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Ohno’s hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1. In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno’s hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and “dosage-sensitive”.

Poor maternal nutrition (restricted- and over-feeding) during gestation may alter leptin and ghrelin, key hormones in energy homeostasis and appetite control. They may also have a regulatory role in maternal metabolic adaptations critical during gestation to ensure optimal offspring growth and development. We hypothesized that restricted- and over-feeding during gestation would alter plasma concentrations of leptin and ghrelin in ewes and their offspring. Pregnant Western White-faced ewes were individually fed 60% (RES; n = 13), 100% (CON; n = 11), or 140% (OVER; n = 13) of NRC requirements for TDN starting on d 30±0.02 of gestation. Blood samples were collected in the morning from fasted pregnant ewes weekly from d 20 of gestation until parturition and from offspring (n = 31) within 24 hours after birth. Leptin and ghrelin concentrations were determined by radioimmunoassay. Data were analyzed using the MIXED procedure in SAS with main effects of treatment, day of gestation and their interaction. Leptin (P < 0.002) and ghrelin (P < 0.015) concentrations were altered in pregnant ewes between treatments within a day of gestation. At d 100 (P = 0.008) and d 128 (P = 0.04), RES ewes (5.392.58 ng/mL; 6.392.50 ng/mL) had decreased leptin concentrations compared with OVER ewes (14.972.48 ng/mL; 13.612.47 ng/mL), with CON ewes intermediate. RES ewes (0.260.04 ng/mL) had increased ghrelin concentrations compared with CON ewes at d 142 (0.150.04 ng/mL; P = 0.042), with OVER ewes intermediate. Leptin (P < 0.002) and ghrelin (P < 0.015) concentrations were altered between days of gestation within a dietary treatment. Leptin concentration increased across gestation in OVER ewes. In RES ewes, leptin concentration decreased and ghrelin concentration increased over gestation. Leptin (P = 0.5) and ghrelin (P = 0.5) concentrations in lambs were not different at birth. Alterations in leptin and ghrelin in ewes during gestation may disrupt critical metabolic adaptations that may contribute to suboptimal offspring growth and development.

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 0.06) 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.

Poor maternal nutrition during gestation can have immediate and life-long negative effects on offspring growth and health. In livestock, this leads to reduced product quality and increased costs of production. Based on previous evidence that both restricted- and overfeeding during gestation decrease offspring muscle growth and alter metabolism postnatally, we hypothesized that poor maternal nutrition during gestation would reduce the growth and development of offspring muscle prenatally, reduce the number of myogenic progenitor cells, and result in changes in the global expression of genes involved in prenatal muscle development and function. Ewes were fed a control (100% NRC)-, restricted (60% NRC)-, or overfed (140% NRC) diet beginning on day 30 of gestation until days 45, 90, and 135 of gestation or until parturition. At each time point fetuses and offspring (referred to as CON, RES, and OVER) were euthanized and longissimus dorsi (LM), semitendinosus (STN), and triceps brachii (TB) were collected at each time point for histological and RNA-Seq analysis. In fetuses and offspring, we did not observe an effect of diet on cross-sectional area (CSA), but CSA increased over time (P < 0.05). At day 90, RES and OVER had reduced secondary:primary muscle fiber ratios in LM (P < 0.05), but not in STN and TB. However, in STN and TB percent PAX7-positive cells were decreased compared with CON (P < 0.05). Maternal diet altered LM mRNA expression of 20 genes (7 genes downregulated in OVER and 2 downregulated in RES compared with CON; 5 downregulated in OVER compared with RES; false discovery rate (FDR)-adj. P < 0.05). A diet by time interaction was not observed for any genes in the RNA-Seq analysis; however, 2,205 genes were differentially expressed over time between days 90 and 135 and birth (FDR-adj. P < 0.05). Specifically, consistent with increased protein accretion, changes in muscle function, and increased metabolic activity during myogenesis, changes in genes involved in cell cycle, metabolic processes, and protein synthesis were observed during fetal myogenesis. In conclusion, poor maternal nutrition during gestation contributes to altered offspring muscle growth during early fetal development which persists throughout the fetal stage. Based on muscle-type-specific effects of maternal diet, it is important to evaluate more than one type of muscle to fully elucidate the effects of maternal diet on offspring muscle development.

