADG) and serum zinc period. The abusive use of zinc supplements in livestock hus- concentration. Zinc concentration in the liver and kidney did bandry is very serious; it results in severe environment con- not differ between treatments. The mRNA expressions of tamination and huge waste of zinc resources [4]. The normal ZnT1, ZIP4, and ZIP5 in CS-Zn treatment were all upregulated form of zinc used as supplement is inorganic, and the bioavail- (p < 0.05) than ZnSO4 or mixture of ZnSO4 and chitosan ability of inorganic zinc is very low. High dietary concentra- groups. ZnT1 abundance was greater (p <0.05)withCS-Zn tion of Zn in inorganic form results in large quantities of zinc as compared with ZnSO4 and mixture of ZnSO4 and chitosan residue in the manure [4]. And zinc accumulates in the soil, treatments, whereas ZIP4 and ZIP5 abundance was higher when it is used as fertilizer. According to a research, Zn con- (p < 0.05) in ZnSO4 group. The results indicate that CS- centrations of pig manure have been substantially increased Zn is more effective in serum zinc accumulation, and it about six times from the early 1990s to 2003, in China [5]. In might regulate zinc homeostasis by affecting zinc transporter general, organic zinc has a similar result of increasing perfor- mRNA expression and absorption mechanism might be dif- mance and reducing scours but supplied with a low concen- ferent with ZnSO4. tration [6], and as a result, the pollution of swine industry would be alleviated. CS-Zn is a cheated complex of Zn and chitosan. Chitosan is Keywords Zinc chitosan chelate . Zinc transporters . mRNA a derivative of chitin, which possess many features, including level . Western blot . Weaned piglets hydrophilicity, biocompatibility, biodegradability, antibacterial properties, and good ion-chelation activity [7]. For its proper- ties, it is applied in food industry, pharmaceuticals industry, * Xinyan Han chemical industry, and agriculture [8]. It was reported that CS- [email protected] Zn complex has high biological activity and better antibacterial activity. [9], and our previous research has found that chitosan- 1 Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Science, Zhejiang Zn chelate increased antioxidant enzyme activity, improved im- University, 866 Yuhangtang Road, Hangzhou 310058, China mune function [10], improved intestinal structure and mucosal Lv et al. function, and decreased apoptosis in ileal mucosal epithelial Table 1 Composition and nutrient levels of basal diet cells in weaned piglets [11]. However, absorption mechanism Ingredient (%) Analyzed compositionb of CS-Zn absorption is unclear and whether the expressions of zinc transporters are regulated by CS-Zn is still unknown. Corn 63 Digestible energy (MJ/kg) 13.7 The intestine is the site of zinc absorption and the major route Soybean meal 16 CP (%) 18.1 of zinc excretion [12], and the most accepted absorptive mech- Extruded soybeans 3 EE (%) 3.26 anism was proposed by Evans. There are two protein families Fish meal 4 Lysine (%) 1.48 taking part in the regulation of zinc homeostasis of mammal, Whey powder 5 Met + Cys (%) 0.88 ZIP (SLC39) family and ZnT (SLC30) family [13, 14]. Bran 2 Ca (%) 0.93 Fourteen members of ZIP family have been found, and the role Soybean oil 1 P (%) 0.79 of ZIP family is that they could combine with zinc ions from Glucose 2 Zn (%) 27.9 extracellular matrix or organelles and transport zinc atoms into Lysine 0.9 the cytoplasm for the sake of improving the cytoplasmic zinc Salt 0.3 level [15, 16]. And there are ten members in ZnT family Limestone 1 acting the opposite function to reduce the zinc level of cy- Calcium hydrophosphate 0.8 toplasm by transporting zinc to extracellular matrix or organ- Vitamin and mineral premixa 1 elles. ZIP4, ZIP5, and ZnT1 are located in the small intestine of pigs, and they play a crucial role in zinc homeostasis [17]. Nutrient content of the diets was based on NRC recommendations (1998) a This study was aimed to investigate whether CS-Zn affects Vitamin-mineral premix provided (per kilogram basic diet): vitamin A, 5000 IU; vitamin D, 2000 IU; vitamin E, 20 IU, vitamin K, 1.0 mg; the expressions of duodenum zinc transporters ZIP4, ZIP5, vitamin B1, 3.0 mg; vitamin B2, 6.5 mg; vitamin B6, 2.4 mg; biotin, and ZnT1 in weaned pigs. 0.4 mg; folic acid, 1.45 mg; pantothenate acid, 23.0 mg; niacin, 18 mg; Fe (as FeSO4·7H2O), 200 mg; Mn (as MnSO4·H2O), 30 mg; Cu (as CuSO4·5H2O) 100 mg b Materials and Methods Analyzed composition except for the digestible energy value Animal and Experimental Design Sample Collection Animals used in this experiment were approved by the prin- After 12-h fast, six pigs from each group were selected at ciples of the Zhejiang University Animal Care and Use random and euthanized with sodium pentobarbital (50 mg/kg Committee. Ninety piglets (Duroc × Landrace × Yorkshire, BW). Serum samples were separated from venous blood, after weaned at 28 days) with average weight 7.3 kg were randomly which was centrifuged (3000×g) at 4 °C for 15 min. Duodenal divided into three treatments, and each treatment was replicat- mucosa was stripped from the seromuscular layer in oxygen- ’ ed three times with ten piglets per replication. Pigs received ated Ringer s solution. Serum and duodenal mucosa were − diets based on corn-soybean meal containing different zinc saved at Eppendorf tubes, respectively, and stored at 70 °C. − sources, and dietary treatments were as follows: (1) basal diet Liver and kidney samples were excised and stored at 70 °C. +ZnSO4 (100 mg/kg Zn), (2) basal diet + mixture of chitosan and ZnSO4 (100 mg/kg Zn, the content of chitosan was equal Zinc Content Analysis to CS-Zn), (3) basal diet + CS-Zn (100 mg/kg Zn). CS-Zn was supplied by the Feed Science Institute of Zhejiang University, The Zn content in the serum, liver, and kidney samples was and the content of Zn was 16.1 %. estimated by an atomic absorption spectrophotometer Pigs were housed in standard pens equipped with a fully (Thermo Scientific M5 AA Spectrometer, USA) after being slatted mesh floor and a nipple waterer. All nutrients of the wet digested with nitric acid via microwave. basal diet met or exceeded NRC (1998) nutrient requirement except for Zn (Table 1), and the content of Zn was mRNA Expression Analysis by Real-Time PCR 27.90 mg/kg. All pigs were given ad libitum access to feed and water. Before the 30-day feeding trial, pigs had a 7-day Real-time PCR was used to determine the messenger RNA adaption period with a basal diet. (mRNA) level of duodenal mucosa samples and, according to Lodemann et al. [18], to sample, isolate RNA, control quality, Assessment of Performance Parameters synthesize complementary DNA (cDNA), and perform real- time PCR. According to the manufacture’s protocol, using All piglets were weighed at the start and the end of feeding TRIzol reagent (HaoJi Biotechnology, Hangzhou, China) ex- trial to calculate the average daily gain (ADG). Feed residuals tracts RNA from the duodenal mucosa. The First-Strand cDNA were recorded daily to assess the daily feed intake (AFDI). Synthesis Kit (HaoJi Biotechnology, Hangzhou, China) was The Effects of CS-Zn on Intestine Zinc Transporters Expressions used to perform reverse transcription. PCR products were re- Table 3 Effect of different zinc sources on growth performance in solved by agarose gel electrophoresis with Power SYBR® piglets Green (Invitrogen, Carlsbad, CA, USA), and the iQ5 software Items ZnSO4 CS + ZnSO4 CS-Zn carried out the qualitative analysis of PCR. Table 2 shows the primers of ZIP4, ZIP5, and ZnT1 used in gene expression. Initial weight (kg) 6.28 ± 0.24 6.24 ± 0. 26 6.26 ± 0. 23 Final weight (kg) 14.81 ± 1.82b 14.94 ± 2.37b 18.41 ± 2.31a b b a Protein Expression Analysis by Western Blot ADG (g/day) 243.69 ± 47.05 248.29 ± 62.07 347.06 ± 61.33 ADFI (g/day) 473.68 ± 5.55b 489.33 ± 11.82b 622.03 ± 19.51a a a b The protein concentration was measured by BCA protein F/G 1.94 ± 0.12 1.97 ± 0.16 1.79 ± 0.07 quantitative kit (HaoJi Biotechnology, Hangzhou, China).
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