Emma N. Bermingham

Global DNA methylation measurement by HPLC using low amounts of DNA.

Epigenetic changes in chromatin structure can influence gene expression without affecting the DNA sequence. The most commonly studied epigenetic modification, DNA methylation, has been implicated in normal tissue development and disease progression, and can be influenced by diet and other environmental factors. Current HPLC methods of determining DNA methylation may require relatively large amounts of DNA (50 μg); as many tissues have low DNA yields, this can be hard to achieve. We isolated DNA from mouse colon and liver in a study investigating post-natal supplementation with selenium and folic acid. After optimizing the methods to account for lower initial DNA amounts, we digested 3 μg of DNA to deoxynucleotide monophosphates, then purified and quantified it. Samples were analyzed by reversed-phase HPLC to determine global DNA methylation levels using commercial nucleotide standards. The HPLC column was cooled to 6(C (reducing run time), and detection was at 280 nm (UV). We showed that methylated cytosine can be accurately and reproducibly measured in as little as 3 μg of DNA using this HPLC analysis method (within-assay CV <2%). We also used this method to detect reduced DNA methylation in liver (P = 0.009) in response to post-natal supplementation with selenium and folate.

Faecal Microbiota of Forage-Fed Horses in New Zealand and the Population Dynamics of Microbial Communities following Dietary Change

The effects of abrupt dietary transition on the faecal microbiota of forage-fed horses over a 3-week period were investigated. Yearling Thoroughbred fillies reared as a cohort were exclusively fed on either an ensiled conserved forage-grain diet (“Group A”; n = 6) or pasture (“Group B”; n = 6) for three weeks prior to the study. After the Day 0 faecal samples were collected, horses of Group A were abruptly transitioned to pasture. Both groups continued to graze similar pasture for three weeks, with faecal samples collected at 4-day intervals. DNA was isolated from the faeces and microbial 16S and 18S rRNA gene amplicons were generated and analysed by pyrosequencing. The faecal bacterial communities of both groups of horses were highly diverse (Simpson’s index of diversity >0.8), with differences between the two groups on Day 0 (P<0.017 adjusted for multiple comparisons). There were differences between Groups A and B in the relative abundances of four genera, BF311 (family Bacteroidaceae; P = 0.003), CF231 (family Paraprevotellaceae; P = 0.004), and currently unclassified members within the order Clostridiales (P = 0.003) and within the family Lachnospiraceae (P = 0.006). The bacterial community of Group A horses became similar to Group B within four days of feeding on pasture, whereas the structure of the archaeal community remained constant pre- and post-dietary change. The community structure of the faecal microbiota (bacteria, archaea and ciliate protozoa) of pasture-fed horses was also identified. The initial differences observed appeared to be linked to recent dietary history, with the bacterial community of the forage-fed horses responding rapidly to abrupt dietary change.

Investigating micronutrients and epigenetic mechanisms in relation to inflammatory bowel disease

Epigenomic regulation, via DNA methylation, histone modification and non-coding RNA, is increasingly recognised as having a key role in normal development and function of an organism, acting to control cellular and tissue growth and differentiation. It is also thought to be involved in many complex diseases now common in the Western world, including cardiovascular disease, type 2 diabetes, obesity and inflammatory bowel disease (IBD). There is a range of evidence to suggest that nutrition plays a vital role in the protection from such diseases. However, there is little information about the role of nutrition on the epigenetic regulation of IBD. This review aims to elucidate the interactions of nutrients and the epigenome in IBD. More specifically, the plasticity of epigenetic modifications that occur due to low selenium and folate levels in the diet during gestation and lactation will be discussed. A better understanding of this plasticity, and of nutrient-epigenome interactions, will have important implications for enhancing human health through foods.

