bluuebunny/crossref_metadata_2025 huggingface dataset:
413 EFFECT OF FLUSHING WITH DIFFERENT DEGRADABILITY PROTEIN SOURCE ON THE PLASMA CONCENTRATIONS OF N-UREA AND PROGESTERONE IN Santa Inês SHEEP
Authors: Lazarin G. , Alves N. , Perez J. , Neto An. , Vale D.
Published: August 2010

Protein excess in diet and or rumen degradable protein (RDP) has been associated with elevated concentrations of N-urea in blood and reduction of the concentration of progesterone (P4) in plasma. The objective of this study was to evaluate the overnutrition (flushing) effects of diets containing different rates of RDP on the plasma concentrations of N-urea and P4 in Santa InÊs ewes. Twelve ewes were assigned in a randomized block design formed by 4 body weight bands. Two isocaloric (3.10 and 2.89 Mcal/day) and isonitrogenous (13.15 and 13.46% crude protein; CP) diets with different proportions of RDP (8.43 and 6.60%) containing soybean meal and corn gluten meal, respectively, and 1 maintenance diet (1.91 Mcal/day; 11.30% CP and 6.31% of RDP) were provided for 28 days before ovulation and during the next estrous cycle. Hence, 3 treatments were compared and 4 replicates were done. On Day 17 after starting the diet treatments, intravaginal sponges containing 60 mg of medroxyprogesterone acetate (MAP-60®, Tecnopec, São Paulo, Brazil) were inserted for 11 days. Nine days after sponge insertion, 240 IU of eCG (Novormon®, Syntex S.A., Buenos Aires, Argentina) and 50 μg of cloprostenol (Prolise®, ARSA S.R.L., Buenos Aires, Argentina) were administered i.m. Blood samples for determination of N-urea were collected on Days 7, 14, 21, and 28 after starting the diets and on Days 3, 6, 9, 12, and 15 after ovulation. Blood samples collected during the postovulatory interval were also analyzed for P4. Data were analyzed by ANOVA and treatment means were compared by orthogonal contrasts, where theF-testwas significant. Contrast 1 =T1 andT2 v. T3, and contrast 2 = T1 v. T2. The N-urea and P4 concentrations in plasma were analyzed according to split plot in time scheme. The sheep dry matter (DM) and CP consumption that were recorded in the flushing diet was higher (P < 0.01) than in ewes receiving the maintenance diet. The DM and CP consumption of ewes did not differ between treatments T1 and T2. The plasma N-urea concentration was not different (P > 0.05) between the ewes that consumed the flushing diet and ewes that received the maintenance diet, but it was higher (P < 0.01) in ewes that were fed the flushing diet containing soybean meal (25.09 mg dL-1) as compared with that of ewes fed the flushing diet containing corn gluten meal (20.81 mgdL-1). The P4 concentration in plasma did not differ (P > 0.05) among animals that received the flushing diet and those that received the maintenance diet, but there was a significant difference (P = 0.05) between flushing diets containing soybean meal (7.28 ngmL-1) compared with those containing corn gluten meal (4.46 ngmL-1). In summary, the nutritional level (flushing v. maintenance) did not alter the concentrations of N-urea and P4 in plasma of ewes, but the supply of a greater protein degradability source did increase their concentrations.

Funded by FAPEMIG, CNPq, and TECNOPEC.
423 PRODUCTION OF CLONED PIGS EXPRESSING APOLIPOPROTEIN E-SPECIFIC SMALL HAIRPIN (shRNA)
Authors: El-Beirouthi M. , Albornoz M. S. , Martinez-Diaz M. A. , Zadworny D. , Agellon L. B. , Bordignon V.
Published: August 2010

Apolipoprotein E (apo E) is a known risk factor for developing premature atherosclerosis and Alzheimer’s syndrome. The aim of this study was to create a pig model with reduced apo E levels using RNA interference (RNAi) and somatic cell nuclear transfer (SCNT) technologies. Three synthetic small interfering RNA targeting the porcine apo E mRNA were designed, and the knockdown efficiency was assessed in cultured porcine granulosa cells by real-time PCR. The observed apo E knockdown efficiency ranged from 45 to 82% compared with control cells, indicating the targeted degradation of apoE mRNA.A small hairpin RNA (shRNA) expressing vector was constructed in PRNAT.U6.Neo (Genscript Corp., Piscataway, NJ, USA) based on the most effective apo E RNAi sequence under the control of polymerase III (U6) promoter, and then introduced into fetal porcine fibroblast cells. Clones were selected by neomycin treatment and green fluorescent protein (GFP) expression. SCNT was performed using IVM oocytes collected from prepubertal gilts. Oocyte maturation and activation and embryo culture were performed as previously described (Nascimento et al. 2009 Reprod. Domest. Anim. in press). Embryos were cultured in vitro for 5 to 6 days, briefly exposed to fluorescent light to confirm GFP expression, and then surgically transferred into the uterus of recipient gilts. The recipient gilts were synchronized by daily oral administration of altrenogest (20 mg day-1; Regu-Mate®, Intervet, Millsboro, MD, USA) for 12 or 13 days, followed by 1000 IU of eCG injected in the last day of altrenogest treatment and 500 IU of hCG 72 h later. Pregnancy diagnosis was performed by ultrasonography at Day 20 to 25 after embryo transfer, and parturition was induced by injecting PGF2? (10 mg of dinoprost tromethamine; Lutalyse®, Pfizer Canada Inc., Montreal, QC, Canada) at Day 115 of pregnancy. Rates of cleavage (74.7%) and development to the blastocyst stage (37.2%) were comparable with that of embryos reconstructed with nontransfected cells from the same cell line. A total of 309 embryos were transferred to 5 recipients, of which 3 became pregnant and farrowed. Seven live and 1 stillborn piglets were delivered naturally. The presence of the introduced plasmid and the expression of the GFP transgene tag were confirmed by PCR in placental and umbilical tissues of all the piglets. Six cloned pigs have survived after weaning and exhibit no obvious morphological defects. The status of apo E gene expression is currently under investigation.

