296 Comparison of Dna Methylation Patterns in Sperm Collected from Brahman Bulls with Differences in Embryo Development
Authors: Ketchum Jaclyn N , Drum Jessica , Willis Caleta , Guy Chloey P , Ortega Sofia , Kerns Karl , Long Charles R , Love Charles , Welsh Thomas H , Randel Ronald D , Perry George A 
Published: November 2023

Abstract
The objective was to determine if methylation patterns in sperm differed between bulls with good vs poor embryo development. Semen collected from sexually mature Brahman bulls was purified from frozen-thawed straws using a gradient [50% (vol/vol) and 90% (vol/vol)] of Isolate (Irvine Scientific, Santa Ana, CA), and used to fertilize abattoir-derived cumulus oocyte complexes (n = 81-100/bull in 2 wells). Number of blastocysts were recorded on d 8, and bulls were classified as Good (GD; &gt;35% blastocyst rate; n = 5) or Poor (PD; &lt; 35% blastocyst rate; n = 4) embryo development. Methylation of sperm DNA was assessed by reduced representation sodium bisulfite sequencing. Differences in methylation were determined with the Dispersion Shrinkage for Sequencing method in conjunction with Wald test. Differentially methylated cytosines and regions located in promoters of genes and gene bodies were analyzed to reveal gene ontology terms associated with them. Proportion of sperm cells that were viable (before and after a three-hour stress test), with inceased reactive oxygen species (ROS), and with damaged chromatin was determined. Statistical analysis (PROC GLIMMIX; SAS 9.4) included the fixed effect of embryo development classification which was considered significant at P &lt; 0.05. Mean separation was performed using LSmeans. The proportion of viable (P &gt; 0.21; GD = 46 ± 5%; PD = 36 ± 5%) sperm cells did not differ before the stress test. Post-stress test, PD bulls had more viable sperm cells (P &lt; 0.02; 37 ± 3%) than GD (25 ± 2%) bulls. There was no difference between GD and PD for ROS (H2O2; P &gt; 0.16 and •O2; P &gt; 0.59). Proportion of sperm cells with damaged chromatin was greater (P &lt; 0.01) in PD (3 ± 0.3%) than GD bulls (1 ± 0.01%). There were 1,742 differentially methylated cytosines and 343 differentially methylated regions when GD bulls were compared with PD bulls. Genes whose promoters were differentially methylated included DDC, GTSF1L, PPP1R27, NTM, HCK, GPR173, and TNFRSF1A. Functionally enriched pathways for differentially methylated promoters included apoptosis and amyotrophic lateral sclerosis while some of the significant biological processes were regulation of apoptotic process involved in morphogenesis, negative regulation of neuron migration, pyrimidine nucleoside catabolic process, and regulation of cardiac muscle tissue development. Differentially methylated gene bodies were significantly related to cellular components such as cell surface, endomembrane system, synapse, and cell periphery while molecular functions included binding, protein binding, and transmembrane receptor protein tyrosine kinase activity. Biological processes impacted by differentially methylated gene bodies included system development, neurogenesis, animal organ development, anatomical structure development, generation of neurons, nervous system development, and multicellular organism development. In summary, differences in embryo development are likely due to differences in methylation patterns impacting developmental processes, as semen characteristics were mainly similar between bulls with good versus poor embryo development.