Effects of poor maternal nutrition during gestation on ewe and offspring plasma concentrations of leptin and ghrelin.

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health. Leptin and ghrelin, key hormones in energy homeostasis and appetite control, may mediate these changes. We hypothesized that restricted- and over-feeding during gestation would alter plasma concentrations of leptin and ghrelin in ewes and offspring. Pregnant ewes (n = 37) were fed 1 of 3 diets starting on d 30 ± 0.02 of gestation until necropsy at d 135 of gestation or parturition: restricted- [RES; 60% National Research Council (NRC) requirements for total digestible nutrients, n = 13], control- (CON; 100% NRC, n = 11), or over-fed (OVER; 140% NRC, n = 13). Blood samples were collected from pregnant ewes at days 20, 30, 44, 72, 100, 128, and 142 of gestation. Offspring blood samples were collected within 24 h after birth (n = 21 CON, 25 RES, 23 OVER). Plasma leptin and ghrelin concentrations were determined by RIA. Ewe data were analyzed using the MIXED procedure in SAS with ewe as the repeated subject. Offspring data were analyzed using the MIXED procedure. Correlations between BW and leptin and ghrelin concentrations were identified using PROC CORR. At d 100, RES (5.39 ± 2.58 ng/mL) had decreased leptin concentrations compared with OVER (14.97 ± 2.48 ng/mL; P = 0.008) and at d 128, RES (6.39 ± 2.50 ng/mL) also had decreased leptin concentrations compared with OVER (13.61 ± 2.47 ng/mL; P = 0.04). At d 142, RES (0.26 ± 0.04 ng/mL) had increased ghrelin concentrations compared with CON (0.15 ± 0.04 ng/mL; P = 0.04). Leptin and ghrelin concentrations were also altered between days of gestation within a dietary treatment. In CON ewes, plasma concentrations of leptin were increased at d 30 (19.28 ± 7.43 ng/mL) compared with d 44 (5.20 ± 3.10 ng/mL; P = 0.03), and the plasma concentrations of ghrelin at d 128 (0.20 ± 0.03 ng/mL) were increased compared with d 30 (0.16 ± 0.03 ng/mL; P = 0.01) and d 100 (0.17 ± 0.03 ng/mL; P = 0.04). Maternal diet did not alter plasma ghrelin or leptin concentrations in the offspring (P > 0.50). There were no strong, significant correlations between ewe BW and leptin (r < 0.33; P > 0.06) or ghrelin (r > -0.47; P > 0.001) concentrations or lamb BW and leptin or ghrelin concentrations (r > -0.32, P > 0.06). Maternal alterations in circulating leptin and ghrelin may program changes in energy balance that could result in increased adiposity in adult offspring. Alterations in energy homeostasis may be a mechanism behind the long-lasting changes in growth, body composition, development, and metabolism in the offspring of poorly nourished ewes.

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.

Ultrasound during mid-gestation: Agreement with physical foetal and placental measurements and use in predicting gestational age in sheep.

To determine the effects of poor maternal nutrition and litter size on foetal growth during mid-gestation, pregnant ewes (n = 82) were fed 100%, 60% or 140% of NRC TDN beginning at day 30.2 ± 0.2 of gestation. Transabdominal ultrasound was performed weekly between day 46.0 ± 0.4 and 86.0 ± 0.7 to monitor foetal heart width (HW), umbilical diameter (UMB), rib width (RW) and placentome outer (OD) and inner diameter (ID). Data were analysed with repeated-measures using the mixed procedure for effects of maternal diet, litter size and gestation, and equations predictive of gestational age were generated using the regression procedure. To determine the agreement of ultrasound measurement and actual size, ewes (n = 20-21) were euthanized at day 45 or 90 to obtain corresponding postmortem measurements for Bland-Altman analysis. The HW, UMB and placentome OD and ID increased with gestation (p < .0001) but were unaffected by maternal diet or litter size (p ≥ .12). Ultrasound underestimated postmortem measurements of HW (14.8%), UMB (7.3%), placentome OD (4.5%) and ID (37.3%) at day 90 of gestation. Ultrasound underestimated RW at day 45 (7.7%) but overestimated RW (23.8%) at day 90, indicating inconsistent bias when reporting RW by ultrasound. Combining the HW, UMB, RW and placentome OD generated the strongest equation predictive of gestational age (R2  = .91). These findings indicate that during mid-gestation, maternal diet or litter size did not affect HW, UMB or placentome diameters and these factors can be used to estimate gestational age.

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

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.

Poor maternal nutrition during gestation alters the expression of genes involved in muscle development and metabolism in lambs.