Supported by a NSERC Discovery Grant to VB.
425 HIGH-LEVEL EXPRESSION OF LEA29Y IN PANCREATIC ISLETS OF TRANSGENIC PIGS
Authors: Klymiuk N. , Baehr A. , Kessler B. , Kurome M. , Wuensch A. , Herbach N. , Wanke R. , Nagashima H. , Wolf E.
Published: August 2010

Among the candidate organs or tissues for pig-to-primate xenotransplantation, pancreatic islets are probably closest to clinical application. Rejection of islet xenografts occurs mainly by cellular mechanisms; that is, T cells. A candidate molecule to protect porcine islets against the attack by human T cells is CTLA-4Ig, which represents the T-cell-inactivating extracellular domain of the human CTLA-4 protein linked to a region of the human immunoglobulin (Ig). This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T-cell proliferation and T-cell-dependent antibody production. The survival of human, rabbit, and porcine islets after transplantation into streptozotocin-treated diabetic mice was found to be prolonged after treatment with CTLA-4Ig. In order to facilitate local protection of pig-to-primate islet xenografts, we generated transgenic pigs expressing LEA29Y, a modification of the original CTLA-4Ig with higher potency, specifically in the pancreatic islets. In LEA29Y, 2 amino acids in the binding region of CTLA-4 are altered. The LEA29Y coding sequence was placed under the control of the 1.3-kb core promoter from the porcine insulin gene (INS), and the polyadenylation signal from the bovine growth hormone gene (GH) was added. The construct was linked with a floxed neomycin resistance cassette and transfected into porcine fetal fibroblasts. The cells were selected and stable clones were pooled and used as donors for nuclear transfer. After electrofusion and activation, embryos were transferred to 2 synchronized gilts; 8 piglets survived to term with 7 of them carrying the transgene. Southern blot analysis suggested that the founder animals contain 1 or 2 independent integration sites. Four founders were autopsied at the age of 3 months to evaluate expression of LEA29Y in the pancreatic islets by immunohistochemistry. The ratio of immunohistochemically stained islet cell profiles to all islet cell profiles in the islet profiles visible in the sections was estimated. The staining intensity was also estimated qualitatively, by grading from weak to strong immunoreactivity (brown color, using DAB as chromogen). Although 2 founders exhibited single LEA29Y-positive islet cells in some pancreatic islet profiles, the other 2 founders showed a high percentage of strongly positive cells in all islet profiles examined, suggesting beta-cell specific expression. Fibroblasts from the latter 2 founders are currently being used for recloning to generate multiple pigs with constitutive expression of LEA29Y in the pancreatic islets. The protective effect of this strategy will be tested by transplanting LEA29Y-expressing porcine islets in diabetic mouse models with a humanized immune system and in diabetic nonhuman primate models.

Supported by the Deutsche Forschungsgemeinschaft (FOR 535).
427 INTEGRATION SITE ANALYSIS IN TRANSGENIC PIGS BY THERMAL ASYMMETRIC INTERLACED (TAIL)-PCR AND JUNCTION PCR
Authors: Kong Q. R. , Liu Z. H.
Published: August 2010

Transgenic animals have been used to study gene function, produce important proteins, xenotransplantation donor, and generate models for the study of human diseases. Recent progress in animal cloning has provided an attractive alternative to improve transgenic efficiency, through the combination of transfection and somatic cell nuclear transfer (SCNT). However, when transgenic animals are produced by SCNT using randomly transfected cells as donor, the integration sites of transgene cannot be predicted. Many methods on the basis of genome walking have been demonstrated to clone transgene integration sites but they are either complicated or inefficient. In the study, we report a PCR-based method, thermal asymmetric interlaced PCR (TAIL-PCR), which relies on a series of 3 nested PCR reactions with transgene specific, designed with melting temperature of about 64, and arbitrary degenerate primers, by control of annealing temperature to efficiently reduce the nonspecific amplification to clone the integration sites in transgenic pigs by SCNT. Junction PCR combined with transgene-specific and integration site primers was performed to confirm the integration sites. Three integration sites were found (1 mapped on chromosome 4; the other 2 met a significant match in the pig expressed sequence tag database) in 2 founder transgenic pigs. Junction PCR resulted in specific amplification bands to identify the integration sites, and segregation of the integration sites was also detected in subsequent progeny by junction PCR analysis. We also used junction PCR combining with transgene-specific 5′ and 3′ integration site primers to analyze zygosity of the integration sites. Besides the specific amplification bands amplifying by transgene specific and integration site primers, bands amplified by 5′ and 3′ integration site primers were obtained to determine the heterozygosity of integration site. In conclusion, this strategy can be efficiently employed to clone transgene integration site and determine zygosity.

