T.G. McEvoy

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.

Fatty acid composition of lipids in immature cattle, pig and sheep oocytes with intact zona pellucida

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 chromatograph. Mean (+/- SEM) fatty acid content in samples of pig oocytes (161 +/- 18 micrograms per 1000 oocytes) was greater than that in cattle (63 +/- 6 micrograms; P < 0.01) and sheep oocytes (89 +/- 7 micrograms; 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 species-specific 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.

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.

Feed and forage toxicants affecting embryo survival and fetal development

Early embryonic and fetal development in mammals is sensitive to deficiencies and excesses of specific nutrients and toxicants. Operating directly and/or indirectly, these deficiencies and excesses can result in embryonic death or, in less severe circumstances, disruption of normal embryo and fetal growth. This paper explores the threats posed by feed and forage toxicants to the developing embryo and their impact on early programming of fetal development. Using significant examples, we consider the relevance of temporal sensitivities during early development in utero, and their implications for the morphology and functional competence of specific organs and tissues.

Consequences of exposure to serum, with or without vitamin E supplementation, in terms of the fatty acid content and viability of bovine blastocysts produced in vitro

To determine whether serum supplementation influenced fatty acid content of bovine blastocysts and whether vitamin E addition to culture medium containing serum could improve development in vitro, cleaved eggs were cultured in synthetic oviduct fluid supplemented with bovine serum albumin (BSA, 0.4% w/v, fraction V) (SVBSA), fetal calf serum (FCS, 10% v/v) (SFCS) or FCS (10% v/v) plus 100 micro M vitamin E (SFCS + E). Blastocyst yields were recorded and fatty acid composition was determined by gas chromatography. Day 7 blastocysts were incubated with [2-(14)C] pyruvate for 3 h and then fixed for cell counts. Yields of good quality blastocysts were greatest from cleaved eggs cultured in serum-free conditions (P < 0.01). In the presence of serum, supplementation with vitamin E increased both total and good quality blastocyst yields (P < 0.01). Presence of serum increased fatty acid content (mean +/- SEM) of blastocysts (SVBSA v. SFCS = 57 +/- 2 v. 74 +/- 2 ng embryo(-1); P < 0.001). In contrast, pyruvate metabolism was greater in blastocysts produced without serum (27 +/- 3 v. 21 +/- 3 picomoles embryo(-1) 3h(-1); P < 0.01) but, on a per cell basis, no differences were detected. Addition of vitamin E to the serum-supplemented formulation did not alter either the fatty acid content (73 +/- 2 ng embryo(-1)) or pyruvate metabolism index (19 +/- 1 pmol embryo(-1) 3h(-1)) of SFCS + E blastocysts. Thus, despite lipid accumulation, supplementary vitamin E improved blastocyst yields in embryos exposed to serum.

Development and de novo protein synthetic activity of bovine embryos produced in vitro in different culture systems

In vitro matured (IVM) and fertilized (IVF) putative Day 1 zygotes (Day 0 = IVF) were allocated randomly to culture in formulations based on Synthetic Oviduct Fluid (SOF) medium and identified on the basis of their contrasting principal supplements, which were 10% v/v steer serum (SS; n = 558) or 4 mg/mL crystalline BSA (SBSA; n = 531) or 3 mg/mL polyvinyl alcohol (SPVA; n = 607) in 9 replicates. SBSA and SPVA also contained 10 microg/mL non-essential amino acids, while the former was further supplemented with 20 microL/mL essential amino acids and the latter with 0.5 mmol/L sodium citrate and 5 ng/mL epidermal growth factor. Zygotes were cultured in 20 microL drops (4 zygotes per drop) until Day 8 in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 39 degrees C and droplets were renewed every 48 hours. The incidence of zygote cleavage was lower (P < 0.05) in SS (mean +/- SEM = 61 +/- 3%) than in SBSA (76 +/- 3%) but not in SPVA (72 +/- 4%) up to Day 3. The SPVA generated a lower yield of blastocysts on Day 7 (12 +/- 2%; P < 0.001) and by Day 8 (21 +/- 4%; P < 0.01) than did SS (33 +/- 3%; 40 +/- 3%) and SBSA (30 +/- 3%; 37 +/- 4%). Cell numbers (n) and diameters (d) of blastocysts on Day 8 were greater (P < 0.001; Replicates 1 to 5) in embryos from SBSA (n, 156 +/- 9; d, 203 +/- 4 microm) than in those from SS (n, 81 +/- 4; d, 177 +/- 3 microm) and SPVA (n, 76 +/- 5; d, 167 +/- 3 microm). Embryos produced in SS incorporated less 3H-phenylalanine into PCA-precipitable protein (replicates 6 to 9; log10 dpm = 3.03 +/- 0.04) than did embryos cultured in SBSA (3.21 +/- 0.03; P < 0.001) or in SPVA (3.14 +/- 0.03; NS). In conclusion, blastocyst yield was poor in SPVA, but the embryos had metabolic activities similar to those of embryos produced in SBSA. Blastocyst yields from SS were not compromised but their capacity for de novo protein synthesis was reduced significantly.

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.

Dietary carbohydrates and amino acids influence oocyte quality in dairy heifers.

The objective of the present experiment was to determine whether increasing plasma insulin by different nutritional regimes affects oocyte quality. Holstein dairy heifers (eight per treatment) were assigned, using a two times two factorial design, to diets containing either low or high dietary leucine and either low or high dietary starch. Each heifer underwent six sessions of ovum pick-up beginning 25 days after introduction of the diets. Oocyte quality was assessed by development to the blastocyst stage in synthetic oviducal fluid following in vitro fertilisation. Feeding diets containing high leucine resulted in significantly higher plasma free leucine and tyrosine concentrations. The high-starch diet significantly increased plasma insulin but not glucagon concentration, whereas high dietary leucine increased plasma glucagon but not insulin. Oocyte cleavage was not influenced by diet. The high-starch diet, which was associated with a high plasma insulin : glucagon ratio, had adverse effects on oocyte quality that were avoided when leucine intake was increased. There was an association between total plasma free amino acid concentration and oocyte cleavage. Therefore, in dairy heifers dietary amino acids and carbohydrates during antral follicle development appear to mediate effects on oocyte quality by different mechanisms. These findings have implications for both diet formulation and feeding regimes.

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCCs) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCCs were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCCs (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCCs suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.