Research On The Mechanism Of Intestinal Development And Nutrients Metabolism Regulated By Long-chain Fatty Acids In Pigeon Squab

In this study, we took American king pigeon as experimental model. Different carbon number and chain-length fatty acids were used to investigate their effects on mRNA expression of genes of fatty acid transporters, enteric peptides and transmitters and cell proliferation and apoptosis in pigeon intestine based on the model of embryonic intestinal organ culture. After then, different lipids were added in parental pigeon’ diet, intestinal development and nutrients’ metabolism were studied in squabs. Therefore, we can have a preliminary conclusion on mechanism of intestinal development and fat metabolism regulated by long-chain fatty acids in pigeon squabs.Experiment1was conducted to clone the genes in fatty acid metabolism and study the effects of different fatty acids on their expression. The results showed that: the full-length of fatty acid translocase (FAT/CD36) was2282bp which encoded a protein of471amino acids; the full-length of intestinal fatty acid-binding protein (I-FABP) was855bp which encoded a protein of132amino acids; the full-length of PPARa (peroxisome proliferator-activated receptor α) and PPARγ were1941bp and1653bp encoding468amino acids and475amino acids, respectively. They all had high identities with other avian corresponding genes. Using quantitative real-time PCR, pigeon FAT/CD36and I-FABP mRNA in duodenum and jejunum increased progressively with development stage. Expression of FAT/CD36was the greatest in the duodenum at28days post-hatch, and the greatest in jejunum at14days post-hatch (P<0.05). I-FABP mRNA was expressed at the highest level at28day post hatch in the duodenum and jejunum (P<0.05). However, expression of FAT/CD36was shown no regular pattern in the ileum, and I-FABP gene expression reached the maximum at the day of hatch but decreased subsequently. The expression of PPARa gene showed highest level in kidey tissue, while PPARy was highest in adipose tissue. The study in vitro showed that low concentration (5μM) of oleic acid, palmitic acid or linoleic acid can significantly increased FAT/CD36and PPARγ mRNA level (P<0.05). However, for linolenic acid or arachidonic acid, the inducement of increases of both two gene expressions needed higher concentration (50μM or250μM)(P<0.05).250 μM palmitic acid was shown to suppress FAT/CD36gene expression (P<0.05). Significant increases in the pigeon I-FABP mRNA level were induced by linoleic acid or arachidonic acid (P<0.05), whereas I-FABP gene expression appeared to be unaffected by oleic acid and a-linolenic acid (P>0.05). The results suggest that FAT/CD36and I-FABP may be representatives of intestine development in pigeon,, and FAT/CD36could be regulated by long-chain fatty acids via PPARy pathway, and I-FABP could be regulated by n-6polyunsaturated fatty acids.Experiment2was conducted to clone the genes of enteric peptides and related transmitters and study their expression patterns. The results showed that:the full length of ghrelin was800bp which encoded116amino acids; the full length of cholecystokinin (CCK) was739bp encoding130amino acids; we also obtained the partial length of neuronal nitric oxide synthase (nNOS),835bp. Using quantitative real-time PCR,gene expression of ghrelin and nNOS were highest in duodenum, jejunum and ileum at8day post-hatch (P<0.05). CCK gene expression in duodenum and ileum were also maximum at8day post-hatch (P<0.05), but no significance was shown in other stages, while in jejunum, its level increased progressively and became highest at28day post-hatch (P<0.05), The results of experiments in vitro were shown that:treatments of5μM oleic acid and higher concentration (50and250μM) of linoleic acid, linolenic acid and arachidonic acid all increased the mRNA level of ghrelin (P<0.05), while in palmitic acid treatments, there were no significances compared with control group (P>0.05). Treatments of5μM linoleic acid, linolenic acid or arachidonic acid, and250μM palmitic acid decreased the CCK gene expression, while in250μM oleic acid or linolenic acid treatments, it increased significantly (P<0.05). nNOS gene expression in treatments of5μM linoleic acid, linolenic acid or palmitic acid and250μM oleic acid or linolenic acid were all decreased significantly (P<0.05). Therefore, Ghrelin、CCK and nNOS may play important roles in early gastrointestinal development of pigeon squabs, and distinct pathways probably existed in three genes’ expression when affected by fatty acids