In ovo feeding of probiotic lactobacilli differentially alters expression of genes involved in the development and immunological maturation of bursa of Fabricius in pre-hatched chicks.

Compelling evidence indicates that immunological maturation of the gut-associated lymphoid tissues, including the bursa of Fabricius, is dependent upon antigenic stimulation post-hatch. In view of these data, the present study investigated the impact of exposing the immune system of chick embryos to antigenic stimuli, via in ovo delivery of poultry-specific lactobacilli, on the expression of genes associated with early bursal development and maturation. Broiler line embryonated eggs were inoculated with 106 and 107 colony-forming units (CFUs) of an individual or a mixture of Lactobacillus species, including L. crispatus (C25), L. animalis (P38), L. acidophilus (P42), and L. reuteri (P43), at embryonic day 18 (ED18). The bursa of Fabricius was collected from pre-hatched chicks (ED20) to measure the expression levels of various immune system genes. The results revealed that L. acidophilus and the mixture of Lactobacillus species at the dose of 106 CFU consistently elicited higher expression of genes responsible for B cell development, differentiation, and survival (B cell activating factor (BAFF), BAFF-receptor (BAFF-R)), and antibody production (interleukin (IL)-10) and diversification (TGF-ß). Similar expression patterns were also noted in T helper (Th) cell-associated cytokine genes, including Th1-type cytokines (interferon (IFN)-γ and IL-12p40), Th2-type cytokines (IL-4 and IL-13) and Th17 cytokine (IL-17). Overall, these results suggest that the supplementation of poultry-specific lactobacilli to chick embryos might be beneficial for accelerating the development and immunological maturation of the bursa of Fabricius. However, further studies are required to determine if the changes in gene expression are associated with the developmental trajectory and phenotypes of bursal cells.

Assessing the Differential Abundance of Maternal Circulating MicroRNAs or Interferon-Stimulated Genes with Early Pregnancy.

Interferon-stimulated genes (ISG) and microRNA (miRNA) present in maternal circulation have been reported to be diagnostic of pregnancy in cattle prior to day (d)30 of gestation. The objective of this study was to assess specific ISG and miRNA abundance on d 18 of gestation. Cattle were subjected to estrous synchronization and artificially inseminated to a single Angus sire. At time of insemination (d 0) and d 18 post-insemination, blood was collected and total RNA isolated. Differential abundance (DA) in specific ISG and miRNA between d 0 and d 18 samples in pregnant (n = 10) and open (n = 10) cows were assessed via RT-qPCR. The relative Ct values were normalized using abundance of cyclophilin or the geometric mean of specific miRNA for the ISG and miRNA genes of interest, respectively. The DA of the ISG were increased due to pregnancy (p < 0.05); however, there was no expected day of gestation by pregnancy interaction. Relative abundance of Bta-miR-16 increased on d18 regardless of pregnancy status (p < 0.05). None of the miRNA evaluated in this study were associated with pregnancy status. These data indicate that certain ISG may serve as early indicators of pregnancy in cattle, but abundance of the miRNA does not.

Effects of Exogenous Glucocorticoid Infusion on Appetitic Center Development in Postnatal Dairy Bull Calves.

The objective of this study was to determine the effects of exogenous glucocorticoid administration on leptin concentrations and brain development markers, such as protein and hypothalamic gene expression, in dairy bull calves. Within 4 h of parturition, Holstein bulls were intravenously infused with either a low cortisol dose (LC; n = 9, 3.5 µg/kg of body weight (BW)), high cortisol dose (HC; n = 9, 7.0 µg/kg BW), or control (CON; n = 9, saline) dose, with a 2nd infusion 24 h postpartum. Jugular blood was collected prior to infusion and daily until the calves were euthanized (day 5). Cerebrospinal fluid (CSF) from the third ventricle and adipose (omental, perirenal, and mesenteric) and hypothalamic tissue were collected. The blood and CSF samples were analyzed for leptin concentrations. The data were analyzed using SAS. Serum (p = 0.013) and CSF (p = 0.005) leptin concentrations in HC- and LC-treated calves were decreased compared with CON-treated calves. Leptin protein expression was decreased (p < 0.044) in perirenal and omental adipose tissue of LC-treated calves compared with CON-treated calves. Gene abundance of brain-derived neurotrophic factor and fibroblast growth factors 1 and 2 were decreased (p < 0.006) in HC- and LC-treated calves compared with CON-treated calves. In summary, cortisol administered to dairy bull calves reduced leptin concentrations, decreased leptin protein expression in perirenal and omental adipose tissue, and altered gene expression in hypothalamic tissue.

