Late-gestation Sheep Research Articles

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone.

Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein–umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.

Tissue-specific responses that constrain glucose oxidation and increase lactate production with the severity of hypoxemia in fetal sheep.

Fetal hypoxemia decreases insulin and increases cortisol and norepinephrine concentrations and may restrict growth by decreasing glucose utilization and altering substrate oxidation. Specifically, we hypothesized that hypoxemia would decrease fetal glucose oxidation and increase lactate and pyruvate production. We tested this by measuring whole body glucose oxidation and lactate production, and molecular pathways in liver, muscle, adipose, and pancreas tissues of fetuses exposed to maternal hypoxemia for 9 days (HOX) compared with control fetal sheep (CON) in late gestation. Fetuses with more severe hypoxemia had lower whole body glucose oxidation rates, and HOX fetuses had increased lactate production from glucose. In muscle and adipose tissue, expression of the glucose transporter GLUT4 was decreased. In muscle, pyruvate kinase (PKM) and lactate dehydrogenase B (LDHB) expression was decreased. In adipose tissue, LDHA and lactate transporter (MCT1) expression was increased. In liver, there was decreased gene expression of PKLR and MPC2 and phosphorylation of PDH, and increased LDHA gene and LDH protein abundance. LDH activity, however, was decreased only in HOX skeletal muscle. There were no differences in basal insulin signaling across tissues, nor differences in pancreatic tissue insulin content, β-cell area, or genes regulating β-cell function. Collectively, these results demonstrate coordinated metabolic responses across tissues in the hypoxemic fetus that limit glucose oxidation and increase lactate and pyruvate production. These responses may be mediated by hypoxemia-induced endocrine responses including increased norepinephrine and cortisol, which inhibit pancreatic insulin secretion resulting in lower insulin concentrations and decreased stimulation of glucose utilization.NEW & NOTEWORTHY Hypoxemia lowered fetal glucose oxidation rates, based on severity of hypoxemia, and increased lactate production. This was supported by tissue-specific metabolic responses that may result from increased norepinephrine and cortisol concentrations, which decrease pancreatic insulin secretion and insulin concentrations and decrease glucose utilization. This highlights the vulnerability of metabolic pathways in the fetus and demonstrates that constrained glucose oxidation may represent an early event in response to sustained hypoxemia and fetal growth restriction.

Lower citrate synthase activity, mitochondrial complex expression, and fewer oxidative myofibers characterize skeletal muscle from growth-restricted fetal sheep.

Skeletal muscle from the late gestation sheep fetus with intrauterine growth restriction (IUGR) has evidence of reduced oxidative metabolism. Using a sheep model of placental insufficiency and IUGR, we tested the hypothesis that by late gestation, IUGR fetal skeletal muscle has reduced capacity for oxidative phosphorylation because of intrinsic deficits in mitochondrial respiration. We measured mitochondrial respiration in permeabilized muscle fibers from biceps femoris (BF) and soleus (SOL) from control and IUGR fetal sheep. Using muscles including BF, SOL, tibialis anterior (TA), and flexor digitorum superficialis (FDS), we measured citrate synthase (CS) activity, mitochondrial complex subunit abundance, fiber type distribution, and gene expression of regulators of mitochondrial biosynthesis. Ex vivo mitochondrial respiration was similar in control and IUGR muscle. However, CS activity was lower in IUGR BF and TA, indicating lower mitochondrial content, and protein expression of individual mitochondrial complex subunits was lower in IUGR TA and BF in a muscle-specific pattern. IUGR TA, BF, and FDS also had lower expression of type I oxidative fibers. Fiber-type shifts that support glycolytic instead of oxidative metabolism may be advantageous for the IUGR fetus in a hypoxic and nutrient-deficient environment, whereas these adaptions may be maladaptive in postnatal life.

