B. W. Hess

Maternal Undernutrition from Early- to Mid-Gestation Leads to Growth Retardation, Cardiac Ventricular Hypertrophy, and Increased Liver Weight in the Fetal Sheep

Abstract Early gestation is critical for placentomal growth, differentiation, and vascularization, as well as fetal organogenesis. The fetal origins of adult disease hypothesis proposes that alterations in fetal nutrition and endocrine status result in developmental adaptations that permanently change structure, physiology, and metabolism, thereby predisposing individuals to cardiovascular, metabolic, and endocrine disease in adult life. Multiparous ewes were fed to 50% (nutrient restricted) or 100% (control fed) of total digestible nutrients from Days 28 to 78 of gestation. All ewes were weighed weekly and diets adjusted for individual weight loss or gain. Ewes were killed on Day 78 of gestation and gravid uteri recovered. Fetal body and organ weights were determined, and numbers, morphologies, diameters, and weights of all placentomes were obtained. From Day 28 to Day 78, restricted ewes lost 7.4% of body weight, while control ewes gained 7.5%. Maternal and fetal blood glucose concentrations were reduced in restricted versus control pregnancies. Fetuses were markedly smaller in the restricted group than in the control group. Further, restricted fetuses exhibited greater right- and left-ventricular and liver weights per unit fetal weight than control fetuses. No treatment differences were observed in any gross placentomal measurement. However, caruncular vascularity was enhanced in conceptuses from nutrient-restricted ewes but only in twin pregnancies. While these alterations in fetal/placental development may be beneficial to early fetal survival in the face of a nutrient restriction, their effects later in gestation as well as in postnatal life need further investigation.

Maternal nutrient restriction affects properties of skeletal muscle in offspring.

Maternal nutrient restriction (NR) affects fetal development with long‐term consequences on postnatal health of offspring, including predisposition to obesity and diabetes. Most studies have been conducted in fetuses in late gestation, and little information is available on the persistent impact of NR from early to mid‐gestation on properties of offspring skeletal muscle, which was the aim of this study. Pregnant ewes were subjected to 50% NR from day 28–78 of gestation and allowed to deliver. The longissimus dorsi muscle was sampled from 8‐month‐old offspring. Maternal NR during early to mid‐gestation decreased the number of myofibres in the offspring and increased the ratio of myosin IIb to other isoforms by 17.6 ± 4.9% (P < 0.05) compared with offspring of ad libitum fed ewes. Activity of carnitine palmitoyltransferase‐1, a key enzyme controlling fatty acid oxidation, was reduced by 24.7 ± 4.5% (P < 0.05) in skeletal muscle of offspring of NR ewes and would contribute to increased fat accumulation observed in offspring of NR ewes. Intramuscular triglyceride content (IMTG) was increased in skeletal muscle of NR lambs, a finding which may be linked to predisposition to diabetes in offspring of NR mothers, since enhanced IMTG predisposes to insulin resistance in skeletal muscle. Proteomic analysis by two‐dimensional gel electrophoresis demonstrated downregulation of several catabolic enzymes in 8‐month‐old offspring of NR ewes. These data demonstrate that the early to mid‐gestation period is important for skeletal muscle development. Impaired muscle development during this stage of gestation affects the number and composition of fibres in offspring which may lead to long‐term physiological consequences, including predisposition to obesity and diabetes.

AMP-activated protein kinase signalling pathways are down regulated and skeletal muscle development impaired in fetuses of obese, over-nourished sheep

