Effects of maternal obesity in an ovine model on metabolic outcomes in F2 adults and F3 neonates.

Accumulating evidence suggests that indications of metabolic syndrome can be inherited through the germline as a result of maternal obesity. We hypothesized that diet-induced maternal obesity during gestation would program metabolic consequences for multiple generations of offspring, even when first, second, and third generation offspring (F1, F2, F3, respectively) were fed only to requirements. Control (CON) and obese (OB) ewes (generation 0; F0) were bred to a single ram to produce the first generation of offspring (F1). From 60 d prior to conception through term, CONF0 ate 100% National Research Council recommendations (NRC), while OBF0 ewes ate 150% NRC. All F1, F2, and F3 ate 100% NRC after weaning. All mature F1 ewes were bred to a single ram to generate CONF2 (n = 6) and OBF2 (n = 10). All mature F2 ewes were bred to a single ram to produce CONF3 (n = 6) and OBF3 (n = 10). OBF2 ewes exhibited greater (P < 0.0001) plasma cortisol than CONF2 throughout gestation. A glucose tolerance test at 90% gestation revealed OBF2 ewes had higher (P < 0.05) insulin response with similar glucose, resulting in greater (P < 0.05) insulin resistance. OBF3 neonates had similar weight, lean mass, and body fat mass to CONF3 neonates. These data suggest that multigenerational programming of adverse metabolic phenotypes occur in association with F0 maternal obesity, yet adiposity may return to CON levels in F3 neonates.

Effects of maternal obesity on maternal and fetal plasma concentrations of adiponectin and expression of adiponectin and its receptor genes in cotyledonary and adipose tissues at mid- and late-gestation in sheep.

Adiponectin potentially influences fetal weight by altering insulin signaling and trans-placental amino acid and glucose transporters. The objective of this study was to determine how maternal obesity influences maternal and fetal plasma concentrations of adiponectin, expression of fetal adiponectin, its receptors, and adipogenic genes at mid- and late-gestation. Blood samples and tissues were collected from obese and control multiparous pregnant ewes at day 75 or 135 of gestation. Although day of gestation or maternal obesity did not influence (P > 0.6) maternal plasma concentrations of adiponectin, fetal weight was increased (P < 0.001) and adiponectin tended to decrease (P = 0.10) at mid-gestation in fetuses from obese ewes. Differences were not apparent at late-gestation (P > 0.70). Relative abundance of adiponectin (P = 0.01), AdipoR2 (P = 0.04) and PPARγ (P = 0.01) mRNA was less at mid-gestation in fetal adipose tissue from obese mothers. By late gestation, maternal obesity tended to associated with a decrease in relative abundance of adiponectin (P = 0.09) and SREBF1 (P = 0.10) mRNA in fetal adipose tissue. Maternal obesity did not influence (P ≥ 0.20) the relative abundance of adiponectin, AdipoR1 and AdipoR2 mRNA in cotyledonary tissue at mid or late- gestation. In conclusion, maternal obesity in sheep influences relative abundance of fetal adipose tissue mRNA for adiponectin and adipogenic, as well as plasma concentrations of total adiponectin. Although adiposity in pregnant ewes did not influence maternal adiponectin, maternal obesity potentially influenced fetal adipogenesis by altering the abundance of adiponectin, PPARγ and SREBF1 mRNA in fetal adipose tissue.

Intergenerational impact of maternal overnutrition and obesity throughout pregnancy in sheep on metabolic syndrome in grandsons and granddaughters.

We previously reported that maternal overnutrition and obesity (MO) throughout pregnancy and lactation in sheep (MOF0) decreases term fetal pancreatic ß-cell numbers and increases perirenal adiposity producing hyperphagia, increased adiposity and insulin resistance in adult female offspring (MOF1) fed ad libitum. Pregnant female MOF1 exhibited increased blood glucose from mid to late gestation vs control F1 (CTRF1) though both groups ate only to NRC recommendations. MOF1 ewes delivered female offspring (F2) who like their MOF1 mothers exhibited increased abdominal adiposity and absent neonatal leptin surge. In the current work, we determined if adult MOF2 exhibited metabolic syndrome components when fed ad libitum. After weaning, MOF2 males (n = 5), MOF2 females (n = 6), CTRF2 males (n = 5), and CTRF2 females (n = 6) were fed to NRC requirements until 19 mo followed by 12-wk ad libitum feeding. Body weight and % fat increased (P < 0.01) in all F2 during this feeding trial. MOF2 males were heavier (P < 0.01) than CTRF2 males and females, and MOF2 females throughout the trial. By wk 8, baseline blood glucose concentrations increased (P < 0.001) in MOF2 females, but not other groups, remaining elevated throughout the trial. Baseline insulin was similar through wk 6, increasing (P < 0.05) at wk 8 in MOF2 females only. MOF2 female insulin returned to CTRF2 female levels during wk 10 and 12. The progressive increase of plasma glucose on wk 8 in association with increased insulin in MOF2 females but not other groups demonstrated a diet-induced increase (P < 0.001) in MOF2 female insulin resistance. The subsequent decline in insulin during wk 10 and 12 despite elevated glucose in MOF2 females is consistent with a decrease in glucose-stimulated pancreatic ß-cell function. These data indicate that ad libitum feeding exceeds the pancreatic secretory response predisposing MOF2 females to hyperglycemia. Furthermore, there was a sex difference where MOF2 males increased body mass and MOF2 females displayed insulin/glucose dysregulation.

