Maternal restricted- and over- feeding during gestation perturb offspring sperm epigenome in sheep.

In brief: Inadequate maternal nutrition during gestation can have immediate and lifelong effects on offspring. This study shows that maternal restricted - and over- nutrition during gestation do not affect semen characteristics in F1 male offspring but alters offspring sperm sncRNA profiles and DNA methylome in sheep. Abstract: There is a growing body of evidence that inadequate maternal nutrition during gestation can have immediate and lifelong effects on offspring. However, little is known about the effects of maternal nutrition during gestation on male offspring reproduction. Here, using a sheep model of maternal restricted - and over - nutrition (60 or 140% of the National Research Council requirements) during gestation, we found that maternal restricted - and over - nutrition do not affect semen characteristics (i.e. volume, sperm concentration, pH, sperm motility, sperm morphology) or scrotal circumference in male F1 offspring. However, using small RNA sequencing analysis, we demonstrated that both restricted - and over - nutrition during gestation induced marked changes in composition and expression of sperm small noncoding RNAs (sncRNAs) subpopulations including in male F1 offspring. Whole-genome bisulfite sequencing analysis further identified specific genomic loci where poor maternal nutrition resulted in alterations in DNA methylation. These findings indicate that maternal restricted - and over - nutrition during gestation induce epigenetic modifications in sperm of F1 offspring sperm in sheep, which may contribute to environmentally influenced phenotypes in ruminants.

Dens Invaginatus in Patients With Cleft Lip and Palate: A Case Series.

Dens invaginatus is a rare developmental anomaly characterized by an infolding of the enamel organ within the crown or root of a tooth, and it is an example of a dental anomaly that has a higher incidence in patients with CL/P. If undiagnosed, dens invaginatus can lead to severe, acute pain and pulpal necrosis since it can permit direct entry of bacteria into the dental pulp. Treatment of dens invaginatus includes prophylactic sealant or composite restoration, endodontic therapy if pulpal involvement has already occurred, or extraction if aberrant tooth morphology precludes endodontic therapy. Few studies report on the incidence of dens invaginatus in patients with CL/P. The purpose of this article is to describe 4 cases of dens invaginatus in patients with CL/P which were encountered in a cleft-craniofacial orthodontic clinic. Each case describes dens invaginatus in a maxillary lateral incisor, and treatments ranged from sealant application to endodontic therapy to extraction. These cases highlight the importance of awareness of this dental anomaly among cleft team providers to facilitate early diagnosis in patients with CL/P.

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

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.

Restricted- and over-feeding during gestation decreases growth of offspring throughout maturity.

To determine the effects of poor maternal nutrition on the growth and metabolism of offspring into maturity, multiparous Dorset ewes pregnant with twins (n = 46) were fed to either 100% (control; n = 13), 60% (restricted; n = 17), or 140% (over; n = 16) of National Research Council requirements from day 30 ± 0.02 of gestation until parturition. Offspring of these ewes are referred to as CON (n = 10 ewes; 12 rams), RES (n = 13 ewes; 21 rams), or OVER (n = 16 ewes; 13 rams), respectively. Lamb body weights (BW) and blood samples were collected weekly from birth (day 0) to day 28 and then every 14 d until day 252. Intravenous glucose tolerance test (infusion of 0.25 g dextrose/kg BW) was performed at day 133 ± 0.25. At day 167 ± 1.42, individual daily intake was recorded over a 77 d feeding period to determine residual feed intake (RFI). Rams were euthanized at day 282 ± 1.82 and body morphometrics, loin eye area (LEA), back fat thickness, and organ weights were collected. The right leg was collected from rams at necropsy and dual-energy x-ray absorptiometry was used to determine bone mineral density (BMD) and length. Averaged from day 0 until day 252, RES and OVER offspring weighed 10.8% and 6.8% less than CON offspring, respectively (P ≤ 0.02). When adjusted for BW, liver and testes weights tended to be increased and decreased, respectively, in RES rams compared with CON rams (P ≤ 0.08). Additionally, RES BMD and bone length were less than CON rams (P ≤ 0.06). Treatment did not influence muscle mass, LEA, or adipose deposition (P ≥ 0.41). Rams (-0.17) were more feed efficient than ewes (0.23; P < 0.01); however, no effect of maternal diet was observed (P ≥ 0.57). At 2 min post glucose infusion, glucose concentrations in OVER offspring were greater than CON and RES offspring (P = 0.04). Concentrations of insulin in CON rams tended to be greater than OVER and RES ewes at 5 min (P ≤ 0.07). No differences were detected in insulin:glucose or area under the curve (AUC) for glucose or insulin (P ≤ 0.29). Maternal diet did not impact offspring triglycerides or cholesterol (P ≤ 0.35). Pre-weaning leptin tended to be 70% greater in OVER offspring than CON (P ≤ 0.07). These data indicate that poor maternal nutrition impairs offspring growth throughout maturity but does not affect RFI. Changes in metabolic factors and glucose tolerance are minimal, highlighting the need to investigate other mechanisms that may contribute to negative impacts of poor maternal diet.