Maternal obesity and over‐nutrition give rise to both obstetric problems and neonatal morbidity. The objective of this study was to evaluate effects of maternal obesity and over‐nutrition on signalling of the AMP‐activated protein kinase (AMPK) pathway in fetal skeletal muscle in an obese pregnant sheep model. Non‐pregnant ewes were assigned to a control group (Con, fed 100% of NRC nutrient recommendations, n= 7) or obesogenic group (OB, fed 150% of National Research Council (NRC) recommendations, n= 7) diet from 60 days before to 75 days after conception (term 150 days) when fetal semitendinosus skeletal muscle (St) was sampled. OB mothers developed severe obesity accompanied by higher maternal and fetal plasma glucose and insulin levels. In fetal St, activity of phosphoinositide‐3 kinase (PI3K) associated with insulin receptor substrate‐1 (IRS‐1) was attenuated (P < 0.05), in agreement with the increased phophorylation of IRS‐1 at serine 1011. Phosphorylation of AMP‐activated protein kinase (AMPK) at Thr 172, acetyl‐CoA carboxylase at Ser 79, tuberous sclerosis 2 at Thr 1462 and eukaryotic translation initiation factor 4E‐binding protein 1 at Thr 37/46 were reduced in OB compared to Con fetal St. No difference in energy status (AMP/ATP ratio) was observed. The expression of protein phosphatase 2C was increased in OB compared to Con fetal St. Plasma tumour necrosis factor α (TNFα) was increased in OB fetuses indicating an increased inflammatory state. Expression of peroxisome proliferator‐activated receptor γ (PPARγ) was higher in OB St, indicating enhanced adipogenesis. The glutathione: glutathione disulphide ratio was also lower, showing increased oxidative stress in OB fetal St. In summary, we have demonstrated decreased signalling of the AMPK system in skeletal muscle of fetuses of OB mothers, which may play a role in altered muscle development and development of insulin resistance in the offspring.

Maternal obesity accelerates fetal pancreatic β-cell but not α-cell development in sheep: prenatal consequences

Maternal obesity affects offspring weight, body composition, and organ function, increasing diabetes and metabolic syndrome risk. We determined effects of maternal obesity and a high-energy diet on fetal pancreatic development. Sixty days prior to breeding, ewes were assigned to control [100% of National Research Council (NRC) recommendations] or obesogenic (OB; 150% NRC) diets. At 75 days gestation, OB ewes exhibited elevated insulin-to-glucose ratios at rest and during a glucose tolerance test, demonstrating insulin resistance compared with control ewes. In fetal studies, ewes ate their respective diets from 60 days before to 75 days after conception when animals were euthanized under general anesthesia. OB and control ewes increased in body weight by approximately 43% and approximately 6%, respectively, from diet initiation until necropsy. Although all organs were heavier in fetuses from OB ewes, only pancreatic weight increased as a percentage of fetal weight. Blood glucose, insulin, and cortisol were elevated in OB ewes and fetuses on day 75. Insulin-positive cells per unit pancreatic area were 50% greater in fetuses from OB ewes as a result of increased beta-cell mitoses rather than decreased programmed cell death. Lambs of OB ewes were born earlier but weighed the same as control lambs; however, their crown-to-rump length was reduced, and their fat mass was increased. We conclude that increased systemic insulin in fetuses from OB ewes results from increased glucose exposure and/or cortisol-induced accelerated fetal beta-cell maturation and may contribute to premature beta-cell function loss and predisposition to obesity and metabolic disease in offspring.

Comparison of acidic and alkaline catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis on conjugated linoleic acid.