Multigenerational impact of maternal overnutrition/obesity in the sheep on the neonatal leptin surge in granddaughters.

BACKGROUND/OBJECTIVES: We have reported that maternal overnutrition/obesity (OB) in sheep resulting from feeding 150% of National Research Council (NRC) requirements throughout gestation leads to maternal hyperglycemia and hyperinsulinemia. Further, newborn lambs born to OB vs control-fed (CON, 100% of NRC) ewes exhibited greater adiposity, increased blood cortisol, insulin and glucose and the elimination of the postnatal leptin spike seen in lambs born to CON ewes. This early postnatal leptin peak is necessary for the development of hypothalamic circuits, which program appetite in later life. This study evaluated the multigenerational impact of OB on insulin:glucose dynamics of mature female F1 offspring fed only to requirements throughout gestation and on their lambs (F2 generation). DESIGN AND METHODS: Adult F1 female offspring born to OB (n=10) or CON (n=7) ewes were utilized. All F1 ewes were subjected to a glucose tolerance test at midgestation and late gestation. Jugular blood was obtained from F2 lambs at birth (day 1) through postnatal day 11, and plasma glucose, insulin, cortisol and leptin concentrations were determined. Dual-energy X-ray absorptiometry was utilized to determine bone mineral density, bone mineral content, lean tissue mass and fat tissue mass. RESULTS: Fasted blood glucose and insulin concentrations were greater (P<0.05) in OBF1 than CONF1 ewes at midgestation and late gestation. Further, after glucose infusion, both glucose and insulin concentrations remained higher in OBF1 ewes (P<0.05) than CONF1 ewes, demonstrating greater insulin resistance. Blood concentrations of glucose, insulin and cortisol and adiposity were higher (P<0.01) in OBF2 lambs than CONF2 lambs at birth. Importantly, OBF2 lambs failed to exhibit the early postnatal leptin peak exhibited by CONF2 lambs. CONCLUSIONS: These data suggest that these OBF2 lambs are predisposed to exhibit the same metabolic alterations as their mothers, suggesting a multigenerational programming effect.

CELL BIOLOGY SYMPOSIUM: Impacts of maternal obesity on placental and gut inflammation and health.

Obesity in pregnant women is a growing public health concern that negatively affects fetal development and has long-term impacts on offspring health. The placenta plays an essential role in nutrient transport to the fetus and supports fetal growth and development. Maternal obesity (MO) induces an exacerbated proinflammatory milieu in the placenta providing an inflammatory environment for fetuses. The gut is one of the largest immune organs and mainly develops during the fetal stage. Maternal obesity and the corresponding inflammatory uteroplacental environment affect gut development, incurring inflammatory responses in the fetal intestine that further prime or program the offspring gut to enhance inflammation and impair intestinal barrier integrity. This review summarizes the impact of MO on inflammatory responses in placenta and fetal intestine and the long-term effects on offspring intestinal health. Because "leaky gut" is one of the main etiological factors for a number of common diseases, including inflammatory bowel diseases, type I diabetes, and related autoimmune diseases, the adverse effect of MO on the overall health of progeny is further discussed.

Maternal obesity downregulates microRNA let-7g expression, a possible mechanism for enhanced adipogenesis during ovine fetal skeletal muscle development.

BACKGROUND: Obesity in women of childbearing age is increasing at an alarming rate. Growing evidence shows that maternal obesity induces detrimental effects on offspring health, including pre-disposition to obesity. We have shown that maternal obesity increases fetal intramuscular adipogenesis at mid-gestation. However, the mechanisms are poorly understood. MicroRNAs (miRNAs) regulate mRNA stability. We hypothesized that maternal obesity alters fetal muscle miRNA expression, thereby influencing intramuscular adipogenesis. METHODS: Non-pregnant ewes received a control diet (Con, fed 100% of National Research Council (NRC) recommendations, n=6) or obesogenic diet (OB; 150% NRC recommendations, n=6) from 60 days before to 75 days after conception when the fetal longissimus dorsi (LD) muscle was sampled and miRNA expression analyzed by miRNA microarray. One of miRNAs with differential expression between Con and OB fetal muscle, let-7g, was further tested for its role in adipogenesis and cell proliferation in C3H10T1/2 cells. RESULTS: A total of 155 miRNAs were found with a signal above 500, among which, three miRNAs, hsa-miR-381, hsa-let-7g and bta-miR-376d, were differentially expressed between Con and OB fetuses, and confirmed by quantitative real-time PCR (QRT-PCR) analyses. Reduced expression of miRNA let-7g, an abundantly expressed miRNA, in OB fetal muscle was correlated with higher expression of its target genes. Overexpression of let-7g in C3H10T1/2 cells reduced their proliferation rate. Expression of adipogenic markers decreased in cells overexpressing let-7g, and the formation of adipocytes was also reduced. Overexpression of let-7g decreased expression of inflammatory cytokines. CONCLUSION: Fetal muscle miRNA expression was altered due to maternal obesity, and let-7g downregulation may enhance intramuscular adipogenesis during fetal muscle development in the setting of maternal obesity.