Poor maternal diet during gestation alters offspring muscle proteome in sheep.

Poor maternal nutrition during gestation can result in reduced offspring muscle growth and altered muscle metabolism. We hypothesized that over- or restricted-nutrition during gestation would alter the longissimus dorsi muscle (LM) proteome of offspring. Pregnant ewes were fed 60% (restricted), 100% (control), or 140% (over) of National Research Council requirements for total digestible nutrients from day 30 of gestation until parturition. Fetal (RES, CON, OVER) LM were collected at days 90 and 135 of gestation, or from offspring within 24 h of birth. Sarcoplasmic proteins were isolated, trypsin digested, and subjected to multiplexed, label-based quantitative mass spectrometry analysis integrating tandem mass tag technology. Differential expression of proteins was identified by ANOVA followed by Tukey's HSD post hoc tests, and regularized regression via the elastic net. Significance was set at P < 0.05. Over-represented pathways containing differentially expressed proteins were identified by Reactome and included metabolism of proteins, immune system, cellular response to stress/external stimuli, developmental biology, and infectious disease. As a result of maternal diet, a total of 312 proteins were differentially expressed (day 90 = 89 proteins; day 135 = 115 proteins; birth = 131 proteins). Expression of eukaryotic initiation factor (EIF) 2S3, EIF3L, and EIF4G2 was lower in OVER fetuses at day 90 of gestation (P < 0.05). Calcineurin A and mitogen-activated protein kinase 1 were greater in RES fetuses at day 90 (P < 0.04). At day 135 of gestation, pyruvate kinase and lactate dehydrogenase A expression were greater in OVER fetuses than CON (P < 0.04). Thioredoxin expression was greater in RES fetuses relative to CON at day 135 (P = 0.05). At birth, proteins of the COP9 signalosome complex were greater in RES offspring relative to OVER (P < 0.05). Together, these data indicate that protein degradation and synthesis, metabolism, and oxidative stress are altered in a time and diet-specific manner, which may contribute to the phenotypic and metabolic changes observed during fetal development and postnatal growth.

Poor maternal diet during gestation results in changes in body composition and metabolism in the offspring. Here, we demonstrate that over- and restricted-feeding during gestation alter global protein expression in the longissimus dorsi muscle of offspring during gestation and just after birth. These protein changes are related to protein synthesis and degradation, stress responses, metabolism, and oxidative stress. Proteins related to the initiation of protein translation were increased in offspring of over-fed dams at mid-gestation, while changes in abundance of enzymes associated with metabolism were altered in late gestation and just after birth. In offspring of restricted-fed ewes, proteins relating to cell signaling were increased at mid-gestation, while again, changes in late gestation and birth were related to metabolism, protein degradation, and stress responses. Together, these may provide a mechanism by which poor maternal diet during gestation alters the poor growth and development that occurs in these offspring.

Maternal nutrient restriction and over-feeding during gestation alter expression of key factors involved in placental development and vascularization.

