Maternal N3 feeding study to determine the impact on offspring DNA methylation, 1730_CN3
Library Preparation:  Genomic DNA was isolated from buffy coats using the Purelink genomic DNA isolation kit (Life Technologies, Carlsbad, CA) and fragmented to 200-400 bp using a Diagenode Bioruptor Sonicator (Denville, NJ). The Methylminer kit (Life Technologies, Carlsbad, CA) was used to isolate fragmented DNA that contained methylated CpG islands.  Fragmented genomic DNA (4 µg) was allowed to bind human MBD2 protein with the bound fraction that contained methylated DNA according to manufacturer’s instructions. Methylation-enriched DNA was purified and a single 2M NaCl fraction was performed to enrich for highly methylated DNA. The enriched DNA was then used to prepare next generation DNA libraries for each individual animal (TruSeq, Illumina, San Diego, CA). Indexed libraries were pooled (3 animals per lane) and 100bp single end reads were generated on an Illumina HiSeq 2000 at the Iowa State University DNA Facility (Ames, IA). An average of 24,017,392 reads were generated per animal, which corresponded to 26.2% of the genome and an average of 3.4x coverage depth over the captured region (Table 1).    Experimental Design:  Briefly, five sows were fed a basal diet and 10 sows a diet enriched with omega-3 PUFA upon confirmation of pregnancy. The two sow diets were: 1) standard corn-soybean meal diet devoid of EPA and DHA (basal) and 2) the basal diet supplemented with a protected source of marine EPA and DHA provided in the form of 0.5% Gromega™ (JBS United Inc., Sheridan, IN). These diets met or exceeded swine National Research Council dietary guidelines for (1998) gestation and lactation nutrient and energy requirements for sows. Upon farrowing, litters were standardized to ten piglets per litter within 24 h of birth, with cross fostering between litters occurring within treatment and only if needed.  At weaning (18-21 days of age), piglets were separated and individually penned over a ten-week nursery period to complete the last phase of the diet trial.    Over this nursery period, pigs from control sows stayed on an EPA and DHA devoid corn-soybean meal control nursery diet (CON, n=5). The ten piglets from the omega-3 fed sows were then split onto two nursery diets. Five piglets were fed the same control nursery diet as the CON piglets, thus only had EPA and DHA enrichment from maternal sources (Mn3). The remaining five littermates were continuously fed an omega-3 PUFA nursery diet supplemented with EPA and DHA in the form of 0.5% Gromega™ throughout gestation, lactation and post-weaning.  A schematic of the experimental design is presented in supplemental figure 1.  In all supplemented diets, Gromega™ was mixed in the basal diet as a substitution to corn and formulated to meet or exceed the nutrient requirements of the respective growth period.