The impact of maternal overnutrition and obesity on hypothalamic-pituitary-adrenal axis response of offspring to stress.

We evaluated the effect of maternal obesity before and throughout gestation on offspring hypothalamic-pituitary-adrenal axis function. Multiparous Rambouillet by Columbia crossbred ewes were fed either 100% of National Research Council (NRC) recommendations (control, C) or 150% of NRC recommendations (obese, OB) from 60 d before mating until lambing. Ten lambs born to OB ewes (five males and five females), and eight lambs born to C ewes (three male and five female) were studied. From delivery to weaning lambs were maintained with their mothers, who were all fed 100% NRC recommendations. After weaning, all lambs were group housed and fed the same diet to meet NRC requirements. At 19 mo of age lambs were placed in individual pens and fed a pelletized diet to meet maintenance requirements. Jugular vein catheters were placed and 2 d later lambs received an intravenous (i.v.) adrenocorticotropic hormone (ACTH) challenge followed by an i.v. corticotropin-releasing hormone (CRH)/arginine vasopressin (AVP) challenge 1 d later. Thirty d later offspring were again catheterized and placed into metabolism crates for 2 d before receiving an isolation stress test. ACTH and cortisol responses to the isolation stress test and CRH/AVP challenge and cortisol responses to ACTH challenge were determined. Cortisol was quantified via radioimmunoassay and ACTH was quantified using an Immulite 1000; both were analyzed using repeated measures using the MIXED procedure of SAS. Offspring from OB ewes had elevated basal plasma ACTH and cortisol compared with C offspring before all three challenges (P < 0.05). Offspring from OB mothers tended (P = 0.06) to have a greater ACTH response after an i.v. CRH/AVP injection than offspring from C mothers (12,340 ± 1,430 vs 8,170 ± 1,570 area under the curve, respectively). Cortisol response to the CRH/AVP and ACTH challenges was not influenced by maternal nutrition (P = 0.46) and averaged 4.77 ± 0.2 µg/dL and 1.94 ± 0.01 µg/dL, respectively. The ACTH response following the isolation stress test was also similar (P = 0.82) for OB and C offspring (147 ± 20 pg/mL), and cortisol response during the isolation stress test was similar between C and OB offspring (P = 0.64, 5.25 ± 0.3 µg/dL). These findings suggest that maternal obesity before and during gestation does not affect stress responses by the offspring, but has an impact on hypothalamic-pituitary-adrenal sensitivity. The lack of differences in cortisol release under the influence of difference concentrations of ACTH during the CRH/AVP challenge could indicate adrenal dysfunction in OB offspring.

Maternal obesity upregulates fatty acid and glucose transporters and increases expression of enzymes mediating fatty acid biosynthesis in fetal adipose tissue depots.

Maternal nutrient restriction leads to alteration in fetal adipose tissue, and offspring from obese mothers have an increased risk of developing obesity. We hypothesized that maternal obesity increases fetal adipogenesis. Multiparous ewes (Columbia/Rambouillet cross 3 to 5 yr of age) carrying twins were assigned to a diet of 100% (Control; CON; n = 4) or 150% (Obese; OB, n = 7) of NRC maintenance requirements from 60 d before conception until necropsy on d 135 of gestation. Maternal and fetal plasma were collected and stored at -80°C for glucose and hormone analyses. Fetal measurements were made at necropsy, and perirenal, pericardial, and subcutaneous adipose tissues were collected from 7 male twin fetuses per group and snap frozen at -80°C. Protein and mRNA expression of fatty acid translocase [cluster of differentiation (CD) 36], fatty acid transport proteins (FATP) 1 and 4, insulin-sensitive glucose transporter (GLUT-4), fatty acid synthase (FASN), and acetyl-coA carboxylase (ACC) was evaluated. Fetal weight was similar, but fetal carcass weight (FCW) was reduced (P < 0.05) in OB versus CON fetuses. Pericardial and perirenal adipose tissue weights were increased (P < 0.05) as a percentage of FCW in OB versus CON fetuses, as was subcutaneous fat thickness (P < 0.001). Average adipocyte diameter was greater (P < 0.01) in the perirenal fat and the pericardial fat (P = 0.06) in OB fetuses compared with CON fetuses. Maternal plasma showed no difference (P > 0.05) in glucose or other hormones, fetal plasma glucose was similar (P = 0.42), and cortisol, IGF-1, and thyroxine were reduced (P ≤ 0.05) in OB fetuses compared with CON fetuses. Protein and mRNA expression of CD 36, FATP 1 and 4, and GLUT-4 were increased (P ≤ 0.05) in all fetal adipose depots in OB versus CON fetuses. The mRNA expression of FASN and ACC was increased (P < 0.05) in OB vs. CON fetuses in all 3 fetal adipose tissue depots. Fatty acid concentrations were increased (P = 0.01) in the perirenal depot of OB versus CON fetuses, and specific fatty acid concentrations were altered (P < 0.05) in subcutaneous and pericardial adipose tissue because of maternal obesity. In conclusion, maternal obesity was associated with increased fetal adiposity, increased fatty acid and glucose transporters, and increased expression of enzymes mediating fatty acid biosynthesis in adipose depots. These alterations, if maintained into the postnatal period, could predispose the offspring to later obesity and metabolic disease.

