| Conjugated linoleic acid can inhibit the fat deposition in piglet, but its mechanism is not clear. Combined with animal nutrition research methods and molecular biological technology, this thesis from individual, cell and molecular level investigates the CLA that inhibit the fat deposition of pigs is caused by induced adipocyte apoptosis, then trys to reveal its possible mechanism in fat deposition.In this experiment,36 landrace × Rongchang piglets about body weight at 13.91±1.53 kg were randomly allotted into 3 groups, each group had 3 repetitions, each repetition had 4 piglets. The piglets of each group were fed one of three diets containing 0% (control group),1% and 2% CLA for 30 days. At the end of experiment, all piglets were weighed after an empty stomach for 12h, and then two middle-weight pigs chosen from each replicate were blood, killed and sampled. Serum divided from blood were used to analyse the changes of biochemical indicators. Sampling the dorsal muscle and leg muscle were used to detect the intramuscular fat content; Dorsal fat thickness was measured, back fat and abdominal fat were weighed. A small piece of back fat was used to observe the morphologic changes of adipocyte apoptosis. In addition, the activity levels of Caspase-3,-8,-9 enzymes were detected in back fat. Another piece of back fat were used to measure the mRNA and protein levels of apoptosis regulators. The main results were as follows:1. CLA had significant effects on average daily feed intake (P<0.05), but no significant effect on feed efficiency (P>0.05). Dietary supplementation with 1% and 2% CLA respectively decreased the average daily gain 13.13%(P<0.05) and 7.95% (P>0.05).2. CLA had significant effects on dorsal fat deposition (P<0.05). And with the level of CLA improved, back fat thickness was reduced. In serum assay, we found that CLA had significant effects on total cholesterol level (P<0.05), but no effects on glucose, triglyceride, LDL-c and HDL-c levels (P>0.05).3. Through the fatty tissue fluorescence staining, we found that supplemention with CLA had significant effects on adipocyte apoptosis and some typical characteristics of apoptosis were significantly observed, for example, chromatin condensation, pyknosis and DNA fragmentation, particularly in 2% CLA group. In Caspase activity assay, CLA significantly increased the activity levels of Caspase-3,-8,-9 enzyme in back fat of piglets(P<0.05). In qPCR and western blotting assays, CLA down-regulated the expression of anti-apoptosis factor Bcl2 mRNA and protein, increased the expression of pro-apoptosis factor Bax and P53 mRNA and protein, while increased the expression of Cyt-C mRNA and released Cyt-C protein from mitochondrial to cytoplasmic. In addition, the expression of Caspase-3 mRNA and the level of Cleaved Caspase-3 protein were increased, which indicated that activated Caspase-3 can promote adipocyte apoptosis On the other hand, CLA increased the expression of TNF-a and TNFR1 mRNA and the level of TNFR1 protein in adipose tissue which induced adipocyte apoptosis through death receptor pathway. These results suggest that mitochondrial pathway and death receptor pathways are involved in the CLA-induced apoptosis of piglet fat.Our study has demonstrated that piglets are fed on CLA for 30 days, the back fat deposition is significantly reduced, which is caused by induced apoptosis of adipose cells, and that both the mitochondrial pathway and the death receptor pathway are involved in this effect. |
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