Adolescent Sheep Research Articles

Abstract 17016: Mitral Valve Prolapse Induces Localized Left Ventricular Fibrosis in a Novel Large Animal Model

Background: Mitral valve prolapse (MVP) has recently been associated with localized left ventricular (LV) fibrosis linked to malignant arrhythmia and sudden cardiac death. However, fibrosis mechanisms remain unknown, with no large animal model to distinguish mechanical and genetic influences. Goals: We tested the hypothesis that surgically induced MVP leads to localized myocardial fibrosis in LV regions mechanically linked to the mitral valve, aiming to establish a reproducible large animal model of MVP as a platform for mechanistic studies. Methods: In 6 adolescent sheep, MVP was created surgically by replacing primary chordae tendineae to P2 with longer artificial chords. Intraoperative echocardiography and LV pressure-volume loops were obtained. After 7 months recovery, cardiac MRI with gadolinium and T1-mapping were done before end-of-study surgery with repeat echo and pressure-volume loops. Three sham surgery sheep were similarly studied. Myocardial samples from valve-linked papillary muscle and inferobasal LV and control septal regions were analyzed by histology. Bulk RNA was sequenced on samples from 3 MVP and 3 sham sheep. Results: All 6 MVP sheep showed pronounced localized LV fibrosis in valve-linked regions but not control regions on MRI, with replacement fibrosis by Masson’s trichrome stain. Sham sheep had no fibrosis, with similar LV function and contractility at end-of-study. RNA sequencing revealed gene expression differences between the two LV regions in MVP sheep and the valve-linked regions of MVP vs sham sheep. Conclusions: In a novel surgically created large animal model of MVP, localized fibrosis is induced in formerly healthy LV myocardium. This suggests a mechanical mechanism is sufficient to create the localized LV fibrosis associated with arrhythmia in MVP patients. This model provides a platform to study fibrosis mechanisms and investigate therapeutic approaches to avoid catastrophic events in MVP patients.

Timing and duration of nutrient restriction and its impacts on placental development and umbilical blood flow in adolescent sheep

We hypothesized that nutrient restriction from day 50–90 of gestation decreases umbilical blood flow and that umbilical blood flow would recover to control values upon realimentation during late gestation (d 90 to 130) or remain reduced in ewes that continued to be nutrient restricted. On d 50 of gestation, young nulliparous whiteface ewes (6–8 mo; n = 41) carrying singletons were randomly assigned to two dietary treatments: 100% of NRC recommendations (CON) or 60% of CON (RES). On d 90 of gestation, ewes either remained on CON or RES until d 130, or CON ewes were RES from d 90 to 130, or RES ewes were realimented to CON from d 90 to 130. This resulted in 4 treatment groups on day 130: CON–CON, CON-RES, RES-RES, RES-CON. Umbilical blood flow and fetal and placental measurements were obtained via ultrasonography every 10 days from day 50–110. Non-survival surgeries were performed on days 50, 90, and 130 (n = 6–7 ewes/group) where uterine artery and umbilical blood flows were measured during surgery via ultrasonography. Conceptus weights were recorded and placentomes collected to determine binucleate cell numbers. The study was conducted as a completely randomized design arrangement with repeated measures. Data were analyzed using the MIXED procedure of SAS. There was a nutritional treatment by day interaction (P < 0.01) with CON ewes having greater umbilical blood flow compared with RES by d 90. Fetal biparietal distance, abdominal width, and kidney area increased (P < 0.05) in CON-RES with all these measurements increasing during late gestation. We partially accept our hypothesis as nutrient restriction during mid gestation decreased umbilical blood flow. However, blood flow did not return to control levels upon realimentation. By d 130, fetal and placental weights were similar between RES-RES and CON–CON. Binucleate cell numbers in the fetal trophoblast were not influenced by nutritional treatments. Our findings suggest that refeeding previously nutrient restricted pregnant adolescent ewes to control levels does not reestablish umbilical blood flow. Adequate placental development during mid gestation could protect the fetus from a decreased umbilical blood flow later in gestation when nutrients were limited by 40%.

