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Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats
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Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats L.F. Wang a,∗ , G.Y. Yang a , G.Q. Yang b , H.S. Zhu a , Y.Y. Wang a , L.Q. Han a , Z.W. Zhao c , Z. Zhang a , C.T. He a a Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China b Modern Experimental Technology and Managing Centre, Henan Agricultural University, Zhengzhou 450002, China c Ping Ding Shan Animal Husbandry Bureau, PingDingShan 467000, Henan, China
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Article history: Received 1 August 2013 Received in revised form 7 June 2014 Accepted 10 June 2014 Available online xxx Keywords: ZIPII zinc transporter Gene expression Distribution Zinc source Dairy goat
a b s t r a c t The expression and function of the zinc transporter genes SLC39A1, SLC39A2, and SLC39A3 in the Zrt/Irt-like proteins (ZIP)II subfamily have been reported in monogastric but not ruminant animals. Thus, the response of these three ZIPII genes to different sources of zinc in ruminants was examined in goats in the present study. A total of 18 Guanzhong dairy goats were randomly divided into three groups of 6 goats each. The goats were fed one of three diets. The control group received a basic diet with no additional zinc. The second and third groups ate the same diet but with 60 mg/kg of zinc sulfate or zinc amino acid chelate (Zn-AA) complex added, respectively. After 7 days of treatment, the goats were sacrificed, 23 tissue samples were collected from each goat, and the expression and distribution of the ZIPII genes were determined with PCR analyses. In addition, seven tissues relevant to digestion or metabolism were examined using real-time PCR to identify the responses of the three genes to different zinc sources. The results showed that the expression of SLC39A1-3 mRNA could be detected in 21 of the 23 tissue types examined in the dairy goats, with no expression detected in heart or testis. The levels of expression for the three genes in the tissues relevant to digestion or metabolism were tissue specific and varied with the zinc source, indicating that the source of zinc affects the SLC39A1-3 mRNA expression. © 2014 Published by Elsevier B.V.
1. Introduction Zinc is a trace nutrient indispensable for life. More than 300 metalloenzymes of six major functional classes require zinc as a key structural component or as a cofactor (Vallee and Auld, 1990). As an important component of zinc finger domains, zinc regulates DNA replication, RNA transcription, and genes expression (Gaither and Eide, 2001). The absorption mechanism of zinc transport in cell and the transporter gene families has been described (Kambe et al., 2004; Hill and Link, 2009; Fukunaka and Kambe, 2010). In the animal feed industry, there are two main forms of zinc additives, organic and inorganic. Although
Abbreviations: CDF, cation diffusion facilitator; CP, crude protein; DM, dry matter; ME, metabolizable energy; NRC, national research council; PCR, polymerase chain reaction; SLC39A1, SLC39A2, and SLC39A3, gene codes for ZIP1, ZIP2, and ZIP3; TMR, total mixed ration; ZIP, Zrt/Irt-like proteins; Zn-AA, zinc amino acid chelate; ZnSO4 , zinc sulfate. ∗ Corresponding author. Tel.: +86 371 63558180; fax: +86 371 63558180. E-mail address: [email protected] (L.F. Wang). http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006 0377-8401/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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Table 1 Nutrient composition and components in the basic diet (DM basis). Ingredient
g/kg
Component
g/kg
Leymus chinensis hay Alfalfa hay Corn Soybean meal Cotton meal Wheat middling powder Corn DDGS Palm meal Dibasic calcium phosphate Salt Premixa Limestone meal Sodium bicarbonate
600 100 135 22 48 10 48 18 6 3 5 0.30 5.40
MEb (MJ/kg) DM CP Znc (mg/kg) Ca P Ca/P Na/K
9.25 900.60 114.60 16.30 3.80 2.50 1.52 1.60
a The premix provided the following diet (per kg): VA 1, 620,000 IU; VD3 324,000 IU; VE 540 IU; VK3 150 mg; FeSO4 ·7H2 O 170 mg; CuSO4 ·5H2 O 70 g; MnSO4 ·5H2 O 290 g; CoCl2 ·6H2 O 510 mg; KI 220 mg; Na2 SeO3 130 mg. b ME, DM, CP were calculated according to the values provided in the Feed Database in China (2011). c The Zn, Ca, P, Na, and K levels in the basic diet were measured values.
studies examining zinc digestive mechanisms vary widely (Evans et al., 1975; Tacnet et al., 1993; Vallee and Falchuk, 1993; Yu et al., 2007), a common understanding is that zinc can only be transformed into an organic form combined with protein for absorption into intestinal mucosal cells and eventual transport into body tissues (Lönnerdal, 2000). On the intestinal epithelial membrane, zinc transport proteins can be categorized into two families: the Zrt/Irt-like proteins (ZIP) protein family and the cation diffusion facilitator (CDF) protein family (Fukunaka and Kambe, 2010; Liuzzi and Cousins, 2004). Both families play important roles in zinc transport, with ZIP family proteins transporting zinc into the cytoplasm and CDF family proteins transporting zinc out of the cytoplasm. These two types of zinc transport are balanced under stable physiological conditions to maintain cellular zinc at specific concentrations but are varied in different tissues of the body and under diverse physiological conditions. Currently, there are at least 14 types of proteins known in the ZIP family in mammals, and these are cataloged into four subfamilies: I, II, LIV-1, and gufA. ZIPII protein family members are significant and include three members ZIP1, ZIP2, and ZIP3 with genes encoded by SLC39A1, SLC39A2, and SLC39A3 (SLC39A1-3), respectively (Kambe et al., 2008). These genes have been identified and shown to have a wide distribution in most tissues or organs (Andrews, 2008; Huang et al., 2006). Although the expression of these genes helped to illustrate the mechanisms of zinc absorption in rodents (Huang et al., 2006; Kambe et al., 2008) and poultry (Huang et al., 2008), the effect of different zinc sources on the expression of these genes in ruminants is scarce. Previously published papers have examined zinc function in ruminants involved in growth, nutrient utilization, the immune response, and carcass traits (Garg et al., 2008; Huerta et al., 2002; Mandal et al., 2007); however, the gene response was not examined, and thus, the characteristics of the response of ZIPII genes to various zinc sources remains unclear. Exploring the ZIPII gene responses to various zinc sources in ruminants will clarify the mechanisms for intracellular changes and will enrich the knowledge of the molecular mechanisms for zinc absorption and transportation. Therefore, the present study examined the expression and distribution of mRNA for SLC39A1-3 in dairy goats fed two sources of dietary zinc and identified the differences in the molecular mechanisms for these genes. 