Comparison of the 7-day CO-Synch and 8-day estradiol-based protocols for estrus synchronization and timed artificial insemination in suckled Bos taurus cows.

Two experiments were conducted to compare, follicle diameter (FD) on Day -1, corpus luteum (CL) area on Day 7, progesterone (P4) concentration on Day 7 and 18, pregnancy per timed artificial insemination (TAI) on Day 30, and pregnancy loss (PL) between Days 30 and 60 after TAI (TAI, Day 0) using two different synchronization protocols. In Experiment 1, Angus cows (n = 1148) were randomly assigned to either 7-d progesterone CO-Synch (7-d CO-Synch) or 8-d progesterone + estradiol (8-d P + ES) synchronization protocols for TAI. On Day -10, cows in the 7-d CO-Synch treatment group (n = 574) received a progesterone-releasing intravaginal device (PIVD; 0.5 g P4) and GnRH (0.105 mg), on Day -3 the PIVD was removed and cows received cloprostenol (0.150 mg), then, on Day 0 (64 h after PIVD removal), cows received GnRH (0.105 mg) and were TAI. On Day -10, cows in the 8-d P + ES treatment group (n = 574) received a PIVD (0.5 g P4) and estradiol benzoate (2.0 mg), on Day -2 the PIVD was removed, and cows received cloprostenol (0.150 mg) and estradiol cypionate (0.5 mg), then, on Day 0 (48 h after PIVD removal), cows were TAI. Pregnancy per TAI was determined on Days 30 and 60. In a subset of cows (7-d CO-Synch, n = 41; 8-d P + ES, n = 40), serum P4 concentration was evaluated on Day 18. In Experiment 2, anestrus (n = 34) and cyclic (n = 34) suckled beef cows were selected and submitted at random on Day -10, to either 7-d CO-Synch or 8-d P + ES treatment groups. Follicle diameter on Day -1, CL area, and serum P4 concentration on Day 7 were determined. In Experiment 1, pregnancy per TAI on Day 30 did not differ (7-d CO-Synch = 48.9%; 8-d P + ES = 45.6%) between treatments but it was greater for cows with BCS ≥5 (P < 0.01). Pregnancy loss between Days 30 and 60 did not differ between treatment groups but tended to be greater in cows with BCS <5.0 (P < 0.1). In a subset of cows, serum P4 concentration on Day 18 did not differ between treatment groups but tended to be lower (P < 0.1) in cows that had PL between Days 30 and 60 compared to cows that had no PL. In Experiment 2, FD tended to be greater (P < 0.1) and CL area was greater (P = 0.05) in anestrus cows from 7-d CO-Synch treatment. In cyclic cows, the treatment did not affect the FD or CL area. In conclusion, there was no difference in pregnancy per TAI on Day 30 and PL between Days 30 and 60 between cows using 7-d CO-Synch + PIVD or 8-d estradiol-based + PIVD protocols for estrus synchronization and TAI.

Effects of postweaning supplementation of immunomodulatory feed ingredient on circulating cytokines and microbial populations in programmed fed beef heifers.