Timing of maternal nutrient restriction during mid- to late-gestation influences net umbilical uptake of glucose and amino acids in adolescent sheep.

Previous research demonstrated that maternal nutrient restriction during mid- to late-gestation influenced net umbilical uptakes of glucose and amino acids in sheep. However, it is unclear how the timing and duration of nutrient restriction during mid- to late-gestation influences net uterine, uteroplacental, and fetal flux of glucose and amino acids. On day 50 of gestation, 41 adolescent ewe lambs carrying singletons were randomly assigned to one of six dietary treatments: 1) 100% of nutrient requirements from days 50 to 90 of gestation (CON; n = 7); 2) 60% of nutrient requirements (RES; n = 7) from days 50 to 90 of gestation; 3) 100% of nutrient requirements from days 50 to 130 of gestation (CON-CON; n = 6); 4) 100% of nutrient requirements from days 50 to 90 of gestation and 60% of nutrient requirements from days 90 to 130 of gestation (CON-RES; n = 7); 5) 60% of nutrient requirements from days 50 to 90 of gestation and 100% of nutrient requirements from days 90 to 130 of gestation (RES-CON; n = 7); or 6) 60% of nutrient requirements from days 50 to 130 of gestation (RES-RES; n = 7). On day 90 (n = 14) and day 130 (n = 27), intraoperative procedures were performed to evaluate uteroplacental blood flows, collect blood samples, and then ewes were euthanized. Net uterine, uteroplacental, and umbilical fluxes of glucose and amino acids were calculated by multiplying blood flow by the arterial-venous concentration difference. Data from days 90 and 130 were analyzed separately using ANOVA in SAS. Maternal nutrient restriction during mid-gestation increased (P = 0.04) net umbilical glucose uptake but, maternal nutrient restriction during late-gestation decreased (P = 0.02) net umbilical glucose uptake. Net umbilical essential amino acid uptake decreased (P = 0.03) with nutrient restriction during mid-gestation; however, net umbilical uptakes of Phe (P = 0.02), Thr (P = 0.05), Met (P = 0.09), and His (P = 0.08) increased or tended to increase after nutrient restriction during late-gestation. These data demonstrate that net umbilical glucose and amino acid uptakes were influenced by the timing of nutrient restriction during mid- to late-gestation. Elevated net umbilical glucose uptake after mid-gestational nutrient restriction was sustained throughout late-gestation, independent of late-gestational feeding level. Long-term adaptations in umbilical glucose uptake may have implications for prenatal and postnatal growth and development of the offspring.

Perinatal complications and maximising lamb survival in an adolescent paradigm characterised by premature delivery and low birthweight.

The competition for nutrients in overnourished and still-growing adolescent sheep negatively impacts gestation length, colostrum supply and lamb birthweight, all of which may affect neonatal morbidity and survival to weaning. Herein perinatal complications and the requirement for supplementary feeding were analysed in relation to gestational-intake, and the degree of premature delivery and prenatal growth-restriction exhibited. Pregnancies were established by embryo transfer and the mean/standard deviation (SD) gestation length and birthweight of the optimally-fed control group (n = 100) was used to define early delivery and reduced birthweight categories (1.5 and 3.0 SDs below the control mean for each aspect). Control lambs were largely delivered at term (94%), and had a normal birthweight (92%), while very preterm (≤139days, 18.5%) and preterm delivery (140-142days, 54.8%), extremely low birthweight (ELBW; females ≤2838g and males ≤3216g, 21.1%) and low birthweight (LBW; females 2839 to ≤4001g and males 3217 to ≤4372g, 32.2%), were common in the overnourished group (n = 270, P<0.001). Accordingly, overnourished dams were more likely to lamb without assistance while the incidence of major dystocia was greater in controls. Initial lamb vigour at birth was independent of gestational-intake, delivery or birthweight category but both ELBW and very premature lambs required more assistance with feeding in the first 24h postnatal, primarily reflecting low colostrum availability. Indeed, relative to normal, ELBW lambs had a greater risk of experiencing mismothering, and enhanced likelihood of requiring supplementary feeding throughout the neonatal period (P<0.001). ELBW lambs also had a greater possibility of respiratory issues at birth (P<0.01) and renal complications (P<0.001), while very preterm delivery was associated with an increased risk of gastrointestinal tract problems (P<0.01). In spite of these complications, all-cause mortality was low (5.4%) suggesting that our proactive neonatal care regime can overcome many of the issues associated with extreme prematurity and low birthweight.

