A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B12 and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult healthrelated phenotypes. E vidence from both epidemiological studies in humans and direct intervention studies in animals indicates that altering key developmental processes in utero can predispose offspring to many late-onset diseases such as dyslipidemia, type II diabetes, and heart disease (1, 2). In this regard, the effects of gross nutrient or protein deficiencies in maternal diet during pregnancy are well documented (3), although little is known about the effects of specific nutrients or the timing and mechanistic basis of nutrient programming (4). Here we investigated the effects of restricting the supply of specific B group vitamins (i.e., vitamin B 12 and folate) and sulfur amino acids (in particular, methionine) from the diet of adult female sheep from 8 weeks preceding until 6 days after conception, within physiological ranges encountered in both sheep (5) and humans (i.e., within the 5th and 95th percentiles) (6, 7). These micronutrients are important intermediates and/or have specific regulatory functions in the linked methionine-folate cycles (5, 7). In rodents, maternal supraphysiological methyl group supply and a low-protein diet (50% control) offered throughout pregnancy altered DNA methylation of candidate genes (agouti, glucocorticoid receptor, and peroxisomal proliferator-activated receptor-␣) (8, 9), but the extent of methylation change in these or more clinically relevant diets is not known. Gametes and preimplantation emb...

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

Cattle, pig and sheep oocytes isolated from healthy cumulus-oocyte complexes were pooled, within species, to provide samples of immature denuded oocytes with intact zona pellucida (n = 1000 per sample) for determination of fatty acid mass and composition in total lipid, constituent phospholipid and triglyceride. Acyl-containing lipid extracts, transmethylated in the presence of a reference penta-decaenoic acid (15:0), yielded fatty acid methyl esters which were analysed by gas chromatography. Mean (± SEM) fatty acid content in samples of pig oocytes (161 ± 18 µg per 1000 oocytes) was greater than that in cattle (63 ± 6 µg; P < 0.01) and sheep oocytes (89 ± 7 µg; P < 0.05). Of 24 fatty acids detected, palmitic (16:0; 25-35%, w/w), stearic (18:0; 14-16%) and oleic (18:1n-9; 22-26%) acids were most prominent in all three species. Saturated fatty acids (mean = 45-55%, w/w) were more abundant than mono-(27-34%) or polyunsaturates (11-21%). Fatty acids of the n-6 series, notably linoleic (18:2n-6; 5-8%, w/w) and arachidonic acid (20:4n-6; 1-3%), were the most abundant polyunsaturates. Phospholipid consistently accounted for a quarter of all fatty acids in the three species, but ruminant oocytes had a lower complement of polyunsaturates (14-19%, w/w) in this fraction than pig oocytes (34%, w/w) which, for example, had a three-to fourfold greater linoleic acid content. An estimated 74 ng of fatty acid was sequestered in the triglyceride fraction of individual pig oocytes compared with 23-25 ng in ruminant oocytes (P < 0.01). It is concluded that the greater fatty acid content of pig oocytes is primarily due to more abundant triglyceride reserves. Furthermore, this speciesspecific difference, and that in respect of polyunsaturated fatty acid reserves, may underlie the contrasting chilling, culture and cryopreservation sensitivities of embryos derived from pig and ruminant (cattle, sheep) oocytes.

Heifers were assigned either low or high (HE) levels of energy intake and low or high concentrations of dietary crude protein. The effect of these diets on the plasma concentrations of insulin, insulin-like growth factor (IGF)-I, and urea on follicular growth and early embryo development is described. We propose that the observed dietary-induced changes in the ovarian IGF system increase bioavailability of intrafollicular IGF, thus increasing the sensitivity of follicles to FSH. These changes, in combination with increased peripheral concentrations of insulin and IGF-I in heifers offered the HE diet, contribute to the observed increase in growth rate of the dominant follicle. In contrast to follicular growth, increased nutrient supply decreased oocyte quality, due in part to increased plasma urea concentrations. Clearly a number of mechanisms are involved in mediating the effects of dietary energy and protein on ovarian function, and the formulation of diets designed to optimize cattle fertility must consider the divergent effects of nutrient supply on follicular growth and oocyte quality.

This review examines the ‘Quiet Embryo Hypothesis’ which proposes that viable preimplantation embryos operate at metabolite or nutrient turnover rates distributed within lower ranges than those of their less viable counterparts. The ‘quieter’ metabolism consistent with this hypothesis is considered in terms of (i) ‘functional’ quietness; the contrasting levels of intrinsic metabolic activity in different cell types as a consequence of their specialized functions, (ii) inter-individual embryo/cell differences in metabolism and (iii) loss of quietness in response to environmental stress. Data are reviewed which indicate that gametes and early embryos function in vivo at a lower temperature than core body temperature, which could encourage the expression of a quiet metabolism. We call for research to determine the optimum temperature for mammalian gamete/embryo culture. The review concludes by examining the key role of reactive oxygen species, which can induce molecular damage, trigger a cellular stress response and lead to a loss of quietness.