Effects of early- to mid-gestational undernutrition with or without protein supplementation on offspring growth, carcass characteristics, and adipocyte size in beef cattle.

Angus × Gelbvieh cows with 2 to 3 previous pregnancies were used to evaluate effects of maternal nutrient restriction on offspring adipose tissue morphology at standard production endpoints. At 45 d after AI to a single sire, pregnancy was confirmed and cows randomly allotted into groups and fed a control (Con, 100% of NRC recommendations), nutrient-restricted (NR, 70% of Con diet), or nutrient-restricted + protein-supplemented (NRP, 70% of Con + essential AA supply to the small intestine equal to Con) diet. At d 185 of gestation, cows were commingled and received the Con diet thereafter. Bull calves were castrated at 2 mo of age. Calves were weaned at 210 d, backgrounded for 28 d, and then placed in the feedlot for 195 d. Steers and heifers were slaughtered at an average 12th-rib fat thickness of 7.6 mm. Adipose tissue from selected depots was collected for adipocyte size analysis. There was no significant difference in BW or BCS between Con, NRP, and NR cows at d 45 of gestation, which averaged 489.7 ± 17.7 kg and 5.35 ± 0.13, respectively. At d 185 of gestation, Con and NRP groups had similar BW (566.1 ± 14.8 and 550.2 ± 14.8 kg) and BCS (6.34 ± 0.27 and 5.59 ± 0.27), but NR cows exhibited reduced (P < 0.05) BW (517.9 ± 14.8 kg) and BCS (4.81 ± 0.27). Among offspring (steers and heifers) at slaughter, there were no significant differences in BW or organ weights among treatment groups. Yield grade was reduced (P < 0.05) and semitendinosus weight/HCW tended (P = 0.09) to be reduced in NR offspring compared with Con and NRP offspring. Average adipocyte diameter was increased (P < 0.05) in subcutaneous, mesenteric, and omental adipose tissue and tended (P = 0.09) to increase in perirenal adipose tissue in NR compared with Con offspring with NRP offspring adipocyte diameter being either intermediate or similar to Con calves. The adipocyte size alterations observed in NR offspring were confirmed by DNA concentration of the adipose tissue depots. There also was an increased mRNA expression (P < 0.05) of fatty acid transporter 1 in subcutaneous adipose tissue from NR offspring compared with Con and NRP offspring. Nutritional restriction during early and mid gestation increased or tended to increase (P < 0.09) adipocyte diameter in all adipose tissue depots in finished steer and heifer calves.

Upregulation of growth signaling and nutrient transporters in cotyledons of early to mid-gestational nutrient restricted ewes.

Multiparous ewes received 100% (control, C, n = 13) or 50% (nutrient restricted, NR, n = 14) of NRC dietary requirements from d28-d78 of gestation. On d78, 5 C and 6 NR ewes were necropsied. The remaining 8 C and 8 NR ewes were fed to 100% of NRC from d78-d135 and necropsied. Maternal blood was collected at both necropsies and at weekly intervals for assay of glucose, insulin and leptin. Fetal blood was collected at d78 and d135 necropsies for assay of glucose and lipids. Cotyledonary (COT) tissue was evaluated for protein and mRNA expression [fatty acid transporter (FATP)1, FATP4, CD36, glucose transporter (GLUT)1 and GLUT3], mRNA expression only [placenta fatty acid binding protein (FABPpm) and lipoprotein lipase (LPL)], or expression of phosphorylated and total protein forms [AMP kinase (AMPK)α, acetyl-CoA carboxylase (ACC), extracellular signal-regulated kinase (Erk)1/2, mammalian target of rapamycin (mTOR) and protein kinase B (Akt)]. On d78, but not d135, placental and fetal weights were reduced (P < 0.05) in NR vs. C ewes. Maternal circulating glucose, insulin and leptin levels were decreased in NR vs. C ewes on d78 (P < 0.05) but similar at d135. Fetal blood glucose and triglyceride levels were lower in NR vs. C ewes (P < 0.05) on d78, but similar on d135. On d78, GLUT1, FATP4, CD36 mRNA and protein expression levels, FABPpm mRNA level, and leptin protein level were all increased (P < 0.05) in COT of NR vs. C ewes. AMPK, ACC, and Erk1/2 activities were also increased (P < 0.05) in NR vs. C COT on d78. In contrast, only FATP4 was increased (P < 0.05) at both the mRNA and protein levels in COT of NR realimented vs. C ewes on d135. These data demonstrate placental adaptation to maternal NR through increasing nutrient transporter production and growth signaling activity.

