| In this dissertation, 5 experiments were conducted to analyze chemical characteristics of organic Fe sources, evaluate the relative bioavailabilities of organic Fe sources with different complex(chelation) strength [quotient of formation(Qf) values], and investigate the absorption traits and mechanisms of inorganic Fe and organic Fe sources with different complex(chelation) strength in the ligated small intestinal segments of broiler chicks.Exp. 1 Studies on the chemical characteristics of organic iron sourcesTwenty four organic Fe sources were collected from China and abroad, and were characterized. Organic Fe sources included 6 Fe-Mets, 10 Fe-Glys, 1 Fe-Lys, 4 Fe-proteinates(Fe-Prot), and 3 Fe-AA. The total Fe content, the solubility in buffers(p H 5.0 0.1 M KH2PO4- K2HPO4 and p H 2.0 0.2 M HCl-KCl) and deionized water were determined using inductively coupled plasma emission spectroscopy. The composition of AA in organic Fe sources was analyzed using high performance liquid chromatography(HPLC). The ferric Fe(Fe3+) contents in Fe sources were determined by a titration method. The complex(chelation) strengths of organic Fe sources were measured by polarographic analysis using a hanging mercury drop electrode. The results showed that, 1) the total Fe contents of the 24 Fe sources varied from 10.2 to 20.1%, and the Fe3+ contents ranged from 0 to 5.66%; 2) molar ratios(total amino acid : Fe) in the 24 organic Fe sources varied from 0.80:1 to 2.48:1, in which only the molar ratio in Fe-Prot 2 was over 2:1, while in Fe-Met 2, Fe-Gly 1, Fe-Gly 10, Fe-Prot 1, Fe-Prot 4 and Fe-AA 3 were below 1:1, in the other 12 organic Fe sources were between 1:1 and 2:1; 3) the difference of the solubilities in solvents was great(0~100%), and the solubilities of the organic Fe sources in p H 2.0, 0.2 M HCl-KCl buffer > in deionized water > in p H 5.0, 0.1 M KH2PO4- K2HPO4 buffer; 4) all 6 Fe-Met products(Fe-Met 1 to 6), 10 Fe-Gly complexes(Fe-Glys 1 to 10), 1 Fe-Lys complex(Fe-Lys), 1 Fe-AA complex(Fe-AA 2), and 1 Fe-proteinate(Fe-Prot 4) with Qf values below than 10, had weak complex strengths, 2 organic Fe-AA complexes(Fe-AA 1 and Fe-AA 3) and 2 Fe-proteinates(Fe-Prot 1 and Fe-Prot 3) with Qf values between 10 to 100 and showed moderate complex strength, 1 Fe-proteinate(Fe-Prot 2) with a Qf value of 8,590, above 1000, had an extremely strong chelation strength. The results from the experiment indicated that the chemical characteristics of the organic Fe sources varied considerably, and these provided a foundation for a further study to investigate the relationships between the Qf values of organic Fe sources and their relative bioavailabilities.Exp. 2 Studies on the relative bioavailabilities of organic iron sources with different complex(chelation) strength for broilersAs the complex(chelation) strengths of organic Fe source is the most important chemical characteristics which correlate to the relative bioavailabilities, 3 organic Fe sources which belong to different categories in complex(chelation) strength were chosen based on the comprehensive chemical characteristics of organic Fe sources. The 3 organic Fe source were Fe-Met 2 with weak complex strength(Fe-Met W,14.7% Fe,Qf = 1.37), Fe-Prot 1 with moderate complex strength(Fe- Prot M,14.2% Fe,Qf = 43.6), Fe-Prot 2 with extremely strong chelation strength(Fe- Prot ES,10.2% Fe,Qf = 8,590), respectively. A total of 1,170 1-d-old Arbor Acres(AA)male broilers were randomly allotted to 6 replicate cages(15 chicks/cage) for each of 13 treatments in a completely randomized design involving a 4 × 3 factorial arrangement of treatments(4 Fe sources × 3 added Fe levels), plus a control with no added Fe. Dietary treatments included a corn-soybean meal basal diet(Control, analyzed Fe content was 55.8 mg/kg), and the basal diet supplemented with 20, 40, or 60 mg Fe/kg as one of Fe sulfate(Fe SO4?7H2O), Fe-Met with weak complex strength(Fe-Met W, Qf = 1.37, 14.7% Fe), Fe-Prot with moderate complex strength(Fe-Prot M, Qf = 43.6, 14.2% Fe), or Fe-Prot with extremely strong chelation strength(Fe-Prot ES, Qf = 8,590, 10.2% Fe). The growth performance, Fe concentrations, hematological indices, activities and m RNA expressions of two Fe-containing enzymes(Catalase, CAT; Succinate Dehydrogenase, SDH) in tissues of broilers at 7, 14 and 21 d of age were determined in the present experiment