Development of a molecular assay to estimate mitochondrial content in cattle tissues

摘要

A qRT-PCR assay was developed to estimate cattle mitochondrial content by counting copies of mtDNA (mitochondrial DNA) relative to nDNA. Out of a set of competing primer pairs designed and tested in cattle tissues it was found that the preferred primer pair (mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 1 or MT-ND1, and wntl-inducible signalling pathway protein 1 or WISP) amplified unique products with high PCR efficiency (>95%). The MT-ND1 and WISP primer pair combination suggested the following rank order of tissues with increasing aerobic capacity in cattle; white fat < liver < skeletal muscle < heart, in line with biological expectation. The assay was then deployed to determine the mitochondrial content of samples of liverand semitendinosus muscle collected from Bos taurus and Bos indicus (n= 15/sub-species), different nutritional states (low metabolisable energy (ME) and low crude protein (CP) intake, high ME and high CP intake and low ME and high CP intake; n=10/diet)and different hormone states (bovine somatotropin and control; n=5/treatment). While we observed a number of trends in mitochondrial content none of the treatment comparisons reached significance (PO.05). The observed trend for high ME intake to increasecattle mitochondrial content in muscle is in line with previous nutritional observations made in the muscle and liver of rodents. Exogenous bovine somatotropin treatment tended to increase mitochondrial content of both muscle and liver with the liver appearing the more responsive of the two tissues.