2. Materials and methods 2.1. Animals, experimental design, and treatments A total of 18 adult Guanzhong dairy goats (40–45 kg of body weight; 2.5–3.0 years of age) were randomly divided into three groups (equal numbers of males and females in each group). Goats 1–6 were in the control group; goats 7–12 were in the group administered zinc sulfate (ZnSO4 group); goats 13–18 were in the group administered zinc amino acid chelate complex (Zn-AA group). All goats were individually housed in dedicated iron cages with enough space for the animals to move around inside. All of the animals received care according to the Guide for the Care and Use of Laboratory Animals by the Chinese Academy of Sciences. Basal rations were formulated to meet the nutrient requirements for goats according to NRC guidelines (2007), except for the zinc additive. The chemical and ingredient composition in the basic diet is listed in Table 1. Analyses of the Zn, Ca, Na, and K in the diet were conducted using atomic absorption spectrophotometry (Z-2000, Hitachi, Japan), and the analysis of P was conducted with colorimetry (T6, Pgeneral, Beijing). ME, DM and CP in the diets were determined according to AOAC (1990) procedures. The control group was fed the basal diet, and the ZnSO4 and Zn-AA groups were assigned ZnSO4 -supplemented and Zn-AA-supplemented diets, respectively. Zinc-supplemented diets were prepared by adding calculated amounts of ZnSO4 (produced in the Tianjin chemical plant, China; ≥98% purity, ≥22.5% zinc) or Zn-AA (donated by Chengdu Aohe Biotechnology Co., LTD. China; complex amino acids, ≥92% chelating rate, ≥25% AA, ≥10% zinc) to the basic diet (the original zinc already in the basal feedstuff was ignored), modifying the zinc level to 60 mg/kg (DM). All the diets were supplied as total mixed ration (TMR). Feed and demineralized water were provided ad libitum throughout the experiment. The duration of the feeding Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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experiment was for 22 d (15 d of transition and adaptation and 7 d of formal testing). For further details, see Wang et al. (2011). 2.2. Collection of tissue samples When the feeding experiment was completed, all goats were sacrificed, and 23 tissues samples (abomasum, duodenum, jejunum, ileum, liver, etc.) were collected into Eppendorf tubes and immediately snap-frozen in liquid nitrogen. The procedures were performed in accordance with a protocol approved by Henan Agricultural University Institutional Animal Care and Use Committee. The samples were stored in liquid nitrogen until used in further analysis. 2.3. cDNA preparation Total RNA was extracted using Trizol Reagent kits (TaKaRa, Dalian, China). Moloney murine leukemia virus (MMLV) reverse transcriptase reagent kits and Oligo primer transcriptase were used to reverse RNA to cDNA. 2.4. Primer design Real-time PCR primers for SLC39A1-3 (HQ322407, HQ615881, HQ322408) were designed according to the mRNA sequences previously submitted to the GenBank: qSLC39A1 (forward: 5 -GGAAC AGTCG GGACC ACCAC CTC-3 , reverse: 5 CCACC GCCAG CCCTT CAAAC ACT-3 ; 194 bp); qSLC39A2 (forward: 5 -GCCCT CATCC TCTTG CTCTC AC-3 , reverse: 5 -GCAGT CCTAC GCCAA ACACC AC-3 ; 145 bp); qSLC39A3 (forward: 5 -CTTCC TCTTC GTCAC CTTCT TTG-3 , reverse: 5 -CCTGT GCATC TTGTG GTGCT CT-3 ; 173 bp). The reference gene was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AJ431207) (forward: 5 -AGCGA GATCC TGCCA ACATC AAG-3 , reverse: 5 -CAGCA GAAGG TGCAG AGATG AT-3 ; 144 bp). 2.5. Tissue distribution of SLC39A1-3 All of the 23 tissue-derived cDNAs were amplified by PCR using a master mix of 25 L containing 5 L of diluted cDNA, 12.5 L of premix Taq (Loading dye mix, TaKaRa, Dalian), 1 L of forward primer (20 M), 1 L of reverse primer (20 M), and 5.5 L of water. The PCR program consisted of pre-denaturation (95 ◦ C for 5 min), 40 cycles of denaturation (95 ◦ C for 30 s), annealing (65 ◦ C for 30 s), and extension (72 ◦ C for 20 s), with a final extension of 10 min. A no-template control was also included with each run. The amplification products were detected with 2.0% agarose gel electrophoresis. 2.6. Real-time PCR of SLC39A1-3 Real-time PCR was conducted using the Eppendorf realplex system (German). The concentration of SLC39A1-3 mRNA in digestive tissues was detected using real-time PCR. For each cDNA, 10 L (final volume) was used containing 4 L of diluted cDNA, 5 L of 2× SYBR Premix Ex Taq (TaKaRa, Dalian), 0.2 L of forward primer, 0.2 L of reverse primer, and 0.6 L of water. The standard cycling conditions consisted of 1 cycle at 95 ◦ C for 2 min, then 40 cycles at 95 ◦ C for 15 s, 65 ◦ C for annealing and extension for 40 s, and 80 ◦ C for extension and for reading the fluorescence for 15 s. The results were normalized to GAPDH. Real-time PCR data were converted using the Ct method and expressed as relative mRNA concentrations (Livak and Schmittgen, 2001). 2.7. Statistical analysis Statistical analyses were performed using SPSS 19.0 software for analysis of variance using a general linear model (GLM). Multiple comparisons with Bonferroni corrections between the groups were conducted when the results of the F-test showed P<0.05. All data are reported as the mean ± SD. Values were considered significant when P<0.05. 3. Results 3.1. SLC39A1-3 tissue distribution The results from the PCR indicated that the three genes (SLC39A1-3) in the control group were detected and identified in 21 of 23 tissues (heart and testis were the exception), and the expression levels of the three genes differed significantly (Fig. 1). All of the digestive tracts and metabolic tissues, such as rumen, reticulum, abomasum, duodenum, jejunum, ileum, cecum, colon, rectum, and liver, exhibited a steady expression for the three genes. 3.2. Gene response to different sources of zinc in digestive and metabolic tissues The expression of the transporter genes in digestive tract and metabolic tissues was closely related to zinc absorption, transportation, and metabolism. The difference in the levels of expression of the three genes from goats fed two different zinc Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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Fig. 1. The distribution of SLC39A1, SLC39A2, and SLC39A3 mRNA in tissue. 1. Abomasum. 2. Proximal duodenum. 3. Middle duodenum. 4. Distal duodenum. 5. Jejunum. 6. Ileum. 7. Liver. 8. Kidney. 9. Heart. 10. Spleen. 11. Lung. 12. Pancreas. 13. Muscle. 14. Brain. 15. Rumen. 16. Reticulum. 17. Omasum. 18. Rectum. 19. Colon. 20. Cecum. 21. Skin. 22. Testis. 23. Mammary gland. 24. Negative control. Notes: The PCR products were detected using 2.0% agarose gel electrophoresis; M, DNA ladder.