Moderate overfeeding of different sources of metabolizable energy and protein. II: Effects on inflammatory status of sheep in late pregnancy and growth trajectory of the offspring

Our objective was to evaluate the effects of moderate overfeeding of different sources of metabolizable energy (ME) and protein (MP) on inflammatory status of sheep in late gestation and short-term growth performance of the offspring. A total of 32 healthy pregnant Zell ewes (2.5 ± 1 years old, 42.19 ± 1.65 kg body weight [BW]) were selected 7 weeks before lambing. After receiving a ten-day adaptation diet, eight ewes were randomly allocated to each of the four treatments including: 1) The adaptation diet that fulfills ME and MP requirements, according to the National Research Council's (NRC, 2007) recommendations for late pregnant ewes (CON); 2) A diet with 10% higher levels of ME than CON, using corn grain for extra ME (HEC); 3) A diet with 10% higher level of ME compared to CON, using flaxseed for additional ME (HEF); and 4) A diet with a 10% higher level of MP than CON, using a rumen-protected lysin and methionine supplement (RP-Lys & Met) to supply extra MP (PRO). After lambing, ewes continued their dietary treatments until 30 days postpartum; however, the content of ingredients were proportionally increased to meet the recommended levels of energy and protein for lactating ewes (NRC, 2007). Blood samples from the ewes were taken on days −21, −14, and −7 before parturition. Colostrum yield and components were determined, and lamb's BW and milk intake (MI) were measured weekly until 28 days of age. Other growth performance indexes were computed accordingly. Blood samples from the offspring were taken on days 15 and 30 of life. The data was analyzed using SAS software (SAS Institute Inc., 9.4). Compared with the CON, HEF-fed ewes were the only group that had lower levels of oxidative stress and blood inflammatory markers at −7 d before parturition (P < 0.05). Concentrations of aminotransferases in CON-fed ewes were greater than other groups in late gestation (P ≤ 0.05). Offspring of HEF- and PRO-fed ewes had higher birth BW than lambs of the CON-fed group (3.71 and 4.11 vs. 3.01 ± 0.24, Kg for HEF, PRO, and CON, respectively). Except for week 2, lambs of CON-fed ewes had lower BW gain compared with the other offspring (P < 0.01). Lambs of PRO-fed ewes had greater levels of plasma immunoglobulin G (IgG, days 15 and 30 of life; P < 0.05) and MI (from week 2; P < 0.001) than other groups. In conclusion, regardless of the source, moderate overfeeding of ME/MP was more beneficial for the immune status of late pregnant dams and the offspring's performance than non-overfed ones.

MATERNAL AND FETAL BUPRENORPHINE PHARMACOKINETICS IN PREGNANT SHEEP DURING TRANSDERMAL PATCH DOSING: Buprenorphine pharmacokinetics in pregnant sheep

BackgroundBuprenorphine is used in the opioid maintenance treatment for opioid dependent patients, including pregnant women. Despite the wide use, limited data exists on buprenorphine pharmacokinetics and fetal exposure during pregnancy. The aim of our study was to determine the buprenorphine pharmacokinetics during transdermal patch dosing to pregnant sheep and, to determine the extent of transplacental transfer of buprenorphine to the fetus. MethodsPregnant sheep in late gestation (n=50) received 20, 25 or 40 µg/h of buprenorphine as a 7-day extended-release transdermal patch. Plasma samples were collected from the ewe and the fetus on days 1 – 6, and buprenorphine and norbuprenorphine concentrations were determined. During the exposure period the sheep had a surgical procedure on the second day, a recovery phase, and an experimental procedure on the sixth day. In the experiment, hypoxia was induced under anesthesia for 18 sheep to investigate if decreased fetal pH would cause ion-trapping of buprenorphine in the fetus. The fetal/maternal plasma concentration ratio was determined on the second and on the sixth exposure day at baseline and during hypoxia. Maternal pharmacokinetics were modelled with a population pharmacokinetic method using the data from this study and our previous intravenous administration study. ResultsThe transdermal patch provided an extended release of buprenorphine throughout the exposure period, but the release rate declined approximately 20 h after patch placement. The median fetal/maternal plasma concentration ratio was 13 – 27 % throughout the exposure period at baseline. A ratio over 100 % was observed for four sheep on the sixth exposure day (102 – 269 %). A minor increase was seen in the median fetal/maternal-ratios during maternal hypoxia. Norbuprenorphine was undetected in all plasma samples. ConclusionsThe low transplacental passage of less than one fourth of the ewe's exposure supports buprenorphine as an alternative to methadone in opioid maintenance therapy during pregnancy.

Brief hypoxia in late gestation sheep causes prolonged disruption of fetal electrographic, breathing behaviours and can result in early labour.