to assess their relative bioavailabilities. The results demonstrated that: 1) transferrin saturation in plasma on 14 d, bone Fe on d 7 and 14, liver Fe on d 7, 14 and 21, kidney Fe on d 14, SDH activities in liver on d 21 and in kidney on d 7 and 21, CAT m RNA levels in kidney and heart on d 14, and SDH m RNA levels in liver and kidney on d 21 increased linearly(P < 0.05) as added Fe levels increased; 2) differences in bioavailabilities among Fe sources were detected(P < 0.05) only for the SDH m RNA levels in liver and kidney of broilers on d 21 based on slope ratios from the multiple linear regression on daily dietary analyzed Fe intake; 3) based on slope ratios from the multiple linear regression of SDH m RNA level in liver or kidney of broilers on d 21 on daily dietary analyzed Fe intake, the bioavailabilities of Fe-Met W, Fe-Prot M, and Fe-Prot ES relative to Fe sulfate(100%) were 129%(P = 0.18), 164%(P < 0.003) and 174%(P < 0.001), or 102%(P = 0.95), 143%(P = 0.09) and 174%(P < 0.004), respectively. The Fe-Prot ES was more available(P < 0.05) than Fe sulfate and Fe-Met W, and there were no difference(P > 0.05) between Fe-Prot M and Fe-Prot ES, or between Fe-Met W and Fe sulfate. These results indicated that the SDH m RNA expression levels in liver and kidney of broilers on d 21 were sensitive criteria for assessing the relative bioavailabilities of Fe sources with different complex(chelation) strength. Based on slope ratios from the above two indices, the bioavailabilities of Fe-Met W, Fe-Prot M, and Fe-Prot ES relative to Fe sulfate(100%) were 116, 154 and 174%, respectively. Under conventional diets supplemented with Fe levels at near Fe requirement of broilers, using more sensitive gene expression indices of Fe-containing enzymes in target tissues, the multiple linear regression slope ratio method could more accurately estimate Fe biovailabilties of Fe sources for broilers.Exp. 3 The Fe absorption traits and molecular mechanisms of inorganic iron sulfate in the ligated small intestinal segments of broilersTwo trials were conducted to study the kinetics of inorganic Fe absorption in the ligated duodenal, jejunal, and ileal loops of broilers and whether the expressions of Fe transporters(Divalent Metal Transporter1, DMT1; and Ferroportin1, FPN1) were involved. Trial 1 was conducted to investigate the Fe absorption curves as the post-perfused time prolonged to determine the appropriate post-perfusion time in the next trial. One hundred Fe-deficient commercial male AA broilers on d 28 were randomly allotted to 5 treatments(5 post-perfusion time points) according to the bodyweight. Each treatment had ten replicates with 2 chicks per replicate. The ligated loops were perfused with solutions containing 25 μg/L(0.45 m M) Fe as Fe SO4·7H2O, and samples were collected at 0, 5, 15, 30 or 60 min post-perfusion. The results showed that Fe absorption increased in an asymptotic response to post-perfusion time within 60 min in the ligated duodenum and jejunum, but a quadratic response was observed in ileum. The Fe absorption rates at 60, 60 and 43 min reached to the maximum in duodenum, jejunum and ileum according to the fitting equations of Fe absorption in duodenum, jejunum and ileum at different post-perfusion time. The Fe absorptions in the duodenum at post-perfusion time 30, 45 and 60 min were significantly(P < 0.006) greater than that in jejunum, and at 60 min, the Fe absorption in duodenum was greater(P < 0.03) than that in ileum. In addition, the Fe absorption at 30 min was more than 85.0% of the maximum absorption in each segment. For this reason, it is appropriate to choose 30 min as the optimal perfusion time in next trial. The trial 2 was carried out to study the Fe absorption kinetics in the ligated intestinal segments of broilers and the m RNA expressions of DMT1 and FPN1, in order to explain Fe absorption mechanisms in broilers. Seventy 28-d-old Fe-deficient broilers were randomly allotted to seven treatments(seven perfusion solutions). Each treatment had 10 replicates with one chick for each replicate. Intestinal loops were perfused with solutions containing 0(0 m M), 6.25(0.11 m M), 12.5(0.22 m M), 25(0.45 m M), 50(0.89 m M), 100(1.76 m M), or 200(3.58 m M) μg/ml Fe as Fe SO4·7H2O. The DMT1 and FPN1 m RNA levels in ligated intestinal segments in the 0 or 25 μg/ml Fe groups were analyzed. The