The objective was to determine the effects of an immunomodulatory feed ingredient following weaning on cytokine expression and fecal microbial populations of heifers. Commercial Angus heifers (n = 72) were weaned (227 ± 7 d of age), blocked by BW (n = 9 blocks), and randomly assigned to one of two pens per block. Pens within weight block (four heifers per pen) were then randomly assigned to treatments. Heifers were fed twice daily from days 0 to 60 (to gain 0.75 kg/d) and top dressed with either 18 g/heifer/d of the immunomodulatory feed ingredient (Celmanax; Arm and Hammer Animal Nutrition, Princeton, NJ; CEL) or corn-germ meal (CON). Blood samples were collected on days 0, 15, 30, 45, 60 and fecal grab samples on day 0 of the feeding trial. After day 60, two heifers per pen (n = 32) were randomly selected for a transportation challenge. Serum samples were collected at hours 0, 4, 8, 12 and fecal grab samples at hours -24, 0, 24 and 7 d postchallenge. Blood samples were analyzed for interferonγ (IFNγ), interleukin-8 (IL-8), and haptoglobin (HP) using commercially available ELISA kits and qRT-PCR for genes of interest associated with cytokine expression. Fecal samples were enumerated for Clostridia and E. coli using selective media (≤5 isolates from each media/sample), tested to determine whether they were Clostridium perfringens or pathogenic E. coli, and then enriched for detection of Salmonella. Data were analyzed via ANOVA. During the feeding trial, HP was reduced (P = 0.018) in CEL compared with CON at days 15, 45, and 60, whereas IFNγ and IL-8 did not differ (P > 0.080) between treatments. All cytokines were decreased (P < 0.001) in CEL compared with CON during the challenge. During the feeding trial, HP mRNA was increased (P = 0.045) in CEL compared with CON at days 30 and 60. Similarly, IFNγ mRNA was increased (P = 0.040) in CEL compared with CON; however, other genes of interest did not differ (P > 0.172). Both C. perfringens and total E. coli counts were decreased (P = 0.036) in CEL compared with CON at 24 h after the start of the transportation challenge. Clostridia and pathogenic E. coli counts did not differ (P = 0.941) between treatments. Total Clostridia and E. coli counts were increased (P < 0.014) 24 h postchallenge. All microbial populations, except pathogenic E. coli, observed decreased (P ≤ 0.009) counts from 24 h to 7 d postchallenge. Overall, Celmanax supplementation decreased circulating cytokines, and altered microbial populations and gene expression, thus, may serve a role in preparing animals to better cope with immunological challenges.

With consumers wanting less antibiotic usage in cattle production, the need for natural feed ingredients that have positive effects on animal health are needed. The feeding of a yeast-based feed product decreased cytokines in the blood and their mRNA expression in white blood cells that act on stimulating an inflammatory response. When an animal has an inflammatory response, their immune system is working harder than necessary. This means they are using energy that could otherwise be used for growth, which decreases efficiency and performance. The feeding of this yeast-based feed ingredient also reduced the amount of harmful bacteria in the feces of the heifers. Having lower amounts of harmful bacteria (such as Salmonella) in the feces decreases the chance of carcass contamination. For consumers, this means less instances of food-borne illnesses.

Discovery and preclinical development of AR453588 as an anti-diabetic glucokinase activator.

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.

Ergot alkaloid exposure during gestation alters. I. Maternal characteristics and placental development of pregnant ewes1.

Tall fescue [Lolium arundinaceum (Scheyreb.) Darbysh] is the primary cool season forage grass in the Southeastern United States. Most tall fescue contains an endophytic fungus (Epichloë coenophiala) that produces ergot alkaloids and upon ingestion induces fescue toxicosis. The objective of this study was to assess how exposure to endophyte-infected (E+; 1.77 mg hd-1 d-1 ergovaline and ergovalinine) or endophyte-free (E-; 0 mg hd-1 d-1 ergovaline and ergovalinine) tall fescue seed fed during 2 stages of gestation (MID, days 35-85/LATE, days 86-133) alters placental development. Thirty-six, fescue naïve Suffolk ewes were randomly assigned to 1 of 4 fescue treatments: E-/E-, E-/E+, E+/E-, or E+/E+. Ewes were individually fed the same amount of E+ or E- seed mixed into total mixed ration during MID and LATE gestation. Terminal surgeries were conducted on day 133 of gestation. Ewes fed E+ fescue seed had elevated (P < 0.001) ergot alkaloid excretion and reduced (P < 0.001) prolactin levels during the periods when fed E+ seed. Ewes switched on day 86 from E- to E+ seed had a 4% reduction (P = 0.005) in DMI during LATE gestation, which translated to a 2% reduction (P = 0.07) in DMI overall. Average daily gain was also reduced (P = 0.049) by 64% for E-/E+ ewes during LATE gestation and tended to be reduced (P = 0.06) by 33% overall. Ewes fed E+ seed during LATE gestation exhibited a 14% and 23% reduction in uterine (P = 0.03) and placentome (P = 0.004) weights, respectively. Caruncle weights were also reduced by 28% (P = 0.003) for E-/E+ ewes compared with E-/E- and E+/E-. Ewes fed E+ seed during both MID and LATE gestation exhibited a 32% reduction in cotyledon (P = 0.01) weights, whereas ewes fed E+ seed only during MID gestation (E+/E-) had improved (P = 0.01) cotyledon weights. The percentage of type A placentomes tended to be greater (P = 0.08) for E+/E+ ewes compared with other treatments. Other placentome types (B, C, or D) did not differ (P > 0.05). Total fetal weight per ewe was reduced (P = 0.01) for ewes fed E+ seed during LATE gestation compared with E-; however, feeding E+ seed during MID gestation did not alter (P = 0.70) total fetal weight per ewe. These results suggest that exposure to ergot alkaloids during LATE (days 86-133) gestation has the greatest impact on placental development by reducing uterine and placentome weights. This, in turn, reduced total fetal weight per ewe by 15% in ewes fed E+ seed during LATE gestation (E-/E+ and E+/E+).