Poor maternal nutrition can negatively affect fetal and placental growth and development. However, the mechanism(s) that contribute to altered placenta growth and function are not well understood. We hypothesized that poor maternal diet would impact signaling through the C-X-C motif chemokine ligand (CXCL) 12-CXCL4 axis and/or placental expression of the insulin-like growth factor (IGF) axis. Using our established sheep model of poor maternal nutrition, we examined the effects of restricted- and over-feeding on ewe placentome gene and protein expression. Specifically, ewes were fed a control (CON; 100%), restricted (RES; 60%), or over (OVER; 140%) diet beginning at day 30.2 ± 0.02 of gestation, and samples were collected at days 45, 90, and 135 of gestation, representing periods of active placentation, peak placental growth, and near term, respectively. Placentomes were separated into cotyledon and caruncle, and samples snap frozen. Protein was determined by western blot and mRNA expression by real-time PCR. Data were analyzed by ANOVA and significance determined at P ≤ 0.05. Ewes fed a RES diet had decreased CXCL12 and vascular endothelial growth factor (VEGF), and increased tumor necrosis factor (TNF)α protein compared with CON ewes in caruncle at day 45 (P ≤0.05). In day 45 cotyledon, CXCR7 protein was increased and mTOR was decreased in RES relative to CON (P ≤0.05). At day 90, CXCR4 and CXCR7 were reduced in RES caruncle compared with CON, whereas VEGF was reduced and mTOR increased in cotyledon of RES ewes relative to CON (P ≤0.05). In OVER caruncle, at day 45 CXCR4 and VEGF were reduced and at day 90 CXCR4, CXCR7, and TNFα were reduced in caruncle compared with CON (P ≤0.05). There was no observed effect of OVER diet on protein abundance in the cotyledon (P > 0.05). Expression of IGF-II mRNA was increased in OVER at day 45 and IGFBP-3 was reduced in RES at day 90 in caruncle relative to CON (P ≤0.05). Maternal diet did not alter placentome diameter or weight (P > 0.05). These findings suggest that restricted- and over-feeding negatively impact protein and mRNA expression of key chemokines and growth factors implicated in proper placenta development and function.

Too little or too much food during gestation can lead to poor growth and health of the resulting offspring. The placenta is an important source of nutrient supply for the fetus and poor maternal diet can impair placenta growth and function. Although placental development and function are well studied, the mechanisms by which maternal diet can affect placental growth and fetal development are not well understood. Based on our previous findings that specific proteins are important regulators of placental growth and function, we used a sheep model of poor maternal nutrition to demonstrate that protein abundance of these factors is altered in the placenta. These findings demonstrate potential mechanism by which maternal diet can affect the placenta and thereby impact fetal growth.

Serum concentrations of insulin-like growth factor-I and insulin-like growth factor binding protein-2 and -3 in eight hoofstock species.

The somatotropic axis, which includes growth hormone, insulin-like growth factor (IGF)-I, and IGF binding proteins (IGFBP), is involved in the regulation of growth and metabolism. Measures of the somatotropic axis can be predictive of nutritional status and growth rate that can be utilized to identify nutritional status of individual animals. Before the somatotropic axis can be a predictive tool, concentrations of hormones of the somatotropic axis need to be established in healthy individuals. To begin to establish these data, we quantified IGF-I, IGFBP-2, and IGFBP-3 in males and females of eight threatened hoofstock species at various ages. Opportunistic blood samples were collected from Bos javanicus (Java banteng), Tragelaphus eurycerus isaaci (bongo), Gazella dama ruficollis (addra gazelle), Taurotragus derbianus gigas (giant eland), Kobus megaceros (Nile lechwe), Hippotragus equines cottoni (roan antelope), Ceratotherium simum simum (white rhinoceros), and Elephas maximus (Asian elephant). Serum IGF-I and IGFBPs were determined by radioimmunoassay and ligand blot, respectively. Generally, IGF-I and IGFBP-3 were greater in males, and IGFBP-2 was greater in females. In banteng (P = 0.08) and male Nile lechwe (P < 0.05), IGF-I increased with age, but decreased in rhinoceros (P = 0.07) and female Nile lechwe (P < 0.05). In banteng, IGFBP-3 was greater (P < 0.01) in males. In elephants (P < 0.05) and antelope (P = 0.08), IGFBP-2 were greater in females. Determination of concentrations of hormones in the somatotropic axis in healthy animals makes it possible to develop models that can identify the nutritional status of these threatened hoofstock species.

Evaluation of an alternative to feeding whole frozen fish in belugas (Delphinapterus leucas).

