پژوهشهای علوم دامی ایران (Apr 2016)
Effects of soy-lecithin, soy-oil and tallow on performance and expression of SREBP-1 gene in broiler chickens
Abstract
Introduction Using vegetable oils and animal fats in poultry diets have beneficial effects for poultry production. They often have higher biological value than expected, increasing dietary metabolizable energy, which usually results in higher growth rates and better feed efficiency. Sterol-regulatory element binding proteins (SREBPs) play a key role in transcriptional regulation of cholesterol metabolism in response to cholesterol levels in the cell. This study was carried out to evaluate the effects of soy-lecithin, soy-oil and tallow on performance and expression of SREBP-1 gene in the liver of broiler chickens. Materials and methods A total of 768 male broiler chickens (Ross 308 strain) were used in a completely randomized design as a 3 × 4 factorial arrangement with 4 replicates and 16 chicks per each. Broiler chickens were fed with three types of fat (soy-lecithin, soy-oil and or tallow) and four levels of fat (0, 1, 2, and 3) from day 1 to day 42. Experimental diets were formulated to be isocaloric and isonitrogenous. At 42 d, liver samples of birds washed with normal saline, put into the liquid nitrogen tank and transferred to -80°C freezer. Relative real-time polymerase chain reaction (RT-PCR) was performed to assess HSP70 gene expression in the heart and liver of broiler chickens. Total RNAs were extracted from the homogenised tissues using high pure RNA isolation kit (Roche, Basel, Switzerland). RNA concentration was assayed by spectrophotometer nano-drop (MD-1000) in wavelength of 260/280 nm. Synthesis of cDNA was done by gene PAK RT universal kit (Fermentas, Hanover, MD, USA), with reverse specific primer and hexanucleotide random primer. Genotype and sequence of the primers of B-actin and SREBP-1 was collected from the National Center for Biotechnology Information (Bethesda, MD, USA). Then, specific primers were designed by primer-5 software and examined by BLAST for checking the specificity of primers. Synthesis of the primers was done by Sigma Company. Qualitative PCR showed that primers designed well and there was no non-specific band or primer dimer (Figures 1 and 2). Optimization of annealing temperature was examined with melting curve by applied biosystems-7300 RT-PCR system. The highest ∆Rn and the lowest Ct were considered to determine the optimum annealing temperature, which was 62°C for both genes. The optimum level of primers was 0.15 µL. Real time PCR was executed in triplicate. Reaction conditions were 45 cycles of a three phase PCR (denaturation at 95°C for 15 s; annealing at 62°C for 30 s; extension at 72°C for 30 s) after an initial denaturation step (95°C for 10 min). In real-time assay, a melt curve analysis, performed at the end of the PCR cycles, will confirm specificity of primer annealing. The thermal profile for melting curve is 95°C for 15 s, 60°C for 1 min; 95°C for 15 s and 60°C for 15 s. The efficiency calibrated model is a more generalized ∆∆Ct model. In this model, Ct is the sign of the first cycle that amplification curve begins to rise. The model considers both Ct of target gene and also Ct of reference gene or housekeeping gene. ∆Ct for each target gene is then calculated by subtracting the Ct number of target gene from that of housekeeping gene for each sample. ∆∆Ct for each gene was calculated by subtracting the ∆Ct of target sample from that of control sample. Results and discussion Soy-lecithin improved birds’ average daily feed intake and average daily body weight gain during the whole experimental period (P< 0.05). Soy oil caused the best feed conversion ratio during the whole period of rearing. Average body weight, average daily gain and average daily feed intake increased as the dietary fat level increased (P< 0.05). Breast, thigh, liver, abdominal fat pad, proventriculus and gizzard, back and neck, duodenum, ceca weights and expression of SREBP-1 were not affected by type or level of supplemental fat. With increasing the dietary fat level the edible carcass and heart weights improved. Fat at the level of 1 percent caused the highest jejunum weight. The highest wing and ileum weights were found by soy lecithin and tallow, respectively. Highest level of serum HDL was shown by chicken fed diet without fat and diet containing soy oil. Conclusion Since dietary soy-lecithin had a similar growth performance compared to soy oil, it can be included as an energy source in broiler chickens diets. Further studies should be done to clear the physiological mechanisms of soy-lecithin on birds’ performance.
