S. M. Pillai

Fetal and organ development at gestational days 45, 90, 135 and at birth of lambs exposed to under- or over-nutrition during gestation

Abstract To determine the effects of poor maternal nutrition on offspring body and organ growth during gestation, pregnant Western White-faced ewes (n = 82) were randomly assigned into a 3 × 4 factorial treatment structure at d 30.2 ± 0.2 of gestation (n = 5 to 7 ewes per treatment). Ewes were individually fed 100% (control), 60% (restricted) or 140% (over) of NRC requirements for TDN. Ewes were euthanized at d 45, 90 or 135 of gestation or underwent parturition (birth) and tissues were collected from the offspring (n = 10 to 15 offspring per treatment). Offspring from control, restricted and overfed ewes are referred to as CON, RES and OVER, respectively. Ewe data were analyzed as a completely randomized design and offspring data were analyzed as a split-plot design using PROC MIXED. Ewe BW did not differ at d 30 (P ≥ 0.43), however restricted ewes weighed less than overfed and overfed were heavier than controls at d 45, and restricted weighed less and overfed were heavier than controls at d 90 and 135 and birth (P ≤ 0.05). Ewe BCS was similar at d 30, 45 and 90 (P ≤ 0.07), however restricted ewes scored lower than control at d 135 and birth (P ≤ 0.05) and over ewes scored higher than control at d 135 (P ≤ 0.05) but not at birth (P = 0.06). A maternal diet by day of gestation interaction indicated that at birth the body weight (BW) of RES offspring was less than CON and OVER (P ≤ 0.04) and heart girth of RES was smaller than CON and OVER (P ≤ 0.004). There was no interaction of maternal diet and day of gestation on crown-rump, fetal, or nose occipital length, or orbit or umbilical diam. (P ≥ 0.31). A main effect of maternal diet indicated that the RES crown-rump length was shorter than CON and OVER (P ≤ 0.05). An interaction was observed for liver, kidney and renal fat (P ≤ 0.02). At d 45 the liver of RES offspring was larger than CON and OVER (P ≤ 0.002), but no differences observed at d 90, 135 or birth (P ≥ 0.07). At d 45, the kidneys of OVER offspring were larger than CON and RES (P ≤ 0.04), but no differences observed at d 90, 135 or birth (P ≥ 0.60). At d 135, OVER had more perirenal fat than CON and RES (P ≤ 0.03), and at birth RES had more perirenal fat than CON and OVER (P ≤ 0.04). There was no interaction observed for offspring heart weight, length or width, kidney length, adrenal gland weight, loin eye area or rib width (P ≥ 0.09). In conclusion, poor maternal nutrition differentially alters offspring body size and organ growth depending on the stage of gestation.

PHYSIOLOGY AND ENDOCRINOLOGY SYMPOSIUM:The effects of poor maternal nutrition during gestation on offspring postnatal growth and metabolism

Poor maternal nutrition during gestation has been linked to poor growth and development, metabolic dysfunction, impaired health, and reduced productivity of offspring in many species. Poor maternal nutrition can be defined as an excess or restriction of overall nutrients or specific macro- or micronutrients in the diet of the mother during gestation. Interestingly, there are several reports that both restricted- and over-feeding during gestation negatively affect offspring postnatal growth with reduced muscle and bone deposition, increased adipose accumulation, and metabolic dysregulation through reduced leptin and insulin sensitivity. Our laboratory and others have used experimental models of restricted- and over-feeding during gestation to evaluate effects on early postnatal growth of offspring. Restricted- and over-feeding during gestation alters body size, circulating growth factors, and metabolic hormones in offspring postnatally. Both restricted- and over-feeding alter muscle growth, increase lipid content in the muscle, and cause changes in expression of myogenic factors. Although the negative effects of poor maternal nutrition on offspring growth have been well characterized in recent years, the mechanisms contributing to these changes are not well established. Our laboratory has focused on elucidating these mechanisms by evaluating changes in gene and protein expression, and stem cell function. Through RNA-Seq analysis, we observed changes in expression of genes involved in protein synthesis, metabolism, cell function, and signal transduction in muscle tissue. We recently reported that satellite cells, muscle stem cells, have altered expression of myogenic factors in offspring from restricted-fed mothers. Bone marrow derived mesenchymal stem cells, multipotent cells that contribute to development and maintenance of several tissues including bone, muscle, and adipose, have a 50% reduction in cell proliferation and altered metabolism in offspring from both restricted- and over-fed mothers. These findings indicate that poor maternal nutrition may alter offspring postnatal growth by programming stem cell populations. In conclusion, poor maternal nutrition during gestation negatively affects offspring postnatal growth, potentially through impaired stem and satellite cell function. Therefore, determining the mechanisms that contribute to fetal programming is critical to identifying effective management interventions for these offspring and improving efficiency of production.