sources was examined in seven tissues: abomasum, proximal duodenum, middle duodenum, distal duodenum, jejunum, ileum, and liver. Their profiles varied with the zinc source and gene type. 3.2.1. SLC39A1 mRNA expression in dairy goats fed different zinc sources The relative levels of expression for SLC39A1 mRNA in seven tissues from dairy goats fed the two zinc sources are shown in Table 2. The SLC39A1 mRNA expression in the various tissues was influenced by the source of zinc. Except for the middle duodenum, the level of the SLC39A1 mRNA expression in tissues from goats fed either zinc source was higher than that in the control group. In the ZnSO4 group, the expression of SLC39A1 mRNA in distal duodenum, jejunum, and ileum was markedly higher than that in the control group (P1 <0.0001, 0.0096, 0.009, respectively). In the Zn-AA group, the SLC39A1 mRNA expression level in abomasum, jejunum, ileum, and liver was markedly higher (P2 =0.0382, 0.0183, 0.0062, 0.002, respectively). However, there was no significant difference in the mRNA expression level for any tissue between the two zinc groups, except for that in the distal duodenum (P3 =0.0008). 3.2.2. SLC39A2 mRNA expression in dairy goats fed different zinc sources The relative levels for the SLC39A2 mRNA expression in the seven tissues from the goats fed two different zinc sources are shown in Table 3. Generally, the SLC39A2 expression level varied with both the tissue and the zinc source. In the middle duodenum, the level of SLC39A2 mRNA expression was higher in both zinc groups than that in the control group, and the level in the ZnSO4 group was markedly higher than that in both the control and Zn-AA groups (P1 <0.0001, P3 <0.0001). Table 2 Relative abundance of SLC39A1 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.00 1.00 1.01 1.09 1.00 1.02 1.00
ZnSO4 (fold)
Zn-AA (fold)
P-values Pg
± ± ± ± ± ± ±
0.14a 0.18a 0.21a 0.43a 0.11a 0.30a 0.08a
1.26 1.40 0.80 2.06 1.67 2.01 1.35
± ± ± ± ± ± ±
0.41ab 0.56a 0.26a 0.10b 0.31b 0.44b 0.29ab
1.66 1.38 0.80 1.35 1.61 2.06 1.67
± ± ± ± ± ± ±
0.55b 0.44a 0.27a 0.09a 0.47b 0.65b 0.36b
0.0390 0.2170 0.2694 <0.0001 0.0055 0.0030 0.0025
P1
P2
P3
0.8484
0.0382
0.3214
<0.0001 0.0096 0.0090 0.1224
0.3118 0.0183 0.0062 0.0020
0.0008 >0.9999 >0.9999 0.1760
The following were assumed for data presented in Tables 2–4: The PCR amplification coefficient was 1, the expression level of reference gene GAPDH mRNA was 1000 copies. The expression of the three genes SLC39A1, SLC39A2, and SLC39A3 was calculated as 1000 copies divided by CtSLC39A1 -CtGAPDH , CtSLC39A2 -CtGAPDH , and CtSLC39A3 -CtGAPDH raised to the 2nd power, respectively. Pg denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05).
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Table 3 Relative abundance of SLC39A2 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.02 1.01 1.07 1.07 1.00 1.00 1.00
± ± ± ± ± ± ±
0.32a 0.22a 0.53a 0.23a 0.18a 0.03a 0.02a
ZnSO4 (fold)
1.22 0.49 5.74 1.69 1.12 1.23 0.44
± ± ± ± ± ± ±
0.28a 0.25b 2.03b 0.78a 0.40a 0.21b 0.16b
Zn-AA (fold)
0.47 0.66 1.45 1.03 1.13 0.75 1.60
± ± ± ± ± ± ±
0.08b 0.35ab 0.45a 0.49a 0.13a 0.09c 0.23c
P-values Pg
P1
P2
P3
0.0003 0.0169 <0.0001 0.1100 0.6459 <0.0001 <0.0001
0.5573 0.0169 <0.0001
0.0051 0.1385 >0.9999
0.0003 0.9235 <0.0001
0.0272 <0.0001
0.0160 <0.0001
<0.0001 <0.0001
P denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05). Table 4 Relative abundance of SLC39A3 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.00 1.01 1.05 1.05 1.00 1.00 1.01
± ± ± ± ± ± ±
0.07a 0.22a 0.45a 0.15a 0.01a 0.14a 0.21a
ZnSO4 (fold)
1.47 0.52 0.92 2.58 0.56 1.61 1.33
± ± ± ± ± ± ±
0.08ab 0.06b 0.22a 0.98b 0.20b 0.88ab 0.33a
Zn-AA (fold)
1.98 0.58 0.39 2.00 0.38 1.97 1.30
± ± ± ± ± ± ±
0.84b 0.18b 0.04b 0.43ab 0.03b 0.63b 0.26a
P-values Pg
P1
P2
P3
0.0120 0.0002 0.0031 0.0025 <0.0001 0.0516 0.1276
0.3498 0.0004 >0.9999 0.0021 <0.0001
0.0102 0.0014 0.0039 0.0560 <0.0001
0.2726 >0.9999 0.0193 0.3846 0.0528
Pg denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05).