Poor maternal nutrition during gestation can result in reduced muscle mass and increased adiposity of the muscle tissue in the offspring. This can have long-lasting consequences on offspring health and productivity. However, the mechanisms by which poor maternal nutrition affects postnatal muscle development are poorly understood. We hypothesized that poor maternal nutrition during gestation would alter expression of key pathways and genes involved in growth, development, and maintenance of the muscle of lambs. For this study, beginning at d 31 ± 1.3 of gestation, ewes were fed 100 (control), 60 (restricted), or 140% (overfed) of the NRC requirements. Within 24 h of birth, lambs were necropsied and semitendinosus muscle tissue was collected for gene expression analysis. Using RNA sequencing (RNA-seq) across dietary treatment groups, 35 and 10 differentially expressed genes were identified using the and reference annotations, respectively. Maternal overfeeding caused changes in the expression of genes involved in regulating muscle protein synthesis and growth as well as metabolism. Alternately, maternal nutrient restriction affected genes that are involved in muscle cell proliferation and signal transduction. That is, despite a similar phenotype, the genes identified differed between offspring born to restricted- or overfed, ewes indicating that the mechanism for the phenotypic changes in muscle are due to different mechanisms.

The effects of poor maternal nutrition during gestation on postnatal growth and development of lambs.

Poor maternal nutrition can affect the growth and development of offspring, which may lead to negative consequences in adult life. We hypothesized that lambs born to poorly nourished ewes would have reduced growth rate and increased fat deposition, with corresponding changes in the somatotropic axis, and leptin, insulin and glucose concentrations. Ewes ( = 36; 12/treatment) were assigned 1 of 3 diets; 100% (CON), 60% (RES), or 140% (OVER) of NRC requirements for TDN at d 31 of gestation until parturition. One lamb per ewe ( = 35; 11 to 12 per treatment) was used; 18 lambs were euthanized at d 1, and 17 were fed the same diet for 3 mo and then euthanized. Lamb crown rump length (CRL), heart girth, BW, and BCS were measured, and blood samples were collected at d 1 and then at weekly intervals until euthanasia. Averaged from d 1 until 3 mo, lambs from OVER ewes were larger compared with lambs born to CON ewes (BW [16.97 vs. 15.44 kg ± 0.60; = 0.09], ADG [0.23 vs. 0.21 ± 0.01 kg/d; = 0.01], and CRL [68.9 vs. 66.1 ± 0.80 cm; = 0.02]). On a BW basis, heart weight from lambs from RES (0.18 kg ± 0.03; = 0.03) ewes was greater than that of CON lambs (0.15 kg ± 0.03). Backfat thickness was reduced in RES lambs (0.11 ± 0.06; ≤ 0.04) compared with CON (0.20 ± 0.06) and OVER (0.26 ± 0.06) lambs. Concentrations of IGF-I at 3 mo and IGFBP-3 from weaning (d 56 of age) to 3 mo of age tended to be greater ( ≤ 0.06) in OVER lambs (334 ± 66 ng/mL and 175 ± 11 arbitrary units [AU], respectively) than CON lambs (149 ± 66 ng/mL and 140 ± 11 AU, respectively). At 3 mo, leptin was greater in OVER lambs compared with RES lambs (1.24 vs. 0.78 ± 0.13 ng/mL; < 0.05). Over time, average insulin concentrations were greater in OVER and RES lambs than CON lambs (0.49 and 0.49 vs. 0.33 ± 0.05 ng/mL; ≤ 0.02). However, concentrations of GH, IGFBP-2, glucose, triglycerides, and total cholesterol were not different ( > 0.10) between treatment groups. During in vivo glucose tolerance test, baseline insulin concentrations were 68% and 85% greater ( 0.01), respectively, in RES and OVER lambs compared with CON lambs. Similarly, the glucose:insulin ratio was greater in RES and OVER lambs compared with CON lambs ( 0.01). Thus, in this experiment, poor maternal nutrition during gestation influenced body size, organ growth, fat accumulation, and concentrations of IGF-I, IGFBP-3, leptin, and insulin of offspring during the first 3 mo of age.

Restricted maternal nutrition alters myogenic regulatory factor expression in satellite cells of ovine offspring.