Effects of Poor Maternal Nutrition during Gestation on Bone Development and Mesenchymal Stem Cell Activity in Offspring

Poor maternal nutrition impairs overall growth and development of offspring. These changes can significantly impact the general health and production efficiency of offspring. Specifically, poor maternal nutrition is known to reduce growth of bone and muscle, and increase adipose tissue. Mesenchymal stem cells (MSC) are multipotent stem cells which contribute to development of these tissues and are responsive to changes in the maternal environment. The main objective was to evaluate the effects of poor maternal nutirtion during gestation on bone and MSC function in offspring. Thirty-six ewes were fed 100%, 60%, or 140% of energy requirements [NRC, 1985] beginning at day 31 ± 1.3 of gestation. Lambs from ewes fed 100% (CON), 60% (RES) and 140% (OVER) were euthanized within 24 hours of birth (1 day; n = 18) or at 3 months of age (n = 15) and bone and MSC samples were collected. Dual X-ray absorptiometry was performed on bones obtained from day 1 and 3 months. Proliferation, differentiation, and metabolic activity were determined in the MSC isolated from lambs at day 1. Data were analyzed using mixed procedure in SAS. Maternal diet negatively affected offspring MSC by reducing proliferation 50% and reducing mitochondrial metabolic activity. Maternal diet did not alter MSC glycolytic activity or differentiation in culture. Maternal diet tended to decrease expression of P2Y purinoreceptor 1, but did not alter expression of other genes involved in MSC proliferation and differentiation. Maternal diet did not alter bone parameters in offspring. In conclusion, poor maternal diet may alter offspring growth through reduced MSC proliferation and metabolism. Further studies evaluating the potential molecular changes associated with altered proliferation and metabolism in MSC due to poor maternal nutrition are warranted.

Gestational restricted- and over-feeding promote maternal and offspring inflammatory responses that are distinct and dependent on diet in sheep

Abstract Inflammation may be a mechanism of maternal programming because it has the capacity to alter the maternal environment and can persist postnatally in offspring tissues. This study evaluated the effects of restricted- and over-feeding on maternal and offspring inflammatory gene expression using reverse transcription (RT)-PCR arrays. Pregnant ewes were fed 60% (Restricted), 100% (Control), or 140% (Over) of National Research Council requirements beginning on day 30.2 ± 0.2 of gestation. Maternal (n = 8–9 ewes per diet) circulating nonesterified fatty acid (NEFA) and expression of 84 inflammatory genes were evaluated at five stages during gestation. Offspring (n = 6 per diet per age) inflammatory gene expression was evaluated in the circulation and liver at day 135 of gestation and birth. Throughout gestation, circulating NEFA increased in Restricted mothers but not Over. Expression of different proinflammatory mediators increased in Over and Restricted mothers, but was diet-dependent. Maternal diet altered offspring systemic and hepatic expression of genes involved in chemotaxis at late gestation and cytokine production at birth, but the offspring response was distinct from the maternal. In the perinatal offspring, maternal nutrient restriction increased hepatic chemokine (CC motif) ligand 16 and tumor necrosis factor expression. Alternately, maternal overnutrition increased offspring systemic expression of factors induced by hypoxia, whereas expression of factors regulating hepatocyte proliferation and differentiation were altered in the liver. Maternal nutrient restriction and overnutrition may differentially predispose offspring to liver dysfunction through an altered hepatic inflammatory microenvironment that contributes to immune and metabolic disturbances postnatally. Summary Sentence Restricted- and over-feeding differentially alter maternal inflammation and metabolism and indirectly promote systemic and hepatic inflammation in the perinatal offspring, which may contribute to liver dysfunction postnatally.

Effects of maternal nutrition on the expression of genomic imprinted genes in ovine fetuses

ABSTRACT Genomic imprinting is an epigenetic phenomenon of differential allelic expression based on parental origin. To date, 263 imprinted genes have been identified among all investigated mammalian species. However, only 21 have been described in sheep, of which 11 are annotated in the current ovine genome. Here, we aim to i) use DNA/RNA high throughput sequencing to identify new monoallelically expressed and imprinted genes in day 135 ovine fetuses and ii) determine whether maternal diet (100%, 60%, or 140% of National Research Council Total Digestible Nutrients) influences expression of imprinted genes. We also reported strategies to solve technical challenges in the data analysis pipeline. We identified 80 monoallelically expressed, 13 new putative imprinted genes, and five known imprinted genes in sheep using the 263 genes stated above as a guide. Sanger sequencing confirmed allelic expression of seven genes, CASD1, COPG2, DIRAS3, INPP5F, PLAGL1, PPP1R9A, and SLC22A18. Among the 13 putative imprinted genes, five were localized in the known sheep imprinting domains of MEST on chromosome 4, DLK1/GTL2 on chromosome 18 and KCNQ1 on chromosome 21, and three were in a novel sheep imprinted cluster on chromosome 4, known in other species as PEG10/SGCE. The expression of DIRAS3, IGF2, PHLDA2, and SLC22A18 was altered by maternal diet, albeit without allelic expression reversal. Together, our results expanded the list of sheep imprinted genes to 34 and demonstrated that while the expression levels of four imprinted genes were changed by maternal diet, the allelic expression patterns were un-changed for all imprinted genes studied.