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In this study, actinin-3 ( ACTN3 ) gene expression was investigated in relation to the feed efficiency phenotype in Bos indicus - Bos taurus crossbred steers. A measure of relative feed efficiency based on residual feed intake relative to predictions from the NRC beef cattle model was analyzed by the use of a mixed linear model that included sire and family nested within sire as fixed effects and age, animal type, sex, condition, and breed as random effects for 173 F 2 Nellore-Angus steers. Based on these residual intake observations, individuals were ranked from most efficient to least efficient. Skeletal muscle samples were analyzed from 54 steers in three groups of 18 (high efficiency, low efficiency, and a statistically average group). ACTN3 , which encodes a muscle-specific structural protein, was previously identified as a candidate gene from a microarray analysis of RNA extracted from muscle samples obtained from a subset of steers from each of these three efficiency groups. The expression of ACTN3 was evaluated by quantitative reverse transcriptase PCR analysis. The expression of ACTN3 in skeletal muscle was 1.6-fold greater in the inefficient steer group than in the efficient group ( p = 0.007). In addition to expression measurements, blocks of SNP haplotypes were assessed for breed or parent of origin effects. A maternal effect was observed for ACTN3 inheritance, indicating that a maternal B. indicus block conferred improved residual feed efficiency relative to the B. taurus copy ( p = 0.03). A SNP haplotype analysis was also conducted for m-calpain ( CAPN2 ) and fibronectin 1 ( FN1 ), and a significant breed effect was observed for both genes, with B. indicus and B. taurus alleles each conferring favorable efficiency when inherited maternally ( p = 0.03 and p = 0.04). Because the ACTN3 structural protein is specific to fast-twitch (type II) muscle fibers and not present in slow-twitch muscle fibers (type I), muscle samples used for expression analysis were also assayed for fiber type ratio (type II/type I). Inefficient animals had a fast fiber type ratio 1.8-fold greater than the efficient animals ( p = 0.027). Because these fiber-types exhibit different metabolic profiles, we hypothesize that animals with a greater proportion of fast-twitch muscle fibers are also less feed efficient., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vaughn, Kochan, Torres, Du, Riley, Gill, Herring, Sanders and Riggs.)