Proteomic profile of pre-implantational ovine embryos produced in vivo.

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Optimizing injection time of GFP plasmid into sheep zygote.

Microinjection of exogenous DNA into the cytoplasm of matured oocytes or zygotes is a promising technique to generate transgenic animals. However, the data about the microinjection time and procedure in sheep are limited and have not treated in detail. To obtain more in-depth information, the Sarda sheep oocytes from abattoir-derived ovaries were subjected to IVM and IVF. Then, the GFP plasmid as a reporter gene was injected into the cytoplasm of MII oocytes (n: 95) and zygotes at different post-insemination intervals (6-8 hpi, n: 120; 8-10 hpi, n: 122; 10-12 hpi, n: 110 and 12-14 hpi, n: 96). There were no significant differences in the cleavage rates between the groups. However, blastocyst rate of injected zygotes at all-time intervals was significantly lower than injected MII oocytes and control group (p < 0.05). Interestingly, the proportion of GFP-positive embryos was higher at 8-10 hpi compared with other injected groups (4 % versus 0 %, p  < 0.01). Among these, the proportion of mosaic embryos was high and two of those embryos developed to the blastocyst stage. In conclusion, we settled on the cytoplasmic microinjection of GFP plasmid at 8-10 hpi as an optimized time point for the production of transgenic sheep and subsequent experiments.

Growth of foetal bones and metabolic profile during gestation in primiparous ewes and multiparous ewes.

Primiparous ewes and multiparous ewes show physiological differences during pregnancy, which can have an impact on the development of their offspring. The objective of this study was to compare the changes in the metabolic profile and in the size of some foetal bones throughout gestation between primiparous and multiparous ewes. Twelve primiparous (PM) ewes and 14 multiparous (MT) ewes were used. According to the dates of lambing, two groups of ewes were formed: Group 1 (G1, n = 6 PM and n = 7 MT) and Group 2 (G2, n = 6 PM and n = 7 MT). The body weight, body condition score, metabolic and foetal morphometric parameters were determined from before conception until the end of gestation. After lambing, the body weight and survival rate during the first 72 hr of life of lambs, as well as the maternal behaviour score were recorded. The PM ewes were lighter (p < .01) and had a greater mobilization of body reserves during gestation, mainly evidenced by a greater serum concentration of NEFAs and lower serum concentration of total proteins (p < .05) compared with the MT ewes. The parity did not affect the foetal morphometric variables. The lambs of MT ewes were heavier at parturition (p = .002) and tended to have a greater survival rate than those lambs of PM ewes (p = .09). In conclusion, PM ewes and MT ewes differ in their metabolic profile throughout the gestation. However, in the present study, we did not find parity differences in the dimensions of foetal bones during growth in gestation.

Shifts in ovine cardiopulmonary microRNA expression in late gestation and the perinatal period.

BACKGROUND: MicroRNAs (miRNAs) have been identified as important contributors to the regulation of early fetal cardiopulmonary development. However, miRNA expression profiles during late gestation and the early neonatal period are not fully elaborated in large mammals such as sheep (ovis aries). The aim of this study was to sequence miRNA from cardiopulmonary tissues in late gestation and neonate sheep to identify changes in miRNA expression. METHODS: Illumina HiSeq next-generation deep sequencing (NGS) was performed on ovine tissues from the left (LV) and right ventricles (RV), lungs and pulmonary artery (PA) of preterm fetuses (128 days), near-term fetuses (140 days) (term = 148 days) and neonatal lambs (5 days). NGS reads were mapped to the sheep genome (OviAri) and published miRNA sequences. RESULTS: Of 1345 cardiopulmonary miRNAs that were sequenced, relatively few major shifts in miRNA expression were detected with increased age from near term to neonates, and were confirmed by quantitative real-time PCR: bta-miR-146a (lung), bta-miR-22-3p (lung, LV), hsa-miR-335* (lung, PA), and miR-210 (lung, PA, LV). CONCLUSIONS: Sequencing of miRNA led to identification of four predominant miRNA in ovine cardiopulmonary tissues which alter expression during late gestation and the early neonatal period, concurrent with important functional changes in heart and lungs.

Developmental competence of in vitro matured ovine oocytes vitrified in solutions with different concentrations of trehalose.