which were different from that in mammals.Experiment3was conducted to study the effects of different long-chain fatty acids on intestinal cell proliferation and apoptosis. The results showed fhat:oleic acid, linoleic acid, linolenic acid and arachidonic acid all can increase the cell proliferation (P<0.05), while in palmitic acid treatment, no significance was observed (P>0.05). Ghrelin receptor antagonist, GHR-6can make the inducement of oleic acid, linoleic acid, linolenic acid and arachidonic acid lower to the control group. Beside this, these four type of fatty acids can also increase the ratio of gene expression value of Bcl-2and Bax (P<0.05), and they also showed effective inhibition of cell apoptosis in Tunel experiment (P<0.05). The result in5μM arachidonic acid treatment was most significant (P<0.05), but in palmitic acid treatment, the cell apoptosis seemed to be enhanced. Meanwhile, GHR-6and SNP (NO donor) increased and decreased the cell apoptosis, respectively. Therefore, oleic acid, linoleic acid, linolenic acid and arachidonic acid all can promote cell proliferation and inhibit cell apoptosis in pigeon intestine, which was reverse in palmitic acid treatment. Polyunsaturated fatty acids may affect cell proliferation via ghrelin pathway, and regulate cell apoptosis by affecting gene expression of Bcl-2and Bax and activity of nNOS.Experiment4was conducted study the influence of supplementation of parental diet with different fat sources on performance intestinal development in pigeon squabs. A total of240pairs of parent White King pigeons (240males and240females) were randomly assigned to4dietary treatments groups, each of which included6replications of10pairs (one pair per cage).480newly hatched squabs were randomly allocated and every two squabs were raised by each pair of parent pigeons. Pigeons were fed a basal diet supplemented with3%lard oil (LO), palm oil (PO), soybean oil (SO), or fish oil (FO), respectively. FO inclusion caused a lower body weight gain of squabs (P<0.05). The SO treatment group presented a greater villus height and duodenal surface area in squabs than that in other three treatment groups. Both villus height and crypt depth were increased in the jejunum of the LO group, as well as in the ileum of the PO group. FO inclusion increased lipase activity in the jejunum content. Addition of SO in parental diet showed a higher alkaline phosphatase activity in duodenum mucosa of squabs (P<0.05). Compared with SO treatment, leucine aminopeptidase activity in duodenum, jejunum and ileum mucosa of squabs were decreased by54.2%,40.1%and53.1%, respectively in PO treatment. LO inclusion in parental diet induced highest percentages of enteric group, Clostridium, Bifidobacterium, Coriobacterium, and Bacteroides-Porphyromonas-Prevotella-group in colorectal content of squabs (P<0.05). The results suggest that fish oil in parental diet causes squab lower body weight gain, but soybean oil present beneficial effects on the villus morphology and digestive function compared with other groups, which is therefore probably a better oil supplementation in parental pigeon diet.Experiment5was conducted to investigate the effects of different lipid resources on nutrients’metabolism in pigeon squabs. The results showed that:the concentration of high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, free fatty acid, triglyceride and total cholesterol in pigeon plasma in LO treatment were all significant higher than those in FO treatment (P<0.05). The contents of fat and cholesterol in pigeon breast muscle in LO treatment were also higher than those in FO treatment. The relative contents of fatty acids, C16:0, C16:1, C18:0and C18:1in pigeon breast muscle in SO treatment were lower than those in LO or PO treatment, while it was significant higher in the relative contents of fatty acids, C18:2n-6, C18:3n-3, C20:1n-9and C20:2n-6(P<0.05). Except arachidonic acid (C20:4n-6), the content of long-chain fatty acids whose number of carbon was more than20in pigeon breast in FO treatment were all highest among four groups. Besides these, the result of GC-MS showed that glycine, propionic acid, phosphoric acid and pentadecane relative contents in pigeon liver tissue was higher in FO treatment, while cholesterol content was significant lower (P<0.05); Glucose, mannose, myo-inositol and pyrimidine content in SO treatment was increased significantly (P<0.05). Therefore, these nine substances measured by GC-MS could be small biomarkers of metabolism in the liver of pigeon when their feed was added with fats.