Brief episodes of severe fetal hypoxia can arise in late gestation as a result of interruption of normal umbilical blood flow Systemic parameters and blood chemistry indicate complete recovery within 1-2 hours, although the long-term effects on fetal brain functions are unknown Fetal sheep were subjected to umbilical cord occlusion (UCO) for 10 min at 131 days of gestation, and then monitored intensively until onset of labour or delivery (<145 days of gestation) Normal patterns of fetal behaviour, including breathing movements, episodes of high and low voltage electorcortical activity, eye movements and postural (neck) muscle activity, were disrupted for 3-10days after the UCO Preterm labour and delivery occurred in a significant number of the pregnancies after UCO compared to the control (sham-UCO) cohort. Complications arising from antepartum events such as impaired umbilical blood flow can cause significant fetal hypoxia. These complications can be unpredictable, as well as difficult to detect, and thus we lack a detailed understanding of the (patho)physiological changes that occur between the antenatal in utero event and birth. In the present study, we assessed the consequences of brief (∼10min) umbilical cord occlusion (UCO) in fetal sheep at ∼0.88 gestation on fetal plasma cortisol concentrations and fetal behaviour [electrocortical (EcoG), electo-oculargram (EOG), nuchal muscle electromyography (EMG) and breathing activities] in the days following UCO. UCO caused a rapid onset of fetal hypoxaemia, hypercapnia, and acidosis; however, by 6h, all blood parameters and cardiovascular status were normalized and not different from the control (Sham-UCO) cohort. Subsequently, the incidence of fetal breathing movements decreased compared to the control group, and abnormal behavioural patterns developed over the days following UCO and leading up to the onset of labour, which included increased high voltage and sub-low voltage ECoG and EOG activities, as well as decreased nuchal EMG activity. Fetuses subjected to UCO went into labour 7.9±3.6 days post-UCO (139.5±3.2 days of gestation) compared to the control group fetuses at 13.6±3.3 days post-sham UCO (144±2.2 days of gestation; P < 0.05), despite comparable increases in fetal plasma cortisol and a similar body weight at birth. Thus, a single transient episode of complete UCO late in gestation in fetal sheep can result in prolonged effects on fetal brain activity and premature labour, suggesting persisting effects on fetal cerebral metabolism.

Pharmacokinetics of buprenorphine in pregnant sheep after intravenous injection.

Buprenorphine is a semi‐synthetic opioid, widely used in the maintenance treatment for opioid‐dependent pregnant women. Limited data exist on the pharmacokinetics of buprenorphine in pregnancy. We conducted a pharmacokinetic study to determine the pharmacokinetics of intravenous buprenorphine in pregnant sheep. Fourteen pregnant sheep in late gestation received 10 µg/kg of buprenorphine as an intravenous bolus injection. Plasma samples were collected up to 48 h after administration. Buprenorphine and its metabolite, norbuprenorphine, were quantified from plasma using a LC/MS/MS method, with lower limits of quantification of 0.01 µg/L and 0.04 µg/L for buprenorphine and norbuprenorphine, respectively. The pharmacokinetic parameters were calculated using noncompartmental analysis. The pharmacokinetic parameters, median (minimum−maximum), were Cmax 4.31 µg/L (1.93–15.5), AUC inf 2.89 h*µg/L (1.72–40.2), CL 3.39 L/h/kg (0.25–6.02), terminal t½ 1.75 h (1.07–31.0), Vss 8.04 L/kg (1.05–49.3). Norbuprenorphine was undetected in all plasma samples. The median clearance in pregnant sheep was higher than previously reported for nonpregnant sheep and human (male) subjects. Our sensitive analytical method was able to detect long terminal half‐lives for six subjects, and a wide between‐subject variability in the study population.Significance statement: Buprenorphine is widely used for the treatment of opioid use disorder in pregnancy. However, limited data exist on the pharmacokinetics of buprenorphine during pregnancy. As this type of study cannot be done in humans due to ethical reasons, we conducted a study in pregnant sheep. This study provides pharmacokinetic data on buprenorphine in pregnant sheep and helps us to understand the pharmacokinetics of the drug in humans.