Ergot alkaloid exposure during gestation alters: II. Uterine and umbilical artery vasoactivity1.

Previous research has shown that livestock exposed to ergot alkaloids results in decreased vasoactivity of gastrointestinal and peripheral vasculature. Little is known regarding the effect ergot alkaloid exposure during gestation may have on vasculature supporting the fetus. The objective of this study was to evaluate contractile responses of uterine and umbilical arteries collected from ewes consuming ergot alkaloids during gestation. On day 35 of gestation, 36 Suffolk ewes (78.24 ± 9.5 kg) were assigned to endophyte-infected (E+) or endophyte-free (E-) tall fescue seed treatments that were fed either throughout or switched on day 86 of gestation, creating four seed treatments E+E+, E+E-, E-E+, and E-E-. Ewes were fed E+ tall fescue seed to provide 1.77 mg of total ergovaline ⋅ hd-1 ⋅ d-1 with E- ewes receiving the same quantity of E- seed. Gestation was terminated on day 133, and sections of uterine artery and umbilical cord were surgically collected. Only collections from 28 ewes (n = 7/treatment) were of sufficient viability to proceed with the contractility experiments. Arteries were cleaned, sliced into 2-mm cross sections, and suspended in multi-myograph chambers containing 5 mL of continuously oxygenated Krebs-Henseleit buffer. Vessels were exposed to increasing concentrations (5 × 10-8 to 1 × 10-4 M) of norepinephrine, serotonin, ergotamine, and ergovaline (5 × 10-9 to 1 × 10-5M; extract of tall fescue seed) in 15-min intervals. Increasing concentrations of norepinephrine generated a contractile response by the uterine artery (P < 0.05), but no response in the umbilical artery. Increasing concentrations of serotonin resulted in negligible responses in uterine preparations, whereas umbilical artery preparations were responsive (P < 0.05) to serotonin. Ewes receiving E+E+ and E-E+ treatments had decreased vasoactivity in umbilical arteries to serotonin with a dextral shift in concentrations where the response curve initiated (P < 0.05). Interestingly, uterine arteries were not responsive to exposure to ergotamine or ergovaline, whereas umbilical arteries were responsive (P < 0.05). Umbilical arteries collected from ewes receiving E-E- and E+E- were more vasoactive to ergot alkaloids (P < 0.05) than other treatments. These findings indicate that maternal blood supply to the placenta appears protected from negative effects of ergot alkaloids; however, umbilical vasculature is not, and this could adversely influence fetal growth.

Novel and differentially abundant microRNAs in sperm cells, seminal plasma, and serum of boars due to porcine reproduction and respiratory syndrome virus infection.