Methanolic reagents containing acidic catalysts, HCl (0.5 M, 1 h, 80° C) or BF(3) (14%, 1 h, 80° C), or alkaline catalysts, KOH (0.2 M, 15-60 min, 50° C) or NaOCH(3) (0.5 M, 15-60 min, 50° C), were compared for use in preparation of fatty acid methyl esters for GC analysis of total lipids from freeze-dried semitendinosus muscle of lambs fed a 3.6% linoleate diet. Lipid preparations were in duplicate and included a total lipid extract, as well as direct transesterification and direct saponification of freeze-dried muscle. For the total lipid extracts, the weight% of 18:2 cis-9, trans-11 (CLA) with BF(3) (1.15) was 14.0% lower (P=0.001) than with either KOH (1.32) or NaOCH(3) (1.36); however, with HCl (1.25) CLA was intermediate (P=0.02). Concentrations of CLA (mg/g tissue) were similar (P ⩾0.44) within acidic or alkaline catalysts, but were 18.1% higher (P ⩽0.01) with KOH (2.56) and NaOCH(3) (2.52) than with HCl (2.01) or BF(3) (2.12). For direct transesterification, weight% of CLA was similar (P=0.55) with KOH (1.34) and NaOCH(3) (1.33), but each was 11.9% greater (P=0.003) than with HCl (1.18) and 19.1% greater (P=0.005) than with BF(3) (1.08). Concentrations of CLA after direct transesterification were greatest (P ⩽0.04) with KOH (3.31), followed by HCl (2.89, P=0.04), BF(3) (2.42, P ⩽0.004), and lowest (P ⩽0.002) with NaOCH(3) (2.21), indicating differences in efficiency of direct transesterification. Weight% of CLA in semitendinosus muscle, ranked highest to lowest, was lambs fed 3.6% linoleate (P ⩽0.003) > lambs fed 3.8% oleate (P ⩽0.01) > lambs fed a non-fat supplemented control diet (P ⩽0.01) when either BF(3) (saponified lipids) or KOH (direct transesterification) was used. Thus, dietary treatment effects on muscle CLA were not affected by catalyst. For the muscle of high-linoleate, high-oleate, and control lambs, CLA was 20.2, 13.9 and 0.0% higher, respectively, with KOH than BF(3), indicating that degradation of CLA by acidic catalysts decreased with lower starting amounts of CLA.

Maternal Nutrient Restriction Reduces Concentrations of Amino Acids and Polyamines in Ovine Maternal and Fetal Plasma and Fetal Fluids

Abstract Amino acids and polyamines are essential for placental and fetal growth, but little is known about their availability in the conceptus in response to maternal undernutrition. We hypothesized that maternal nutrient restriction reduces concentrations of amino acids and polyamines in the ovine conceptus. This hypothesis was tested in nutrient-restricted ewes between Days 28 and 78 (experiment 1) and between Days 28 and 135 (experiment 2) of gestation. In both experiments, ewes were assigned randomly on Day 28 of gestation to a control group fed 100% of National Research Council (NRC) nutrient requirements and to an nutrient-restricted group fed 50% of NRC requirements. Every 7 days beginning on Day 28 of gestation, ewes were weighed and rations adjusted for changes in body weight. On Day 78 of gestation, blood samples were obtained from the uterine artery and umbilical vein for analysis. In experiment 2, nutrient-restricted ewes on Day 78 of gestation either continued to be fed 50% of NRC requirements or were realimented to 100% of NRC requirements until Day 135. Fetal weight was reduced in nutrient-restricted ewes at both Day 78 (32%) and Day 135 (15%) compared with controls. Nutritional restriction markedly reduced (P < 0.05) concentrations of total α-amino acids (particularly serine, arginine-family amino acids, and branched-chain amino acids) and polyamines in maternal and fetal plasma and in fetal allantoic and amniotic fluids at both mid and late gestation. Realimentation of nutrient-restricted ewes increased (P < 0.05) concentrations of total α-amino acids and polyamines in all the measured compartments and prevented intrauterine growth retardation. These novel findings demonstrate that 50% global nutrient restriction decreases concentrations of amino acids and polyamines in the ovine conceptus that could adversely impact key fetal functions. The results have important implications for understanding the mechanisms responsible for both intrauterine growth retardation and developmental origins of adult disease.

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.

AMP-activated protein kinase and adipogenesis in sheep fetal skeletal muscle and 3T3-L1 cells.

