In their experiment, thirty-six pregnant Suffolk ewes (78.24 kg ± 9.5) were randomly assigned to dietary treatments of endophyte-free tall fescue seed (E−; 0.0 µg ergovaline + ergovalinine/ewe per day) or endophyte-infected tall fescue seed (E+; 1,722 µg ergovaline + ergovalinine/ewe per day) at specific stages of gestation (d 35–85 or d 86–133) in a 2 × 2 factorial arrangement of treatments. Fetal and maternal necropsies were performed at d 133 of gestation. Semitendinosus (ST) muscle was removed from each fetus and immediately frozen in liquid nitrogen for storage at −80°C. Total cellular RNA was extracted using the mirVana miRNA Isolation Kit (Ambion, Austin, TX). Quality analysis of RNA was performed using an Agilent 2100 Bioanalyzer, with a RNA integrity number threshold of 7.0. The translational control RNA from 3 fetuses per treatment from the ST was used for miRNA sequencing and data analysis (LC Sciences, Houston, TX).
MicroRNA sequencing yielded 113,252,743 reads with 92,177,228 mappable to the ovine reference genome. Of the mappable reads, 27% were specific to the Ovis aries genome and 18% were specific to mammals. There were 4,242 unique miRNA identified by sequencing, which included 208 that were specific to the Ovis aries genome and 676 that were mammalian but novel to Ovis aries. Known MyomiR (miR-1, miR133a, miR133b, miR206, miR-208b, miR-486, and miR-499) in skeletal muscle were present in our samples but not (P > 0.05) differentially expressed due to treatment. miR-148b, miR-300-3p, miR-431-3p, miR-299-3p, and miR-541-5p were upregulated (P < 0.05) in E+/E+ compared with E−/E− fetal ST muscles. miR-652, miR-628, miR-2427, miR-22-3p, miR-8118-p5, miR-376d, and miR-677 were downregulated (P < 0.05) in E+/E+ versus E−/E−. Skeletal muscle miR-148b has been shown to reduce glucose uptake in response to insulin in humans and miR-541 promotes vascular smooth muscle cell proliferation.
From the data presented in this study, research concludes that exposure to ergot alkaloids in utero alters miRNA expression in fetal skeletal muscle.