Feeding fish to captive piscivores can be challenging owing to cost, availability, variability in nutrient, and caloric composition, as well as handling and storage concerns. This trial evaluated the response of three belugas to being fed Fish Analog, an alternative to frozen fish. Body condition, gut transit time, serum chemistry and metabolic hormone analytes, immune function, and behavioral motivation were the dependent variables. Belugas (n=3) were fed various levels of Fish Analog (0-50%) over a 6-month period, and follow-up studies were conducted to further examine several dependent variables. When provided in gradually increasing amounts, belugas consumed the Fish Analog, with only minor fecal consistency changes and without behavioral responses indicative of gastric discomfort. Axillary girth and blubber thickness were positively correlated, and did not differ significantly with changes in the percentage of Fish Analog fed. Individual animal variation in initial passage time, some serum chemistry analytes, and immune function differences were noted following feeding of Fish Analog. Feeding Fish Analog reduced blood n9 fatty acids compared with captive belugas fed no Fish Analog. Feeding a DHA-enriched Fish Analog increased several n3 fatty acids, including eicosapentaenoic acid, but not DHA, compared with whales fed no Fish Analog or non-DHA-enriched Fish Analog. Fish Analog was shown to be a viable alternative to feeding fish at up to 50% of the dietary caloric density.

Understanding gestational and feed management practices of New England sheep producers.

Several sources of information are available to producers for guidance in managing their breeding flocks; however, it is unknown if sheep producers utilize any or all of these resources. Because maternal diet during gestation can have immediate and long-lasting negative effects on growth and health of offspring, it is important for producers to insure they are providing appropriate nutritional management to ewes during breeding and gestation. Historically, New England sheep producers have not been included in USDA surveys of sheep producers, and therefore, there is a lack of information about how New England producers manage their flocks, especially in terms of nutrition and gestation. The objective was to determine flock size, breeds, pregnancy detection methods, and feeding management practices of New England sheep producers. To meet this objective, a 12-question survey was developed and disseminated to New England sheep producers 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% have flock sizes of 11 to 50 sheep, whereas 15.6% had 10 or less and 23% had greater than 50 sheep. Most respondents (63.5%) maintain one breed of sheep; however, larger flocks (>50 sheep) are more likely to maintain multiple breeds (P < 0.05). The largest percentage (40.6%) use their sheep for both meat and fiber production, 38.5% for meat only, and 20.8% manage sheep for fiber only. Spring (January to May) is the primary (59.4%) lambing season. The majority (76.0%) of New England sheep producers do not have their feed chemically analyzed for nutrient composition, which presents an opportunity for improving feeding management. There were associations (P < 0.05) between flock size and flock purpose, flock size and number of breeds owned, flock size and feed type, feed type and feed analysis, feed type and source of feed information, and source of feed information and state. In conclusion, New England sheep producers have flocks of varying size and purpose, and would likely benefit from outreach education on the value of diet analysis and formulation for their breeding flocks, especially during gestation. Furthermore, findings of this survey may represent the management needs of smaller flocks throughout the United States.

Maternal Under- and Over-Nutrition during Gestation Causes Islet Hypertrophy and Sex-Specific Changes to Pancreas DNA Methylation in Fetal Sheep.

The mechanisms by which fetal programming predisposes offspring to reduced ß-cell function later in life are poorly understood. We hypothesized that maternal under- and over-nutrition during gestation would negatively affect offspring pancreas development and alter DNA methylation patterns. Pregnant ewes (n = 78) were fed 100, 60, or 140% of NRC requirements beginning at d 30.2 ± 0.2 of gestation. The fetuses are referred to as CON, RES, and OVER, respectively. Fetal pancreas tissue was collected at d 90 or 135 of gestation or within 24 h of birth. Tissue was preserved for histological (n = 8 to 9 offspring per treatment per time point) and DNA methylation analyses (n = 3 to 4 fetuses per treatment per sex). At d 135, OVER exhibited an increased islet size, reduced islet number, and greater insulin positive area compared with CON (p ≤ 0.03). An increased islet size was also observed at d 135 in RES (p ≤ 0.03) compared with CON. Cellular proliferation was reduced at birth in OVER vs. CON (p = 0.01). In the RES vs. CON females, 62% of the differentially methylated regions (DMRs) were hypomethylated (p ≤ 0.001). In the RES vs. CON males, 93% of the DMRs were hypermethylated (p ≤ 0.001). In OVER, 66 and 80% of the DMRs were hypermethylated in the female and male offspring compared with CON (p ≤ 0.001). In conclusion, changes to maternal diet during pregnancy affects the islet hypertrophy and cellular proliferation of the offspring at early post-natal time points. Additionally, changes in DNA methylation patterns appear to be in a diet-specific and sex-dependent manner.

Maternal Overnutrition During Gestation in Sheep Alters Autophagy Associated Pathways in Offspring Heart.