By contrast, in the proximal duodenum, the SLC39A2 mRNA expression level was significantly lower in both zinc groups than that in the control (P1 =0.0169, P2 =0.1385). No significant difference was detected for the three groups in the distal duodenum or jejunum. The levels of the SLC39A2 mRNA expression in the abomasum and ileum were higher in the ZnSO4 group (P1 =0.5573, 0.0272, respectively) but lower in the Zn-AA group (P2 =0.0051, 0.016, respectively) compared with those in the control group, and therefore, the difference in the levels of expression was significant between the two zinc groups (P3 =0.0003, <0.0001, respectively). In the liver, the levels of SLC39A2 mRNA expression for the two zinc groups were opposite those for the abomasum and ileum. Thus, for the liver, the expression was markedly lower in the ZnSO4 group (P1 <0.0001) and markedly higher in the Zn-AA group (P2 <0.0001) than that in the control group, and the difference between the two zinc groups was significantly different (P3 <0.0001). 3.2.3. SLC39A3 mRNA expression in dairy goats fed different zinc sources The relative levels of SLC39A3 mRNA expression in seven tissues from the goats fed two different sources of zinc are shown in Table 4. The expression for both zinc groups in the abomasums, distal duodenum, ileum, and liver was higher than that in the control group, but the expression for both zinc groups in the proximal and middle duodenum and in the jejunum was lower than that in the control group. In addition, the decreased expression levels for both zinc groups observed in the proximal duodenum and jejunum were significantly different from that in the control (P1 =0.0004, <0.0001; P2 =0.0014, <0.0001, respectively), whereas no difference was detected in the level of expression between the two zinc groups for any tissue, except for in the middle duodenum (P3 =0.0193). 4. Discussion The effect of dietary zinc on the expression of zinc transporter genes in tissues was studied previously (Huang et al., 2006, 2008). The present study focused on the response of three genes in the ZIPII family to two dietary sources of zinc. The findings showed that the two zinc sources variably influenced the expression of SLC39A1-3 in different tissues. The SLC39A1-3 genes were detected in most tissues under normal physiological conditions (Fig. 1), but their expression levels and distribution differed markedly. The results showed that the relative expression levels of SLC39A2 mRNA were generally very low compared to those of SLC39A1 and SLC39A3 mRNA, consistent with a previous study (Kambe et al., 2008). In addition, SLC39A1 and SLC39A3 were abundantly expressed in digestive tissues, indicating that ZIP1 and ZIP3 were the primary transporters responsible for zinc absorption. Because SLC39A2 expression in the duodenum and jejunum was very low, it can be speculated that ZIP2 is not the main transporter in the gastrointestinal tract for the two zinc sources used in the present study. The results of the present study supported those from previous studies suggesting that SLC39A2 expression Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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is cell-specific in the digestive tract (Kambe et al., 2008); however, the pattern of SLC39A2 expression in other organs was beyond the scope of the present study. Studies examining the distribution of SLC39A1-3 in different tissues are relatively sparse. Some studies, such as Huang et al. (2008) and Wang et al. (2011), reported high levels of SLC39A1 expression in various tissues and cells. One report suggested that the SLC39A1 expression exhibited little change when mice were fed zinc-deficient diets (Lee et al., 2002). Another study detected no different phenotypes in SLC39A1-knockout mice fed normal zinc diets, but the embryo survival rate of pregnant mice significantly decreased with the low zinc diet (Lee et al., 2002). The results of these studies suggest that the function of SLC39A1 is similar in other animals, such as goat, and that the low SLC39A1 expression resulting from low zinc levels is alleviated by a zinc-supplemented diet. In ruminants, zinc is absorbed primarily in the small intestine, and the large intestine has only a weak ability to absorb zinc (Lu, 2004). However, the expression of SLC39A1-3 in the colon and cecum observed in the present study indicated that they might be active in zinc absorption, which supports the findings from our previous studies (Wang et al., 2011; Yang et al., 2011). Although the expression and distribution of zinc transporters are related to other factors, such as age, physical condition and so on, the large intestine has been shown to absorb zinc, and the degree of absorbance is related to the source of the zinc. The diverse response of the SLC39A2 mRNA expression to the zinc source (Table 3) indicated that various zinc transiting pathways exist for different zinc sources in the goat digestive tract, supporting the results of previous studies (Evans et al., 1975; Tacnet et al., 1993; Vallee and Falchuk, 1993; Yang et al., 2011). However, the effect of transiting pathways on zinc absorption efficiency was not studied. Further analysis found that the two zinc sources more strongly influenced the expression of SLC39A1 and SLC39A2 than that of SLC39A3 in many tissues, demonstrating that SLC39A1 and SLC39A2 were more easily influenced by zinc sources than SLC39A3, consistent with the results of a previous study (Lee et al., 2002). In addition, the marked difference in the expression of SLC39A2 mRNA in the liver for the two zinc groups suggested that ZIP2 was a mutable transporter for zinc metabolism. 5. Conclusions In summary, the present study detected the expression and distribution of three genes in the ZIPII transporter family in most tissues of dairy goats. The responses of the expressed genes to two dietary sources of zinc were successfully determined in the digestive tract and metabolic tissues. The relative gene expression was found to vary with both the tissue type and zinc source. Conflict of interest statement This study focus on the expression of SLC39A1-3 in dairy goats under two zinc sources, this kind of study on ruminants has rare similar reports up to now. We do not have any interest conflicts with others. Acknowledgements This work was supported by a grant from the National Basic Research Program of China (973 Planning Program) under the contract of 2011CB100802. We thank professor Ming Li in College of Animal Science and Veterinary Medicine, Henan Agricultural University for his generous support. References Andrews, G.K., 2008. Regulation and function of Zip4, the acrodermatitis enteropathica gene. Biochem. Soc. Trans. 36, 1242–1246. AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC, USA. Evans, G.W., Grace, C.I., Votava, H.J., 1975. A proposed mechanism for zinc absorption in the rat. Am. J. Phys. 228, 501–505. Fukunaka, A., Kambe, T., 2010. 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Interaction of dietary zinc and growth implants on weight gain, carcass traits and zinc in tissues of growing beef steers and heifers. Anim. Feed Sci. Technol. 