| Four experiments for this thesis were conducted to investigate the potential mechanisms of the manganese absorption as different manganese sources in the small intestines of broilers by the methods of the ligated intestinal segments and practical feeding.The first experiment was conducted to clone and sequence the completed cDNAs of chicken DMT1 (divalent metal transporter 1) in order to investigate the mechanisms of Mn absorption as different manganese sources in the different intestinal segments of broilers and compare them with each other. DMT1 plays an important role in the process of divalent manganese absorption in broiler intestine. To identify the entire nucleotide sequence of DMT1 cDNA in chickens, 1 289 bp and 1 092 bp 3'-end of DMT1 cDNA fragments from broilers intestinal mucosa were amplified by 3'RACE (rapid amplification of cDNA ends). The amplified fragments were subcloned into the vector and sequenced. The specific primers using in the 3'RACE were designed from the conserved sequences of DMT1 proteins of different animal species. The 5'-end of the DMT1 cDNA fragment was amplified using 5'RACE procedure. The specific primers using in the 5'RACE were designed from 3'-end of chicken DMT1 cDNA sequenced. The 5'-end of DMT1 cDNA fragment was 907 bp, including 242 bp of overlapped region with 3'-end of DMT1 cDNA fragment. The chicken DMT1 isoform I cDNA is 1 972 bp and contains a 1 695 bp open reading frame (ORF) encoding a 564-amino-acid protein, and the chicken DMT1 isoform II cDNA is 1 775 bp and contains a 1 593 bp ORF encoding a 530-amino-acid protein. The chicken DMT1 isoform I cDNA differs from the chicken isoform II cDNA in the 3'-translated region and untranslated region. The molecular weights of chicken DMT1 isoform I and isoform II deduced from the DMT1 cDNAs were 61.56 kDa and 57.90 kDa respectively, and the predicted isoelectric points of chicken DMT1 isoform I and isoform II were 5.51 and 5.75, respectively. Analyses of hydrophobicity and transmembrane regions in amino acid sequences showed that DMT1 proteins were highly hydrophobic with characteristics of the integral membrane protein. Hydropathy profile analysis of chicken DMT1 proteins identified potential 12 highly hydrophobic putative transmembrane (TM) domains in the two amino acid sequences.The amino acid sequences of the two chicken DMT1 proteins had three potential N-linked glycosylation signals (N-X-S/T) (position 278,332, and 345), and two of them (positions 332 and 345) clustered in the fourth extracellular loop flanked by the two TM domains delineated by residues 292-309 and 355-374. The homologies of amino acid sequences of DMT1 protein deduced from the DMT1 isoform I between chicken, human, mouse, rat, and monkey were 80%, 82%, 82, and 78%, respectively, while the homologies of amino acid sequences of DMT1 protein deduced from the DMT1 isoform II between chicken, human, mouse, rat, and monkey were 83%, 84%, 84%, and 81%, respectively. Lastly, the chicken's entire DMT1 cDNAs were amplified by End-to-End PCR with terminal primers designed on the entire nucleotide sequence of chicken DMT1, and the entire cDNAs copies were cloned into pMD-18T vector. The forward and reverse full-length cDNA clones were successfully obtained.The second experiment was conducted to study the mechanisms of the inorganic manganese absorption in ligated small intestinal segments of AA broilers, and be compared with each other. This study was composed of two trials. The trial 1 was a primary study to determine the sampling time to investigate the kinetic of manganese absorption in ligated small intestinal segments of the broilers, according to the changes of the percentages of manganese absorption with the time. The trial 2 was conducted to study the kinetics of manganese absorption in different small intestinal segments of AA broilers, according to the change of manganese absorption rates with the different concentrations of manganese. The levels of divalent metal transporter 1 (DMT1) mRNA in treatment (added 2.18 mmol/L Mn) and control groups were determined in order to study the role of DMT1 in the process of manganese absorption in different small intestinal segments of AA broilers. In the trial 1, the sampling time of four treatments was 5, 15, 30, and 40 min, respectively. Because the samples collected in the different time could be obtained from the same intestinal