results demonstrated that, the duodenum may be the main Fe absorption site in the intestine of broilers, Fe absorption in duodenum and jejunum depended on a saturated carrier-mediated process, whereas Fe absorption in the ileum occurred with a non-saturated diffusion process. Moreover, the DMT1 and FPN1 m RNA levels in the duodenum were greater(P < 0.0001) than that in jejunum, which were greater(P < 0.009) than that in ileum, further indicating that Fe absorptions in the duodenum and jejunum occurred mainly by a saturated carrier-mediated process, however, in ileum it mainly depended on a unsaturated free diffusion process, and the Fe absorptions in the duodenum and jejunum may relate to the expressions of DMT1 and FPN1.Exp. 4 Traits of Fe absorption of Fe sources with different complex(chelation) strength in the ligated small intestinal segments of broilersThe experiment was conducted by a 2 ╳ 8 two factorial completely randomized design. The two perfused Fe concentrations were 200 μg/ml(3.58 m M) and 400 μg/ml(7.16 m M). The eight Fe sources were inorganic ferrous sulfate(Fe SO4ï¹'7H2O), a mixture of ferrous sulfate and methionine(Fe SO4ï¹'7H2O + Met), a mixture of inorganic ferrous sulfate and glycine(Fe SO4ï¹'7H2O + Gly), a Fe methionine complex(Fe-Met), a Fe glycine complex(Fe-Gly), a Fe-Met with weak complex strength(Fe-Met W, Qf = 1.37), a Fe proteinate with complex strength(Fe-Prot M, Qf = 43.6) and a Fe proteinate with extremely strong chelation strength(Fe-Prot ES, Qf = 8,590), respectively. One hundred and sixty Fe-deficient commercial male AA broilers on d 28 were randomly allotted to 16 treatments(2 perfused Fe concentrations ╳ 8 perfused Fe source) according to the bodyweight. Each treatment had ten replicates with 1 chick per replicate. The samples were collected at 30 min post-perfusion. The results showed that: 1) Fe absorption in Fe-supplemented 7.16 m M group was greater(P < 0.0001) than that in Fe-supplemented 3.58 m M group, Fe absorption in duodenum was significantly higher(P < 0.003) than those in jejunum and ileum, and there was no significant difference(P > 0.10) in Fe absorption between jejunum and ileum, the above results provided a further evidence for the duodenum is the main Fe absorption site in broilers; 2) Fe source affected(P < 0.0001) Fe absorption in the small intestine. There was a tendency that adding Met or Gly to inorganic Fe sulfate solutions improved Fe absorption(P = 0.10, 0.02 respectively), but there were no differences(P > 0.05) between the mixture of inorganic Fe sulfate with Met or Gly and the complexes of Fe and Met or Gly. Iron absorption in moderate and extremely strong complex(chelation) strength was significantly greater(P < 0.01) than that in inorganic ferrous sulfate. There were no differences(P > 0.05) among the groups of a mixture of inorganic ferrous sulfate and Met, a mixture of ferrous sulfate and Gly, Fe-Met, Fe-Gly, a Fe-Met with weak chelation strength in Fe absorption. And no differences were detected(P > 0.05) between the Fe-Prot M with the moderate chelation strength and the Fe-Prot ES with extremely chelation strength. The numerical order of each group in Fe absorption from high to low were Fe-Prot ES with extremely strong chelation strength > Fe-Prot M with moderate complex strength > Fe-Met > Fe-Gly > the mixture of inorganic Fe sulfate and Gly > the mixture of inorganic ferrous sulfate and Met > Fe-Met with weak complex strength > inorganic ferrous sulfate. These results further indicated that, the duodenum is the main Fe absorption site, Fe absorption of organic Fe sources were better than that in inorganic Fe, and was related to their Qf values. The Fe absorption of organic Fe source with extremely strong chelation strength was the best, the next was the organic Fe source with moderate complex strength, the Fe absorption in the organic Fe source with weak complex strength or in inorganic Fe sulfate were the lowest. The Fe absorption from the simple mixture of inorganic Fe source and Met or Gly was same as that from Fe-Met or Fe-Gly, and they were similar in improving Fe absorption.Exp. 5 Mechanisms of Fe absorption difference of Fe sources with different chelation strength in ligated small intestine of broilersThis experiment was carried out, based on the results of previous experiments, to study the Fe absorption kinetics from Fe