Maternal obesity in ewes results in reduced fetal pancreatic ß-cell numbers in late gestation and decreased circulating insulin concentration at term.

About 30% of U.S. women of reproductive age are obese, a condition linked to offspring obesity and diabetes. This study utilized an ovine model of maternal obesity in which ewes are overfed to induce obesity at conception and throughout gestation. At mid-gestation, fetuses from these obese ewes are macrosomic, hyperglycemic, and hyperinsulinemic, and they exhibited markedly increased pancreatic weight and ß-cell numbers compared with fetuses of ewes fed to requirements. This study was conducted to establish fetal pancreatic phenotype and function in late gestation and at term in this ovine model. Multiparous ewes were fed a control (C, 100% National Research Council [NRC] recommendations) or obesogenic (OB, 150% NRC) diet from 60 days before conception to necropsy at day 135 of gestation or to lambing. No differences were observed in fetal size or weight on day 135 or in lamb birth weights between C and OB ewes. In contrast to our previously published results at mid-gestation, pancreatic weights (P < 0.01) and ß-cell numbers (P < 0.05) of OB fetuses were markedly lower than those from C fetuses, whereas the ß-cell apoptotic rate was increased (P < 0.05) in day 135 OB versus C fetuses. At birth, blood insulin concentration was lower (P < 0.05) and glucose level was higher (P < 0.05) in newborn lambs from OB versus C ewes. These data demonstrate differential impacts of maternal obesity on fetal pancreatic growth and ß-cell numbers during early and late gestation. During the first half of gestation there was a marked increase in pancreatic growth, ß-cell proliferation, and insulin secretion, followed by a reduction in pancreatic growth and ß-cell numbers in late gestation, resulting in reduced circulating insulin at term. It is speculated that the failure of the pancreas to return to a normal cellular composition and function postnatally could result in glucose/insulin dysregulation, leading to obesity, glucose intolerance, and diabetes in postnatal life.

Evidence for similar changes in offspring phenotype following either maternal undernutrition or overnutrition: potential impact on fetal epigenetic mechanisms.

The goal of this review is to shed light on the role of maternal malnutrition in inducing epigenetic changes in gene expression, leading to alterations in fetal growth and development, and to altered postnatal phenotype and the development of metabolic disease. We present evidence supporting the concept that both maternal undernutrition and overnutrition can induce the same cadre of fetal organ and tissue abnormalities and lead to the same postnatal metabolic changes in the resulting offspring. Furthermore, we present evidence that in both overnourished and undernourished ovine pregnancies, fetuses experience a period of nutrient restriction as a result of alterations in placental delivery of maternal nutrients into the fetal compartment. We argue that this bout of reduced fetal nutrition in undernourished and overnourished pregnancies leads to the development of a thrifty phenotype in which the fetus attempts to alter the function of its tissues and organs to maximise its chances of survival in a postnatal environment that is deficient in nutrients. Importantly, we present evidence to support the concept that these phenotypic changes in offspring quality resulting from maternal malnutrition are transmitted to subsequent generations, independent of their maternal nutritional inputs.

Hypothalamic expression of genes for appetite regulators and estrogen α, estrogen ß and leptin receptors in obese dams and their fetuses.

Under- and over-nutrition during gestation may influence fetal hypothalamic development resulting in individuals predisposed to adverse health effects. This study examined fetuses from obese and control ewes to determine whether dam obesity alters hypothalamic expression of fetal appetite regulatory genes. A second objective was to contrast the expression of appetite regulatory genes in ewes that become the most obese to those that remained in moderate body condition on the same energy-rich diet. Multiparous, western white-faced ewes were weighed and individually fed 100% (control) or 150% (obese) of National Research Council requirements from day 60 before mating until day 75 of gestation. At day 75 of gestation, fetuses were collected and weighed. Hypothalamic tissue from fetal lambs and dams was collected and frozen for mRNA extraction. Dam obesity (P ≥ 0.16), fetal sex (P ≥ 0.44) or their interaction (P ≥ 0.42) did not affect the relative expression of fetal hypothalamic regulators of appetite, including neuropeptide Y, agouti-related protein, pro-opiomelanocortin, cocaine- and amphetamine-regulated transcript and receptors for leptin. Maternal obesity at day 75 of gestation in ewes did not affect developmental mechanisms responsible for the expression of fetal appetite regulatory genes and would not be expected to predispose offspring to adult-onset obesity through disrupted appetite regulation at this developmental time point. In the ewe, appetite regulatory genes did not differ (P > 0.20) with ewe adiposity; however, expression of estrogen receptor α, but not ß (P = 0.37), in the medial basal hypothalamus was greater (P = 0.04) in obese than in control ewes.

Maternal obesity and increased nutrient intake before and during gestation in the ewe results in altered growth, adiposity, and glucose tolerance in adult offspring.