A Chronic Fetal Leucine Infusion Potentiates Fetal Insulin Secretion and Increases Pancreatic Islet Size, Vascularity, and β Cells in Late-Gestation Sheep

Background:Infusion of a complete amino acid mixture into normal late-gestation fetal sheep potentiates glucose-stimulated insulin secretion (GSIS). Leucine acutely stimulates insulin secretion in late-gestation fetal sheep and isolated fetal sheep islets in vitro. Objectives:We hypothesized that a 9-d leucine infusion would potentiate GSIS in fetal sheep. Methods:Columbia-Rambouillet fetal sheep at 126 days of gestation received a 9-d leucine infusion to achieve a 50%–100% increase in leucine concentrations or a control infusion. At the end of the infusion we measured GSIS, pancreatic morphology, and expression of pancreatic mRNAs. Pancreatic islet endothelial cells (ECs) were isolated from fetal sheep and incubated with supplemental leucine or vascular endothelial growth factor A (VEGFA) followed by collection of mRNA. Data measured at multiple time points were compared with a repeated-measures 2-factor ANOVA. Data measured at 1 time point were compared using Student's t test or the Mann–Whitney test. Results:Glucose-stimulated insulin concentrations were 80% higher in leucine-infused (LEU) fetuses than in controls (P < 0.05). In the pancreas, LEU fetuses had a higher proportion of islets >5000 μm2 than controls (75% more islets >5000 μm2; P < 0.05) and a larger proportion of the pancreas that stained for β cells (12% greater; P < 0.05). Pancreatic and pancreatic islet vascularity were both 25% greater in LEU fetuses (P < 0.05). PancreaticVEGFAand hepatocyte growth factor (HGF) mRNA expressions were 38% and 200% greater in LEU fetuses than in controls (P < 0.05), respectively. In isolated islet ECs,HGFmRNA was 20% and 50% higher after incubation in ce:supplemental leucine (P < 0.05) or VEGFA (P < 0.01), respectively. Conclusions:A 9-d leucine infusion potentiates fetal GSIS, demonstrating that glucose and leucine act synergistically to stimulate insulin secretion in fetal sheep. A greater proportion of the pancreas being comprised of β cells and higher pancreatic vascularity contributed to the higher GSIS.

Maternal and fetal cardiovascular and metabolic effects of intra-operative uterine handling under general anesthesia during pregnancy in sheep

A cohort study of 6,500,000 human pregnancies showed an increased risk of adverse fetal outcomes following abdominal but not non-abdominal surgery under general anesthesia. This may be the consequence of uterine handling during abdominal surgery. However, there are no data on any effects on the cardiometabolic physiology of the fetus or mother in response to uterine manipulation in otherwise healthy pregnancy. Consequently, 9 sheep in late gestation were anesthetized with isofluorane and maternal and fetal catheters and flow probes were implanted to determine cardiovascular and metabolic changes during uterine handling. Uterine handling led to an acute increase in uterine artery vascular resistance, fetal peripheral vasoconstriction, a reduction in oxygen delivery to the femoral circulation, worsening fetal acidosis. There was no evidence of systemic fetal hypoxia, or changes in fetal heart rate, carotid blood flow or carotid oxygen delivery. Therefore, the data support that uterine handling during abdominal surgery under general anesthesia can impact adversely on fetal cardiometabolic health. This may provide a potential explanation linking adverse fetal outcomes in abdominal compared with non-abdominal surgery during pregnancy. The data have important implications for human fetal surgery where the uterus is handled, as operative procedures during late gestation under general maternal anesthesia become more prevalent.

PSIV-8 Effects of restricted maternal nutrition and re-alimentation on fetal muscle development from mid to late gestation in sheep

