The effects of gender and muscle type on the mRNA levels of the calpain proteolytic system and beef tenderness during post-mortem aging, Livestock Science, http://dx.doi.org/10. 1016/j.livsci.2016.01.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The objective of this study was to determine the effects of gender and muscle type on the mRNA levels of the calpain system and the tenderization of beef meat. TheLongissimus thoracis (LT) and the Semimembranosus (SM) were sampled from each bull, steer and heifer after routine slaughter (Six animals per group). The mRNA levels of µ-calpain, m-calpain, calpain-3 and calpastatin were quantified using real-time PCR. Concurrently, tenderness was determined following the WarnerBratzler Shearforce (WBSF) procedure and rate of tenderization during post-mortem storage was calculated from the WBSF values of 7d and 35d aged steaks. The results show that bulls had significantly lower (P < 0.01) WBSF values than heifers which were accompanied by higher (P < 0.01) levels of µ-calpain and calpain-3 mRNA but similar levels of calpastatin as compared to heifers. There was a significantly higher (P < 0.05) calpastatin expression in steers, as compared to heifers. However, µ-calpain expression was lower (P < 0.05) in heifers whose meat was significantly tougher (P < 0.05) than that of steers. Steer meat was slightly tougher than that of bulls, while steers had had a tendency to express higher levels of calpastatin but similar µ-calpain and calpain-3 mRNA. The LT had lower (P < 0.05) WBSF values than the SM but these muscles tenderised at the same rate, and 2 had similar mRNA levels for all investigated genes. M-calpain mRNA levels were not significantly affected by muscle and gender (P > 0.05). Moreover, calpain 3 was negatively correlated to 7d WBSF values (P < 0.05). Despite the small sample size, these results suggest that variations in beef tenderness could be modulated through the differential expression of the members of the calpain system, specifically, µ-calpain, calpain 3 and calpastatin.

The objectives of this study were to evaluate the relative expression stability of five candidate reference genes in the semimembranosus (SM) and longissimus thoracis (LT) of beef steers, heifers and young bulls. The mRNA levels of Beta-Actin, eukaryotic initiation factor-2B Subunit 2, glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase-A and succinate dehydrogenase complex-subunit A were quantified using real-time polymerase chain reaction (qPCR). The combined analysis using the geNorm algorithm revealed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic initiation factor-2B subunit 2 (EIF2B2) were the most stable gene pairs. However, individual experimental conditions showed that succinate dehydrogenase complex-subunit A was most stably expressed in bulls and heifers SM, and in bulls and steers LT. Glyceraldehyde-3-phosphate dehydrogenase was most stably expressed in bull SM, steer SM, bull LT and heifer LT. The expression stability ranking order differed between experimental conditions, but all genes had low expression variability. Therefore, using the two most stable reference genes, namely GAPDH and EIF2B2, would result in more accurate normalizations for quantitative real-time PCR studies in the SM and LT muscles of beef cattle. The need for prior evaluation of candidate reference genes in different muscles and sex groups of beef cattle is thus emphasized by the present results.