Supranutritional Maternal Organic Selenium Supplementation During Different Trimesters of Pregnancy in Beef Cattle

We used an RNA-Seq transcriptomic approach to investigate Longissimus dorsi muscle gene expression profiles of calves from cows receiving weekly Se-yeast boluses at supranutritional concentrations during different trimesters of gestation. Pregnant beef cows received, except the control group (CTR), weekly supranutritional selenium-yeast boluses (105 mg Se/wk) during the first (TR1), second (TR2), or third (TR3) trimester of gestation. Within 12 to 48 h of birth, muscle samples were collected from the Longissimus dorsi using a Bergstrom biopsy needle (inner diameter: 5 mm). Muscle biopsies were placed into sterile vials, stored on ice and frozen at – 80 ºC until total RNA isolation was performed. After sequencing and read quality control, we identified 3,048 unique differentially expressed genes (DEGs) across all group comparisons (FDR < 0.05 and |log2FC| > 1.5). Furthermore, we predicted 237 unique transcription factors that putatively regulate the DEGs. Our findings suggest a beneficial effect of supranutritional maternal organic Se supplementation during late gestation on Se-status and muscle development and function of newborn calves. Angus-cross cows that had calved at least once previously were assigned to one of four groups at conception (control and groups 1, 2, and 3 corresponding to trimester of Se-treatment: CTR, TR1, TR2, and TR3, respectively), using a randomized complete block design. Except in the CTR group, cows received Se-supplementation during their corresponding pregnancy trimester in the form of three Se-yeast (Phibro Selenium Yeast 2000, Prince Agri Products, Inc., Quincy IL) boluses per week for 13 weeks, equaling 105 mg Se/wk throughout their treatment trimester. Thus, cows received Se-yeast during the first (TR1), second (TR2), or third (TR3) trimester of gestation. In addition, cows had access to a mineral supplement containing 120 mg/kg Se (US FDA regulations) from Na selenite. Within each group of cows, ten were chosen for repeated measures based on uniform genetic background (black color), medium size, and middle aged (5 – 9 years). The resulting calf group demographics from these cows were as follows: CTR group n = 10 (3 females, 7 males), TR1 n = 9 (2 females, 7 males), TR2 n = 11 (3 females, 8 males), TR3 n = 10 (6 females, 4 males). Within 12 to 48 h of birth, muscle samples were collected from the Longissimus dorsi using a Bergstrom biopsy needle (inner diameter: 5 mm). Muscle biopsies were placed into sterile vials, stored on ice and frozen at – 80 ºC until total RNA isolation was performed. Total RNA was isolated using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Germantown, MA), following the manufacturer's protocol. After RNA quality control, strand-specific libraries (poly-A enrichment) were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs, Ipswich, MA). Paired-end sequencing (150-bp reads) at a depth of 20 M reads/sample was carried out on the Illumina NovaSeq 600 platform. RNA isolation, library preparation, and RNA sequencing were performed by Novogene Co. (Nanjing, China). Read quality control was performed with FastQC v0.11.8 (https://bit.ly/3pCUvar) and MultiQC v1.9 (https://multiqc.info/) software. After QC, clean reads (38 samples) were mapped to the Bos taurus reference genome (ARS-UCD 1.2) using the STAR aligner v. 2.7.3a (https://rb.gy/dlgdva). The –quantMode GeneCounts flag from STAR and the annotation information (release 100, Ensembl) were used to determine the number of raw counts per gene in each sample. MultiQC was used for post-mapping quality control. Furthermore, exploratory data analyses were based on Principal Component Analysis (PCA) and clustering using the R-packages NOISeq v.2.31.0 (10.18129/B9.bioc.NOISeq), edgeR v.3.30.3 (10.18129/B9.bioc.edgeR), and factoextra v.1.0.7 (https://bit.ly/3s2fZPA). The gene expression normalization procedure was performed through the DESeq2 v.1.28.1 (10.18129/B9.bioc.DESeq2) using the VST function. The DESeq2 design model included the dam’s age, offspring sex, and treatment group. Differentially expressed genes (DEGs) were identified between the treatments considering the t-test method proposed by Reverter et al. and implemented in the R-package CeTF v.1.0.7 (10.18129/B9.bioc.CeTF. The differences between the treatments, i.e., TR1 vs. CTR, TR2 vs. CTR, TR3 vs. CTR, were tested, and DEGs were considered significant at an FDR adjusted P-value < 0.05 and |log2 FC| > 1.5. The treatments were used as the reference group, and DEGs were classified as up or downregulated based on the log2 fold change direction. Gene annotation was performed using biomaRt v.2.38.0 (10.18129/B9.bioc.biomaRt) based on the B. taurus Ensembl database.