Abstract Inadequate maternal nutrition during gestation negatively impacts offspring muscle development, which may be attenuated by re-alimentation. We hypothesized that restricted maternal nutrition would alter fetal muscle fiber cross-sectional area (CSA) and the percent of Pax7(+) progenitor cells. To test this, 48 primiparous ewes, pregnant with singletons, were fed 100% of NRC requirements (CON) between days 25 and 50 of gestation. At day 50 of gestation, seven ewes were euthanized for fetal sample collection. The remaining ewes were fed either CON or 60% NRC requirements (RES). At day 90 of gestation, seven ewes per diet were euthanized for fetal sample collection. The remaining ewes were maintained on their current diet (CON-CON, RES-RES) or switched to the alternate diet (CON-RES, RES-CON). At day 130 of gestation, all ewes (n = 6 or 7 ewes/diet) were euthanized, and fetal samples were collected. On days 90 and 130, fetal longissimus (LM) and triceps brachii (TB) were collected and immunostained for enumeration of Pax7(+) cells and muscle fiber CSA. The percent of Pax7(+) cells was determined by dividing the number of Pax7(+) cells by the total number of cells. At day 90 of gestation, the percent of Pax7(+) cells was less (P &amp;lt; 0.01) in the TB of RES compared with CON. At day 130, fiber CSA was greater (P &amp;lt; 0.04) in RES-CON compared with CON-CON, CON-RES, and RES-CON in the TB, and was greater (P &amp;lt; 0.02) in RES-CON compared with RES-RES and CON-RES in the LM. An effect of maternal nutrition was not observed for the percent of Pax7(+) cells in the LM, or for CSA at day 90 in either muscle. Thus, restricted maternal nutrition during early to mid-gestation alters the percent of Pax7(+) cells at mid-gestation in a muscle specific manner. Re-alimentation during late gestation increased fiber CSA, demonstrating that re-alimentation alters fetal muscle development.

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.

Altered autonomic control of heart rate variability in the chronically hypoxic fetus.

Key points Fetal heart rate variability (FHRV) has long been recognised as a powerful predictor of fetal wellbeing, and a decrease in FHRV is associated with fetal compromise. However, the mechanisms by which FHRV is reduced in the chronically hypoxic fetus have yet to be established.The sympathetic and parasympathetic influences on heart rate mature at different rates throughout fetal life, and can be assessed by time domain and power spectral analysis of FHRV.In this study of chronically instrumented fetal sheep in late gestation, we analysed FHRV daily over a 16 day period towards term, and compared changes between fetuses of control and chronically hypoxic pregnancy.We show that FHRV in sheep is reduced by chronic hypoxia, predominantly due to dysregulation of the sympathetic control of the fetal heart rate. This presents a potential mechanism by which a reduction in indices of FHRV predicts fetuses at increased risk of neonatal morbidity and mortality in humans.Reduction in overall FHRV may therefore provide a biomarker that autonomic dysregulation of fetal heart rate control has taken place in a fetus where uteroplacental dysfunction is suspected. Although fetal heart rate variability (FHRV) has long been recognised as a powerful predictor of fetal wellbeing, the mechanisms by which it is reduced in the chronically hypoxic fetus have yet to be established. In particular, the physiological mechanism underlying the reduction of short term variation (STV) in fetal compromise remains unclear. In this study, we present a longitudinal study of the development of autonomic control of FHRV, assessed by indirect indices, time domain and power spectral analysis, in normoxic and chronically hypoxic, chronically catheterised, singleton fetal sheep over the last third of gestation. We used isobaric chambers able to maintain pregnant sheep for prolonged periods in hypoxic conditions (stable fetal femoral arterial PO2 10–12 mmHg), and a customised wireless data acquisition system to record beat‐to‐beat variation in the fetal heart rate. We determined in vivo longitudinal changes in overall FHRV and the sympathetic and parasympathetic contribution to FHRV in hypoxic (n = 6) and normoxic (n = 6) ovine fetuses with advancing gestational age. Normoxic fetuses show gestational age‐related increases in overall indices of FHRV, and in the sympathetic nervous system contribution to FHRV (P < 0.001). Conversely, gestational age‐related increases in overall FHRV were impaired by exposure to chronic hypoxia, and there was evidence of suppression of the sympathetic nervous system control of FHRV after 72 h of exposure to hypoxia (P < 0.001). This demonstrates that exposure to late gestation isolated chronic fetal hypoxia has the potential to alter the development of the autonomic nervous system control of FHRV in sheep. This presents a potential mechanism by which a reduction in indices of FHRV in human fetuses affected by uteroplacental dysfunction can predict fetuses at increased risk.

177 Effects of Metabolizable Protein Level on Mammary Gland Vascularity, Proliferation, and Alveoli Size during Late Gestation in Sheep.

177 Effects of Metabolizable Protein Level on Mammary Gland Vascularity, Proliferation, and Alveoli Size during Late Gestation in Sheep.