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.

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.

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.

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

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 decreases postnatal muscle growth, alters muscle lipid accumulation and alters metabolism. However, the mechanisms that drive these adverse effects remain poorly understood. Oxidative stress is associated with lipid and protein oxidative damage, inflammation, and metabolic function modifications, which may occur in the muscle of offspring born to ewes exposed to poor nutrition during gestation. Therefore, we hypothesized that measures of oxidative stress in offspring muscle would be increased by poor maternal nutrition during gestation. To test this, 36 multiparous ewes (25 Dorsets, 7 Shropshires, and 4 Southdowns) received diets containing 60% (restricted), 100% (control), or 140% (over) of NRC requirements for TDN from d 31 ± 1.3 of gestation until parturition. Offspring were necropsied within 24 h of birth or maintained on a control diet for 3 mo. Longissimus muscle samples were collected from each lamb and used for malondialdehyde (MDA) and protein carbonyl concentration analyses (n = 3 to 6 per maternal diet per age) to determine muscle lipid and protein oxidation, respectively. Data were analyzed for effects of maternal diet, age of the offspring, and their interaction using the glimmix procedure in SAS. There was a tendency for an effect of maternal diet on MDA concentration (P = 0.10) where offspring from over-fed ewes had greater MDA concentrations than offspring from restricted-fed ewes (over = 2.1 ± 0.13 µg/mg protein, restricted = 1.6 ± 0.15 µg/mg protein; P = 0.04). There was no effect of offspring age on MDA concentrations (P = 0.12). There was no effect of treatment or age on protein carbonyl concentrations in offspring muscle (P > 0.10). These results suggest that maternal over-nutrition during gestation may have greater effects on lipid oxidation than protein oxidation, which may contribute to poor postnatal muscle growth in offspring.

Poor maternal nutrition affects the development and growth of a variety of tissues and organs in offspring. However, research on the effects of poor maternal nutrition on offspring brain development is limited. There is evidence that fetal brain is influenced by maternal diet in the last third of gestation, where brain growth is at its peak. Therefore, we hypothesized that maternal restricted- and over-feeding during gestation would decrease the expression of genes involved in offspring brain development. To investigate, pregnant Western White-faced ewes (n = 16) were individually housed and fed a control- (100% NRC), restricted- (60% NRC), or over-fed (140% NRC) diet starting at d 30.2 ± 0.2 of gestation. At d 135 of gestation, ewes and fetuses (n = 6 control, 12 restricted, and 9 over) were euthanized and brain tissue collected from offspring. Samples were snap frozen and stored at -80°C. Brain tissue RNA was extracted using a Qiagen RNeasy kit, reverse transcribed, and real-time PCR was performed. The mRNA expression of three genes involved in brain development and neurogenesis was quantified, including Brain Derived Neurotrophic Factor (BDNF), Katanin Regulatory Subunit B1 (KATNB1), and Microcephalin (MCPH1). Glyceraldehyde 3-phosphate dehydrogenase was used as the housekeeping gene. Data were analyzed using PROC MIXED in SAS with the main effect as maternal diet. Significance was determined at P ≤ 0.05. No effect of maternal diet on gene expression was observed for BDNF (P = 0.66), KATNB1 (P = 0.36), or MCPH1 (P = 0.36). Lack of changes could be due to age of offspring as others have shown that poor maternal diet affects mRNA expression of genes relating to neurodevelopment postnatally. Additional research is necessary to determine whether poor maternal nutrition affects other genes relating to brain development and cognition in offspring as well as at different time points.

Greater maternal milk production during gestation can alter dairy calf growth and metabolism, thereby impacting productivity and health of the offspring. However, the effects on offspring immunity and health are not well established. We hypothesized that increased maternal milk production would negatively affect factors related to health and immunity in offspring. Multiparous Holstein cows (n = 35) were separated into two groups (21,947 kg, high; 15,937 kg, low) based on their milk production during gestation. Calves (n = 10 per group per gender) born to these cows are referred to as HIGH and LOW. Within 24 h of birth, calf body measurements and whole blood were collected. Circulating insulin and haptoglobin concentrations were determined by ELISA. Serum blood biochemistry was performed at the University of Missouri Veterinary Diagnostic Laboratory. Data were analyzed using PROC GLM in SAS with milk production as a covariate. Statistical significance was considered at P≤ 0.05 and tendencies at 0.05