This study aimed to determine the optimum concentration of trehalose in solutions used for vitrification of in vitro matured (IVM) ovine oocytes. IVM oocytes were randomly divided into four experimental (vitrified) and one control (fresh) groups. Experimental groups were treated with different concentrations (0.0, 0.25, 0.5 and 1.0 M) of trehalose. After warming, some viable oocytes were exposed to 0.25% pronase to test zona pellucida hardening, whereas the others were fertilized and cultured in vitro for 8 days to evaluate their developmental competence. Blastocysts quality was assessed by differential staining and TUNEL test. Survival and developmental rates of oocytes vitrified in the presence of 0.5 M trehalose were significantly higher than those of the other vitrified groups. Furthermore, there was a significant difference between fresh and vitrified groups in total blastocyst rate. Analysis of blastocysts quality also revealed a significant difference between the group treated with 0.5 M trehalose and other groups in terms of apoptotic index. Furthermore,zona pellucida digestion time period was longer in trehalose-free (0.0 M) group compared to other groups. In conclusion, we found that IVM ovine oocytes vitrified in solutions containing 0.5 M trehalose are fertilization-competent and are able to produce good-quality blastocysts with an apoptotic index comparable to that of the fresh oocytes. Therefore, 0.5 M may be considered the optimum concentration of trehalose to be used in solutions prepared for vitrification of oocytes.

Qualitative and quantitative ultrasound attributes of maternal-foetal structures in pregnant ewes.

The aim of this study was to examine foetal organs and placental tissue to establish a correlation between the changes in the composition of these structures associated with their maturation and the ultrasonographic characteristics of the images. Twenty-four pregnant ewes were included in the study. Ultrasonography assessments were performed in B-mode, from the ninth gestational week until parturition. The lungs, liver and kidneys of foetuses and placentomes were located in transverse and longitudinal sections to evaluate the echogenicity (hypoechoic, isoechoic, hyperechoic or mixed) and echotexture (homogeneous and heterogeneous) of the tissues of interest. For quantitative evaluation of the ultrasonographic characteristics, it was performed a computerized image analysis using a commercial software (Image ProPlus® ). Mean numerical pixel values (NPVs), pixel heterogeneity (standard deviation of NPVs) and minimum and maximum pixel values were measured by selecting five circular regions of interest in each assessed tissue. All evaluated tissues presented significant variations in the NPVs, except for the liver. Pulmonary NPVmean, NPVmin and NPVmax decreased gradually through gestational weeks. The renal parameters gradually decreased with the advancement of the gestational weeks until the 17th week and later stabilized. The placentome NPVmean, NPVmin and NPVmax decreased gradually over the course of weeks. The hepatic tissue did not show echogenicity and echotexture variations and presented medium echogenicity and homogeneous echotexture throughout the experimental period. It was concluded that pixels numerical evaluation of maternal-foetal tissues was applicable and allowed the identification of quantitative ultrasonographic characteristics showing changes in echogenicity related to gestational age.

Cathepsin B inhibitor improves developmental competency and cryo-tolerance of in vitro ovine embryos.

BACKGROUND: Cathepsin B is a lysosomal cysteine protease involved in apoptosis and oocytes which have lower developmental competence show higher expression of Cathepsin B. Furthermore, expression of Cathepsin B show a decreasing trend from oocyte toward blastocyst stage. RESULTS: Present study assessed the effect of cathepsin B inhibitor, E-64, on developmental competency and cryo-survival of pre-implantation ovine IVF derived embryos. Cathepsin B inhibitor was added during day 3 to 8 of development. One µM E-64 was defined as the optimal concentration required for improving blastocyst rate. This concentration also reduced DNA fragmentation and BAX as apoptotic markers while increasing total cell number per blastocyst and improving anti-apoptotic marker, the BCL2. We further showed that addition of 1.0 µM of E-64 during day 3 to 8 of development improved re-expansion and hatching rates of blastocysts post vitrification. E-64 also reduced rate of DNA fragmentation and BAX expression and increased total cell number per blastocyst and BCL2 expression post vitrification. However, addition of E-64 post vitrification reduced the hatching rate. CONCLUSION: Therefore, it can be concluded that inhibition of cathepsin B in IVC, not only improves quality and quantity of blastocysts but also improves the cryo-survival of in vitro derived blastocysts.

