Thirty-six Suffolk ewes (78.24 kg ± 9.5) estimated to be carrying twins at d 35 of gestation were randomly assigned to 1 of 2 treatments—endophyte-free tall fescue seed (E−; 0.0 µg ergovaline + ergovalinine/g) or endophyte-infected tall fescue seed (E+; 4.14 µg ergovaline + ergovalinine/g)—from d 35 through 85 and d 86 through 133 of gestation, creating 4 unique dietary treatments: E−E−, E−E+, E+E−, and E+E+. Endophyte-infected tall fescue seed was fed at a level to provide 1,772 µg of ergovaline + ergovalinine/ewe per day for E+ treatments, and an equal weight of endophyte-free seed (0.0 µg ergovaline + ergovalinine) was fed for E− treatments. Ewes were pair fed across treatments in order to maintain equal DMI. Fetal and maternal necropsies were performed at d 133 of gestation. Placental samples were weighed, frozen in liquid nitrogen, and stored at −80°C. Total RNA was extracted from cotyledon placental tissue using TRIzol and a PureLink RNA Mini Kit (ThermoFisher Scientific). Total RNA was quantified and quality was assessed using an Agilent 2100 Bioanalyzer. Ribonucleic acid integrity numbers of >7 were considered sufficient quality. A subsample (n = 4) was selected for both the E+E+ and E−E− treatment groups, and total cotyledon RNA was submitted for gene expression profiling via RNA sequencing. Numerous miRNA transcripts were upregulated (P < 0.01; n = 15) in E+E+ compared with E−E− and included miR-21, miR-221, miR-99a, miR-152, miR-381, miR-30a, miR-143, and miR-122. miR-21 exhibited a 9.89-fold increase in expression level for E+E+ compared with E−E−.
miR-21 has previously been implicated as a potential biomarker for fetal hypoxia due to upregulation in both placental and plasma/serum samples. Similar increases in miR-21 and miR-122 levels have been correlated to other pathological diseases of the placenta including placental insufficiency, IUGR, and preeclampsia.