Poor maternal nutrition inhibits muscle development and postnatal muscle growth. Satellite cells are myogenic precursor cells that contribute to postnatal muscle growth, and their activity can be evaluated by the expression of several transcription factors. Paired-box (Pax)7 is expressed in quiescent and active satellite cells. MyoD is expressed in activated and proliferating satellite cells and myogenin is expressed in terminally differentiating cells. Disruption in the expression pattern or timing of expression of myogenic regulatory factors negatively affects muscle development and growth. We hypothesized that poor maternal nutrition during gestation would alter the in vitro temporal expression of MyoD and myogenin in satellite cells from offspring at birth and 3 months of age. Ewes were fed 100% or 60% of NRC requirements from day 31±1.3 of gestation. Lambs from control-fed (CON) or restricted-fed (RES) ewes were euthanized within 24 h of birth (birth; n=5) or were fed a control diet until 3 months of age (n=5). Satellite cells isolated from the semitendinosus muscle were used for gene expression analysis or cultured for 24, 48 or 72 h and immunostained for Pax7, MyoD or myogenin. Fusion index was calculated from a subset of cells allowed to differentiate. Compared with CON, temporal expression of MyoD and myogenin was altered in cultured satellite cells isolated from RES lambs at birth. The percent of cells expressing MyoD was greater in RES than CON (P=0.03) after 24 h in culture. After 48 h of culture, there was a greater percent of cells expressing myogenin in RES compared with CON (P0.05). In satellite cells from RES lambs at 3 months of age, the percent of cells expressing MyoD and myogenin were greater than CON after 72 h in culture (P<0.05). Fusion index was reduced in RES lambs at 3 months of age compared with CON (P<0.001). Restricted nutrition during gestation alters the temporal expression of myogenic regulatory factors in satellite cells of the offspring, which may reduce the pool of myoblasts, decrease myoblast fusion and contribute to the poor postnatal muscle growth previously observed in these animals.

Poor maternal nutrition during gestation in sheep reduces circulating concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-3 in offspring.

To determine if poor maternal nutrition alters growth, body composition, circulating growth factors, and expression of genes involved in the development of muscle and adipose of offspring, 24 Dorset and Shropshire ewes were fed either 100% (control fed), 60% (restricted fed), or 126% (over fed) of National Research Council requirements. Diets began at day 116 ± 6 of gestation until parturition. At parturition, 1 lamb from each control fed (CON), restricted fed (RES), and over fed (OVER) ewe was necropsied within 24 h of birth (1 d; n = 3/treatment) or reared on a control diet for 3 mo (CON = 5, RES = 5, and OVER = 3/treatment) and then euthanized. Body weights and blood samples were collected from lambs from 1 d to 3 mo. Organ weights, back fat thickness, loin eye area, and tissue samples (quadriceps, adipose, and liver) were collected at 1 d and 3 mo of age. The RES lambs weighed 16% less than CON (P = 0.01) between 1 d and 3 mo of age. In RES, there was a tendency for reduced heart girth at 1 d and 3 mo (P < 0.07) and back fat was reduced 36% at 3 mo (P = 0.03). Heart weight was 30% greater in OVER at 1 d when compared with RES lambs (P = 0.02). Serum IGF-I and IGFBP-3 were reduced in RES and OVER lambs (P < 0.05). Leptin tended to be greater in OVER lambs compared with CON at 1 d and 3 mo (P ≤ 0.08). Triiodothyronine was reduced in RES at 1 d (P = 0.05) and triglycerides tended to be greater in OVER at 3 mo (P = 0.07). In liver, there was a tendency for increased expression of IGF-I in OVER (P = 0.06) and decreased IGFBP-3 in RES (P = 0.09) compared with CON lambs at 1 d. In adipose tissue, adiponectin expression was decreased in RES (P = 0.05) at 3 mo. At 1 d of age, muscle expression of IGF-I tended to increase in RES (P = 0.06). In conclusion, poor maternal nutrition during gestation reduced growth rate in offspring which may be because of reduced circulating IGF-I and IGFBP-3 and decreased expression of IGFBP-3 in the liver.

Short communication: Expression of T-box 2 and 3 in the bovine mammary gland.

To increase our understanding of the mechanisms by which growth hormone (GH) and insulin-like growth factor (IGF)-I influence bovine mammary gland development, the potential roles of T-box2 (TBX2) and T-box3 (TBX3) were investigated. Although no information regarding expression of either transcription factor in the bovine mammary gland exists, it is known that TBX3 and its closely related family member, TBX2, are required for mammary gland development in humans and mice. Additionally, TBX3 mutations in humans and mice lead to ulnar mammary syndrome. Evidence is present in bone that TBX3 is required for proliferation and its expression is regulated by GH, an important regulator of mammary gland development and milk production. We hypothesized that TBX2 and TBX3 are expressed in the bovine mammary gland and that GH, IGF-I, or both increase TBX2 and TBX3 expression in bovine mammary epithelial cells (MEC). Bovine mammary gland tissue, MAC-T cells, primary MEC, and fibroblasts were obtained and TBX2 and TBX3 expression was determined by real-time reverse transcription PCR. In addition, TBX2 and TBX3 expression was examined in cells treated with 100 or 500 ng/mL of GH or 100 or 200 ng/mL of IGF-I for 24 or 48 h. Both TBX2 and TBX3 were expressed in bovine mammary tissue. Surprisingly, expression of TBX2 was only detected in mammary fibroblast cells, whereas TBX3 was expressed in all 3 cell types. Growth hormone did not alter TBX3 expression in MAC-T cells or MEC. However, IGF-I increased TBX3 expression in MAC-T, but not in primary MEC. We did not observe a change in TBX2 or TBX3 expression in fibroblasts treated with GH and IGF. Therefore, we concluded that (1) TBX2 and TBX3 are expressed in bovine mammary gland, (2) their expression is cell-type specific, and (3) IGF-I stimulates TBX3 expression in MAC-T cells.