We evaluated the effects of preconception and gestational obesity in the ewe on offspring growth, metabolism, and glucose homeostasis. From 60 d before conception through parturition, multiparous ewes were fed 100% (control; n = 8) or 150% (obese, OB; n = 10) of NRC (1985) recommendations. Ewes on the OB diet increased BW by 30% from diet initiation to mating (P = 0.03) and by 52% by d 135 of gestation (P = 0.04), whereas control ewes increased BW by 7% (P = 0.65) from diet initiation to d 135 of gestation. Lambs were weaned at 120 d of age and were maintained as a group. At 19.5 ± 0.5 mo of age, offspring from control and OB ewes were individually penned and subjected to a 12-wk ad libitum feeding challenge. At the beginning and end of the feeding challenge, dual x-ray absorptiometry was used to determine percentage of body fat, and a frequently sampled intravenous glucose tolerance test (FSIGT) with minimal model analysis was used to assess insulin and glucose homeostasis. At the beginning of the feeding challenge, BW and percentage of body fat were similar for control and OB offspring, averaging 69.0 ± 1.5 kg and 5.3 ± 0.5%, respectively. At the initial FSIGT, glucose effectiveness and insulin sensitivity were reduced (P < 0.05) in offspring from OB compared with control ewes. During the feeding challenge, plasma concentrations of leptin were increased (P < 0.05) in offspring from OB compared with control ewes. Fasted plasma glucose before the feeding challenge tended to be greater (P = 0.06) in the OB offspring compared with the control offspring (83.3 ± 1.4 vs. 79.0 ± 1.6 mg/dL, respectively). At the end of the feeding challenge, fasted plasma glucose and insulin were increased (P < 0.05) in the OB offspring compared with the control offspring (84.0 ± 1.4 vs. 79.5 ± 1.5 mg/dL and 30.1 ± 2.1 vs. 23.4 ± 2.2 µIU/mL, respectively). During the feeding challenge, offspring from OB ewes consumed approximately 10% more feed (P < 0.05) and tended to have increased BW gain (approximately 14%; P = 0.08) compared with offspring from control ewes. At the final dual x-ray absorptiometry scan, percentage of body fat was greater (P < 0.05) for offspring from OB ewes than for offspring from control ewes (16.5 ± 1.2 vs. 10.8 ± 1.1%). At the final FSIGT, offspring from OB ewes had a decreased (P ≤ 0.05) acute insulin response to glucose, disposition index, and glucose effectiveness, and tended (P = 0.10) to have a decreased insulin sensitivity compared with offspring from control ewes. Maternal obesity induced before and during gestation leads to alterations in appetite, glucose and insulin regulation, and adiposity of mature offspring.

Effects of stage of gestation and nutrient restriction during early to mid-gestation on maternal and fetal visceral organ mass and indices of jejunal growth and vascularity in beef cows.

The objectives were to evaluate effects of maternal nutrient restriction and stage of gestation on maternal and fetal visceral organ mass and indices of jejunal growth and vascularity in beef cows. Thirty multiparous beef cows (BW = 571 +/- 63 kg; BCS = 5.4 +/- 0.7) carrying female fetuses (d 30 of gestation) were allocated to receive a diet of native grass hay (CON; 12.1% CP, 70.7% IVDMD, DM basis) to meet NRC recommendations for BW gain during early gestation or a nutrient-restricted diet of millet straw (NR; 9.9% CP, 54.5% IVDMD, DM basis) to provide 68.1% of NE(m) and 86.7% of MP estimated requirements. On d 125 of gestation, 10 CON and 10 NR cows were killed and necropsied. Five remaining CON cows received the CON diet, and 5 NR cows were realimented with a concentrate supplement (13.2% CP, 77.6% IVDMD, DM basis) and the CON hay to achieve a BCS similar to CON cows by d 220 of gestation. Remaining cows were necropsied on d 245 of gestation. Cow BW and eviscerated BW (EBW) were less (P < 0.01) for NR than CON at d 125 but did not differ (P > 0.63) at d 245. Cows fed the CON diet had greater (P < 0.09) total gastrointestinal (GI) tract, omasal, and pancreatic weights. Stomach complex, ruminal, and liver weights were greater for CON than NR cows (P < 0.09) on d 125. Total GI, stomach complex, and pancreatic weights increased (P < 0.001) with day of gestation. Restricted cows had decreased (P = 0.09) duodenal RNA:DNA compared with CON. Duodenal DNA was less (P = 0.01) and jejunal RNA:DNA (P = 0.09) was greater for cows at d 125 vs. 245. Cow jejunal capillary area density increased with day of gestation (P = 0.02). Fetal BW and EBW were unaffected by dietary treatment (P > or = 0.32). Total GI tract and all components increased in mass with day of gestation (P < 0.001). Fetuses from NR dams had greater (P = 0.003) reticular mass at d 245 than CON fetuses. Fetuses from NR cows had greater (P = 0.02) percent jejunal proliferation at d 125 and greater (P = 0.03) total intestinal vascularity (mL) at d 245. Fetal jejunal DNA decreased (P = 0.09), RNA:DNA increased (P = 0.05), and total jejunal proliferating cells increased (P < 0.001) with day of gestation. Jejunal capillary area density, number density, and surface density were greater (P < 0.008) during late gestation. Results indicate that maternal and fetal intestines undergo changes during gestation, which can be affected by nutrient restriction and may partially explain differences observed in fetal development and postnatal performance.

