Kevin D. Sinclair

Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture

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.

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status

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 health-related phenotypes.

Effect of Dietary Energy and Protein on Bovine Follicular Dynamics and Embryo Production In Vitro: Associations with the Ovarian Insulin-Like Growth Factor System

Abstract 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.

Impact of Nutrition on Oocyte Quality: Cumulative Effects of Body Composition and Diet Leading to Hyperinsulinemia in Cattle

Abstract The present study sought to assess the combined effects of body composition and diet (level of feeding) on the postfertilization developmental potential of oocytes recovered from heifers using ultrasound-guided transvaginal follicular aspiration and to relate oocyte quality to the metabolic status of these animals. By collecting oocytes on repeated occasions spanning several weeks, it was possible to assess the cumulative effects of changes in nutritional status on oocyte quality over this period. Twenty-four heifers of low and moderate body condition were placed on one of two levels of feeding (equivalent to once or twice the maintenance requirements of these animals). Oocytes were recovered at two defined time points within each of three successive estrous cycles and were matured, fertilized, and cultured to the blastocyst stage in vitro. The results show that the effect of feeding level on oocyte quality is dependent on the body condition of the animal, with the high level of feeding being beneficial to oocytes from animals of low body condition but detrimental to oocytes from animals of moderately high body condition. Furthermore, the effects of high levels of feeding on oocyte quality were cumulative, with blastocyst yields for relatively fat heifers on twice the maintenance requirement deteriorating with time relative to yields for relatively thin heifers on the same level of feeding. Finally, a significant proportion of the moderately fat animals on the high level of feeding were hyperinsulinemic, and we show, to our knowledge for the first time in ruminants, that this condition is associated with impaired oocyte quality.

Nutritional effects on foetal growth

The emphasis in nutritional studies on foetal growth has now moved from the last trimester of pregnancy, when most of the increase in foetal size takes place, to earlier stages of pregnancy that coincide with foetal organogenesis and tissue hyperplasia. At these stages absolute nutrient requirements for foetal growth are small but foetal metabolic activity and specific growth rate are high. It is thus a time when nutrient supply interacts with maternal factors such as size, body condition and degree of maturity to influence placental growth and set the subsequent pattern of nutrient partitioning between the gravid uterus and maternal body. Throughout pregnancy the maternal diet controls foetal growth both directly, by supplying essential nutrients and indirectly, by altering the expression of the maternal and foetal endocrine mechanisms that regulate the uptake and utilization of these nutrients by the conceptus. Nutritional effects on the endocrine environment of the embryo during the early stages of cell division can alter the subsequent foetal growth trajectory and size at birth; so too can current in vitro systems for oocyte maturation and embryo culture up to the blastocyst stage. There is increasing evidence that subtle alterations in nutrient supply during critical periods of embryonic and foetal life can impart a legacy of growth and developmental changes that affect neonatal survival and adult performance. Identifying the specific nutrients that programme these effects and understanding their mode of action should provide new management strategies for ensuring that nutritional regimens from oocyte to newborn are such that they maximize neonatal viability and enable animals to express their true genetic potential for production.

THE EFFECTS OF AGE AT SLAUGHTER, GENOTYPE AND FINISHING SYSTEM ON THE BIOCHEMICAL PROPERTIES, MUSCLE FIBRE TYPE CHARACTERISTICS AND EATING QUALITY OF BULL BEEF FROM SUCKLED CALVES

Muscle fibre characteristics and biochemical properties of muscle recovered from young bulls of two genotypes (Aberdeen Angus × and Charolais ×), reared on two different diets (silage-based and barley-based) and slaughtered at varying ages between 10 and 19 months of age were established. These analyses were restricted to samples ofm. longissimus lumborum (LI) recovered at 48 h post mortem, vacuum packed and stored at 2°C for 14 days. Biochemical measurements included intramuscular fat content, intramuscular collagen content and its solubility, haem pigment concentration, sarcomere length and myofibril fragmentation. Muscle fibre type was classified according to the contractile nature of thefibres and their metabolic properties. Intramuscular fat content increased ( P v. 295·8 mg/g dry matter, P P v. 3·59 mg/g for Angus × and Charolais × bulls; P × 0·01) and diet (3·97 v. 3·62 mg/g for bulls given barley and silage; P P P P v. 49·3%; P v. 18·9%; P P P

Assisted Reproductive Technology, Epigenetics, and Long-Term Health: A Developmental Time Bomb Still Ticking