Term vs. preterm cord blood cells for the prevention of preterm brain injury.

BACKGROUNDWhite matter brain injury in preterm infants can induce neurodevelopmental deficits. Umbilical cord blood (UCB) cells demonstrate neuroprotective properties, but it is unknown whether cells obtained from preterm cord blood (PCB) vs. term cord blood (TCB) have similar efficacy. This study compared the ability of TCB vs. PCB cells to reduce white matter injury in preterm fetal sheep.METHODSHypoxia-ischemia (HI) was induced in fetal sheep (0.7 gestation) by 25 min umbilical cord occlusion. Allogeneic UCB cells from term or preterm sheep, or saline, were administered to the fetus at 12 h after HI. The fetal brain was collected at 10-day post HI for assessment of white matter neuropathology.RESULTSHI (n=7) induced cell death and microglial activation and reduced total oligodendrocytes and CNPase+myelin protein in the periventricular white matter and internal capsule when compared with control (n=10). Administration of TCB or PCB cells normalized white matter density and reduced cell death and microgliosis (P<0.05). PCB prevented upregulation of plasma tumor necrosis factor (TNF)-a, whereas TCB increased anti-inflammatory interleukin (IL)-10 (P<0.05). TCB, but not PCB, reduced circulating oxidative stress.CONCLUSIONSTCB and PCB cells reduced preterm HI-induced white matter injury, primarily via anti-inflammatory actions. The secondary mechanisms of neuroprotection appear different following TCB vs. PCB administration.

Bisphenol A glucuronide deconjugation is a determining factor of fetal exposure to bisphenol A.

Previous studies in experimental animals have shown that maternal exposure to bisphenol A (BPA) during late pregnancy leads to high plasma concentrations of BPA glucuronide (BPAG) in fetus compared to mother due to the inability of BPAG to cross the placental barrier. A recent in vitro study has reported that BPAG can exert adipogenic effect underlining the need for characterization of the fetal disposition of BPAG. Experiments were conducted in chronically catheterized fetal sheep to determine the contribution of BPAG hydrolysis to BPA to the elimination of BPAG from the fetal compartment and its resulting effect on the overall fetal exposure to free BPA. Serial sampling of fetal arterial blood, amniotic fluid, maternal venous blood and urine was performed following separate single doses of BPA and BPAG administered intravenously to eight fetal/maternal pairs after cesarean section, and repeated BPAG doses given to two fetal sheep. On average 67% of the BPA entering the fetal circulation was rapidly eliminated through fetal to maternal clearance, with a very short half-life (20 min), while the remaining fraction (24%) was glucuronoconjugated. BPA conjugation-deconjugation cycling was responsible for a 43% increase of the overall fetal exposure to free BPA. A very specific pattern of fetal exposure to free BPA was observed due to its highly increased persistence with a hydrolysis-dependent plasma terminal free BPA half-life of several tens of hours. These findings suggest that although the high fetal to maternal clearance of free BPA protects the fetus from transient increases in free BPA plasma concentrations associated with maternal BPA intake, low but sustained basal free BPA concentrations are maintained in the fetus through BPA conjugation-deconjugation cycling. The potential health implications of these low but sustained basal concentrations of free BPA in fetal plasma should be addressed especially when considering time-dependent effects.

Patterns of gene expression in the sheep heart during the perinatal period revealed by transcriptomic modeling.

Septa from sheep hearts at 130 days gestation, term, and 14-day-old lambs were used to model the changes in gene expression patterns during the perinatal period using Agilent 15k ovine microarrays. We used Bioconductor for R to model five major patterns of coexpressed genes. Gene ontology and transcription factor analyses using Webgestalt modeled the biological significances and transcription factors of the gene expression patterns. Modeling indicated a decreased expression of genes associated with anatomical development and differentiation during this period, whereas those associated with increased protein synthesis and growth associated with maturation of the endoplasmic reticulum rose to term but did not further increase from the near term expression. Expression of genes associated with cell responsiveness, for example, immune responses, decreased at term but expression returned by postnatal day 14. Changes in genes related to metabolism showed differential substrate-associated patterns: those related to carbohydrate metabolism rose to term and remained stable thereafter, whereas those associated with fatty acid oxidation facility rose throughout the period. The timing of many of these maturational processes was earlier in relation to birth than in the rodent. The importance of the transcription factors, estrogen-related receptors, and v-myc avian myelocytomatosis viral oncogene homolog was also highlighted in the pattern of gene expression during development of the perinatal sheep heart.

