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Title: USING RT-PCR IN A REAL TIME MODE TO DETERMINE EXPRESSION OF LIPOGENIC GENES IN BROILERS

Author
item Rosebrough, Robert
item Russell, Beverly
item Poch, Stephen
item Richards, Mark

Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2006
Publication Date: 3/29/2007
Citation: Rosebrough, R.W., Russell, B.A., Poch, S.M., Richards, M.P. 2007. Using RT-PCR in a real time mode to determine expression of lipogenic genes in broilers. Comparative Biochemistry and Physiology. Part A. 147:215-222.

Interpretive Summary: Excess fat production by the modern broiler chicken presents a two-fold problem. The consumer has health concerns about the link between cardiovascular disease and dietary fat. The producer would like to produce more lean meat rather than fat condemned at the processing plant. Historically shifts in metabolism have resulted in dietary fat being merely shunted to replace that synthesized from other feed ingredients. We have found that altering feeding regimens and dietary crude protein in the broiler will cause permanent changes in fat synthesis and storage, such that dietary fat will not be shunted to body fat stores. The present study was designed to determine if dietary protein elicited changes in intermediary metabolism and if changes resulted from alterations in the expression of genes coding for certain regulatory proteins. Although increasing dietary protein decreased fat synthesis by the broiler, gene expression did not accompany this decrease unless the diet contained a very high level of protein. Modest increases in dietary protein will decrease lipid synthesis without affecting gene expression.

Technical Abstract: Broiler chickens growing from 7 to 28 days of age were fed 12 or 30% protein diets and then switched to the diets containing the opposite level of protein. Birds were killed on days 28, 29, 30 and 31. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME), isocitrate dehydrogenase (ICD) aspartate aminotransferase (AAT) activities, the expression of the genes for ME, fatty acid synthase (FAS) and acetyl coenzyme carboxylase (ACC). Gene expression was determined with a combined RT-PCR using SYBR green as a fluorescent probe monitored in a real time mode. SYBR Green binds all double stranded DNA, emitting a fluorescent signal on binding, and can be used for quantification of many targets. IVL and ME activity were inversely related to dietary protein levels (12 to 30%) and to acute changes in either level. In contrast, both ICD and AAT activities were increased by any increase in dietary protein. Lipogenic gene expression was inversely related to protein level, whether fed on an acute or chronic basis. It appears that real time RT-PCR is an acceptable method of estimating gene expression in birds. In addition, further work will focus on primer sizes that might further optimize RT-PCR as an instrument for studying the regulation of avian lipid metabolism. Results of the present study demonstrate a continued role for protein in the regulation of broiler metabolism. It should be pointed out; however, that metabolic regulation at the gene level only occurs when feeding very high levels of dietary protein.