Live birthrates following assisted reproduction account for 1 to 3% of pregnancies in developed countries, and these figures seem set to rise. Concerns regarding the safe use of assisted reproductive technology (ART) for the treatment of infertility have been voiced for several years, yet, to date, the vast majority of children conceived using these techniques are apparently normal. Controversy surrounding reports of epigenetic alterations to genomic imprinting following human ART in recent years has fueled the ongoing debate. In contrast, both the incidence and severity of such anomalies are more apparent following ART in comparative animal species. The reasons for this are not known. By and large, the confounding effects of infertility and advanced maternal age do not apply to animal studies, which report better pregnancy rates following embryo transfer. Perhaps the incidence of imprinting disorders is increased when procedures such as ovarian stimulation, in vitro maturation, or both are used in conjunction with extended periods of embryo culture; this frequently occurs in animal but rarely in human ART. The focus of attention on imprinting, however, may have served to distract the scientific community from more subtle epigenetic modifications to nonimprinted loci in gametes and the preimplantation embryo, with health-related consequences that do not manifest until adulthood. Accumulating evidence from animal studies indicates that such effects, not yet apparent in human subjects, exist; and this may ultimately transpire to be the true developmental legacy of human ART. This article discusses these issues in the context of epigenetic and developmental abnormalities following ART in animals.

Large offspring syndrome and other consequences of ruminant embryo culture in vitro: Relevance to blastocyst culture in human ART

In vitro production of embryos from domestic animals is used to augment conventional genetic improvement programmes in agriculture and to facilitate advances in gene transfer and cloning. However, production of embryos in vitro exposes them to hazards not normally encountered in vivo and, as a result, there have been unforeseen consequences including the large offspring syndrome. This syndrome is manifest as abnormal growth and development at fetal, neonatal and later stages after transfer of embryos cultured in vitro for up to 1 week after fertilization. Our embryo culture and fetal development studies have begun to characterize many of the genetic, metabolic and developmental features associated with the syndrome. This review considers the findings of these studies in the context of blastocyst production in vitro, emphasizing the impact of culture strategies on ruminant (cattle and sheep) embryo composition and developmental competence. The need to alter in vitro production strategies to safeguard oocytes and embryos during culture is discussed. Finally, the implications of experiences gained in domestic animal studies are considered in the context of current options for human embryo culture. The need for an appreciation of the sensitivity of the embryo to its environment and the possible short- and long-term consequences of inappropriate in vitro production strategies are considered.

Effect of dietary-induced changes in plasma insulin concentrations during the early post partum period on pregnancy rate in dairy cows

Dietary stimulation of insulin in post partum dairy cows has been found to enhance ovarian follicle development but to impair oocyte developmental competence. The objective of this study was to test the hypothesis that pregnancy rate would be improved by feeding a diet to stimulate higher insulin (H) until cows resumed ovarian cyclic activity after parturition, and then feeding a diet to lower insulin (L) during the mating period. Each diet was fed to 30 post partum dairy cows until their first rise in milk progesterone, when 15 cows in each group were transferred to the other diet (treatments HL and LH) and 15 cows in each group remained on their original diet (treatments HH and LL) until 120 days post partum. Treatments did not affect dry matter intake, milk yield and metabolisable energy balance. Plasma insulin concentration was elevated in cows fed on H compared with cows fed on L. Treatment did not affect days to first progesterone rise, first oestrus or first insemination. At 120 days post partum, 27% of cows on each of treatments HH, LL and LH were pregnant, but 60% of cows on treatment HL were pregnant (P=0.021). These findings support the concept that physiological relationships between insulin and the reproductive system vary according to stage of the reproductive cycle, and suggest that pregnancy rate can be enhanced by a two-diet strategy tailored to optimise responses before and after the first post partum ovulation.

Dietary omega-3 and -6 polyunsaturated fatty acids affect the composition and development of sheep granulosa cells, oocytes and embryos

The evidence that omega-3 (n-3) and -6 (n-6) polyunsaturated fatty acids (PUFAs) have differential effects on ovarian function, oocytes and embryo quality is inconsistent. We report on the effects of n-3 versus n-6 PUFA-enriched diets fed to 36 ewes over a 6-week period, prior to ovarian stimulation and follicular aspiration, on ovarian steroidogenic parameters and embryo quality. Follicle number and size were unaltered by diet, but follicular-fluid progesterone concentrations were greater in n-3 PUFA-fed ewes than in n-6 PUFA-fed ewes. The percentage of saturated FAs (mostly stearic acid) was greater in oocytes than in either granulosa cells or plasma, indicating selective uptake and/or de novo synthesis of saturated FAs at the expense of PUFAs by oocytes. High-density lipoproteins (HDLs) fractionated from sera of these ewes increased granulosa cell proliferation and steroidogenesis relative to the FA-free BSA control during culture, but there was no differential effect of n-3 and n-6 PUFAs on either oestradiol or progesterone production. HDL was ineffective in delivering FAs to embryos during culture, although n-6 PUFA HDL reduced embryo development. All blastocysts, irrespective of the treatment, contained high levels of unsaturated FAs, in particular linoleic acid. Transcripts for HDL and low-density lipoprotein (LDL) receptors (SCARB1 and LDLR) and stearoyl-CoA desaturase (SCD) are reported in sheep embryos. HDL reduced the expression of transcripts for LDLR and SCD relative to the BSA control. The data support a differential effect of n-3 and n-6 PUFAs on ovarian steroidogenesis and pre-implantation development, the latter in the absence of a net uptake of FAs.