Metabolic engineering of propanediol pathways.

Microbial fermentation is an important technology for the conversion of renewable resources to chemicals. In this paper, we describe the application of metabolic engineering for the development of two new fermentation processes: the microbial conversion of sugars to 1,3-propanediol (1,3-PD) and 1,2-propanediol (1,2-PD). A variety of naturally occurring organisms ferment glycerol to 1,3-PD, but no natural organisms ferment sugars directly to 1,3-PD. We first describe the fed-batch fermentation of glycerol to 1,3-PD by Klebsiella pneumoniae. We then present various approaches for the conversion of sugars to 1,3-PD, including mixed-culture fermentation, cofermentation of glycerol and glucose, and metabolic engineering of a "sugars to 1,3-PD" pathway in a single organism. Initial results are reported for the expression of genes from the K. pneumoniae 1,3-PD pathway in Saccharomyces cerevisiae. The best naturally occurring organism for the fermentation of sugars to 1,2-PD is Thermoanaerobacterium thermosaccharolyticum. We describe the fermentation of several different sugars to 1,2-PD by this organism in batch and continuous culture. We report that Escherichia coli strains engineered to express either aldose reductase or glycerol dehydrogenase convert glucose to (R)-1,2-PD. We then analyze the ultimate potential of fermentation processes for the production of propanediols. Linear optimization studies indicate that, under aerobic conditions, propanediol yields that approach the theoretical maximum are possible and CO2 is the primary coproduct. Without the need to produce acetate, final product titers in the range of 100 g/L should be possible; the high titers and low coproduct levels should make product recovery and purification straightforward. The examples given in this paper illustrate the importance of metabolic engineering for fermentation process development in general.

Structural and functional analysis of chicken U4 small nuclear RNA genes.

Two distinct chicken U4 RNA genes have been cloned and characterized. They are closely linked within 465 base pairs of each other and have the same transcriptional orientation. The downstream U4 homology is a true gene, based on the criteria that it is colinear with chicken U4B RNA and is expressed when injected into Xenopus laevis oocytes. The upstream U4 homology, however, contains seven base substitutions relative to U4B RNA. This sequence may be a nonexpressed pseudogene, but the pattern of base substitutions suggests that it more probably encodes a variant yet functional U4 RNA product not yet characterized at the RNA level. In support of this, the two U4 genes have regions of homology with each other in their 5'-flanking DNA at two positions known to be essential for the efficient expression of vertebrate U1 and U2 small nuclear RNA genes. In the case of U1 and U2 RNA genes, the more distal region (located near position-200 with respect to the RNA cap site) is known to function as a transcriptional enhancer. Although this region is highly conserved in overall structure and sequence among U1 and U2 RNA genes, it is much less conserved in the chicken U4 RNA genes reported here. Interestingly, short sequence elements present in the -200 region of the U4 RNA genes are inverted (i.e., on the complementary strand) relative to their usual orientation upstream of U1 and U2 RNA genes. Thus, the -200 region of the U4 RNA genes may represent a natural evolutionary occurrence of an enhancer sequence inversion.

Prevention of monkey sperm penetration of zona-free hamster ova by sperm antibody obtained from vasectomized cynomolgus monkeys.

Incubation of antisperm sera from vasectomized cynomolgus monkeys (Macaca fascicularis) with monkey sperm caused a fourfold reduction of sperm attachment to and penetration of zona-free Golden hamster ova. The attachment of sperm to hamster ova was reduced from 64% to 11%, and the penetration of ova was reduced from 20% to 5%. Sperm antibodies block sperm attachment to the vitelline membrane, thus preventing ovum penetration. This blockage may be one of the reasons for low fertility rates observed following reanastomosis of the vas deferens in those vasectomized males that show high levels of circulating antisperm antibody.

Sex differences in moral internalization and values.

The subjects were fifth- and seventh-grade white middle-class children and their parents. The major moral internalization indices pertain to internal moral judgment, guilt intensity, and fear of punishment. The findings support the prevalent view that consideration for others is more salient in females. They also suggest, with considerable consistency (especially in adults) that moral transgressions are more likely to be associated with guilt in females and fear in males. No sex differences in internal moral judgment were obtained. Evidence was presented suggesting that the differences in children may be due partly to different discipline and affection patterns. It was also suggested that the results for adults as well as children might be explained by differential sex-role socialization as well as by increasing pressures on males over the life cycle to achieve and succeed, which may often conflict with concerns about the welfare of others.