Genetic potential for residual feed intake and diet fed during early- to mid-gestation influences post-natal DNA methylation of imprinted genes in muscle and liver tissues in beef cattle.

Discovery of epigenetic modifications associated with feed efficiency or other economically important traits would increase our understanding of the molecular mechanisms underlying these traits. In combination with known genetic markers, this would provide opportunity to improve genomic selection accuracy in cattle breeding programs. It would also allow cattle to be managed to improve favorable gene expression. The objective of this study was to identify variation in DNA methylation between beef cattle of differential pre-natal nutrition and divergent genetic potential for residual feed intake (RFI). Purebred Angus offspring with the genetic potential for either high (HRFI) or low (LRFI) RFI were prenatally exposed to either a restricted maternal diet of 0.5 kg/d average daily gain (ADG) or a moderate maternal diet of 0.7 kg/d ADG from 30 to 150 d of gestation. We performed DNA methylation analysis of differentially methylated regions (DMR) of imprinted genes (Insulin-like growth factor 2 (IGF2) DMR2, IGF2/H19 imprinting control region (ICR) and IGF2 receptor (IGF2R) DMR2) using post-natal samples of longissimus dorsi (LD) muscle taken from male and female calves at birth and weaning, and of LD muscle, semimembranosus (SM) muscle, and liver samples collected from steers at slaughter (17 months of age). Interestingly, for all three DMR investigated in liver, LRFI steers had higher levels of methylation than HRFI steers. In LD muscle, IGF2/H19 ICR methylation differences for heifers at birth were due to pre-natal diet, while for steers at birth they were mostly the result of genetic potential for RFI with LRFI steers again having higher levels of methylation than HRFI steers. While results from repeated measures analysis of DNA methylation in steers grouped by RFI revealed few differences, in steers grouped by diet, we found higher methylation levels of IGF2 DMR2 and IGF2R DMR2 in LD muscle of restricted diet steers at weaning and slaughter than at birth, as well as increased methylation in LD muscle of restricted diet steers compared with moderate diet steers at weaning and/or slaughter. Our results suggest that differential pre-natal nutrition, and divergent genetic potential for RFI, induces tissue- and sex-specific alterations in post-natal IGF2 and IGF2R methylation patterns and that these patterns can vary with age in Angus beef cattle.

Inactivation of Escherichia coli O157:H7 in Minute Steaks Cooked under Selected Conditions.

A national survey was conducted in Canada to determine consumer cooking practices for minute steaks (thin, mechanically tenderized beef cutlets). Results indicate that most Canadians prefer cooking minute steaks by pan frying and to a medium level of doneness. To identify safe cooking conditions, retail minute steaks (∼125 g), inoculated at three sites per steak with a five-strain cocktail of nontoxigenic Escherichia coli O157:H7 (6.1 log CFU per site), were cooked on a hot plate (200°C), mimicking a pan-frying scenario. The steaks (n = 5) were cooked for 4, 6, 8, or 10 min with turning over (flipping) up to four times at equal time intervals; or to 63 or 71°C at the thickest area with or without a tinfoil lid. When cooked for 4 min, E. coli O157:H7 was recovered from all inoculation sites, and the mean reductions at various sites (1.2 to 3.4 log CFU per site) were not different (P > 0.05), irrespective of the flipping frequency. When cooked for 6 min with flipping once or twice, or for 8 min with flipping once, E. coli O157:H7 was recovered from most sites; the mean reductions (3.8 to 5.3 log CFU per site) were not different (P > 0.05), but they were higher (P < 0.05) than those for steaks cooked for 4 min. When cooked for 10, 8, or 6 min with flipping once, twice, or three times, respectively, E. coli O157:H7 was eliminated from most sites, but sites with <5-log reductions were found. Reductions of E. coli O157:H7 by >5 log at all inoculation sites were attained when the steaks were cooked for 10 or 8 min with two or more or three or more flippings, respectively, or for 6 min with four flippings. When flipped twice during cooking to 63 or 71°C, E. coli O157:H7 was recovered from three or fewer sites; however, >5-log reductions throughout the steaks were only attained for the latter temperature, irrespective of whether the hot plate was covered with the tinfoil lid. Thus, turning over minute steaks twice during cooking to 71°C or flipping two, three, or four times with a cooking time of 10, 8, or 6 min could achieve 5-log reductions throughout the steaks.

Spatial and Temporal Distribution of Escherichia coli on Beef Trimmings Obtained from a Beef Packing Plant.

The objective of this study was to determine the immediate source of Escherichia coli on beef trimmings produced at a large packing plant by analyzing the E. coli on trimmings at various locations of a combo bin filled on the same day and of bins filled on different days. Ten 2,000-lb (907-kg) combo bins (B1 through B10) of trimmings were obtained from a large plant on 6 days over a period of 5 weeks. Thin slices of beef with a total area of approximately 100 cm(2) were excised from five locations (four corners and the center) at each of four levels of the bins: the top surface and 30, 60, and 90 cm below the top. The samples were enriched for E. coli in modified tryptone soya broth supplemented with 20 mg/liter novobiocin. The positive enrichment cultures, as determined by PCR, were plated on E. coli/coliform count plates for recovery of E. coli. Selected E. coli isolates were genotyped using multiple-locus variable-number tandem repeat analysis (MLVA). Of the 200 enrichment cultures, 43 were positive by PCR for E. coli, and 32 of these cultures yielded E. coli isolates. Two bins did not yield any positive enrichment cultures, and three PCR-positive bins did not yield any E. coli isolates. MLVA of 165 E. coli isolates (30, 62, 56, 5, and 12 from B6 through B10, respectively) revealed nine distinct genotypes. MLVA types 263 and 89 were most prevalent overall and on individual days, accounting for 49.1 and 37.6% of the total isolates, respectively. These two genotypes were also found at multiple locations within a bin. All nine genotypes belonged to the phylogenetic group A0 of E. coli, suggesting an animal origin. The finding that the trimmings carried very few E. coli indicates an overall effective control over contamination of beef with E. coli at this processing plant. The lack of strain diversity of the E. coli on trimmings suggests that most E. coli isolates may have come from common sources, most likely equipment used in the fabrication process.

Magel2, a Prader-Willi syndrome candidate gene, modulates the activities of circadian rhythm proteins in cultured cells.

BACKGROUND: The Magel2 gene is most highly expressed in the suprachiasmatic nucleus of the hypothalamus, where its expression cycles in a circadian pattern comparable to that of clock-controlled genes. Mice lacking the Magel2 gene have hypothalamic dysfunction, including circadian defects that include reduced and fragmented total activity, excessive activity during the subjective day, but they have a normal circadian period. Magel2 is a member of the MAGE family of proteins that have various roles in cellular function, but the specific function of Magel2 is unknown. METHODS: We used a variety of cell-based assays to determine whether Magel2 modifies the properties of core circadian rhythm proteins. RESULTS: Magel2 represses the activity of the Clock:Bmal1 heterodimer in a Per2-luciferase assay. Magel2 interacts with Bmal1 and with Per2 as measured by co-immunoprecipitation in co-transfected cells, and exhibits a subcellular distribution consistent with these interactions when visualized by immunofluorescence. As well, Magel2 induces the redistribution of the subcellular localization of Clock towards the cytoplasm, in contrast to the nucleus-directed effect of Bmal1 on Clock subcellular localization. CONCLUSION: Consistent with the blunted circadian rhythm observed in Magel2-null mice, these data suggest that Magel2 normally promotes negative feedback regulation of the cellular circadian cycle, through interactions with key core circadian rhythm proteins.