sources with different complex(chelation) strength in the duodenum of broilers by in situ ligated loops, and investigate the mechanisms of Fe absorption difference in the duodenum of broilers. A 4 × 5 factorial arrangement of treatments was used involving 4 Fe sources and 5 perfused Fe levels. The 4 Fe sources involved in this experiment were the inorganic Fe sulfate and 3 organic Fe source chosen in Exp. 2. The 3 organic Fe sources were Fe-Met W(weak complex strength, Qf = 1.37), Fe-Prot M(moderate complex strength, Qf = 43.6) and Fe-Prot ES(extremely strong chelation strength, Qf = 8,590), respectively. The 5 perfused Fe levels were 50(0.89 m M), 100(1.76 m M), 200(3.58 m M), 400(7.16 m M), and 800(14.33 m M) μg/ml, respectively. Meanwhile, a group without adding Fe was set as the control group. Two hundred and ten Fe-deficient commercial male AA broilers on d 28 were randomly allotted to 21 treatments(4 perfused Fe sources ╳ 5 Fe perfused concentrations + 1 control group) according to the bodyweight. Each treatment had ten replicates with 1 chick per replicate. The samples were collected at 30 min post-perfusion. The results showed that: 1) Fe sources significantly affected(P < 0.0001) Fe absorption rates. The Fe absorption rate in organic Fe source with moderate or extremely strong complex(chelation) strength were higher(P < 0.05) than that in inorganic Fe sulfate ferrous. No differences(P > 0.05) were found between organic Fe source with weak complex strength and inorganic ferrous sulfate or between organic Fe source with extremely strong chelation strength and organic Fe source with moderate complex strength; 2) the kinetic curves showed that the Fe absorption of organic Fe sources in duodenum depended on a saturated carrier-mediated process, and the maximum absorption rate(Jmax) value of organic Fe source with extremely strong chelation strength >(P < 0.05) that of organic Fe source with moderate complex strength >(P < 0.05) that of organic Fe source with weak complex strength and of inorganic Fe source. However, there were no differences among Fe sources in Michaelis constant(Km); 3) compared with the control broilers, added Fe to the perfusate inhibited(P < 0.0003) the DMT1 m RNA expression by about 50%, while improved(P < 0.001) FPN1 m RNA expression by 2~3 times in the duodenum of broilers when Fe was added to the perfusate with different forms. There were no differences(P > 0.14) among Fe sources in inhibiting DMT1 or improving FPN1 m RNA expression; 4) added Fe with different complex(chelation) strength did not influence the protein expression of DMT1 and FPN1(P > 0.20). These results showed that although Fe absorption of Fe sources with different complex(chelation) strength in the ligated duodenum of broilers are a saturated carrier-depend process, and the organic Fe source with greater Qf values would be more available. Beside DMT1 and FPN1, there may be other transporters in duodenum of broiles which participate in Fe transport of Fe sources with different complex(chelation) strength.In summary, the chemical characteristics such as Qf values of 24 organic Fe sources varied greatly. The SDH m RNA levels in liver and kidney of broilers at 21 d of age were sensitive criteria for assessing the relative bioavailabilities of Fe sources with different complex(chelation) strength. Duodenum is the main site of Fe absorption in broilers, and Fe absorption in duodenum and jejunum, depended on a carrier-mediated process, whereas Fe absorption in the ileum occurred with a non-saturated diffusion process. The relative bioavailabilities of organic Fe source and their absorption in intestine was closely related to their Qf values, and organic Fe sources with greater Qf values showed higher Fe absorption and Fe bioavailabilities. Besides DMT1 and FPN1, there may be other transporters in duodenum of broiles which participate in Fe transport of Fe sources with different complex(chelation) strength, but it needs to be further studied. These above results could be a great theoretical and practical significances for developing and utilizing new and highly bioavailable organic Fe additives with optimal chelation strength, so as to promote the growth of broilers healthily and efficiently, and minimize the environmental pollution of Fe excreted by animals. |
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