The effect of early to mid-gestational nutrient restriction on female offspring fertility and hypothalamic-pituitary-adrenal axis response to stress.

Primiparous ewes born as singletons to Rambouillet x Columbia crossbred ewes fed either 100% of NRC recommendations (control, Con; n = 7) or 50% of NRC (nutrient restricted, NR; n = 7) from d 28 through 78 postmating were utilized for this study. At 1 yr of age, a subset of ewes born to Con (n = 4) and NR (n = 4) mothers received jugular catheters and were subjected to a corticotrophin-releasing hormone (CRH)/arginine vasopressin (AVP) challenge, an ACTH challenge, and an isolation stress test, in which ACTH and cortisol responses were determined. A week after these challenges, estrus was monitored twice daily in all ewes from Con (n = 7) and NR mothers (n = 7). Once estrus was observed (d 0), daily blood samples were collected from ewes for progesterone through the subsequent estrus. Estrous detection and daily blood sampling were repeated during an estrous cycle in the next year, ewes were hand mated at the second estrus, and pregnancy was determined by delivery of a live lamb(s). Ewes from NR mothers tended (P = 0.10) to have a greater peak ACTH response after an intravenous CRH/AVP injection than ewes from Con mothers. The cortisol response of ewes to a CRH/AVP or ACTH challenge was not influenced by maternal nutrition. In contrast, ewes from Con mothers tended (P = 0.10) to release more ACTH in response to the isolation stress test and showed a greater (P = 0.04) cortisol release than ewes from NR mothers. Ewes from NR mothers exhibited decreased (P < 0.05) plasma progesterone in both yr 1 and 2 of the study compared with ewes from Con mothers. Furthermore, fewer (P < 0.0001) ewes from NR mothers produced a lamb (1 of 7) than ewes from Con mothers (7 of 7) during yr 2 of the study. These findings indicate that maternal undernutrition during early gestation may affect stress responses by the offspring, but has limited impact on hypothalamo-pituitary-adrenal sensitivity. Furthermore, offspring of NR ewes exhibited reduced progesterone secretion during the luteal phase of their estrous cycles and a markedly reduced fertility compared with offspring from Con ewes.

Maternal obesity up-regulates inflammatory signaling pathways and enhances cytokine expression in the mid-gestation sheep placenta.

Obesity in pregnant women is a growing public health concern. The placenta is a source of cytokines which can induce maternal gestational insulin resistance and alter nutrient transport to the fetus. Obesity induces placental inflammation at term, but the impact of obesity on placental inflammation earlier in pregnancy has not been defined. Using sheep as an experimental model, we hypothesized that maternal obesity (MO) would induce inflammation in the cotyledonary (COT) tissue of the placentome by mid-gestation. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obese (OB, 150% of NRC) group from 60 days before conception to 75 day of gestation (dG), when ewes were necropsied and placental COT tissue collected for analyses. Free fatty acids content, triglyceride and cholesterol content were higher (P < 0.05) in the fetal plasma of OB compared to C ewes on day 75. MO increased mRNA levels of toll-like receptor (TLR) 2 (P < 0.05) and TLR4 (P = 0.06), macrophage markers cluster of differentiation (CD)11b (P = 0.06), CD14 and CD68 (P < 0.05), and proinflammatory cytokines tumor necrosis factor (TNF)alpha (P < 0.01), interleukin (IL)-6 (P < 0.05), IL-8(P < 0.01) and IL-18 (P = 0.06), in COT tissue. Inflammatory c-Jun N-terminal kinase (JNK)/c-Jun and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) signaling pathways were up-regulated (P < 0.05) in COT of OB ewes. In conclusion, MO enhanced the placental inflammatory response in OB ewes at mid-gestation, possibly as a result of increased TLR4 and free fatty acids.

Fetal programming of skeletal muscle development in ruminant animals.

Enhancing skeletal muscle growth is crucial for animal agriculture because skeletal muscle provides meat for human consumption. An increasing body of evidence shows that the level of maternal nutrition alters fetal skeletal muscle development, with long-term effects on offspring growth and performance. Fetal skeletal muscle development mainly involves myogenesis (i.e., muscle cell development), but also involves adipogenesis (i.e., adipocyte development) and fibrogenesis (i.e., fibroblast development). These tissues in fetal muscle are mainly derived from mesenchymal stem cells (MSC). Shifting the commitment of MSC from myogenesis to adipogenesis increases intramuscular fat (i.e., marbling), improving the quality grade of meats. Strong experimental evidence indicates that Wingless and Int (Wnt)/beta-catenin signaling regulates MSC differentiation. Upregulation of Wnt/beta-catenin promotes myogenesis, and downregulation enhances adipogenesis. A lack of nutrients in early to midgestation reduces the formation of secondary muscle fibers in ruminant animals. Nutrient deficiency during mid- to late gestation decreases the number of intramuscular adipocytes and muscle fiber sizes. Knowledge of this regulatory mechanism will allow the development of strategies to enhance muscle growth and marbling in offspring, especially in the setting of nutrient deficiency.