Competitive inhibition of amino acid transport in human preovulatory ovarian follicles

ABSTRACTTo date we have yet to examine whether amino acid (AA) transport in human ovarian follicles is affected by competitive inhibition. In contrast, transplacental transfer of AAs in late-gestation sheep is characterized by reciprocal competition. This phenomenon has been described by algebraic equations of umbilical uptake of AAs based on maternal arterial concentrations. In the present translational study at a university teaching hospital, we verified whether these equations apply to the transport of AAs from blood to follicular fluid (FF) in human preovulatory follicles. For this purpose we used our data on AA concentrations in blood and FF measured earlier by high-performance liquid chromatography in specimens from 14 patients undergoing oocyte retrieval for in vitro fertilization after controlled ovarian stimulation. The main outcome measure was statistical significance of Spearman correlation coefficients for measured versus calculated concentrations of 8 AAs: isoleucine, leucine, valine, phenylalanine, methionine, threonine, lysine, and arginine. Equations for umbilical uptake provided a highly accurate description of blood-to-FF transport for 7 AAs with the exception of lysine: R ≥ 0.899 (p < 0.0001) for the branched-chain AAs, R = 0.829 (p = 0.0003) for threonine, R = 0.754 (p = 0.0019) for arginine, and R = 0.631 (p = 0.0156) for phenylalanine and methionine. We conclude that these equations indicate competitive inhibition between the AAs studied. Our study strongly suggests that many AA transport systems operating in the placenta should also be active in the cells of the preovulatory follicle. Future studies on AA fluxes in human ovarian follicles must consider possible competitive inhibition.Abbreviations: AA: amino acid; FF: follicular fluid; HPLC: high-performance liquid chromatography

Coxiella burnetii seropositivity and associated risk factors in sheep in Ontario, Canada

Coxiella burnetii is a zoonotic bacterium that can cause abortion in sheep in late gestation, as well as the delivery of stillborn, and non-viable lambs (Rodolakis, 2006). A cross-sectional study was conducted in Ontario, Canada, to investigate C. burnetii exposure in sheep. Between August 2010 and January 2012, sera from 2363 reproductively active ewes from 72 farms were tested for C. burnetii specific antibodies using the CHEKIT Q fever ELISA Test kit (IDEXX Laboratories). Overall, exposure was common; sheep-level seroprevalence was 14.7% (347/2363, 95% CI: 13.3–16.2), and was higher in dairy sheep (24.3%, 181/744) than meat sheep (10.2%, 166/1619) (p<0.0001). At the farm-level, 48.6% (35/72, 95% CI: 37.2–60.1) of farms had at least one seropositive sheep. A mixed multivariable logistic model that controlled for farm-level clustering, identified risk factors associated (p<0.05) with sheep seropositivity. Increasing female flock size (logarithmic scale) was associated with increased odds of seropositivity. By way of illustration, increasing the female flock size from 100 to 200 increased the odds of seropositivity by 2.26 times (95% CI: 1.5–3.5). Sheep that lambed in an airspace separate from the flock had 11.3 times (95% CI: 2.9–43.6) the odds of seropositivity relative to other sheep. The practice of loaning sheep that returned to the farm increased odds of seropositivity by 8.1 times (95% CI: 1.8–33.6). Lambing pen hygiene practices also influenced odds of seropositivity. Relative to sheep from farms where all lambing pen hygiene measures were practiced after lambing (i.e., adding bedding, removing birth materials and disinfection), sheep from farms that only added bedding, or those that just added bedding and removed birthing materials had 5.9 times (95% CI: 1.1–32.1) and 9.0 times (95% CI: 2.2–36.9) the odds of seropositivity, respectively. These results can be used to inform prevention and control strategies with the aim of reducing C. burnetii exposure in sheep.

Regulation of microRNA during cardiomyocyte maturation in sheep.