Marbling, or i.m. fat, is an important factor determining beef quality. Both adipogenesis and hypertrophy of existing adipocytes contribute to enhanced marbling. We hypothesized that the fetal stage is important for the formation of i.m. adipocytes and that AMP-activated protein kinase (AMPK) has a key role in adipogenesis during this stage. The objective of this study was to assess the role of AMPK in adipogenesis in fetal sheep muscle and 3T3-L1 cells. Nonpregnant ewes were randomly assigned to a control (Con, 100% of NRC recommendations, n = 7) or overfed (OF, 150% of NRC, n = 7) diet from 60 d before to 75 d after conception, when the ewes were killed. The fetal LM was collected at necropsy for biochemical analyses. The activity of AMPK was less in the fetal muscle of OF sheep. The expression of peroxisome proliferator-activated receptor (PPAR)gamma, a marker of adipogenesis, was greater in OF fetal muscle compared with Con fetal muscle. To further show the role of AMPK in adipogenesis, we used 3T3-L1 cells. The 3T3-L1 cells were incubated in a standard adipogenic medium for 24 h and 10 d. Activation of AMPK by 5-aminoimidazole-4-car-boxamide-1-beta-d-ribonucleoside dramatically inhibited the expression of PPARgamma and reduced the presence of adipocytes after 10 d of differentiation. Inhibition of AMPK by compound C enhanced the expression of PPARgamma. In conclusion, these data show that AMPK activity is inversely related to adipogenesis in fetal sheep muscle and 3T3-L1 cells.

Maternal obesity induces sustained inflammation in both fetal and offspring large intestine of sheep

Background: Both maternal obesity and inflammatory bowel diseases (IBDs) are increasing. It was hypothesized that maternal obesity induces an inflammatory response in the fetal large intestine, predisposing offspring to IBDs. Methods: Nonpregnant ewes were assigned to a control (Con, 100% of National Research Council [NRC] recommendations) or obesogenic (OB, 150% of NRC) diet from 60 days before conception. The large intestine was sampled from fetuses at 135 days (term 150 days) after conception and from offspring lambs at 22.5 ± 0.5 months of age. Results: Maternal obesity enhanced mRNA expression tumor necrosis factor (TNF)&agr;, interleukin (IL)1&agr;, IL1&bgr;, IL6, IL8, and monocyte/macrophage chemotactic protein‐1 (MCP1), as well as macrophage markers, CD11b, CD14, and CD68 in fetal gut. mRNA expression of Toll‐like receptor (TLR) 2 and TLR4 was increased in OB versus Con fetuses; correspondingly, inflammatory NF‐&kgr;B and JNK signaling pathways were also upregulated. Both mRNA expression and protein content of transforming growth factor (TGF) &bgr; was increased. The IL‐17A mRNA expression and protein content was higher in OB compared to Con samples, which was associated with fibrosis in the large intestine of OB fetuses. Similar inflammatory responses and enhanced fibrosis were detected in OB compared to Con offspring. Conclusions: Maternal obesity induced inflammation and enhanced expression of proinflammatory cytokines in fetal and offspring large intestine, which correlated with increased TGF&bgr; and IL17 expression. These data show that maternal obesity may predispose offspring gut to IBDs. (Inflamm Bowel Dis 2010)

Nutrition during mid to late gestation affects growth, adipose tissue deposition, and tenderness in cross-bred beef steers

The objective of this study was to examine whether the plane of nutrition of cows at a critical time for fetal skeletal muscle and adipose tissue development would affect meat quality and carcass composition of offspring. To alter maternal nutrition, beef cows were placed on improved pasture (IP) or native range (NR) pasture from 120 to 150 through 180 to 210days of gestation. Esophageal extrusa samples collected from cows grazing IP varied from 11.1% crude protein of organic matter early in the test period to 6.0% crude protein of organic matter at the end of the grazing period; whereas, extrusa samples of cows grazing NR ranged from 6.5% crude protein of organic matter during early grazing to 5.4% crude protein of organic matter at the end of the grazing period. Steers were slaughtered and carcass characteristics were collected. Warner-Bratzler shear force was performed on longissumus steaks, western blotting was used to measure proteolysis, and myosin isoform typing was performed. Improved pasture steers had heavier live and hot carcass weights. Tenderness was greater in IP compared to NR steers. No difference in calpastatin content and troponin-T degradation was observed between treatments. The 12th rib fat thickness was greater for IP than for NR steers. Subcutaneous adipose tissue of IP steers tended to have a greater number of cells per field of view than NR steers. Data show improving nutritional status of cows during mid to late gestation affects tenderness, adipose tissue deposition and growth in steers.