Comprehensive Characterisation of Left Ventricular Myocardial Function by Cardiac Magnetic Resonance Feature Tracking in an Adolescent Sheep Model of Myocardial Infarction

Myocardial deformation quantification by cardiac magnetic resonance (CMR) feature tracking (FT) may prove useful in presentations of regional dysfunction following ischemic damage such as a myocardial infarction (MI). Our group previously documented regional left ventricular (LV) dysfunction in the adolescent sheep model of MI, albeit without quantitative measures [1]. Comprehensive measures by CMR-FT are important for tracking progression of injury and can be used to test efficacy of MI interventions.

Early pregnancy weight gain and fat accrual predict pregnancy outcome in growing adolescent sheep.

The competition for nutrients when pregnancy coincides with continuing growth in biologically immature adolescent girls increases their risk of preterm delivery and low birthweight and is partly replicated in the overnourished adolescent sheep paradigm. Although overfeeding to promote rapid maternal growth robustly leads to a reduction in average birthweight relative to slow-growing control-fed adolescents of equivalent age, the extent of prenatal compromise is variable. This retrospective analysis of a large cohort of identically managed pregnancies determined whether maternal anthropometry predicts the severity of fetal growth-restriction (FGR) in growing adolescents. Singleton pregnancies were established by embryo transfer in adolescents subsequently control-fed (n = 96) or overnourished. The latter pregnancies were classified as non-FGR (n = 116) or FGR (n = 96) if lamb birthweight was above or below the optimally fed control mean minus 2SD. A similar approach categorised placental growth-restriction (PlGR) and preterm delivery. Gestation length, placental mass and lamb birthweight were FGR < non-FGR < control (post hoc P < 0.01). Relative to the non-FGR group, overnourished dams with FGR were marginally leaner and lighter at conception (P = 0.023/P = 0.014) and had greater gestational weight gain (GWG) during the first-third of pregnancy (P < 0.001). GWG during this early period was also higher in PlGR compared with non-PlGR, and in very preterm vs term deliveries (P < 0.01). Likewise maternal leptin concentrations (fat accrual biomarker) were FGR > non-FGR by day 60, and changes in leptin throughout pregnancy predicted attenuated fetal cotyledon mass and birthweight (P = 0.01 to <0.001). The anthropometric antecedents of FGR in still-growing adolescent sheep originate in early pregnancy coincident with early placental development.

Identification of Novel miRNAs Involved in Cardiac Repair Following Infarction in Fetal and Adolescent Sheep Hearts.

AimsAnimal models have been used to show that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific times in development. For example, specific miRNAs are critical for regulating the response to myocardial infarction (MI) and improving the response to injury. Manipulating these miRNAs in small animal models provides beneficial effects post-MI; however it is not known if these miRNAs are regulated similarly in large mammals. Studying a large animal where the timing of heart development in relation to birth is similar to humans may provide insights to better understand the capacity to repair a developing mammalian heart and its application to the adult heart.MethodsWe used a sheep model of MI that included permanent ligation of the left anterior descending (LAD) coronary artery. Surgery was performed on fetuses (at 105 days gestation when all cardiomyocytes are mononucleated and proliferative) and adolescent sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). A microarray was utilized to determine the expression of known miRNAs within the damaged and undamaged tissue regions in fetal and adolescent hearts after MI.Results73 miRNAs were up-regulated and 58 miRNAs were down-regulated significantly within the fetal infarct compared to remote cardiac samples. From adolescent hearts 69 non-redundant miRNAs were up-regulated and 63 miRNAs were down-regulated significantly in the infarct area compared to remote samples. Opposite differential expression profiles of 10 miRNAs within tissue regions (Infarct area, Border zone and Remote area of the left ventricle) occurred between the fetuses and adolescent sheep. These included miR-558 and miR-1538, which when suppressed using LNA anti-miRNAs in cell culture, increased cardiomyoblast proliferation.ConclusionThere were significant differences in miRNA responses in fetal and adolescent sheep hearts following a MI, suggesting that the modulation of novel miRNA expression may have therapeutic potential, by promoting proliferation or repair in a damaged heart.