The objectives of this study were to identify and determine relative abundance of miRNAs in boar sperm, seminal plasma (SP), and serum pre- and post-viral infection. Functional enrichment analyses on predicted targets of miRNAs of interest were performed. Boars (n = 6) were inoculated with porcine reproductive and respiratory syndrome virus (PRRSv) strain 1-8-4 (Day 0). Semen and serum were collected on Day -2 and 6. Sperm and SP were separated and aliquots were flash frozen and stored at -80 °C. Serum was frozen and stored at -80 °C. Total RNA was isolated from sperm and SP samples and subjected to RNA sequencing. Microarray analysis was performed using the Day -2 and 6 RNA samples from serum, sperm and SP. Potential miRNA targets were predicted using miRanda 3.3a and targets were then analyzed for enrichment of Gene Ontology) and InterPro terms and were considered to be enriched if P < 0.01 using the Bonferroni correction. Microarray analyses resulted in 83, 13, and 10 miRNAs with differences in abundances in sperm, serum, and SP, respectively, when comparing Day -2 and 6. Results from enrichment analyses indicated that the predicted targets of 35, nine, and five miRNAs with differences in abundances for sperm, SP, and serum, respectively, that have functions and/or conserved protein domains that are enriched when compared to the pig genome. Enriched terms for P2X purinoceptors were identified for sperm, SP and serum. Enriched terms for cell adhesion were identified for sperm and serum transcripts. Enriched terms for cell signaling were identified for sperm and SP transcripts.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 µM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 µmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Constraining the Equation of State of Superhadronic Matter from Heavy-Ion Collisions.

The equation of state of QCD matter for temperatures near and above the quark-hadron transition (∼165 MeV) is inferred within a Bayesian framework through the comparison of data from the Relativistic Heavy Ion Collider and from the Large Hadron Collider to theoretical models. State-of-the-art statistical techniques are applied to simultaneously analyze multiple classes of observables while varying 14 independent model parameters. The resulting posterior distribution over possible equations of state is consistent with results from lattice gauge theory.

The bull sperm microRNAome and the effect of fescue toxicosis on sperm microRNA expression.

Tall fescue [Schedonorus phoenix (Scop.) Holub] accounts for nearly 16 million hectares of pasture in the Southeastern and Mid-Atlantic U.S. due to its heat, drought, and pest resistance, conferred to the plant by its symbiotic relationship with the endophyte Neotyphodium coenophialum. The endophyte produces ergot alkaloids that have negative effects on the growth and reproduction of animals, resulting in the syndrome known as fescue toxicosis. The objectives of our study were to identify microRNA (miRNA) present in bovine sperm and to evaluate the effects of fescue toxicosis on sperm miRNA expression. Angus bulls were assigned to treatments of either toxic or non-toxic fescue seed diets. Semen was collected and subjected to RNA isolation. Three samples from each treatment group were chosen and pooled for deep sequencing. To compare miRNA expression between treatment groups, a microarray was designed and conducted. For each of the top ten expressed miRNA, target prediction analysis was conducted using TargetScan. Gene ontology enrichment was assessed using the Database for Annotation, Visualization and Integrated Discovery. Sequencing results elucidated the presence of 1,582 unique small RNA present in sperm. Of those sequences, 382 were known Bos taurus miRNA, 22 were known but novel to Bos taurus, and 816 were predicted candidate miRNA that did not map to any currently reported miRNA. Of the sequences chosen for microarray, twenty-two showed significant differential expression between treatment groups. Gene pathways of interest included: regulation of transcription, embryonic development (including blastocyst formation), Wnt and Hedgehog signaling, oocyte meiosis, and kinase and phosphatase activity. MicroRNA present in mature sperm appears to not only be left over from spermatogenic processes, but may actually serve important regulatory roles in fertilization and early developmental processes. Further, our results indicate the possibility that environmental changes may impact the expression of specific miRNA.

Exposure to ergot alkaloids during gestation reduces fetal growth in sheep.

Tall fescue [Lolium arundinaceum (Schreb.) Darbysh; Schedonorus phoenix (Scop.) Holub] is the primary cool season perennial grass in the eastern U.S. Most tall fescue contains an endophyte (Neotyphodium coenophialum), which produces ergot alkaloids that cause vasoconstriction and could restrict blood flow to the fetus in pregnant animals. The objective of this study was to examine fetal growth during maternal exposure to ergot alkaloids during gestation. Pregnant ewes (n = 16) were randomly assigned to one of two dietary treatments: (1) endophyte-infected (N. coenophialum) tall fescue seed (E+; 0.8 ug of ergovaline /g diet DM) and (2) endophyte-free tall fescue seed (E-; 0.0 ug of ergovaline/g diet DM). Birth weight of lambs was reduced by 37% for E+ compared to E-. Organ and muscle weights were also lighter for E+ than E-. Exposure to ergot alkaloids in utero reduces fetal growth and muscle development.

Fully dynamical simulation of central nuclear collisions.

