Dietary levels of methionine plus cystine and chelated copper on the chemical composition of eggs and yolk cholesterol of 49-week-old brown laying hens

Abstract

The Colombian Creole sheep breed has a high economic and social importance for Colombia. Both males and females of this breed are multipurpose animals, and evaluating the production and meat quality of both sexes is important for small farmers in Colombia. This requires the use of tools that help to evaluate critical production points, such as Real-time quantitative polymerase chain reaction (RT-qPCR), which is a widely used molecular tool for the relative quantification of candidate genes in various tissues. For its correct use, the use of housekeeping genes with stable expression, so-called “reference genes”, is required. However, recent studies have shown that the expression of these reference genes can vary among tissues and can be modulated by breed, sex, or external stimuli. Likewise, there is little information regarding the expression of these genes in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep. In this study, the stability in the expression of seven reference genes (ACTB, YWHAZ, SDHA, GAPDH, TUBB2A, B2M, and PGK1) in the Longissimus thoracis et lumborum muscle of male and female Colombian Creole sheep was compared since they are used in RT-qPCR studies to determine the most stable ones for this breed. Twelve animals, six males and six females, with a body weight of 26 ± 4 kg and 12 ± 3 months of age, were used under grazing conditions. Biopsies of the Longissimus thoracis et lumborum muscle were taken, from which RNA was extracted and cDNA was synthesized. Expression was determined using RT-qPCR, and its stability was analyzed by computational algorithms using the geNorm, Normfinder, and BestKeeper software packages, which were integrated using the RefFinder software package. The results indicate that GAPDH, ACTB, and SDHA have the highest stability, whereas the most variable expression was found for B2M. These data provide the basis for more precise results in RT-qPCR studies of gene expression in the muscle of Colombian Creole sheep.