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health pre- and post-natally. Overfeeding during gestation or maternal obesity (MO) results in altered metabolism and imbalanced endocrine hormones in animals and humans which will have long-lasting and detrimental effects on offspring growth and health. In this study, we examined the effects of overnutrition during gestation on autophagy associated pathways in offspring heart muscles at two gestational and one early postnatal time point (n = 5 for treated and untreated male and female heart respectively at each time point). Two-way ANOVA was used to analyze the interaction between treatment and sex at each time point. Our results revealed significant interactions of maternal diet by developmental stages for offspring autophagy signaling. Overfeeding did not affect the autophagy signaling at mid-gestation day 90 (GD90) in both male and female offspring while the inflammatory cytokines were increased in GD90 MO male offsrping; however, overfeeding during gestation significantly increased autophagy signaling, but not inflammation level at a later developmental stage (GD135 and day 1 after birth) in both males and females. We also identified a sexual dimorphic response in which female progeny were more profoundly influenced by maternal diet than male progeny regardless of developmental stages. We also determined the cortisol concentrations in male and female hearts at three developmental stages. We did not observe cortisol changes between males and females or between overfeeding and control groups. Our exploratory studies imply that MO alters autophagy associated pathways in both male and female at later developmental stages with more profound effects in female. This finding need be confirmed with larger sample numbers in the future. Our results suggest that targeting on autophagy pathway could be a strategy for correction of adverse effects in offspring of over-fed ewes.

Mid- to late-gestational maternal nutrient restriction followed by realimentation alters development and lipid composition of liver and skeletal muscles in ovine fetuses.

Maternal nutrient restriction during gestation adversely affects offspring growth and development of liver and skeletal muscle tissues. Realimentation following nutrient restriction may alleviate these negative impacts on development but may alter metabolism and tissue composition. Forty-eight ewes, pregnant with singletons, were fed to meet 100% National Research Council (NRC) recommendations starting at the beginning of gestation. On day 50 of gestation, seven ewes were euthanized (BASE), and fetal liver, skeletal muscles, and blood samples were collected. The remaining animals were fed either 100% of NRC recommendations (CON) or 60% NRC recommendations (RES), a subset were euthanized at day 90 of gestation (n = 7/treatment), and fetal samples were collected. Remaining ewes were maintained on the current diet (CON-CON, n = 6; RES-RES, n = 7) or switched to the alternate diet (CON-RES, RES-CON; n = 7/treatment). On day 130 of gestation, the remaining ewes were euthanized, and fetal samples were collected. At day 130 of gestation, maternal nutrient restriction during late-gestation (RES-RES and CON-RES) decreased fetal liver weight (P < 0.01) and cross-sectional area in triceps brachii (P = 0.01; TB), longissimus dorsi (P = 0.02; LM), and semitendinosus (P = 0.05; STN) muscles. Maternal nutrient restriction during mid-gestation increased hepatocyte vacuole size at day 130 of gestation. Late-gestational maternal nutrient restriction increased mRNA expression of insulin-like growth factor (IGF) binding protein-1 (P < 0.01), glycogen synthase 2 (P = 0.01; GYS2), and pyruvate dehydrogenase kinase 1 (P < 0.01; PDHK1) in the liver and IGF receptor 1 (P = 0.05) in the LM. Lipid concentration in the LM was decreased by late-gestational nutrient restriction (P = 0.01) and increased by mid-gestational nutrient restriction in STN (P = 0.03) and TB (P < 0.01). Principal component analysis of lipidomics data demonstrated clustering of principal components by day of gestation and elastic net regression identified 50, 44, and 29 lipids that classified the treatments in the fetal liver, LM, and blood, respectively. In conclusion, restricting maternal nutrition impacts fetal liver and muscle morphology, gene expression, and lipid metabolism, whereas realimentation attenuated some of these effects. Therefore, realimentation may be a viable strategy to reduce the impacts of nutrient restriction, but can lead to alterations in lipid metabolism in sheep.

Re-alimentation in harbor seal pups: effects on the somatotropic axis and growth rate.