BackgroundThere is a limited capacity to repair damage in the mammalian heart after birth, which is primarily due to the inability of cardiomyocytes to proliferate after birth. This is in contrast to zebrafish and salamander, in which cardiomyocytes retain the ability to proliferate throughout life and can regenerate their heart after significant damage. Recent studies in zebrafish and rodents implicate microRNA (miRNA) in the regulation of genes responsible for cardiac cell cycle progression and regeneration, in particular, miR-133a, the miR-15 family, miR-199a and miR-590. However, the significance of these miRNA and miRNA in general in the regulation of cardiomyocyte proliferation in large mammals, including humans, where the timing of heart development relative to birth is very different than in rodents, is unclear. To determine the involvement of miRNA in the down-regulation of cardiomyocyte proliferation occurring before birth in large mammals, we investigated miRNA and target gene expression in sheep hearts before and after birth. The experimental approach included targeted transcriptional profiling of miRNA and target mRNA previously identified in rodent studies as well as genome-wide miRNA profiling using microarrays.ResultsThe cardiac expression of miR-133a increased and its target gene IGF1R decreased with increasing age, reaching their respective maximum and minimum abundance when the majority of ovine cardiomyocytes were quiescent. The expression of the miR-15 family members was variable with age, however, four of their target genes decreased with age. These latter profiles are inconsistent with the direct involvement of this family of miRNA in cardiomyocyte quiescence in late gestation sheep. The expression patterns of ‘pro-proliferative’ miR-199a and miR-590 were also inconsistent with their involvement in cardiomyocyte quiescence. Consequently, miRNA microarray analysis was undertaken, which identified six discrete clusters of miRNA with characteristic developmental profiles. The functions of predicted target genes for the miRNA in four of the six clusters were enriched for aspects of cell division and regulation of cell proliferation suggesting a potential role of these miRNA in regulating cardiomyocyte proliferation.ConclusionThe results of this study show that the expression of miR-133a and one of its target genes is consistent with it being involved in the suppression of cardiomyocyte proliferation, which occurs across the last third of gestation in sheep. The expression patterns of the miR-15 family, miR-199a and miR-590 were inconsistent with direct involvement in the regulation cardiomyocyte proliferation in sheep, despite studies in rodents demonstrating that their manipulation can influence the degree of cardiomyocyte proliferation. miRNA microarray analysis suggests a coordinated and potentially more complex role of multiple miRNA in the regulation of cardiomyocyte quiescence and highlights significant differences between species that may reflect their substantial differences in the timing of this developmental process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1693-z) contains supplementary material, which is available to authorized users.

Long-Term Gestational Hypoxia Modulates Expression of Key Genes Governing Mitochondrial Function in the Perirenal Adipose of the Late Gestation Sheep Fetus.

We previously reported that long-term hypoxia (LTH) increases expression of brown adipose tissue (BAT) genes in the perirenal adipose in the ovine fetus. The mechanisms with which hypoxia mediates the enhanced BAT phenotype are unresolved. This study was designed to examine the effects of LTH on (1) the expression of endothelial cell nitric oxide synthase (eNOS) and (2) indicators of mitochondrial biogenesis (transcription factors mitochondrial transcription factor A (mtTFA), nuclear respiratory factor (NRF) 1, and NRF-2; cytochrome c oxidase (COX) I, II, and IV and mitochondrial DNA content). Pregnant ewes were maintained at high altitude (3820 m) from ∼40 to 137 to 140 days of gestation and perirenal adipose was collected from normoxic control and LTH fetuses. There was no effect of LTH on fetal body weight or perirenal adipose mass. Long-term hypoxia increased (P < .05) perirenal eNOS and phospho-eNOS, messenger RNA (mRNA) for NRF1, NRF-2, mtTFA as well as COX-I, COX-II, and COX-IV mRNA. In contrast, mRNA for 2 markers for cellular proliferation (Ki67 and proliferating cell nuclear antigen [PCNA]) was lower in perirenal adipose from LTH fetuses compared to controls (P < .05), while mitochondrial to nuclear DNA ratio did not differ between groups. In conclusion, nitric oxide may function as a mechanism via which LTH enhances the BAT phenotype in fetal sheep prior to birth. Although there is an apparent increase in genes supporting mitochondrial function and adaptive thermogenesis in response to LTH, there does not appear to be an increased mitochondrial biogenesis per se. Such adaptive changes may provide a mechanism for the prominence of the BAT phenotype observed in the late gestation LTH fetus.

Injury of the developing cerebellum: a brief review of the effects of endotoxin and asphyxial challenges in the late gestation sheep fetus.