We present a fully dynamical simulation of central nuclear collisions around midrapidity at LHC energies. Unlike previous treatments, we simulate all phases of the collision, including the equilibration of the system. For the simulation, we use numerical relativity solutions to anti-de Sitter space/conformal field theory for the preequilibrium stage, viscous hydrodynamics for the plasma equilibrium stage, and kinetic theory for the low-density hadronic stage. Our preequilibrium stage provides initial conditions for hydrodynamics, resulting in sizable radial flow. The resulting light particle spectra reproduce the measurements from the ALICE experiment at all transverse momenta.

Isolation of total RNA and detection procedures for miRNA present in bovine-cultured adipocytes and adipose tissues.

Micro-ribonucleic acids (miRNA) regulate gene expression posttranscriptionally by altering translation of protein(s) encoded by specific messenger RNA. Therefore the ability to detect and quantify the expression levels of specific miRNA present within a cell or tissue is necessary to thoroughly examine cellular physiology and gene expression. Here we describe procedures that allow for the isolation and quantification of miRNA in bovine adipocytes and adipose tissue.

Identifying the charge carriers of the quark-gluon plasma.

Charge correlations in lattice gauge calculations suggest that up, down, and strange charges move independently in the quark-gluon plasma, and that the density of such charges is similar to what is expected from simple thermal arguments. Here, we show how specific elements of the charge-charge correlation matrix in the quark-gluon plasma survive hadronization and become manifest in final-state charge-charge correlation measurements.

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality.

Brief report: chimeric pigs produced from induced pluripotent stem cells demonstrate germline transmission and no evidence of tumor formation in young pigs.

The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in-depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSC-derived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression, further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals.

Differential expression of porcine sperm microRNAs and their association with sperm morphology and motility.

MicroRNAs (miRNAs) are involved in nearly every biological process examined to date, but little is known of the identity or function of miRNA in sperm cells or their potential involvement in spermatogenesis. The objective was to identify differences in miRNA expression between normal porcine sperm samples and those exhibiting high percentages of morphological abnormalities or low motility. Quantitative RT-PCR was performed on sperm RNA to compare expression levels of 10 specific miRNAs that are predicted to target genes that code for proteins involved in spermatogenesis, sperm structure, motility, or metabolism. There were increases in the expression of four miRNAs, let-7a, -7d, -7e, and miR-22, in the abnormal group (P < 0.05), whereas miR-15b was decreased compared to controls (P < 0.05). Two miRNAs, let-7d and let-7e, were increased in the low motility group when compared to controls (P < 0.05). Bioinformatic analyses revealed that messenger RNA targets of the differentially expressed miRNAs encode proteins previously described to play roles in sperm function. Although the precise role of miRNA in sperm remains to be determined, their changes as associated with morphology and motility signify a critical biological function. Perhaps they are remnants of spermatogenesis, stored for a later role in fertilization, or are delivered to the oocyte to influence early embryonic development. Although there is no single cause of male infertility, the identification of miRNAs associated with sperm motility, structural integrity, or metabolism could lead to the development of a microarray or real time-based diagnostic assay to provide an assessment of male fertility status.

A novel coenzyme A:diacylglycerol acyltransferase 1 inhibitor stimulates lipid metabolism in muscle and lowers weight in animal models of obesity.

Obesity is characterized by the accumulation of triacylglycerol in adipocytes. Coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of two known DGAT enzymes that catalyze the final and only committed step in triacylglycerol synthesis. In this report, we describe the pharmacological effects of a novel selective DGAT1 inhibitor, Compound-A. This compound inhibited triacylglycerol synthesis in both adipocytes and skeletal myotubes, and increased fatty acid oxidation in skeletal myotubes at 1 µM. The repeated administration of Compound-A to diet-induced obese C57BL/6J and genetically obese KKA(y) mice (3-30 mg/kg for 3-4 weeks) significantly decreased the visceral fat pad weights and the hepatic lipid contents compared to controls without affecting food intake. In addition, fatty acid oxidation in skeletal muscle tissues was increased by the treatment of Compound-A in both mice strains. This is the first report demonstrating that a small synthetic DGAT1 inhibitor increases fatty acid oxidation in skeletal muscle in vitro and ex vivo. These results suggest that DGAT1 inhibition is a promising therapeutic approach for the treatment of obesity and lipid abnormalities such as hepatic steatosis.