The metabolic hormones, growth hormone (GH) and insulin-like growth factor (IGF)-I, together with IGF binding proteins (IGFBP), have been well studied in domestic species and are the primary components of the somatotropic axis. This hormone axis is responsive to nutrient intake, associated with growth rate, and accretion of protein and adipose. However, this relationship has not been evaluated in species that rely heavily on adipose stores for survival, such as pinnipeds. The primary objectives of this research were to investigate the response of the somatotropic axis to reduced nutrient intake and re-alimentation in rehabilitated harbor seal pups, and to assess if these hormones are related to nutritional status and growth rate in harbor seals. Stranded harbor seal pups (n=24) arrived at the rehabilitation facility very thin after fasting for several days (nutritional nadir). Throughout rehabilitation nutrient intake increased and pups gained mass and body condition. Concentrations of GH and IGFBP-2 decreased with re-alimentation, while IGF-I and IGFBP-3 concentrations increased. Overall, GH and IGFBP-2 were negatively associated and IGF-I and IGFBP-3 were positively associated with growth rate and increased body condition of harbor sea pups. Further, the magnitude of the growth response was related to the magnitude in response of the somatotropic axis to varied levels of intake. These data suggest that multiple components of the somatotropic axis may be used to assess the energy status of individuals and may also provide information on the level of feed intake that is predictive of growth rate.

Cell-based meat: the need to assess holistically.

Proof-of-principle for large-scale engineering of edible muscle tissue, in vitro, was established with the product's introduction in 2013. Subsequent research and commentary on the potential for cell-based meat to be a viable food option and potential alternative to conventional meat have been significant. While some of this has focused on the biology and engineering required to optimize the manufacturing process, a majority of debate has focused on cultural, environmental, and regulatory considerations. Animal scientists and others with expertise in muscle and cell biology, physiology, and meat science have contributed to the knowledge base that has made cell-based meat possible and will continue to have a role in the future of the new product. Importantly, the successful introduction of cell-based meat that looks and tastes like conventional meat at a comparable price has the potential to displace and/or complement conventional meat in the marketplace.

Poor maternal nutrition during gestation in sheep alters prenatal muscle growth and development in offspring.

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.

Changes in glucocorticoids, IGF-I and thyroid hormones as indicators of nutritional stress and subsequent refeeding in Steller sea lions (Eumetopias jubatus).

Physiological responses to changes in energy balance are tightly regulated by the endocrine system through glucocorticoids, IGF-I and thyroid hormones. Changes in these hormones were studied in eight captive female Steller sea lions that experienced changes in food intake, body mass, body composition, and blood metabolites during summer and winter. During a period of energy restriction, one group of sea lions was fed reduced amounts of Pacific herring and another was fed an isocaloric diet of walleye pollock, after which both groups returned to their pre-experimental diets of herring. Cortisol was negatively and IGF-I was positively associated with changes in body mass during periods of energy restriction (mass loss associated with increase in cortisol and decrease in IGF-I) and refeeding (body mass maintenance associated with stable hormone concentrations in summer and compensatory growth linked to decrease in cortisol and increase in IGF-I in winter). Cortisol and IGF-I were also correlated with changes in lipid and lean mass, respectively. Consequently, these two hormones likely make adequate biomarkers for nutritional stress in sea lions, and when combined provide indication of the energetic strategy (lipid vs lean mass catabolism) animals adopt to cope with changes in nutrient intake. Unlike type of diet fed to the sea lions, age of the animals also impacted hormonal responses, with younger animals showing more intense hormonal changes to nutritional stress. Thyroid hormones, however, were not linked to any physiological changes observed in this study.

CELL BIOLOGY SYMPOSIUM: METABOLIC RESPONSES TO STRESS: FROM ANIMAL TO CELL: Poor maternal nutrition during gestation: effects on offspring whole-body and tissue-specific metabolism in livestock species1,2.

Poor maternal nutrition, both restricted-feeding and overfeeding, during gestation can negatively affect offspring growth, body composition, and metabolism. The effects are observed as early as the prenatal period and often persist through postnatal growth and adulthood. There is evidence of multigenerational effects demonstrating the long-term negative impacts on livestock production. We and others have demonstrated that poor maternal nutrition impairs muscle growth, increases adipose tissue, and negatively affects liver function. In addition to altered growth, changes in key metabolic factors, increased glucose concentrations, insulin insensitivity, and hyperleptinemia are observed during the postnatal period. Furthermore, there is recent evidence of altered metabolism in specific tissues (e.g., muscle, adipose, and liver) and stem cells. The systemic and local changes in metabolism demonstrate the importance of determining the mechanism(s) by which maternal diet programs offspring growth and metabolism in an effort to develop novel management practices to improve the efficiency of growth and health in these offspring.