Poor maternal nutrition during gestation alters offspring muscle composition, fiber number, and postnatal growth. Additionally, poor maternal nutrition impacts offspring whole body and stem cell metabolism. We hypothesized that over- or restricted-feeding ewes during gestation would alter the offspring longissimus muscle metabolome. Pregnant Western White-faced ewes (n = 47) were individually fed 60% (restricted), 100% (control), or 140% (over) of NRC requirements for TDN starting at d 30.2 ± 0.2 of gestation. At days 90 and 135 of gestation, ewes were euthanized for fetal muscle collection. Another group of ewes were allowed to lamb and offspring were necropsied within 24 h of birth. Mass spectrophotometry of longissimus muscle samples (n = 8 fetuses per treatment per time point) identified 612 metabolites. Data were analyzed by ANOVA for main effects of treatment, time point, and their interaction. Compared with controls, maternal over-feeding altered metabolites in 63 pathways and maternal restricted-feeding altered metabolites in 56 pathways (P < 0.05). Maternal over-feeding decreased concentrations of 1 phosphatidylcholine (PC) metabolite at d 90, 2 PC metabolites at d 135, and 4 PC metabolites at birth compared with controls (P < 0.05). Additionally, at d 135, maternal overfeeding increased 1 phosphatidylethanolamine (PE) metabolite while reducing 10 PE metabolites at birth compared with controls (P < 0.05). Offspring from overfed ewes had decreased concentrations of 12 lysolipids at birth compared with controls (P < 0.05). Maternal restricted-feeding increased expression of reduced glutathione 3.07-fold at d 90 (P = 0.008), whereas at d 135, oxidized glutathione was decreased 0.21-fold compared with control (P = 0.03). Thus, maternal over-feeding may promote increased lipid oxidation in offspring muscle, which may predispose offspring to altered lipid utilization and storage postnatally. Maternal restricted-feeding resulted in alterations in glutathione metabolism, potentially indicative of changes to redox status in these offspring.

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; 0.10) 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.

Maternal nutrition during gestation impacts offspring BW, body composition, and metabolism, thereby affecting productivity. Several sources of information are available to producers for guidance in managing their breeding flocks; however, it is unknown if sheep producers in New England utilize these resources. In previous USDA surveys of sheep producers, New England was not included, thus leaving a gap of knowledge. Our objective was to conduct a survey of New England sheep producers to determine flock size, breeds, pregnancy detection methods, feeding management practices, and producer learning styles. In particular, we wanted to determine if flock size influenced management practices, and if flock purpose (eg., meat, fiber) and feed type (eg., hay) influenced feed management. A 12-question survey was developed and disseminated via Qualtrics using e-mail survey links, with a 33.2% response rate (n = 96 responses). Data were analyzed using SPSS. Of the respondents, 61.5% had flocks sizes of 11 to 50 sheep. Most producers (63.5%) maintain 1 breed of sheep; however, larger flocks (> 50 sheep) are more likely to maintain multiple breeds (P < 0.05). The majority (79.2%) use their sheep for meat production, 61.5% for fiber, and 37.1% manage sheep for both. Spring (January to May) is the primary (59.4%) lambing season. The majority (76.0%) of New England sheep producers do not analyze their feed, which presents an opportunity for improved outreach. There were correlations (P < 0.05) between flock size and flock purpose, flock size and number of breeds owned, flock size and feed type, feed types and number of feed types used, feed type and feed analysis, and source of feed information. In conclusion, New England sheep producers have flocks of varying size and purpose, and would likely benefit from outreach education on the benefits of diet analysis and formulation for their breeding flocks.

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.

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.

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.

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.

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.