segment, only one group of the birds was enough for the four treatment groups. Twelve male AA broilers at the age of 28 day-old were randomly assigned to six replicates of two birds each. In each replicate, the intestines of one animal were perfused with physiological saline without manganese supplementation, in order to deduct the endogenous manganese, while the intestines of the other were perfused with the saline containing 120μg/ml Mn as MnSO4·7H2O. The duodenum, jejunum and ileum of each animal were used as one replicate of the corresponding intestinal segments. There were six replicate observations at each sampling time. In the trial 2 there were eight treatments of manganese concentrations, which were 0, 7.5, 15, 30, 60, 120, 240, and 480 ug/ml MnSO4·7H2O. Eighty male AA broilers at the age of 28 day-old were randomly allotted to one of the eight treatments for ten replicates of one bird each, respectively. The duodenum, jejunum and ileum of each bird were used as one replicate of corresponding intestinal segments. The perfusates were collected at 5 min after perfusion in ligated intestinal segments. Because the percentage of Mn absorption was above 80% of the maximium percentage of Mn absorption in ligated intestinal segments of AA broilers, the mucosae samples were collected at 30 min after perfusion. The results from the trial 1 showed that the amount of Mn absorption was asymptotic with respect to the time after perfusion with 2.18 mmol/L Mn as MnSO4 in duodenal, jejunal, and ileal segments of AA broilers. The percentage of Mn absorption was increased linearly with time within 5 min after perfusion in different intestinal segments. So, 5 min after perfusion were adopted to study the kinetics of Mn absorption in ligated intestinal segments of AA broilers. The kinetic curves of Mn absorption in the trial 2 showed that the best fitting model of Mn absorption in duodenum and jejunum was a carrier-mediated model; while was a non-saturable diffusion model in the ileum of broilers. The maximum absorption rate (Jmax) in ligated duodenal segments was higher (P<0.1) than that in the jejunum (94.08 vs 81.17 nmol/cm/min) at 5 min after perfusion. There was no significant difference (P=0.85) with the Michaelis-Menten constant (Km) values between the duodenum and jejunum (3.41 vs 3.60 mmol/L); the diffusive constant (P) (means±SE) was 2.42×10-2±5.22×10-4 cm2/min. The kinetics parameters of Mn absorption in different small intestinal segments were compared and the results suggested that at least one carrier involved in the inorganic Mn absorption in small intestine of the broilers. The DMT1 mRNA levels in different intestinal segments showed that: the DMT1 mRNA levels in the duodenum and jejunum were higher (P<0.001) than that in the ileum of the broilers; there was no significant difference(P=0.2112)in the DMT1 mRNA levels between the duodenum and jejunum of the control group. The DMT1 mRNA levels in the duodenum and jejunum were also higher (P<0.0175) than that in the ileum of the AA broilers in treatment group (perfused with 2.18 mM Mn). In comparision with the control group, the DMT1 mRNA levels decreased significantly (P<0.001) in different intestinal segments of Mn treatment group. Manganese absorption was a carrier-mediated process in the duodenum and jejunum, while a non-saturable diffusion process was found in the ileum of the broilers. At least one carrier involved in the carrier mediated process of inorganic manganese absorption in the duodenum and jejunum of broilers. The level of DMT1 mRNA in the duodenum and jejunum was higher than that in the ileum of the broilers. The DMT1 mRNA levels in different intestinal segments of the broilers were downregulated when manganese was added to ligated intestinal segments, which suggested that DMT1 play an important role in the process of Mn absorption in the duodenum and jejunum of broilers.The third experiment was conducted to study the kinetics of manganese absorption as different manganese sources in small intestine of the broilers, by the changing of manganese absorption rates with different concentrations of manganese. The levels of DMT1 mRNA in the treatment (added 2.18 mmol/L Mn) and control groups were determined in order to study mechanisms of inorganic and organic manganese absorption in small intestine of the broilers. The factorial arrangement of treatments (3×7) was