Ovine prenatal growth-restriction and sex influence fetal adipose tissue phenotype and impact postnatal lipid metabolism and adiposity in vivo from birth until adulthood.

Adipose tissue development begins in utero and is a key target of developmental programming. Here the influence of nutritionally-mediated prenatal growth-restriction on perirenal adipose tissue (PAT) gene expression and adipocyte phenotype in late fetal life was investigated in both sexes in an ovine model. Likewise circulating leptin concentrations and non-esterified fatty acid (NEFA) and glycerol responses to glucose challenge were determined in relation to offspring adiposity at key stages from birth to mid-adult life. In both studies' singleton-bearing adolescent sheep were fed control or high nutrient intakes to induce normal or growth-restricted pregnancies, respectively. Fetal growth-restriction at day 130 of gestation (32% lighter) was characterised by greater body-weight-specific PAT mass and higher PAT expression of peroxisome proliferator-activated receptor gamma (PPARɤ), glycerol-3-phosphate dehydrogenase, hormone sensitive lipase (HSL), insulin-like growth factor 1 receptor, and uncoupling protein 1. Independent of prenatal growth, females had a greater body-weight-specific PAT mass, more multilocular adipocytes, higher leptin and lower insulin-like growth factor 1 mRNA than males. Growth-restricted offspring of both sexes (42% lighter at birth) were characterised by higher plasma NEFA concentrations across the life-course (post-fasting and after glucose challenge at 7, 32, 60, 85 and 106 weeks of age) consistent with reduced adipose tissue insulin sensitivity. Circulating plasma leptin correlated with body fat percentage (females>males) and restricted compared with normal females had more body fat and increased abundance of PPARɤ, HSL, leptin and adiponectin mRNA in PAT at necropsy (109 weeks). Therefore, prenatal nutrient supply and sex both influence adipose tissue development with consequences for lipid metabolism and body composition persisting throughout the life-course.

Differential impacts of late gestational over-and undernutrition on adipose tissue traits and associated visceral obesity risk upon exposure to a postnatal high-fat diet in adolescent sheep.

We hypothesized that late gestation malnutrition differentially affects expandability of adipose tissues to predispose for early postnatal visceral adiposity. Twin‐lambs born to dams fed HIGH (150%/110% of required energy/protein, respectively), NORM (100% of requirements) or LOW (50% of NORM) diets during the last trimester were used. Postnatally, lambs were raised on moderate (CONV) or high‐carbohydrate‐high‐fat (HCHF) diets. Adipose tissues were sampled at autopsy at 6 months of age (~puberty) to characterize cellularity, adipocyte cross‐sectional area and gene expression patterns. HIGH and LOW compared to NORM lambs had reduced intrinsic (under CONV diet) cellularity in subcutaneous and mesenteric (particularly LOW), and reduced obesity‐induced (under HCHF diet) hyperplasia in subcutaneous, mesenteric and perirenal (particularly HIGH) adipose tissues. This corresponded with more pronounced HCHF diet‐induced hypertrophy in mesenteric (particularly LOW), perirenal (particularly HIGH) and subcutaneous (particularly HIGH) adipose tissues, and tissue‐specific reductions in mRNA expressions for lipid metabolism, angiogenesis and adipose development. Gene expression for inflammation and lipid metabolism markers were increased and decreased, respectively, in HCHF lambs (HCHF lambs became obese) in all tissues. Both prenatal over‐ and undernutrition predisposed for abdominal adiposity and extreme perirenal hypertrophy due to reduced intrinsic (observed under CONV diet) cellularity and impaired ability of subcutaneous, mesenteric and perirenal adipose tissues to expand by hyperplasia rather than hypertrophy on an obesogenic (HCHF) diet.

Differential gene responses 3 days following infarction in the fetal and adolescent sheep heart.

