Effect Of Manganese On Activities And Gene Expressions Of Key Enzymes In Fat Synthesis In Primary Cultured Hepatocytes Of Broilers

This thesis was composed of all the results from three experiment with Arbor Acres (AA) broiler at the age of 21-24 days old to study the effects of different levels and sources of manganese (Mn) on activitives and gene expressions of Fatty Acid Synthase (FAS) and Malic Dehydrogenase (MDH), and which were the key enzymes of fat anabolism the effects of Mn on fat synthesis from the cellular and molecular level.Experiment 1 was conducted to establish a stable and reliable culture method of primary chicken hepatocytes in vitro to study the metabolism of the chicken hepatocytes and its molecular mechanisms, with which conducted the methodological foundation for the following two experiments. The hepatocytes were isolated from liver of a male healthy broiler at the age of 21-24 days old, using the modified two-step perfusion method. The cells gained were cultured in vitro in the Leibovitz's L-15 medium of highly scattered and purified. The total hepatocytes yield and their viability were (2±1)×109 and 95 %, respectively. The cells in the medium were able to maintained normal morphology with the primary monolayer culture, which could meet the requirements of the following two experiments.Experiment 2 was conducted to study the effects of different levels of Mn (Mn from inorganic chloride) on the activities and gene expression of the fat synthesis key enzymes in hepatocytes from broiler (21-24 days old) in vitro were cultured. The broilers from 1 day old to surgery time were fed a Mn-free corn-soybean meal (Concentration of Mn in the diet analyzed was 19.69 mg/kg ), which meet the NRC(1994) Nutrient Requirements of 1 to 3 weeks old broiler recommendation but Mn, and the birds were in a critical Mn deficiency. This experiment included two tests. In test 1, a non–toxic rang and the appropriate levels of Mn in the mudium for culturing the hepatocytes were determined, according to the lactate dehydrogenase (LDH) leakage of the hepatocytes after adding different level of Mn. In Test 2, the effects of the different levels of Mn on activity and gene expression of fatty acid synthase were tested too, according to the result of the Test 1. In the Test 1, as the single factor completely randomized design, five treatment groups (added 0, 0.02, 0.2, 0.5 and 1 mM Mn in the medium) were set with 6 replicates for each treatment (which means this experiment was repeated 6 times), and the hepatocytes in each group were cultured for 24 and 48h. The results of Test 1 showed that different level of Mn in the hepatocytes culturing for 24 and 48h significantly decreased LDH leakage (p<0.0001), and the LDH activity of hepatocytes culture medium showed a significant decrease in the level as the quadratic. The appropriate level of Mn was 0.56-0.57 mM, and Mn in 1 mM was not toxic to the hepatocytes. Therefore the Mn level in Test 2were set 0, 0.25, 0.5 and 0.75 mM. The results in Test 2 showed that medium with different level of Mn in hepatocytes culturing for 24h, the activity and mRNA expression of FAS and MDH had decrease without significance (p>0.24), however the cells culturing for 48h, different levels of Mn in the medium significantly reduced FAS and MDH mRNA relative expression level (p≤0.0009),and FAS and MDH mRNA levels decrease with the increase in the level of the Mn, while the activity of MDH and FAS had decrease without significance (p>0.17). Experiment 3 was conducted to study the effects of different levels and forms of Mn on LDH leakage, the activity and gene expression of FAS and MDH in hepatocytes from broilers (21-24 days old were tested). The broilers used in this experiment were fed the same diet as that in the Experiment 2 described. In this experiment, a 2×2 factorial randomly design involving, and two forms of Mn were amino acid manganese with moderate complex strength (MnAA) and inorganic manganese chloride. The levels of Mn were 0.25 mM and 0.5 mM, and all those groups shared a common control group for which Mn was not added. The hepatocytes in these 5 treatment groups with 6 replicates for each were cultured for 48h. The results showed that adding the Mn groups significantly reduced the LDH leakage in hepatocytes, and significantly reduce FAS and MDH mRNA relative expression (p<0.0001) as well as FAS activity in hepatocytes (p<0.004), and different Mn levels had significant effect on the MDH mRNA, however the source, level and their interaction of Mn in hepatocytes had not significant effect on LDH leakage, activity and mRNA relative expression of FAS (p>0.13).This thesis revealed that different levels and forms of Mn supplementation could have the effect on the activity and gene expression of FAS and MDH at the cellular level.This find was rarely reported before. The results of this thesis enriched and developed the nutrient theoretical knowledge of Mn. And it would be of a certain theoretical and practical significances.