Down-regulation of growth signaling pathways linked to a reduced cotyledonary vascularity in placentomes of over-nourished, obese pregnant ewes.

Both protein kinase B (Akt) and extracellular signal-regulated kinase 1/2 (ERK1/2) are down-stream components of the insulin/insulin like growth factor-1 (IGF-1) signaling pathway. AMP-activated protein kinase (AMPK) is known to sensitize cells to insulin/IGF-1 signaling. The objective of this study was to assess the activity of AMPK and its role in the observed down-regulation of insulin/IGF-1 signaling in cotyledonary (COT) arteries supplying the placental component of the ewe placentome. Nonpregnant ewes were randomly assigned to a control (C, 100% of NRC recommendations) or obesogenic (OB, 150% of NRC) diet from 60 days before conception until necropsy on day 75 of gestation (n=5/group) or until lambing (n=5/group). At necropsy on day 75 of gestation, the smallest terminal arteries that entered the COT tissues (0.5-1.0 mm in diameter) were collected for analyses. Fetal weights were approximately 20% greater (P<0.05) on OB than C ewes, but birth weights of lambs were similar across dietary groups. Fetal plasma concentrations of glucose, insulin and IGF-1 were higher (P<0.05) in the blood of fetuses from OB than C ewes. Total AMPK and phosphorylated AMPK at Thr 172 (the active form) were reduced (P<0.05) by 19.7+/-8.4% and 25.9+/-7.7%, respectively in the COT arterial tissues of OB ewes. Total acetyl-CoA carboxylase (ACC), a down-stream target of AMPK, and its phosphorylated form were also reduced (P<0.05) by 32.9+/-9.2% and 45.4+/-14.6%, respectively. The phosphorylation of IRS-1 at Ser 789, a site phosphorylated by AMPK, was 24.5+/-9.0% lower (P<0.05) in COT arteries of OB than C ewes. No alteration in total insulin receptor, total IGF-1 receptor or their phosphorylated forms was observed, down-stream insulin signaling was down-regulated in COT arteries of OB ewes, which may have resulted in the observed decrease in COT vascular development in OB ewes.

Effects of early gestational undernutrition on fetal growth, organ development, and placentomal composition in the bovine.

Fetal intrauterine growth restriction (IUGR) is known to negatively affect offspring health postnatally. This study evaluated the impacts of early gestational undernutrition followed by realimentation on bovine fetal and placental growth. Thirty multiparous beef cows bred to a single sire and gestating female fetuses were fed to meet NRC recommendations (control; n = 15) or fed below NRC recommendations (68.1% of NE(m) and 86.7% of MP recommendations; nutrient restricted, NR; n = 15) from d 30 to 125 of gestation. On d 125 of gestation, 10 control and 10 NR cows were necropsied. The remaining 5 NR cows were realimented to achieve similar BW and BCS with the remaining 5 control cows by d 190 of gestation; both groups were necropsied at d 245 of gestation. Fetal weight at d 125 of gestation was 948 +/- 14 g (n = 10) for control cows; however, fetal weights of NR cows fell into 2 distinct groups: NR non-IUGR cows had fetal weights similar to control cows (974 +/- 20 g, n = 6), whereas fetal weights of NR IUGR cows were reduced (773 +/- 23 g, n = 4; P < 0.01). Fetal brain weight as a percentage of fetal weight was increased (approximately 11%; P < 0.01) in the NR IUGR fetuses compared with fetuses from the other 2 groups, which were similar. Fetal heart weight as a percentage of fetal weight also tended to be increased (approximately 10%; P = 0.08) in NR IUGR fetuses compared with control fetuses. Nutrient-restricted IUGR cows exhibited reduced (P < 0.01) cotyledonary weights compared with NR non-IUGR and control cows, which were similar (192 +/- 27 vs. 309 +/- 22, and 337 +/- 17 g, respectively). Total placentome surface area also tended to be reduced (P = 0.07) in NR IUGR cows compared with NR non-IUGR and control cows, which again were similar (685.0 +/- 45.6 vs. 828.7 +/- 37.2 and 790.7 +/- 28.9 mm(2), respectively). On d 245 of gestation, fetal weights and caruncle weight were similar for NR and control cows; cotyledonary weights, however, were reduced in NR vs. control cows (1,430 +/- 133 vs. 2,137 +/- 133 g, P < 0.01). Decreased fetal growth in NR IUGR cows on d 125 of gestation was associated with decreased cotyledonary weights and reduced placentomal surface areas. The return of NR cows to a BW and BCS similar to that of control cows through realimentation beginning on d 126 resulted in similar fetal weights of NR and control cows by d 245 of gestation. Thus, a bout of fetal IUGR may go undetected if cows undernourished during early gestation receive feed supplementation in the second half of gestation to assure normal birth weight.