There are critical molecular mechanisms that can be activated to induce myocardial repair, and in humans this is most efficient during fetal development. The timing of heart development in relation to birth and the size/electrophysiology of the heart are similar in humans and sheep, providing a model to investigate the repair capacity of the mammalian heart and how this can be applied to adult heart repair. Myocardial infarction was induced by ligation of the left anterior descending coronary artery in fetal (105 days gestation when cardiomyocytes are proliferative) and adolescent sheep (6 mo of age when all cardiomyocytes have switched to an adult phenotype). An ovine gene microarray was used to compare gene expression in sham and infarcted (remote, border and infarct areas) cardiac tissue from fetal and adolescent hearts. The gene response to myocardial infarction was less pronounced in fetal compared with adolescent sheep hearts and there were unique gene responses at each age. There were also region-specific changes in gene expression between each age, in the infarct tissue, tissue bordering the infarct, and tissue remote from the infarction. In total, there were 880 genes that responded to MI uniquely in the adolescent samples compared with 170 genes in the fetal response, as well as 742 overlap genes that showed concordant direction of change responses to infarction at both ages. In response to myocardial infarction, there were specific changes in genes within pathways of mitochondrial oxidation, muscle contraction, and hematopoietic cell lineages, suggesting that the control of energy utilization and immune function are critical for effective heart repair. The more restricted gene response in the fetus may be an important factor in its enhanced capacity for cardiac repair.

Nutrition and fetal growth: paradoxical effects in the overnourished adolescent sheep

Inappropriate maternal nutrient intake at key developmental timepoints during ovine pregnancy has a profound influence on the outcome of pregnancy and aspects of postnatal productivity. However, the responses to alterations in maternal nutrition in adult sheep are often highly variable and inconsistent between studies. The growing adolescent sheep provides a new, robust and nutritionally sensitive paradigm with which to study the causes, consequences and reversibility of prenatal growth restriction. Overnourishing the adolescent dam to promote rapid maternal growth throughout pregnancy results in a major restriction in placental mass, and leads to a significant decrease in birthweight relative to moderately fed, normally growing adolescents of equivalent gynaecological age. Maternal insulin and IGF-I concentrations are increased from an early stage of gestation in overnourished adolescent dams and these hormones ensure that the anabolic drive required to promote maternal tissue synthesis is initiated at a time when the nutrient requirements of the gravid uterus are low. The major restriction in fetal growth in rapidly growing dams occurs irrespective of high concentrations of essential nutrients in the maternal circulation and suggests that the small size or altered metabolic and transport capacity of the placenta is the primary constraint to fetal growth. The decrease in placental weight in the overnourished animals reflects a significant reduction in both fetal cotyledon number and mean cotyledon weight. The role of nutritionally mediated alterations in progesterone and the components of the IGF system in this early pregnancy placental phenomenon are being investigated. Nutritional switch-over studies have demonstrated that reducing maternal nutrient intake at the end of the first third of pregnancy can stimulate placental growth and enhance pregnancy outcome, but increasing nutrient intake at this time has a deleterious effect on placental development and fetal growth.

Differential Response to Injury in Fetal and Adolescent Sheep Hearts in the Immediate Post-myocardial Infarction Period

Aim: Characterizing the response to myocardial infarction (MI) in the regenerative sheep fetus heart compared to the post-natal non-regenerative adolescent heart may reveal key morphological and molecular differences that equate to the response to MI in humans. We hypothesized that the immediate response to injury in (a) infarct compared with sham, and (b) infarct, border, and remote tissue, in the fetal sheep heart would be fundamentally different to the adolescent, allowing for repair after damage.Methods: We used a sheep model of MI induced by ligating the left anterior descending coronary artery. Surgery was performed on fetuses (105 days) and adolescent sheep (6 months). Sheep were randomly separated into MI (n = 5) or Sham (n = 5) surgery groups at both ages. We used magnetic resonance imaging (MRI), histological/immunohistochemical staining, and qRT-PCR to assess the morphological and molecular differences between the different age groups in response to infarction.Results: Magnetic resonance imaging showed no difference in fetuses for key functional parameters; however there was a significant decrease in left ventricular ejection fraction and cardiac output in the adolescent sheep heart at 3 days post-infarction. There was no significant difference in functional parameters between MRI sessions at Day 0 and Day 3 after surgery. Expression of genes involved in glucose transport and fatty acid metabolism, inflammatory cytokines as well as growth factors and cell cycle regulators remained largely unchanged in the infarcted compared to sham ventricular tissue in the fetus, but were significantly dysregulated in the adolescent sheep. Different cardiac tissue region-specific gene expression profiles were observed between the fetal and adolescent sheep.Conclusion: Fetuses demonstrated a resistance to cardiac damage not observed in the adolescent animals. The manipulation of specific gene expression profiles to a fetal-like state may provide a therapeutic strategy to treat patients following an infarction.