95, 15–32. Kambe, T., Geiser, J., Lahner, B., David, E.S., Andrews, G.K., 2008. Slc39a1 to 3(subfamily II) Zip genes in mice have unique cell-specific functions during adaptation to zinc deficiency. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R1474–R1481. Kambe, T., Yamaguchi-Iwai, Y., Sasaki, R., Nagao, M., 2004. Overview of mammalian zinc transporters. Cell. Mol. Life Sci. 61, 49–68. Lee, J.Y., Cole, T.B., Palmiter, R.D., Suh, W.S., Koh, J.Y., 2002. Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish mutant APP transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 99, 7705–7710. Liuzzi, J.P., Cousins, R.J., 2004. Mammalian zinc transporters. Annu. Rev. Nutr. 24, 151–172. Livak, K.J., Schmittgen, T.D., 2001. 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Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats L.F. Wang a,∗ , G.Y. Yang a , G.Q. Yang b , H.S. Zhu a , Y.Y. Wang a , L.Q. Han a , Z.W. Zhao c , Z. Zhang a , C.T. He a a Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China b Modern Experimental Technology and Managing Centre, Henan Agricultural University, Zhengzhou 450002, China c Ping Ding Shan Animal Husbandry Bureau, PingDingShan 467000, Henan, China
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Article history: Received 1 August 2013 Received in revised form 7 June 2014 Accepted 10 June 2014 Available online xxx Keywords: ZIPII zinc transporter Gene expression Distribution Zinc source Dairy goat
a b s t r a c t The expression and function of the zinc transporter genes SLC39A1, SLC39A2, and SLC39A3 in the Zrt/Irt-like proteins (ZIP)II subfamily have been reported in monogastric but not ruminant animals. Thus, the response of these three ZIPII genes to different sources of zinc in ruminants was examined in goats in the present study. A total of 18 Guanzhong dairy goats were randomly divided into three groups of 6 goats each. The goats were fed one of three diets. The control group received a basic diet with no additional zinc. The second and third groups ate the same diet but with 60 mg/kg of zinc sulfate or zinc amino acid chelate (Zn-AA) complex added, respectively. After 7 days of treatment, the goats were sacrificed, 23 tissue samples were collected from each goat, and the expression and distribution of the ZIPII genes were determined with PCR analyses. In addition, seven tissues relevant to digestion or metabolism were examined using real-time PCR to identify the responses of the three genes to different zinc sources. The results showed that the expression of SLC39A1-3 mRNA could be detected in 21 of the 23 tissue types examined in the dairy goats, with no expression detected in heart or testis. The levels of expression for the three genes in the tissues relevant to digestion or metabolism were tissue specific and varied with the zinc source, indicating that the source of zinc affects the SLC39A1-3 mRNA expression. © 2014 Published by Elsevier B.V.
1. Introduction Zinc is a trace nutrient indispensable for life. More than 300 metalloenzymes of six major functional classes require zinc as a key structural component or as a cofactor (Vallee and Auld, 1990). As an important component of zinc finger domains, zinc regulates DNA replication, RNA transcription, and genes expression (Gaither and Eide, 2001). The absorption mechanism of zinc transport in cell and the transporter gene families has been described (Kambe et al., 2004; Hill and Link, 2009; Fukunaka and Kambe, 2010). In the animal feed industry, there are two main forms of zinc additives, organic and inorganic. Although
Abbreviations: CDF, cation diffusion facilitator; CP, crude protein; DM, dry matter; ME, metabolizable energy; NRC, national research council; PCR, polymerase chain reaction; SLC39A1, SLC39A2, and SLC39A3, gene codes for ZIP1, ZIP2, and ZIP3; TMR, total mixed ration; ZIP, Zrt/Irt-like proteins; Zn-AA, zinc amino acid chelate; ZnSO4 , zinc sulfate. ∗ Corresponding author. Tel.: +86 371 63558180; fax: +86 371 63558180. E-mail address: [email protected] (L.F. Wang). http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006 0377-8401/© 2014 Published by Elsevier B.V.
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Table 1 Nutrient composition and components in the basic diet (DM basis). Ingredient
g/kg
Component
g/kg
Leymus chinensis hay Alfalfa hay Corn Soybean meal Cotton meal Wheat middling powder Corn DDGS Palm meal Dibasic calcium phosphate Salt Premixa Limestone meal Sodium bicarbonate
600 100 135 22 48 10 48 18 6 3 5 0.30 5.40
MEb (MJ/kg) DM CP Znc (mg/kg) Ca P Ca/P Na/K
9.25 900.60 114.60 16.30 3.80 2.50 1.52 1.60
a The premix provided the following diet (per kg): VA 1, 620,000 IU; VD3 324,000 IU; VE 540 IU; VK3 150 mg; FeSO4 ·7H2 O 170 mg; CuSO4 ·5H2 O 70 g; MnSO4 ·5H2 O 290 g; CoCl2 ·6H2 O 510 mg; KI 220 mg; Na2 SeO3 130 mg. b ME, DM, CP were calculated according to the values provided in the Feed Database in China (2011). c The Zn, Ca, P, Na, and K levels in the basic diet were measured values.
studies examining zinc digestive mechanisms vary widely (Evans et al., 1975; Tacnet et al., 1993; Vallee and Falchuk, 1993; Yu et al., 2007), a common understanding is that zinc can only be transformed into an organic form combined with protein for absorption into intestinal mucosal cells and eventual transport into body tissues (Lönnerdal, 2000). On the intestinal epithelial membrane, zinc transport proteins can be categorized into two families: the Zrt/Irt-like proteins (ZIP) protein family and the cation diffusion facilitator (CDF) protein family (Fukunaka and Kambe, 2010; Liuzzi and Cousins, 2004). Both families play important roles in zinc transport, with ZIP family proteins transporting zinc into the cytoplasm and CDF family proteins transporting zinc out of the cytoplasm. These two types of zinc transport are balanced under stable physiological conditions to maintain cellular zinc at specific concentrations but are varied in different tissues of the body and under diverse physiological conditions. Currently, there are at least 14 types of proteins known in the ZIP family in mammals, and these are cataloged into four subfamilies: I, II, LIV-1, and gufA. ZIPII protein family members are significant and include three members ZIP1, ZIP2, and ZIP3 with genes encoded by SLC39A1, SLC39A2, and SLC39A3 (SLC39A1-3), respectively (Kambe et al., 2008). These genes have been identified and shown to have a wide distribution in most tissues or organs (Andrews, 2008; Huang et al., 2006). Although the expression of these genes helped to illustrate the mechanisms of zinc absorption in rodents (Huang et al., 2006; Kambe et al., 2008) and poultry (Huang et al., 2008), the effect of different zinc sources on the expression of these genes in ruminants is scarce. Previously published papers have examined zinc function in ruminants involved in growth, nutrient utilization, the immune response, and carcass traits (Garg et al., 2008; Huerta et al., 2002; Mandal et al., 2007); however, the gene response was not examined, and thus, the characteristics of the response of ZIPII genes to various zinc sources remains unclear. Exploring the ZIPII gene responses to various zinc sources in ruminants will clarify the mechanisms for intracellular changes and will enrich the knowledge of the molecular mechanisms for zinc absorption and transportation. Therefore, the present study examined the expression and distribution of mRNA for SLC39A1-3 in dairy goats fed two sources of dietary zinc and identified the differences in the molecular mechanisms for these genes. 