Maternal Restricted- and Over-Feeding During Gestation Result in Distinct Lipid and Amino Acid Metabolite Profiles in the Longissimus Muscle of the Offspring.

Maternal over- and restricted-feeding during gestation have similar negative consequences for the offspring, including decreased muscularity, increased adiposity, and altered metabolism. Our objective was to determine the effects of poor maternal nutrition during gestation (over- and restricted-feeding) on the offspring muscle metabolite profile. Pregnant ewes (n = 47) were fed 60% (RES), 100% (CON), or 140% (OVER) of NRC requirements starting at day 30.2 ± 0.2 of gestation. Offspring sample collection occurred at days 90 and 135 of gestation, and within 24 h of birth. C2C12 myoblasts were cultured in serum collected from offspring at birth (n = 18; 6 offspring per treatment) for analysis of oxidative and glycolytic capacity. Unbiased metabolite analysis of longissimus muscle samples (n = 72; 8 fetuses per treatment per time point) was performed using mass spectrometry. Data were analyzed by ANOVA for main effects of treatment, time point, and their interaction. Cells cultured in serum from RES offspring exhibited increased proton leak 49% (p = 0.01) compared with CON, but no other variables of mitochondrial respiration or glycolytic function were altered. Mass spectrometry identified 612 metabolites. Principle component analysis identified day of gestation as the primary driver of metabolic change; however, maternal diet also altered the lipid and amino acid profiles in offspring. The abundance of 53 amino acid metabolites and 89 lipid metabolites was altered in RES compared with CON (p ≤ 0.05), including phospholipids, sphingolipids, and ceramides within the lipid metabolism pathway and metabolites involved in glutamate, histidine, and glutathione metabolism. Similarly, abundance of 63 amino acid metabolites and 70 lipid metabolites was altered in OVER compared with CON (p ≤ 0.05). These include metabolites involved in glutamate, histidine, lysine, and tryptophan metabolism and phosphatidylethanolamine, lysophospholipids, and fatty acids involved in lipid metabolism. Further, the amino acid and lipid profiles diverged between RES and OVER, with 69 amino acid and 118 lipid metabolites differing (p ≤ 0.05) between groups. Therefore, maternal diet affects metabolite abundance in offspring longissimus muscle, specifically metabolites involved in lipid and amino metabolism. These changes may impact post-natal skeletal muscle metabolism, possibly altering energy efficiency and long-term health.

Dosage Compensation and Gene Expression of the X Chromosome in Sheep.

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".

Comparison of the somatotropic axis in free-ranging and rehabilitated harbor seal pups (Phoca vitulina).

The somatotropic axis, including growth hormone (GH), insulin-like growth factor (IGF)-I, and IGF binding proteins (IGFBP), is a bridge between growth physiology, developmental age, and nutritional status in domestic animals. However, the importance of the somatotropic axis in nutrition, growth, and development of harbor seals has not been previously explored. Given the difficulty of conducting longitudinal studies in free-ranging harbor seals, this study focused on the potential use of harbor seals in rehabilitation facilities as a model for free-ranging seals. The purpose of this research was to compare concentrations of components of the somatotropic axis in free-ranging versus rehabilitated harbor seal pups. The hypothesis was that measurements of the somatotropic axis will be similar between individuals of comparable age and nutritional status (fasting versus feeding). To investigate this hypothesis, harbor seal pups (n=8) brought to The Marine Mammal Center (Sausalito, California, USA) or Mystic Aquarium (Mystic, Connecticut, U.S.A.) were initially assessed and determined to be healthy but abandoned. All pups were less than 2 wk of age upon arrival at rehabilitation facilities. Standard length was assessed at the time of arrival and again at release. Body mass was measured every week and blood samples were collected from each pup at 0, 4, and 8 wk of rehabilitation. Blood was collected and morphometrics assessed in free-ranging harbor seal pups (n=8) from the Gulf of Maine. Sera were analyzed for GH, IGF-I, and IGFBP concentrations. Concentrations of GH, IGF-I, and IGFBP-2 and -3 in rehabilitated pups were within a similar range compared with free-ranging pups when considered in the context of presumed nutrient intake. These data suggest that rehabilitated harbor seals may provide a useful model to investigate the effects of nutrient intake on growth and development of harbor seals, and will provide insight into phocid endocrinology and metabolism.