Superovulatory response and embryo development in ewes treated with two doses of bovine somatotropin.

This study evaluated whether the administration of 50 and 100mg bovine somatotropin (bST) at the start of synchronization and at the time of natural mating in ewes improves the ovulation rate, embryonic development and pregnancy rate of transferred embryos. Forty-eight donors were assigned to three treatments: the bST-100 treatment (n=15) received 100mg bST at the start of synchronization and at natural mating, the bST-50 treatment (n=15) received 50mg bST on the same schedule as the previous group, and the control (n=18) did not receive any bST. Two embryos were transferred to each recipient (n=121): 35 received embryos from bST-100; 50 received embryos from bST-50, and 36 received embryos from the control. The superovulatory rate, percentage of recovered structures, cleavage rate, percentage of transferable embryos, embryo quality and development and pregnancy rate were analyzed using the GENMOD procedure of SAS. The number of corpora lutea and the cell number were analyzed using the GLM procedure of SAS. The insulin and IGF-1 concentrations were analyzed with ANOVA for repeated measures. The bST application did not affect the superovulatory rate, number of corpora lutea and recovered structures (P>0.05). The numbers of transferable embryos and embryos reaching the blastocyst were higher (P≤0.01) in the bST-50 (96.4±3.6% and 69.0±7.8%) than the bST-100 (93.0±4.5% and 27.2±38.9%) and control (87.7±5.4% and 50.4±6.4%) groups. The insulin and IGF-1 concentrations were higher (P<0.05) in the bST-treated groups, but the insulin concentration was higher (P<0.05) in the bST-100 group than in the bST-50 group. The pregnancy rate was similar (P=0.21) in ewes receiving embryos from the two treatments [bST-50, (70.0%); bST-100, (62.5%), and control, (56.6%)]. The administration of 50mg bST at the start of synchronization and at natural mating in superovulated ewes was concluded to enhance the proportion and development of transferable embryos. However, bST did not affect the pregnancy rate of transferred embryos.

Influence of birth weight and gender on lipid status and adipose tissue gene expression in lambs.

Intrauterine growth restriction (IUGR) is a risk factor for obesity, particularly when offspring are born into an unrestricted nutritional environment. In this study, we investigated the impact of IUGR and gender on circulating lipids and on expression of adipogenic, lipogenic and adipokine genes in perirenal adipose tissue. Singleton lambs born to overnourished adolescent dams were normal birth weight (N) or IUGR (32% lower birth weight due to placental insufficiency). IUGR lambs exhibited increased fractional growth rates but remained smaller than N lambs at necropsy (d77). At 48 days, fasting plasma triglycerides, non-esterified fatty acids and glycerol were elevated predominantly in IUGR males. Body fat content was independent of prenatal growth but higher in females than in males. In perirenal fat, relative to male lambs, females had larger adipocytes; higher lipoprotein lipase, fatty acid synthase and leptin and lower IGF1, IGF2, IGF1R, IGF2R and hormone-sensitive lipase mRNA expression levels, and all were independent of prenatal growth category; peroxisome proliferator-activated receptor gamma and glycerol-3-phosphate dehydrogenase (G3PDH) mRNA expression were not affected by IUGR or gender. Adiposity indices were inversely related to G3PDH mRNA expression, and for the population as a whole the expression of IGF system genes in perirenal fat was negatively correlated with plasma leptin, fat mass and adipocyte size, and positively correlated with circulating IGF1 levels. Higher plasma lipid levels in IUGR males may predict later adverse metabolic health and obesity, but in early postnatal life gender has the dominant influence on adipose tissue gene expression, reflecting the already established sexual dimorphism in body composition.

Effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver.