Dietary format alters fecal bacterial populations in the domestic cat (Felis catus)

The effects of short‐term (5‐week) exposure to wet or dry diets on fecal bacterial populations in the cat were investigated. Sixteen mixed‐sex, neutered, domestic short‐haired cats (mean age = 6 years; mean bodyweight = 3.4 kg) were randomly allocated to wet or dry diets in a crossover design. Fecal bacterial DNA was isolated and bacterial 16S rRNA gene amplicons generated and analyzed by 454 Titanium pyrosequencing. Cats fed dry diets had higher abundances (P < 0.05) of Actinobacteria (16.5% vs. 0.1%) and lower abundances of Fusobacteria (0.3% vs. 23.1%) and Proteobacteria (0.4% vs. 1.1%) compared with cats fed the wet diet. Of the 46 genera identified, 30 were affected (P < 0.05) by diet, with higher abundances of Lactobacillus (31.8% vs. 0.1%), Megasphaera (23.0% vs. 0.0%), and Olsenella (16.4% vs. 0.0%), and lower abundances of Bacteroides (0.6% vs. 5.7%) and Blautia (0.3% vs. 2.3%) in cats fed the dry diet compared with cats fed the wet diet. These results demonstrate that short‐term dietary exposure to diet leads to large shifts in fecal bacterial populations that have the potential to affect the ability of the cat to process macronutrients in the diet.

Energy requirements of adult cats

A meta-analysis was carried out in order to establish the energy requirements of adult cats. Publications that identified cat body weight (BW) were used to generate allometric relationships between energy requirements and BW of healthy adult cats, using log-log linear regression. Energy requirements were expressed in kcal/kg BW to be consistent with those reported by the National Research Council. Mean maintenance energy requirements were 55.1 (se 1.2) kcal/kg BW (115 treatment groups). Three allometric equations were identified to predict the energy requirements for maintenance of BW in the cat based on BW: light (53.7 kcal/kg BW- 1.061), normal (46.8 kcal/kg BW- 1.115) and heavy (131.8 kcal/kg BW- 0 .366). When reported on lean mass, the allometric equation revealed maintenance requirements were 58.4 kcal/kg lean mass- 1.140 (adjusted R2 0.694; thirty-six treatment groups). The present review suggests that values for maintenance energy requirements based on BW alone may not be an accurate prediction and more detailed information on the age, sex and neuter status, BW and composition would enhance the ability to interpret the maintenance energy requirements of cats.

Post-weaning selenium and folate supplementation affects gene and protein expression and global DNA methylation in mice fed high-fat diets

BackgroundConsumption of high-fat diets has negative impacts on health and well-being, some of which may be epigenetically regulated. Selenium and folate are two compounds which influence epigenetic mechanisms. We investigated the hypothesis that post-weaning supplementation with adequate levels of selenium and folate in offspring of female mice fed a high-fat, low selenium and folate diet during gestation and lactation will lead to epigenetic changes of potential importance for long-term health.MethodsFemale offspring of mothers fed the experimental diet were either maintained on this diet (HF-low-low), or weaned onto a high-fat diet with sufficient levels of selenium and folate (HF-low-suf), for 8 weeks. Gene and protein expression, DNA methylation, and histone modifications were measured in colon and liver of female offspring.ResultsAdequate levels of selenium and folate post-weaning affected gene expression in colon and liver of offspring, including decreasing Slc2a4 gene expression. Protein expression was only altered in the liver. There was no effect of adequate levels of selenium and folate on global histone modifications in the liver. Global liver DNA methylation was decreased in mice switched to adequate levels of selenium and folate, but there was no effect on methylation of specific CpG sites within the Slc2a4 gene in liver.ConclusionsPost-weaning supplementation with adequate levels of selenium and folate in female offspring of mice fed high-fat diets inadequate in selenium and folate during gestation and lactation can alter global DNA methylation in liver. This may be one factor through which the negative effects of a poor diet during early life can be ameliorated. Further research is required to establish what role epigenetic changes play in mediating observed changes in gene and protein expression, and the relevance of these changes to health.

Nematodes and nutrient partitioning

Subclinical infection of sheep with gastrointestinal nematodes results in the diversion of nutrients from growth and development towards the repair of damaged intestinal tissues and to sustain the metabolic shifts (nutritional, hormonal and immune) occuring in tissues affected by the parasites. These metabolic effects include decreased nitrogen retention; increased amino acid utilisation and oxidation in the gastrointestinal tract; increased endogenous protein loss and altered protein synthesis in the gastrointestinal tract; increased amino acid utilisation and protein synthesis in the liver and activation of the immune system. These observations strongly suggest that there is competition between the key tissues involved in parasitism and that metabolic decisions are made resulting in the re-prioritisation of nutrient utilisation between the tissues in this inter-organ system. Nutritional status of the host can influence the pathogenesis of parasitic infection and well-nourished animals generally withstand parasitism better than those less adequately fed. Recent studies have focused on the effect of restricting intake on the acquisition of immunity in sheep selected either for resistance or susceptibility to nematodes. The major outcome of these studies was the increase of peripheral eosinophil counts in resistant animals infected with Trichostrongylus colubriformis when fed on an ad libitum diet compared with similar animals fed the same diet at a maintenance level. This was inversely correlated to the numbers of adult parasites recovered. Intake seems to be the primary determinant of the expression of immunity in sheep selected for resistance against nematodes.