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

4 Introduction 6 Review of Literature 8 Mesenchymal Stem Cells 8 Establishment 8 Differentiation of MSC into Bone, Muscle, and Adipose Tissue 8 Roles in the Adult 9 Poor Maternal Nutrition 10 Effects on Mesenchymal Stem Cells 10 Effects on Bone, Muscle, and Adipose Development 11 Effects on Whole Body Function 13 Fetal Programming and Maternal Diet 13 Metabolism 17 Normal Metabolic Physiology 17 Cell Differentiation Status and Metabolic Preference 18 Regulation of Metabolic Status: Role of Oxygen/Nutrient Sensing Pathways ....21 Reactive Oxidative Species: Physiological and Pathological Levels 23 Altered Metabolic Physiology 25 Conclusion 27 Materials and Methods 29 Results 36

Deviations from proper gene dosage of the autosome range from severe to lethal consequences in mammals. Eutherian males (XY), however, have reduced gene dosage compared with females (XX) due to a single X and deteriorating Y chromosome. This dosage imbalance is resolved through X chromosome dosage compensation, according to Ohno’s hypothesis: X-linked gene expression is doubled in both males and females to balance expression of the X chromosome and autosomes. To compensate for doubled X chromosome expression in females, X chromosome inactivation (XCI) inactivates a single X chromosome in each cell. Although these mechanisms have been well studied in mice and humans, controversies exist due to the analysis and interpretation of RNA sequencing data. Here we described X chromosome dosage compensation in the sheep. Twelve ewes were fed 100% (control), 60% (restricted), or 140% (overfed) of the National Research Council requirements for a ewe pregnant with twins (NRC, 1985; Nutrient Requirements of Sheep, 6th ed.). Day 135 brain, lung, and kidney tissues were collected from fetuses of the control, restricted, and overfed groups (n = 7, 4, and 4; respectively). RNA seq libraries were prepared using the Illumina TruSeq stranded mRNA kit and sequenced on the NextSEqn 500 (Illumina Inc., San Diego, CA, USA). Two additional RNA-seq datasets were downloaded from Sequence Read Archive (SRA), including Day 14 embryos (PRJNA254105), and adult and juvenile heart, brain, liver, muscle, rumen, and female- and male-specific tissues (PRJEB6169). The RNA-seq data were trimmed and mapped to the ovine reference genome assembly Oar_v4.0 using Hisat2 (version 2.0.5; https://ccb.jhu.edu/software/hisat2/index.shtml) aligner. The mRNA level of each gene was estimated by transformed transcripts per kilobase million (TPM) and was quantified using IsoEM (version 1.1.4; http://dna.engr.uconn.edu/). The relative expression of X to autosomal (A)(RXE) was calculated using RXE = log2(X expression) – log2(A expression) with an average of 486 X-linked genes and 13,001 autosomal genes after TPM >1 filtering. RXE ≥0 (or X:A ratio ≥ 1)

Background Maternal over and restricted nutrition has negative consequences on the muscle of offspring by reducing muscle fiber number and altering regulators of muscle growth. To determine if over and restricted maternal nutrition affected muscle growth and gene and protein expression in offspring, 36 pregnant ewes were fed 60%, 100% or 140% of National Research Council requirements from d 31 ± 1.3 of gestation until parturition. Lambs from control-fed (CON), restricted-fed (RES) or over-fed (OVER) ewes were necropsied within 1 d of birth (n = 18) or maintained on a control diet for 3 mo (n = 15). Semitendinosus muscle was collected for immunohistochemistry, and protein and gene expression analysis. Results Compared with CON, muscle fiber cross-sectional area (CSA) increased in RES (58%) and OVER (47%) lambs at 1 d of age (P 0.3). Phosphorylated Akt (ser473) was increased in RES at 3 mo compared with CON (P = 0.006). Conclusions In conclusion, maternal over and restricted nutrient intake alters muscle lipid content and growth of offspring, possibly through altered gene and protein expression.

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.

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.

Two varieties of peach, one of nectarine, and one of plum were sampled periodically over a 3-yr period in the San Joaquin Valley of California to determine the relative seasonal densities of the predaceous and phytophagous mites present on the leaves. Predators collected included the phytoseiid Neoseiulus caudiglans (Schuster), Amblyseius hibisci (Chant), Typhloseiopsis citri (Garman and McGregor), Typhlodromus occidentalis Nesbitt, and a stigmaeid, Zetzellia mali (Ewing). Phytophagous mites included Tetranychus urticae Koch, T. pacificus McGregor, and Panonychus ulmin (Koch) (Tetranychidae), and Aculus cornutus (Banks) and Diptacus gigantorhynchus (Nalepa) (Eriophyidae). Relative densities of these species were not constant, but varied irregularly from year to year. Seasonal trends of each species were generally similar on the different tree hosts.

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.