This study investigated the effects of maternal undernutrition during late pregnancy on the development and function of ovine fetal liver. Eighteen ewes with singleton fetuses were allocated to three groups at d 90 of pregnancy: Restricted Group 1 (RG1, 0.175MJMEkgBW(-0.75)d(-1), n=6), Restricted Group 2 (RG2, 0.33MJMEkgBW(-0.75)d(-1), n=6) and a Control Group (CG, ad libitum, 0.67MJMEkgBW(-0.75)d(-1), n=6). Fetuses were recovered at slaughter on d 140. Fetuses in the RG1 group exhibited decreased (P<0.05) liver weight, total antioxidant capacity (T-AOC), superoxide dismutase activity (SOD), cholinesterase (CHE), total protein (TP), globulin (GLB), and alanine transaminase (ALT). In addition, intermediate changes were found in the RG2 fetuses, including decreased liver weight, T-AOC and CHE (P<0.05). In contrast, increases in fetal hepatic collagen fibers and reticular fibers, glutathione peroxidase (GSH-Px), malondialdehyde (MDA), nitric oxide (NO), nitric oxide synthase (NOs), monoamine oxidase (MAO), albumin (ALB)/GLB, aspartate transaminase (AST), and AST/ALT were found in the RG1 fetuses (P<0.05). The RG2 fetuses had increased fetal hepatic collagen fibers, NOs and MAO (P<0.05) relative to the control fetuses. These results indicate that impaired fetal hepatic growth, fibrosis, antioxidant imbalance and dysfunction were associated with maternal undernutrition.

Dose-dependent effect of melatonin on postwarming development of vitrified ovine embryos.

After cryopreservation, embryos become sensitive to the oxidative stress, resulting in lipid peroxidation, membrane injury, and structural destruction. The present study aimed to assess the effect of increasing concentration of melatonin during postwarming culture on embryo's ability to restore its functions after cryopreservation. In vitro-produced blastocysts were vitrified, warmed, and cultured in vitro in TCM 199 with 5 different supplementations: control (CTR): 10% fetal calf serum; bovine serum albumin (BSA): 0.04% (wt/vol) BSA; and MEL(-3), MEL(-6), MEL(-9): BSA plus melatonin 10(-3), 10(-6), and 10(-9) M. The medium with the highest melatonin concentration had the highest trolox equivalent antioxidant capacity, whose values were comparable with those determined in plasma sampled from adult ewes (8.7 ± 2.4 mM). The other media had lower trolox equivalent antioxidant capacity values (P < 0.01), below the range of the plasma. At the same time, embryos cultured with the highest melatonin concentration reported a lower in vitro viability, as evaluated by lower re-expansion and hatching rates, and lower total cell number compared with the other groups (P < 0.05). Their metabolic status was also affected, as evidenced by higher oxidative and apoptotic index and lower ATP concentration. The beneficial effects of melatonin on embryo development during postwarming culture were observed only at low concentration (10(-9) M). These results suggest that melatonin at high concentration may exert some degree of toxic activity on pre-implantation embryos. Thus, the dose at which the embryos are exposed is pivotal to obtain the desiderate effect.

Fetal programming by co-twin rivalry in sheep.

Fetal rivalry for space and nutrients compromises intrauterine environment and fetal growth, this leading to further consequences during adult life (i.e., fetal programming). Focusing on sheep, relevant fetal programming effects have been revealed on body composition and growth although little is known about their potential impact on the reproductive performance of adult ewes. This research focused on the analysis of fetal programming-related effects on 41,475 litter size (LS) records from 7,177 purebred Ripollesa ewes. Fetal programming sources of variation accounted for the linear and quadratic effect of absolute birth BW (ABBW), relative birth BW (RBBW) of twin-born ewes (i.e., both magnitude and direction of the birth BW difference between the ewe and its co-twin), and sex of twin ewe's littermate (SLM). More specifically, data were analyzed under a threshold mixed model and the statistical relevance of models accounting for different combinations of ABBW, RBBW, and SLM effects was compared by Bayes factors (BF; i.e., the ratio between the posterior probability of 2 competing models). The model accounting for RBBW and discarding both ABBW and SLM effects was clearly preferred; its posterior probability was 35.2 to 362.3 times higher than from remaining models and provided very strong (31.6 < BF < 100) and decisive evidences (BF > 100) supporting the relevance of RBBW and the negligibility of both ABBW and SLM. Single-born ewes were included as reference group and they reached a predicted LS of 1.189 lambs per lambing. Twin-born ewes being >600 g lighter than their co-twins suffered from an impaired reproductive ability with 1.162 lambs per lambing (95% credible interval [95CI], 1.147 to 1.179), and this estimate increased until ewes were 151 to 300 g lighter than their co-twins (1.226 lambs per lambing; 95CI, 1.208 to 1.244). Remaining categories (i.e., ewes being heavier or equal than their co-twins) did not provide significant differences and showed an enhanced reproductive ability of approximately 1.23 lambs per lambing. These significant differences in LS highlighted the influence of fetal programming in sheep under rangeland conditions, which implies decisive economic consequences worldwide. Moreover, these results could contribute additional information on twin biology, which could be useful in other mammalian species such as humans.