2. Materials and methods 2.1. Animals, experimental design, and treatments A total of 18 adult Guanzhong dairy goats (40–45 kg of body weight; 2.5–3.0 years of age) were randomly divided into three groups (equal numbers of males and females in each group). Goats 1–6 were in the control group; goats 7–12 were in the group administered zinc sulfate (ZnSO4 group); goats 13–18 were in the group administered zinc amino acid chelate complex (Zn-AA group). All goats were individually housed in dedicated iron cages with enough space for the animals to move around inside. All of the animals received care according to the Guide for the Care and Use of Laboratory Animals by the Chinese Academy of Sciences. Basal rations were formulated to meet the nutrient requirements for goats according to NRC guidelines (2007), except for the zinc additive. The chemical and ingredient composition in the basic diet is listed in Table 1. Analyses of the Zn, Ca, Na, and K in the diet were conducted using atomic absorption spectrophotometry (Z-2000, Hitachi, Japan), and the analysis of P was conducted with colorimetry (T6, Pgeneral, Beijing). ME, DM and CP in the diets were determined according to AOAC (1990) procedures. The control group was fed the basal diet, and the ZnSO4 and Zn-AA groups were assigned ZnSO4 -supplemented and Zn-AA-supplemented diets, respectively. Zinc-supplemented diets were prepared by adding calculated amounts of ZnSO4 (produced in the Tianjin chemical plant, China; ≥98% purity, ≥22.5% zinc) or Zn-AA (donated by Chengdu Aohe Biotechnology Co., LTD. China; complex amino acids, ≥92% chelating rate, ≥25% AA, ≥10% zinc) to the basic diet (the original zinc already in the basal feedstuff was ignored), modifying the zinc level to 60 mg/kg (DM). All the diets were supplied as total mixed ration (TMR). Feed and demineralized water were provided ad libitum throughout the experiment. The duration of the feeding Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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experiment was for 22 d (15 d of transition and adaptation and 7 d of formal testing). For further details, see Wang et al. (2011). 2.2. Collection of tissue samples When the feeding experiment was completed, all goats were sacrificed, and 23 tissues samples (abomasum, duodenum, jejunum, ileum, liver, etc.) were collected into Eppendorf tubes and immediately snap-frozen in liquid nitrogen. The procedures were performed in accordance with a protocol approved by Henan Agricultural University Institutional Animal Care and Use Committee. The samples were stored in liquid nitrogen until used in further analysis. 2.3. cDNA preparation Total RNA was extracted using Trizol Reagent kits (TaKaRa, Dalian, China). Moloney murine leukemia virus (MMLV) reverse transcriptase reagent kits and Oligo primer transcriptase were used to reverse RNA to cDNA. 2.4. Primer design Real-time PCR primers for SLC39A1-3 (HQ322407, HQ615881, HQ322408) were designed according to the mRNA sequences previously submitted to the GenBank: qSLC39A1 (forward: 5 -GGAAC AGTCG GGACC ACCAC CTC-3 , reverse: 5 CCACC GCCAG CCCTT CAAAC ACT-3 ; 194 bp); qSLC39A2 (forward: 5 -GCCCT CATCC TCTTG CTCTC AC-3 , reverse: 5 -GCAGT CCTAC GCCAA ACACC AC-3 ; 145 bp); qSLC39A3 (forward: 5 -CTTCC TCTTC GTCAC CTTCT TTG-3 , reverse: 5 -CCTGT GCATC TTGTG GTGCT CT-3 ; 173 bp). The reference gene was glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AJ431207) (forward: 5 -AGCGA GATCC TGCCA ACATC AAG-3 , reverse: 5 -CAGCA GAAGG TGCAG AGATG AT-3 ; 144 bp). 2.5. Tissue distribution of SLC39A1-3 All of the 23 tissue-derived cDNAs were amplified by PCR using a master mix of 25 L containing 5 L of diluted cDNA, 12.5 L of premix Taq (Loading dye mix, TaKaRa, Dalian), 1 L of forward primer (20 M), 1 L of reverse primer (20 M), and 5.5 L of water. The PCR program consisted of pre-denaturation (95 ◦ C for 5 min), 40 cycles of denaturation (95 ◦ C for 30 s), annealing (65 ◦ C for 30 s), and extension (72 ◦ C for 20 s), with a final extension of 10 min. A no-template control was also included with each run. The amplification products were detected with 2.0% agarose gel electrophoresis. 2.6. Real-time PCR of SLC39A1-3 Real-time PCR was conducted using the Eppendorf realplex system (German). The concentration of SLC39A1-3 mRNA in digestive tissues was detected using real-time PCR. For each cDNA, 10 L (final volume) was used containing 4 L of diluted cDNA, 5 L of 2× SYBR Premix Ex Taq (TaKaRa, Dalian), 0.2 L of forward primer, 0.2 L of reverse primer, and 0.6 L of water. The standard cycling conditions consisted of 1 cycle at 95 ◦ C for 2 min, then 40 cycles at 95 ◦ C for 15 s, 65 ◦ C for annealing and extension for 40 s, and 80 ◦ C for extension and for reading the fluorescence for 15 s. The results were normalized to GAPDH. Real-time PCR data were converted using the Ct method and expressed as relative mRNA concentrations (Livak and Schmittgen, 2001). 2.7. Statistical analysis Statistical analyses were performed using SPSS 19.0 software for analysis of variance using a general linear model (GLM). Multiple comparisons with Bonferroni corrections between the groups were conducted when the results of the F-test showed P<0.05. All data are reported as the mean ± SD. Values were considered significant when P<0.05. 3. Results 3.1. SLC39A1-3 tissue distribution The results from the PCR indicated that the three genes (SLC39A1-3) in the control group were detected and identified in 21 of 23 tissues (heart and testis were the exception), and the expression levels of the three genes differed significantly (Fig. 1). All of the digestive tracts and metabolic tissues, such as rumen, reticulum, abomasum, duodenum, jejunum, ileum, cecum, colon, rectum, and liver, exhibited a steady expression for the three genes. 3.2. Gene response to different sources of zinc in digestive and metabolic tissues The expression of the transporter genes in digestive tract and metabolic tissues was closely related to zinc absorption, transportation, and metabolism. The difference in the levels of expression of the three genes from goats fed two different zinc Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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Fig. 1. The distribution of SLC39A1, SLC39A2, and SLC39A3 mRNA in tissue. 1. Abomasum. 2. Proximal duodenum. 3. Middle duodenum. 4. Distal duodenum. 5. Jejunum. 6. Ileum. 7. Liver. 8. Kidney. 9. Heart. 10. Spleen. 11. Lung. 12. Pancreas. 13. Muscle. 14. Brain. 15. Rumen. 16. Reticulum. 17. Omasum. 18. Rectum. 19. Colon. 20. Cecum. 21. Skin. 22. Testis. 23. Mammary gland. 24. Negative control. Notes: The PCR products were detected using 2.0% agarose gel electrophoresis; M, DNA ladder.