used involving three manganese sources and seven manganese concentrations. The three manganese sources were MnSO4, Mn-AA B (moderate complex strength) and Bioplex Mn (strong complex strength). There were seven treatments of manganese concentrations, which were 0.13, 0.27, 0.54, 1.09, 2.18, 4.37, and 8.74 mmol/L MnSO4·7H2O. An additional group without supplementing Mn to media was used as a control. Because Mn absorption was linear with the time within 30 min after perfusion in ligated duodenal loops, the sampling time was at 30 min after perfusion. Two hundred twenty male AA broilers at the age of 28 day-old were randomly allotted to one of 22 treatments for ten replicates of one bird each. The duodenum of one bird was used as one replicate. The results showed that the best fitting models of Mn absorption of different Mn sources were the carrier-mediated models in the duodenum of the broilers. The maximum absorption rates (Jmax) and the Michaelis-Menten constant (Km) values in Mn-AA B (moderate complex organic manganese) and Bioplex Mn (strong complex organic manganese) groups were higher (P<0.001) than that in MnSO4 group. There was no significant difference (P>0.66) in the maximum absorption rate (Jmax) and the Michaelis-Menten constant (Km) values between the Mn-AA B and Bioplex Mn groups. The kinetics parameters of Mn absorption among different manganese source groups were compared and the results suggested that at least one carrier involved in the organic Mn absorption in small intestine of the broilers. The DMT1 mRNA level in the duodenum of the broilers decreased significantly (P=0.0001) when manganese was added to. In comparision with the control, the level of the total DMT1 mRNA was increased by 80.99% (P<0.0001), 65.58% (P<0.0001), and 58.20% (P=0.0003) in MnSO4, Mn-AA B and Bioplex Mn groups respectively. The level of the total DMT1 mRNA was increased by 81.1% (P=0.0095) and 119.9% (P=0.0011) in Mn-AA B and Bioplex Mn groups than that of the MnSO4 group. However, there was no significant difference (P=0.4018) between Mn-AA B and Bioplex Mn groups. The level of DMT1-Isoform I mRNA in the duodenum was low, and there was no significant difference (P=0.4828) among the four treatments groups. The DMT1-Isoform II mRNA level in the duodenum of the broilers decreased significantly (P=0.0001) when manganese was added to. In comparision with the control, the level of the DMT1-Isoform II mRNA was increased by 83.01% (P<0.0001), 66.84% (P<0.0001), and 59.17% (P=0.0001) in MnSO4, Mn-AA B and Bioplex Mn groups respectively. The level of the total DMT1 mRNA was increased by 95.1% (P=0.0006) and 140.2% (P=0.0004) in Mn-AA B and Bioplex Mn groups than that of the MnSO4 group.The results suggested that the mechanisms of inorganic and organic manganese absorption were the carrier-mediated process in the duodenum of the broilers. At least one carrier involved in the carrier-mediated process of the organic manganese absorption in the duodenum of the broilers. The DMT1 mRNA was mostly expressed as DMT1-Isoform II. The DMT1 mRNA level in ligated duodenal segment of broilers decreased when manganese was added to, and the levels of DMT1 mRNA were higher in organic Mn groups than that of inorganic manganese.The fourth experiment was conducted to compare the effect of manganese sources on the manganese absorption in diets by testing of the contents of Mn in plasma from portal vein, and to investigate the mechanisms of the manganese absorption as different manganese sources by the effect of manganese sources on the DMT1 mRNA level in small intestine of the broilers simultaneously. There are four treatment groups, which were Mn-unsupplemented control, MnSO4, Mn-AA B (moderate complex strength) and Bioplex Mn (strong complex strength) group. Two hundred fivty-six male broilers at the age of 14 day-old were randomly allotted to one of the four treatments for eight replicates of eight birds each. The results showed that: (1) Significant differences (P<0.0001) were observed in Mn content of plasma from portal vein among treatment groups of different manganese sources at 7 and 14 day of this experiment. At the seventh day, Mn content of plasma was increased by 59.7% (P=0.0029), 112.4% (P<0.0001), and 133.1% (P<0.0001) in MnSO4, Mn-AA B and Bioplex Mn groups in comparison with that of the control group. Mn content of plasma in Mn-AA B group and Bioplex Mn