Using type-2 fuzzy logic systems for spike detection in the hypoxic ischemic EEG of the preterm fetal sheep.

Perinatal hypoxia is a major cause of brain injury in preterm babies. Thus, neuro-protective treatments play a pivotal role during the first 6-8 hours post hypoxic-ischemic insult. However, at present it is not possible to determine which infants are suffering from hypoxic ischemia. Recent investigations suggest that there are high frequency micro-scale transients exist in the first 6-8 hours of a hypoxic ischemic EEG which could be utilized as the useful benchmarks for the prediction of hypoxia. Type-2 Fuzzy Logic Systems (Type-2 FLS) have the capability to handle inherent uncertainties in nonlinear signals. This paper describes the application of a Type-2 FLS to detect spikes in the preterm fetal sheep electroencephalogram (EEG) after asphyxia in utero. The Type-2 FLS differentiates each detected event in terms of its spikiness and specifies the potential events based on their degree of similarity to an EEG expert definition of a standard spike. An adaptive thresholding method has been employed in order to increase the spike detection ability of the purposed system. The sensitivity and selectivity verify enhanced performance of the Type-2 FLS for spike detection in fetal sheep EEG signals with a 98.1% and 93.7% respectively which are significantly improved in comparison to our previous methods.

Impacts of arginine nutrition on embryonic and fetal development in mammals.

Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % L-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.

The entotympanic in late fetal Artiodactyla (Mammalia).

The entotympanic is a neomorphic component of the bulla tympanica of placental mammals. Ontogenetically, its rostral component seems to be derived from the tubal cartilage, whereas its caudal component is normally connected with the sheath of the tympanohyal; the present study indicates additional sources of the caudal entotympanic. The entotympanics develop in late fetal or early postnatal life as cartilaginous structures, but in most taxa they ossifiy endochondrally as "os bullae". This skeletal element is absent only in a few placental orders, among them the Artiodactyla. Because it is present in their sister taxa within the Scrotifera, it is likely to be reduced secondarily in the even-toed mammals. The study of histological serial sections of late fetal stages of several artiodactyl species shows that vestigial cartilaginous homologues of the entotympanics are invariably present, contrary to statements in the literature. In a few perinatal stages even secondary ossifications or calcifications of the entotympanic cartilages can be observed. The tubal cartilage of artiodactyls also continues into an anterior tegmen tympani (new term) that forms the floor of the fossa muscularis major.

2011 and 2012 Early Careers Achievement Awards: Placental programming: how the maternal environment can impact placental function.

Proper establishment of the placenta is important for fetal survival; however, placental adaptations to inadequate maternal nutrition or other stressors are imperative for fetal growth to be optimal. The effects of maternal nutritional status and activity level on placental vascular function and uteroplacental blood flows are important to understand as improper placental function leads to reduced growth of the fetus. In environments where fetal growth can be compromised, potential therapeutics may augment placental function and delivery of nutrients to improve offspring performance during postnatal life. Factors that could enhance placental function include supplementation of specific nutrients, such as protein, hormone supplements, such as indolamines, and increased activity levels of the dam. To understand the mechanism of how the maternal environment can impact uterine or umbilical blood flows, assessment of placental vascular reactivity has been studied in several large animal models. As we begin to understand how the maternal environment impacts uterine and umbilical blood flows and other uteroplacental hemodynamic parameters, development of management methods and therapeutics for proper fetal growth can be achieved.