sources was examined in seven tissues: abomasum, proximal duodenum, middle duodenum, distal duodenum, jejunum, ileum, and liver. Their profiles varied with the zinc source and gene type. 3.2.1. SLC39A1 mRNA expression in dairy goats fed different zinc sources The relative levels of expression for SLC39A1 mRNA in seven tissues from dairy goats fed the two zinc sources are shown in Table 2. The SLC39A1 mRNA expression in the various tissues was influenced by the source of zinc. Except for the middle duodenum, the level of the SLC39A1 mRNA expression in tissues from goats fed either zinc source was higher than that in the control group. In the ZnSO4 group, the expression of SLC39A1 mRNA in distal duodenum, jejunum, and ileum was markedly higher than that in the control group (P1 <0.0001, 0.0096, 0.009, respectively). In the Zn-AA group, the SLC39A1 mRNA expression level in abomasum, jejunum, ileum, and liver was markedly higher (P2 =0.0382, 0.0183, 0.0062, 0.002, respectively). However, there was no significant difference in the mRNA expression level for any tissue between the two zinc groups, except for that in the distal duodenum (P3 =0.0008). 3.2.2. SLC39A2 mRNA expression in dairy goats fed different zinc sources The relative levels for the SLC39A2 mRNA expression in the seven tissues from the goats fed two different zinc sources are shown in Table 3. Generally, the SLC39A2 expression level varied with both the tissue and the zinc source. In the middle duodenum, the level of SLC39A2 mRNA expression was higher in both zinc groups than that in the control group, and the level in the ZnSO4 group was markedly higher than that in both the control and Zn-AA groups (P1 <0.0001, P3 <0.0001). Table 2 Relative abundance of SLC39A1 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.00 1.00 1.01 1.09 1.00 1.02 1.00
ZnSO4 (fold)
Zn-AA (fold)
P-values Pg
± ± ± ± ± ± ±
0.14a 0.18a 0.21a 0.43a 0.11a 0.30a 0.08a
1.26 1.40 0.80 2.06 1.67 2.01 1.35
± ± ± ± ± ± ±
0.41ab 0.56a 0.26a 0.10b 0.31b 0.44b 0.29ab
1.66 1.38 0.80 1.35 1.61 2.06 1.67
± ± ± ± ± ± ±
0.55b 0.44a 0.27a 0.09a 0.47b 0.65b 0.36b
0.0390 0.2170 0.2694 <0.0001 0.0055 0.0030 0.0025
P1
P2
P3
0.8484
0.0382
0.3214
<0.0001 0.0096 0.0090 0.1224
0.3118 0.0183 0.0062 0.0020
0.0008 >0.9999 >0.9999 0.1760
The following were assumed for data presented in Tables 2–4: The PCR amplification coefficient was 1, the expression level of reference gene GAPDH mRNA was 1000 copies. The expression of the three genes SLC39A1, SLC39A2, and SLC39A3 was calculated as 1000 copies divided by CtSLC39A1 -CtGAPDH , CtSLC39A2 -CtGAPDH , and CtSLC39A3 -CtGAPDH raised to the 2nd power, respectively. Pg denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05).
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Table 3 Relative abundance of SLC39A2 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.02 1.01 1.07 1.07 1.00 1.00 1.00
± ± ± ± ± ± ±
0.32a 0.22a 0.53a 0.23a 0.18a 0.03a 0.02a
ZnSO4 (fold)
1.22 0.49 5.74 1.69 1.12 1.23 0.44
± ± ± ± ± ± ±
0.28a 0.25b 2.03b 0.78a 0.40a 0.21b 0.16b
Zn-AA (fold)
0.47 0.66 1.45 1.03 1.13 0.75 1.60
± ± ± ± ± ± ±
0.08b 0.35ab 0.45a 0.49a 0.13a 0.09c 0.23c
P-values Pg
P1
P2
P3
0.0003 0.0169 <0.0001 0.1100 0.6459 <0.0001 <0.0001
0.5573 0.0169 <0.0001
0.0051 0.1385 >0.9999
0.0003 0.9235 <0.0001
0.0272 <0.0001
0.0160 <0.0001
<0.0001 <0.0001
P denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05). Table 4 Relative abundance of SLC39A3 mRNA in the main digestive and metabolic tissues of dairy goats fed different sources of zinc. Tissues
Control (fold)
Abomasum Proximal duodenum Middle duodenum Distal duodenum Jejunum Ileum Liver
1.00 1.01 1.05 1.05 1.00 1.00 1.01
± ± ± ± ± ± ±
0.07a 0.22a 0.45a 0.15a 0.01a 0.14a 0.21a
ZnSO4 (fold)
1.47 0.52 0.92 2.58 0.56 1.61 1.33
± ± ± ± ± ± ±
0.08ab 0.06b 0.22a 0.98b 0.20b 0.88ab 0.33a
Zn-AA (fold)
1.98 0.58 0.39 2.00 0.38 1.97 1.30
± ± ± ± ± ± ±
0.84b 0.18b 0.04b 0.43ab 0.03b 0.63b 0.26a
P-values Pg
P1
P2
P3
0.0120 0.0002 0.0031 0.0025 <0.0001 0.0516 0.1276
0.3498 0.0004 >0.9999 0.0021 <0.0001
0.0102 0.0014 0.0039 0.0560 <0.0001
0.2726 >0.9999 0.0193 0.3846 0.0528
Pg denotes the P-value of F-test in GLM; P1 represents the difference between ZnSO4 group and control; P2 represents the difference between Zn-AA group and control; P3 represents the difference between ZnSO4 group and Zn-AA group. The values in the same row with different lowercase letters are significantly different (P<0.05), whereas those with same lowercase letter are not significantly different (P>0.05).