The vulnerability of the fetal and newborn brain to events in utero or at birth that cause damage arising from perturbations of cerebral blood flow and metabolism, such as the accumulation of free radicals and excitatory transmitters to neurotoxic levels, has received considerable attention over the last few decades. Attention has usually been on the damage to cerebral structures, particularly, periventricular white matter. The rapid growth of the cerebellum in the latter half of fetal life in species with long gestations, such as the human and sheep, suggests that this may be a particularly important time for the development of cerebellar structure and function. In this short review, we summarize data from recent studies with fetal sheep showing that the developing cerebellum is particularly sensitive to infectious processes, chronic hypoxia and asphyxia. The data demonstrates that the cerebellum should be further studied in insults of this nature as it responds differently to the remainder of the brain. Damage to this region of the brain has implications not only for the development of motor control and posture, but also for higher cognitive processes and the subsequent development of complex behaviours, such as learning, memory and attention.

Intrafetal glucose infusion alters glucocorticoid signaling and reduces surfactant protein mRNA expression in the lung of the late-gestation sheep fetus.

Increased circulating fetal glucose and insulin concentrations are potential inhibitors of fetal lung maturation and may contribute to the pathogenesis of respiratory distress syndrome (RDS) in infants of diabetic mothers. In this study, we examined the effect of intrafetal glucose infusion on mRNA expression of glucose transporters, insulin-like growth factor signaling, glucocorticoid regulatory genes, and surfactant proteins in the lung of the late-gestation sheep fetus. The numerical density of the cells responsible for producing surfactant was determined using immunohistochemistry. Glucose infusion for 10 days did not affect mRNA expression of glucose transporters or IGFs but did decrease IGF-1R expression. There was reduced mRNA expression of the glucocorticoid-converting enzyme HSD11B-1 and the glucocorticoid receptor, potentially reducing glucocorticoid responsiveness in the fetal lung. Furthermore, surfactant protein (SFTP) mRNA expression was reduced in the lung following glucose infusion, while the number of SFTP-B-positive cells remained unchanged. These findings suggest the presence of a glucocorticoid-mediated mechanism regulating delayed maturation of the surfactant system in the sheep fetus following glucose infusion and provide evidence for the link between abnormal glycemic control during pregnancy and the increased risk of RDS in infants of uncontrolled diabetic mothers.

Exposure to rosiglitazone, a PPAR-γ agonist, in late gestation reduces the abundance of factors regulating cardiac metabolism and cardiomyocyte size in the sheep fetus

It is unknown whether cardiomyocyte hypertrophy and the transition to fatty acid oxidation as the main source of energy after birth is dependent on the maturation of the cardiomyocytes' metabolic system, or on the limitation of substrate availability before birth. This study aimed to investigate whether intrafetal administration of a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, rosiglitazone, during late gestation can stimulate the expression of factors regulating cardiac growth and metabolism in preparation for birth, and the consequences of cardiac contractility in the fetal sheep at ∼140 days gestation. The mRNA expression and protein abundance of key factors regulating growth and metabolism were quantified using quantitative RT-PCR and Western blot analysis, respectively. Cardiac contractility was determined by measuring the Ca(2+) sensitivity and maximum Ca(2+)-activated force of skinned cardiomyocyte bundles. Rosiglitazone-treated fetuses had a lower cardiac abundance of insulin-signaling molecules, including insulin receptor-β, insulin receptor substrate-1 (IRS-1), phospho-IRS-1 (Tyr-895), phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, PI3K catalytic subunit p110α, phospho-3-phosphoinositide-dependent protein kinase 1 (Ser-241), protein kinase B (Akt-1), phospho-Akt (Ser-273), PKCζ, phospho-PKCζ(Thr-410), Akt substrate 160 kDa (AS160), phospho-AS160 (Thr-642), and glucose transporter type-4. Additionally, cardiac abundance of regulators of fatty acid β-oxidation, including adiponectin receptor 1, AMPKα, phospho-AMPKα (Thr-172), phospho-acetyl CoA carboxylase (Ser-79), carnitine palmitoyltransferase-1, and PGC-1α was lower in the rosiglitazone-treated group. Rosiglitazone administration also resulted in a decrease in cardiomyocyte size. Rosiglitazone administration in the late-gestation sheep fetus resulted in a decreased abundance of factors regulating cardiac glucose uptake, fatty acid β-oxidation, and cardiomyocyte size. These findings suggest that activation of PPAR-γ using rosiglitazone does not promote the maturation of cardiomyocytes; rather, it may decrease cardiac metabolism and compromise cardiac health later in life.