# Differential Expression of MicroRNAs in Dark-Cutting Meat from Beef Carcasses
## 1 Introduction  
Minimizing stress and ensuring the overall well-being of food animals are necessary and important goals for all livestock producers. Such practices inevitably enhance production operations since reduction of both physiological and environmental stressors is known to improve beef carcass quality [1]. Environmental, psychological, and physical stress significantly increase the costly occurrence of dark-cutting beef, also known as dark, firm, and dry (DFD). Despite efforts to reduce handling and transport stress and improve management practices, DFD beef remains an industry problem. The attributes of DFDbeef and its presumed causes have been described in great detail since the 1940s, and [PERSON] [2] reported that its occurrence was noted as early as 1774. Although it is known that DFD meat can result from several factors including long-term stress and depleted glycogen stores, some aspects of the physiological mechanisms that cause DFD phenotypes remain poorly understood. In fact, in an extensive literature review, [PERSON] and colleagues [3] suggested that DFD incidence could be potentially managed with better understanding of the impact and complex interactions of animal phenotype and stress factors with management practices. Six decades earlier, [PERSON] [2] noted that effective prevention of DFD beef would depend on \"concomitant progress of genetics and neuro-endocrinology.\" In considering unexplored approaches and advances in genetics, unrelated research in other mammals has shown how profoundly microRNAs (miRNAs) can regulate the response to stress factors in fully developed tissues (e.g., [4; 5]). We hypothesized that certain miRNA signatures expressed in skeletal muscle in response to environmental factors and stresses may regulate physiological networks that result in dark-cutting beef in certain predisposed animals. Better understanding of these phenotypes may enable actionable strategies--including both pre- and post-harvest opportunities--to reduce the economic impact of DFD incidence. The appearance of DFD beef deviates from the bright, cherry red color desired by consumers, and may vary from slight discoloration to dark purple to nearly black in color [6]. In cattle that experience anemtoren stress, muscle glycogen stores can become depleted. After harvest, a process of postmortem glycogen utilization typically occurs in the muscle tissue, resulting in lactic acid production and subsequent pH decline. In stressed animals, however, carcass pH remains high, resulting in de-oxygenated myoglobin and darker color [2; 7; 8]. [PERSON] [2] also noted that certain ratings, especially green grass, could cause undesirable color in beef. Grass-fed and grain-fed cattle have been shown to differ energetically, resulting in darker lean [9]. Mitochondrial respiration rate can also influence the DFD phenotype [10]. Occurrence of DFD has been shown to be a seasonal problem with great economic impact [8; 11]. In 1946, [PERSON] [6] noted a one percent dark cutter incidence in a 1939 survey and estimated Chicago packer economic loss at approximately $1 M at that time. Inflation-adjusted estimates would put that loss at $20.2 M in 2022. In the 2016 United States Beef Quality Audit, overall presence of dark-cutting carcasses was 1.9% [12]. The estimated incidence in the U.S. has fluctuated slightly over the past 20 years with 3.2% in 2011 [13], 1.9% in 2005 [14], and 2.3% in 2000 [15]. Current industry incidence of DFD at the largest U.S. beef packing plants is estimated at approximately 1%. Even at 1%, with an estimated discount averaging $281/head in 2020, the annual economic impact on the US beef industry, with 32.8 million cattle processed, is estimated at $95 to $100 million, demonstrating that dark cutting remains a significant problem. Moreover, this issue is not limited to the U.S. In Australia, for example, a more than 7% incidence of DFD can occur at certain times of year, particularly at the end of summer [11].