Impact of donor and recipient adiposity on placental and fetal growth in adolescent sheep.

The influence of maternal obesity during oocyte development and its putative interaction with nutrient reserves at conception on pregnancy outcome were examined in an adolescent sheep model. Donor ewes were nutritionally managed to achieve contrasting adiposity (control (CD)/obese (ObD)) for 6 weeks prior to superovulation and inseminated by a non-obese sire. Morulae from 6 CD and 7 ObD were transferred in singleton into adolescent recipients of identical age but differing adiposity, classified as relatively fat or thin respectively. Thereafter, all were overnourished to promote rapid growth/adiposity (2 × 2 design, 13/14 pregnancies/group). A fifth recipient group of intermediate adiposity received embryos from another 5 CD, was offered a moderate intake to maintain adiposity throughout gestation and acted as controls for normal pregnancy outcome (optimally treated control (OTC), 19 pregnancies). Donor obesity did not influence ovulation, fertilisation or recovery rates or impact embryo morphology. Gestation length and colostrum yield were unaffected by donor or recipient adiposity and were reduced relative to OTC. Total fetal cotyledon and lamb birth weights were independent of initial donor adiposity but reduced in relatively thin vs relatively fat recipients and lower than those in the OTC group. In spite of high placental efficiency, the incidence of fetal growth restriction was greatest in the thin recipients. Thus, maternal adiposity at conception, but not pre-conception maternal obesity, modestly influences the feto-placental growth trajectory, whereas comparison with the OTC indicates that high gestational intakes to promote rapid maternal growth remain the dominant negative influence on pregnancy outcome in young adolescents. These findings inform dietary advice for pregnant adolescent girls.

Late gestation under- and overnutrition have differential impacts when combined with a post-natal obesogenic diet on glucose-lactate-insulin adaptations during metabolic challenges in adolescent sheep.

To determine whether late gestation under- and overnutrition programme metabolic plasticity in a similar way, and whether metabolic responses to an obesogenic diet in early post-natal life depend on the foetal nutrition history. In a 3 × 2 factorial design, twin-pregnant ewes were for the last 6 weeks of gestation (term = 147 days) assigned to HIGH (N = 13; 150 and 110% of energy and protein requirements, respectively), NORM (N = 9; 100% of requirements) or LOW (N = 14; 50% of requirements) diets. The twin offspring were raised on high-carbohydrate-high-fat (HCHF; N = 35) or conventional (CONV; N = 35) diets from 3 days to 6 months of age (around puberty). Then intravenous glucose (GTT; overnight fasted), insulin (ITT; fed) and propionate (gluconeogenetic precursor; PTT; both fed and fasted) tolerance tests were conducted to evaluate (hepatic) metabolic plasticity. Prenatal malnutrition differentially impacted adaptations of particularly plasma lactate followed by glucose, cholesterol and insulin. This was most clearly expressed during PTT in fasted lambs and much less during ITT and GTT. In fasted lambs, propionate induced more dramatic increases in lactate than glucose, and HIGH lambs became more hyperglycaemic, hyperlactataemic and secreted less insulin compared to the hypercholesterolaemic LOW lambs. Propionate-induced insulin secretion was virtually abolished in fasted HCHF lambs, but upregulated in fasted compared to fed CONV lambs. HCHF lambs had the greatest glucose-induced insulin secretory responses. Prenatal malnutrition differentially programmed glucose-lactate metabolic pathways and cholesterol homeostasis. Prenatal overnutrition predisposed for hyperglycaemia and hyperlactataemia, whereas undernutrition predisposed for hypercholesterolaemia upon exposure to an obesogenic diet. Prenatal overnutrition (not undernutrition) interfered with pancreatic insulin secretion by non-glucose-dependent mechanisms.

