| Neuropeptide Y(NPY),one of the most abundant neuropeptides in vertebrates,is widely distributed in the vertebrate center and peripheral nervous system.There are 5 tyrosine residues in 36 amino acid residues of NPY mature peptide.NPY was named as neuropeptide tyrosine due to its richness of tyrosine.NPY is a member of the pancreatic polypeptide family.NPY acts as a neurotransmitter in the brain and in the autonomic nervous system.In addition,NPY was reported to have several functions,including the enhancement of food intake and storage of energy,and reducing anxiety and stress.So far,NPY was proved to be the factor showing the strongest activity on food intake in vertebrates.In this study,the cDNA of NPY mature peptide of Epinephelus coioides(ecNPY)was amplified and cloned into the expression vector pET-SUMO.The recombinant ec-NPY(r-ecNPY)was successfully expressed in E.coli BL21(DE3)as the soluble protein.The activities of purified r-ecNPY protein or synthetic ecNPY(s-ecNPY)were tested using in vitro culture system in which the tissue fragments of hypothalamus and pituitary of carp were mixed and cultured by stimulation ecNPY.The expression of growth hormone(GH),gonadotropic hormone(GtH)and preprosomatostatin 2(PSS-2)was studied with quantitative RT-PCR at transcriptional level.The results showed that 100 nM r-ecNPY up-regulated GH expression significantly(P<0.05).The expression of PSS-2 mRNA was significantly down-regulated by r-ecNPY or s-ecNPY(P<0.05),except for the dosage of 10 nM of s-ecNPY.Both the r-ecNPY and s-ecNPY did not change the expression of GtH.The regulatory effect of r-ecNPY protein on the expression of GH and PSS-2 gene in carp provided evidence to show that r-ecNPY prepared in this study possesses biological activity.Then the r-ecNPY was added to the feed power to make feed pellets for the carp at 3 concentrations,0.15,0.3 and 0.5 ?g r-ecNPY/g feed respectively.The feeding trail was carried out for 6 weeks.The daily food intake was monitored and the weekly feeding level was calculated.At the third week,feeding level of control group,Group-0.15(0.15 ?g r-ec NPY/g feed),Group-0.3(0.3 ?g r-ecNPY/g feed)and Group-0.5(0.5 ?g r-ecNPY/g feed)was 4.079%,4.198%,4.284% and 4.42% respectively.Group-0.5 showed a significant increase in the weekly feeding level compared to the control group(P <0.05).The result demonstrates that the recombinant grouper showed a modest activity to increase the food intake of carp.After the feeding experiment,the growth and nutritional parameters of carp were calculated.The specific growth rate(SGR)in control group,Group-0.15,Group-0.3 and Group-0.5 was 1.5%,1.45%,1.42% and 1.45% respectively.The feeding level in control group,Group-0.15,Group-0.3 and Group-0.5 was 3.94%,3.98%,4.05% and 4.11% respectively.No statistical difference was found in SGR and feeding level between fish in groups treated with r-ec NPY and fish in control group.The protein content of fish in control group,Group-0.15,Group-0.3 and Group-0.5 was 491.1,446.4,546.3 and 489.6 g/kg respectively.The crude lipid content of fish in control group,Group-0.15,Group-0.3 and Group-0.5 was 151.6,141.9,135.1 and 115.9 g/kg.No statistical difference was found in protein content,crude lipid content,ratio of liver/body or body/viscera between fish in groups treated with r-ecNPY and fish in control group.Accordingly,feeding and growth enhancing effects were not observed in carp fed with 0.15,0.3 or 0.5 ?g /g of r-ecNPY for 6 weeks.It needs further study to explore whether the dosage of NPY in feed is not high enough for stimulation of food intake or NPY was degraded through carp's digestion. |
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