Selenium-Enriched Foods Are More Effective at Increasing Glutathione Peroxidase (GPx) Activity Compared with Selenomethionine: A Meta-Analysis

Selenium may play a beneficial role in multi-factorial illnesses with genetic and environmental linkages via epigenetic regulation in part via glutathione peroxidase (GPx) activity. A meta-analysis was undertaken to quantify the effects of dietary selenium supplementation on the activity of overall GPx activity in different tissues and animal species and to compare the effectiveness of different forms of dietary selenium. GPx activity response was affected by both the dose and form of selenium (p < 0.001). There were differences between tissues on the effects of selenium supplementation on GPx activity (p < 0.001); however, there was no evidence in the data of differences between animal species (p = 0.95). The interactions between dose and tissue, animal species and form were significant (p < 0.001). Tissues particularly sensitive to changes in selenium supply include red blood cells, kidney and muscle. The meta-analysis identified that for animal species selenium-enriched foods were more effective than selenomethionine at increasing GPx activity.

Seasonal and age effects on energy requirements in domestic short‐hair cats (Felis catus) in a temperate environment

There is little information known about the energy requirements of cats in temperature climates. Energy requirement of domestic short-haired cats was determined using three groups of mixed gender - old kept outside (approximately 9.9 years of age; 4.8 kg; n = 9), young kept outside (approximately 3.1 years of age; 3.9 kg; n = 8) or young kept inside (approximately 3.1 years of age; 3.9 kg; n = 8). Cats were housed individually for 5 weeks during summer (18.5 ± 0.5 °C) and winter (8.5 ± 0.4 °C) and were fed a commercially available maintenance diet ad libitum. In both periods, energy expenditure was determined from the rates of (2) H and (18) O elimination for blood H2 O over a 12 day period, from a doubly labelled water bolus (2) H2 O (0.7 g/kg BW) and H2 (18) O (0.13 g/kg BW) administered intravenously. During the summer period, macronutrient digestibility was determined. Older cats had a reduction (p < 0.05) in apparent digestibility of dry matter (approximately 9%), energy (approximately 8%) and protein (6%). There was a significant effect of age and season on energy intake and energy expenditure. While lean mass was affected by age and season, there was no effect of age or season on energy expenditure when expressed as a proportion of lean mass. Possible seasonal differences in nutrient digestibility may explain these results.

Energy Requirements of Adult Dogs: A Meta-Analysis

A meta-analysis was conducted to determine the maintenance energy requirements of adult dogs. Suitable publications were first identified, and then used to generate relationships amongst energy requirements, husbandry, activity level, methodology, sex, neuter status, dog size, and age in healthy adult dogs. Allometric equations for maintenance energy requirements were determined using log-log linear regression. So that the resulting equations could readily be compared with equations reported by the National Research Council, maintenance energy requirements in the current study were determined in kcal/kg0.75 body weight (BW). Ultimately, the data of 70 treatment groups from 29 publications were used, and mean (± standard deviation) maintenance energy requirements were 142.8±55.3 kcal.kgBW−0.75.day−1. The corresponding allometric equation was 81.5 kcal.kgBW−0.93.day−1 (adjusted R2 = 0.64; 70 treatment groups). Type of husbandry had a significant effect on maintenance energy requirements (P<0.001): requirements were greatest in racing dogs, followed by working dogs and hunting dogs, whilst the energy requirements of pet dogs and kennel dogs were least. Maintenance energy requirements were less in neutered compared with sexually intact dogs (P<0.001), but there was no effect of sex. Further, reported activity level tended to effect the maintenance energy requirement of the dog (P = 0.09). This review suggests that estimating maintenance energy requirements based on BW alone may not be accurate, but that predictions that factor in husbandry, neuter status and, possibly, activity level might be superior. Additionally, more information on the nutrient requirements of older dogs, and those at the extremes of body size (i.e. giant and toy breeds) is needed.