Post-operative echocardiographic evaluation of bioprosthetic mitral valve implantation in sheep.

The ovine model is generally considered to be the best for testing bioprosthetic heart valve durability. Although echocardiography is the method of choice for the interim evaluation of the valve, literature on sheep echocardiography is scarce. Within the context of a study on treatment of pericardial heart valve prostheses, 19 adolescent sheep underwent transthoracic echocardiography six days after mitral implantation of bioprosthetic valves. Echocardiographic examination was performed under mild anesthesia and animals were put in a right lateral decubitus position. Four images were obtained: right parasternal long axis four and five chamber views, right parasternal long axis view with left ventricular outflow, and right parasternal short axis view through the mitral valve. We measured aortic annulus and velocity time integral over the aortic valve to determine stroke volume, cardiac output and cardiac index. The mitral valve was evaluated through color Doppler imaging for valvular and paravalvular leakages. Pulsed wave spectral Doppler was used for the measurement of velocities, pressures and velocity time integrals. For the evaluation of valve stenosis deceleration time and pressure half-time were determined. Effective orifice area of the mitral valve was derived. And, although not measured, other structures could clearly be visualized: right and left ventricle and atrium, wall thicknesses, tricuspid valve. This study shows that echocardiography in sheep is feasible, and that right parasternal images, obtained in animals in a right lateral decubitus position, are well qualified for the interim evaluation of bioprosthetic valves implanted in the mitral position. Besides the implanted valve, other cardiac structures like atria and ventricles can be visualized and evaluated.

Placental restriction of fetal growth reduces cutaneous responses to antigen after sensitization in sheep

Prenatal and early childhood exposures are implicated as causes of allergy, but the effects of intrauterine growth restriction on immune function and allergy are poorly defined. We therefore evaluated effects of experimental restriction of fetal growth on immune function and allergic sensitization in adolescent sheep. Immune function (circulating total red and white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils, and the antibody response to Clostridial vaccination) and responses to house dust mite (HDM) allergen and ovalbumin (OVA) antigen sensitization (specific total Ig, IgG1, and IgE antibodies, and cutaneous hypersensitivity) were investigated in adolescent sheep from placentally restricted (PR, n = 23) and control (n = 40) pregnancies. Increases in circulating HDM-specific IgE (P = 0.007) and OVA-specific IgE (P = 0.038) were greater in PR than control progeny. PR did not alter total Ig, IgG1, or IgM responses to either antigen. PR increased OVA-specific but not HDM-specific IgA responses in females only (P = 0.023). Multiple birth increased Ig responses to OVA in a sex-specific manner. PR decreased the proportion of positive cutaneous hypersensitivity responders to OVA at 24 h (P = 0.030) but had no effect on cutaneous responses to HDM. Acute wheal responses to intradermal histamine correlated positively with birth weight in singletons (P = 0.023). Intrauterine growth restriction may suppress inflammatory responses in skin downstream of IgE induction, without impairment in antibody responses to a nonpolysaccharide vaccine. Discord between cutaneous and IgE responses following sensitization suggests new mechanisms for prenatal allergy programming.

1.3 DNA Methylation of Somatotropic Axis Genes in Relation to Postnatal Growth and Metabolism Following Ad.VEGF Treatment of Growth-Restricted Sheep Fetuses

IntroductionWe previously showed that adenovirus (Ad) mediated overexpression of vascular endothelial growth factor (VEGF) in the uteroplacental circulation increases fetal growth velocity in the overnourished adolescent sheep paradigm of fetal...