By contrast, in the proximal duodenum, the SLC39A2 mRNA expression level was significantly lower in both zinc groups than that in the control (P1 =0.0169, P2 =0.1385). No significant difference was detected for the three groups in the distal duodenum or jejunum. The levels of the SLC39A2 mRNA expression in the abomasum and ileum were higher in the ZnSO4 group (P1 =0.5573, 0.0272, respectively) but lower in the Zn-AA group (P2 =0.0051, 0.016, respectively) compared with those in the control group, and therefore, the difference in the levels of expression was significant between the two zinc groups (P3 =0.0003, <0.0001, respectively). In the liver, the levels of SLC39A2 mRNA expression for the two zinc groups were opposite those for the abomasum and ileum. Thus, for the liver, the expression was markedly lower in the ZnSO4 group (P1 <0.0001) and markedly higher in the Zn-AA group (P2 <0.0001) than that in the control group, and the difference between the two zinc groups was significantly different (P3 <0.0001). 3.2.3. SLC39A3 mRNA expression in dairy goats fed different zinc sources The relative levels of SLC39A3 mRNA expression in seven tissues from the goats fed two different sources of zinc are shown in Table 4. The expression for both zinc groups in the abomasums, distal duodenum, ileum, and liver was higher than that in the control group, but the expression for both zinc groups in the proximal and middle duodenum and in the jejunum was lower than that in the control group. In addition, the decreased expression levels for both zinc groups observed in the proximal duodenum and jejunum were significantly different from that in the control (P1 =0.0004, <0.0001; P2 =0.0014, <0.0001, respectively), whereas no difference was detected in the level of expression between the two zinc groups for any tissue, except for in the middle duodenum (P3 =0.0193). 4. Discussion The effect of dietary zinc on the expression of zinc transporter genes in tissues was studied previously (Huang et al., 2006, 2008). The present study focused on the response of three genes in the ZIPII family to two dietary sources of zinc. The findings showed that the two zinc sources variably influenced the expression of SLC39A1-3 in different tissues. The SLC39A1-3 genes were detected in most tissues under normal physiological conditions (Fig. 1), but their expression levels and distribution differed markedly. The results showed that the relative expression levels of SLC39A2 mRNA were generally very low compared to those of SLC39A1 and SLC39A3 mRNA, consistent with a previous study (Kambe et al., 2008). In addition, SLC39A1 and SLC39A3 were abundantly expressed in digestive tissues, indicating that ZIP1 and ZIP3 were the primary transporters responsible for zinc absorption. Because SLC39A2 expression in the duodenum and jejunum was very low, it can be speculated that ZIP2 is not the main transporter in the gastrointestinal tract for the two zinc sources used in the present study. The results of the present study supported those from previous studies suggesting that SLC39A2 expression Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006
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is cell-specific in the digestive tract (Kambe et al., 2008); however, the pattern of SLC39A2 expression in other organs was beyond the scope of the present study. Studies examining the distribution of SLC39A1-3 in different tissues are relatively sparse. Some studies, such as Huang et al. (2008) and Wang et al. (2011), reported high levels of SLC39A1 expression in various tissues and cells. One report suggested that the SLC39A1 expression exhibited little change when mice were fed zinc-deficient diets (Lee et al., 2002). Another study detected no different phenotypes in SLC39A1-knockout mice fed normal zinc diets, but the embryo survival rate of pregnant mice significantly decreased with the low zinc diet (Lee et al., 2002). The results of these studies suggest that the function of SLC39A1 is similar in other animals, such as goat, and that the low SLC39A1 expression resulting from low zinc levels is alleviated by a zinc-supplemented diet. In ruminants, zinc is absorbed primarily in the small intestine, and the large intestine has only a weak ability to absorb zinc (Lu, 2004). However, the expression of SLC39A1-3 in the colon and cecum observed in the present study indicated that they might be active in zinc absorption, which supports the findings from our previous studies (Wang et al., 2011; Yang et al., 2011). Although the expression and distribution of zinc transporters are related to other factors, such as age, physical condition and so on, the large intestine has been shown to absorb zinc, and the degree of absorbance is related to the source of the zinc. The diverse response of the SLC39A2 mRNA expression to the zinc source (Table 3) indicated that various zinc transiting pathways exist for different zinc sources in the goat digestive tract, supporting the results of previous studies (Evans et al., 1975; Tacnet et al., 1993; Vallee and Falchuk, 1993; Yang et al., 2011). However, the effect of transiting pathways on zinc absorption efficiency was not studied. Further analysis found that the two zinc sources more strongly influenced the expression of SLC39A1 and SLC39A2 than that of SLC39A3 in many tissues, demonstrating that SLC39A1 and SLC39A2 were more easily influenced by zinc sources than SLC39A3, consistent with the results of a previous study (Lee et al., 2002). In addition, the marked difference in the expression of SLC39A2 mRNA in the liver for the two zinc groups suggested that ZIP2 was a mutable transporter for zinc metabolism. 5. Conclusions In summary, the present study detected the expression and distribution of three genes in the ZIPII transporter family in most tissues of dairy goats. The responses of the expressed genes to two dietary sources of zinc were successfully determined in the digestive tract and metabolic tissues. The relative gene expression was found to vary with both the tissue type and zinc source. Conflict of interest statement This study focus on the expression of SLC39A1-3 in dairy goats under two zinc sources, this kind of study on ruminants has rare similar reports up to now. We do not have any interest conflicts with others. Acknowledgements This work was supported by a grant from the National Basic Research Program of China (973 Planning Program) under the contract of 2011CB100802. We thank professor Ming Li in College of Animal Science and Veterinary Medicine, Henan Agricultural University for his generous support. References Andrews, G.K., 2008. Regulation and function of Zip4, the acrodermatitis enteropathica gene. Biochem. Soc. Trans. 36, 1242–1246. AOAC, 1990. Official Methods of Analysis, 15th ed. Association of Official Analytical Chemists, Washington, DC, USA. Evans, G.W., Grace, C.I., Votava, H.J., 1975. A proposed mechanism for zinc absorption in the rat. Am. J. Phys. 228, 501–505. Fukunaka, A., Kambe, T., 2010. 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Please cite this article in press as: Wang, L.F., et al., Effect of zinc source on the expression of ZIPII transporter genes in Guanzhong dairy goats. Anim. Feed Sci. Tech. (2014), http://dx.doi.org/10.1016/j.anifeedsci.2014.06.006