group was 32.9% (P=0.0075) and 45.9% (P=0.0004) higher than MnSO4 group. Mn content of plasma in Bioplex Mn group was 9.7% higher than Mn-AA B group, while there was no significant difference (P=0.2669). At 14 day, Mn content of plasma was increased by 31.0% (P=0.0184), 78.3% (P<0.0001), and 128.6% (P<0.0001) in MnSO4, Mn-AA B and Bioplex Mn groups respectively, in comparison with that of the control group. Mn content of plasma in Mn-AA B group and Bioplex Mn group was 36.1% (P<0.0001) and 74.5% (P<0.0001) higher than that of the MnSO4 group. Mn content of plasma in Bioplex Mn group was 28.2% higher (P=0.0004) than that of the Mn-AA B group. (2) The levels of the total DMT1 mRNA in the duodenum and jejunum of broilers were highe(rP<0.01)than that in the ileum, and the level of the total DMT1 mRNA in the dedunum of broilers was highe(rP<0.01)than that in the jejunum regardless of treatments. In comparision with the control, the level of the total DMT1 mRNA in the duodenum and jejunum of broilers increased significantly (P<0.0001) by adding the different manganese source in the diet. There was no significant difference(P=0.1793)in the levels of the total DMT1 mRNA in the ileum among the four treatment groups. The level of the total DMT1 mRNA in the duodenum was increased by 44.2% (P=0.2530), 224.3% (P<0.0001), and 286.8% (P<0.0001) in MnSO4, Mn-AA B and Bioplex Mn groups respectively, as compared with the control. The level of the total DMT1 mRNA in the duodenum was increased by 124.7% (P<0.0001) and 168.1% (P<0.0001) in Mn-AA B and Bioplex Mn groups than that of the MnSO4 group. The DMT1 mRNA level in the duodenum of broilers in Bioplex Mn group was higher (P=0.0995) than that in Mn-AA B group. The level of the total DMT1 mRNA in the jejunum was increased by 47.3% (P=0.0935), 150.7% (P<0.0001), and 149.7% (P<0.0001) in MnSO4, Mn-AA B and Bioplex Mn groups respectively, in comparision with the control group. The level of the total DMT1 mRNA in the jejunum was increased by 70.1% (P=0.0051) and 69.4% (P=0.0065) in Mn-AA B and Bioplex Mn groups than that of the MnSO4 group, while there was no significant difference (P=0.9318) between Mn-AA B and Bioplex Mn groups. (3) No difference(P>0.28)was found in the level of the DMT1-Isoform I mRNA in the corresponding small intestinal segments among the four treatment groups. The level of the total DMT1-Isoform I mRNA in the duodenum and jejunum was higher (P<0.01) than that in the ileum. The results suggested that the DMT1 mRNA was mostly expressed in the duodenum and jejunum of the practical feeding broiler; he level of DMT1 mRNA in the duodenum and jejunum increased by adding the different manganese source in the diet, while there was no significant effect on the level of DMT1 mRNA in the ileum. The level of DMT1 mRNA in the duodenum and jejunum of the birds fed with the complexed organic manganese was higher than that of the birds fed with inorganic manganese; the level of DMT1 mRNA in the duodenum and jejunum of the birds fed with strong complexed organic manganese was higher than that fed with inorganic manganese.In conclusion, inorganic manganese absorption was a carrier-mediated process in the duodenum and jejunum of the broilers, while was a non-saturable diffusion process in the ileum of broilers. The level of DMT1 mRNA in the duodenum and jejunum was higher than that in the ileum of the broilers. Organic manganese absorption was a carrier mediated process in the duodenum of the broilers, and at least one carrier involved in the carrier mediated process of organic manganese. The level of DMT1 mRNA was regulated by adding different manganese sources to the ligated intestinal segments or the diet, which suggested that organic manganese was transported by DMT1 in small intestine of broilers. The level of DMT1 mRNA in the duodenum and jejunum of the birds fed with the complexed organic manganese was higher than that of the birds fed with inorganic manganese; the level of DMT1 mRNA in the duodenum and jejunum of the birds fed with strong complexed organic manganese was higher than that fed with inorganic manganese This results and findings in this thesis would be of very important theoretical and practical significances for developing and utilizing new and highly absorptive organic Mn supplements with moderate or strong complex strengths.
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