Fetoplacental biometry and umbilical artery Doppler velocimetry in the overnourished adolescent model of fetal growth restriction

The purpose of this study was to evaluate ultrasonographically fetal growth trajectories, placental biometry, and umbilical artery (UA) Doppler indices in growth-restricted pregnancies of overnourished adolescent ewes and normally developing pregnancies of control-fed ewes. Singleton pregnancies were established using embryo transfer in 42 adolescent ewes that were overnourished (n = 27) or control-fed (n = 15) and were scanned at weekly intervals from 83-126 days' gestation and necropsied at 131 days' gestation (term = 145 days). Ultrasonographic placental measurements were reduced and UA Doppler indices were increased from 83 days' gestation; measurements of fetal abdominal circumference and femur length, renal volume and tibia length, and biparietal diameter were reduced from 98, 105, and 112 days' gestation, respectively, in overnourished vs control-intake pregnancies. Overnourishment of adolescent sheep dams produced late-onset asymmetric fetal growth restriction that was commensurate with brain sparing. Ultrasonographic placental biometry was already reduced and UA Doppler indices increased by mid gestation in overnourished pregnancies, preceding reduced fetal growth velocity and indicating an early nutritionally mediated insult on placental development.

Testing the plasticity of insulin secretion and β‐cell function in vivo: responses to chronic hyperglycaemia in the sheep

Plasticity of insulin secretion is essential to maintain the action of insulin during insulin resistance and to prevent diabetes. Investigation of the plasticity of insulin secretion and its regulation is challenging, and the objective of this study was to develop a novel large-animal-based model. The effect of chronic moderate hyperglycaemia on the plasticity of insulin secretion, β-cell mass and function was determined in sheep. Adolescent sheep (120 days old) were infused with 25% glucose for 16 days to increase blood glucose by 50% (n = 10), and control animals (n = 9) were infused with saline. Glucose- and arginine-stimulated insulin secretion, insulin sensitivity and glucose effectiveness were measured in vivo before and during treatment (days 10-14), and β-cell mass was measured at the end of treatment. Hyperglycaemia increased blood glucose (+53%) and plasma insulin (+403%; each P < 0.003) and did not alter whole-body insulin sensitivity. Hyperglycaemia increased glucose-stimulated insulin secretion (particularly second phase; five-fold) and arginine-stimulated insulin secretion (particularly first phase; four-fold). Hyperglycaemia reduced β-cell mass (∼50%, P = 0.038) and increased glucose- and arginine-stimulated insulin secretion relative to β-cell mass five-fold (P = 0.060) and 20-fold (P = 0.007), respectively. Chronic hyperglycaemia therefore induces marked adaptation and upregulation of glucose- and arginine-stimulated insulin secretion by enhancing β-cell function rather than increasing β-cell mass in the sheep, consistent with long-term adaptations seen in humans. This marked plasticity of insulin secretion in response to moderate hyperglycaemia provides a novel model for the investigation of factors affecting its capacity and underlying determinants.

Cloning and ontogenetic expression of the uncoupling protein 1 gene UCP1 in sheep

The uncoupling protein 1 (UCP1) is an indicator of brown adipocytes and is involved in the control of body temperature and regulation of energy balance. It abundantly expresses in newborns and has important functions in adults. However, little information was known on UCP1 gene expression in young and adolescent sheep. In this study, we cloned and identified the full-length DNA and cDNA sequences of the ovine UCP1 gene, which were 6659bp and 1621bp, respectively, and predicted the location of the gene on chromosome 17. Forty-eight animals with an equal number of males and females each for both Guangling Large Tail sheep (GLT) and Small Tail sheep Han (STH) sheep were used to study the ontogenetic expression of UCP1 mRNA in eight adipose tissues by quantitative real-time polymerase chain reaction (PCR). The results showed that the mRNA was expressed in all tissues studied and at all stages from 2 to 12months of age. Nevertheless, the mRNA in perirenal fat was expressed significantly higher than that in other tissues and lower in superficial fat than in deep deposits. The highest expression was observed in animals at 2 months of age and then decreased gradually with age. Global expression in GLT was significantly higher than that in STH. Interactions between tissue and breed and age also influenced the mRNA expression significantly. In addition, the mRNA expression was associated with the single nucleotide polymorphism (SNP) haplotypes detected in the cDNA of the gene.