133 DNA methylation level of vulnerable genomic loci associated with large offspring syndrome

Calves derived from in vitro-produced (IVP) embryos are, on average, heavier than those produced by AI. Large/abnormal offspring syndrome (LOS/AOS) is an extreme overgrowth condition, sometimes seen in IVP calves, which is characterised by high birthweight, macrosomia, abdominal wall defects, organomegaly, and difficulty to stand and suckle after birth. The extreme weight of LOS calves can result in dystocia necessitating cesarean section that affects the survival and performance of the calf and dam. Even though the incidence of LOS/AOS is unknown, we have reported that this abnormality can negatively impact the bottom line of the industry and farmer (~$29 000/case). Using a bioinformatic approach, we previously reported 25 genomic regions susceptible to alterations in the level of DNA methylation (henceforth referred to as vulnerable loci) in LOS fetuses and calves. Our overall goal is to determine if these vulnerable loci could serve as molecular markers for the diagnosis of LOS during pregnancy. The immediate goal of this project is to develop bisulfite PCR assays for each of these loci to corroborate the bioinformatically identified altered levels of DNA methylation in LOS. To do this, muscle and intercotyledon of Day 105 female control (AI; n = 4; 416.5 ± 36.01 g) and female IVP (n = 8) conceptuses were used. IVP fetuses weighing >97 percentile of control weight were categorized as LOS. The weight of the IVP normal weight fetuses = 389.33 ± 64.07 g, while the weight of the LOS fetuses = 674 ± 186.39 g. Bisulfite primers were designed to amplify the first 402 base pairs (bp) of the first vulnerable locus (1400 bp), which is located on chromosome 4:102 068 961–102 070 360 (NC_037331.1; ARS-UCD1.3). It should be noted that no gene is annotated at that location in the current bovine genome assembly. The sequence of the forward primer is 5′-GAG GGA TGT TGT AAA TAA ATA ATA TTG G-3′ and the reverse primer is 5′-AAA CAA ACT TCT TTA CTA ACT AAA ACT T-3′. DNA was isolated, bisulfite mutagenized (ED DNA Methylation-Gold Kit, Zymo Research), and PCR was performed. The PCR amplicon was resolved in a 2% agarose gel and purified using Wizard SV gel and PCR Clean-Up System (Promega) before Sanger sequencing. Twenty-one CpGs were identified in the region of interest, and their methylation status was evaluated. From those CpGs, 20 (95.24%) were methylated in all samples, and 1 CpG was a polymorphic site. Therefore, the first section of vulnerable locus one does not appear to be useful for determining LOS. Currently, we are developing bisulfite assays for the rest of the vulnerable locus one and the other 24 loci. Results will be presented.