This work was supported by grant from the State Transgenic Research Programme of China (Grant No. 2008ZX08006-002).
433 NEW TRANSGENIC PIG DIABETES MODEL EXPRESSING THE MUTANT Insulinc93S FOR THE STUDY OF THE PANCREATIC BETA CELL DYSFUNCTION
Authors: Renner S. , Klymiuk N. , Wuensch A. , Kessler B. , Kurome M. , Nagashima H. , Aigner B. , Wolf E.
Published: August 2010

We previously established a mutant mouse line showing diabetes which was caused by a point mutation in the Insulin 2 (Ins2) gene. The point mutation leads to the amino acid exchange C95S and the loss of the A6-A11 intrachain disulfide bond of insulin. Male heterozygous Ins2C95S mutant mice develop progressive diabetes mellitus with strong reduction of the total pancreatic islet volume and the total beta cell volume together with severe alterations of the beta cell structure. As pigs share many anatomical and physiological characteristics with humans, we aimed to establish a transgenic pig model expressing the mutant insulin by additive gene transfer for the subsequent study of beta cell dysfunction in diabetes mellitus. The transgene consisted of German Landrace insulin sequences including 1.3 kb of the insulin promoter and 1 kb insulin gene sequences with the 3 exons and the T to A (C93S) point mutation in exon 3 analogous to the mutant mouse insulin. A floxed neomycin resistance gene at the 3′ end of the transgene was used as a selection marker. After successful transfection of male fetal fibroblasts of both the German Landrace and the Schwaebisch- Haellisch pig breeds, pooled transgenic fibroblasts were used for somatic cell nuclear transfer (SCNT). Five hundred three reconstructed pig embryos were generated and endoscopically transferred to 5 synchronized recipients. One pregnancy with German Landrace genetic background and one pregnancy with Schwaebisch-Haellisch genetic background of the cloned embryos developed to term, which gave rise to 3 living offspring from each of the 2 pregnancies; 5 of the 6 piglets were transgenic. Southern blot analysis showed different transgene signal patterns in all animals examined. Transgenic pigs of both litters revealed unaltered fasting blood glucose levels up to an age of 8 months. However, disturbed intravenous glucose tolerance and reduced insulin secretion were detected in 1 transgenic pig of the first litter at 8 months of age. The area under the glucose curve of this transgenic pig was 75% larger (22 136 v. 12617) and the area under the insulin curve 53% smaller (1250 v. 2670) compared with the control. Ongoing analyses comprise glucose tolerance tests in the second litter as well as pathohistologic analysis of the pancreata of both litters. Cells from suitable transgenic founders will be used for recloning to establish a new transgenic pig model expressing the mutant InsulinC93S for an in-depth study of pancreatic beta cell dysfunction in diabetes mellitus.

Supported by the Deutsche Forschungsgemeinschaft (GRK1029).
434 REPLICATION COMPETENT LENTIVIRUS (RCL) ANALYSIS IN RECIPIENT ANIMALS OF TRANSGENIC EMBRYOS PRODUCED BY LENTIVIRAL TRANSFER
Authors: Tessanne K. , Yao J. , Cornetta K. , Westhusin M. , Spencer T. , Long C.
Published: August 2010

Lentiviral vectors have become a useful tool for gene therapies and the expression of small hairpin (sh)RNAs to target genes both in vitro and in vivo. This is due primarily to their ability to integrate transgenes into both dividing and nondividing cells, as well as the lack of silencing in the germ cell line. However, the retroviral basis for these recombinant, replication-incompetent viruses has prompted investigation into their safety for use in therapeutics and transgenic animal production. Concerns are that recombination with wild-type viruses or endogenous retroviral elements may allow the integrated provirus genome to become replication competent. In order to investigate this, transgenic embryos produced by lentiviral-mediated gene transfer were transferred into recipient animals. The lentiviral plasmids used in this experiment contained a self-inactivating 3′ untranslated region as well as a green fluorescent protein (GFP) reporter gene (Miyoshi et al. 1998 J.Virol. 72, 8150-8157). Recombinant lentivirus was produced through cotransfection of HEK293T cells with the lentiviral transfer plasmid as well as a packaging plasmid and a plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), which was used to pseudotype viral particles. Two methods were used for production of transgenic embryos. The first was lentiviral transduction of bovine fetal fibroblasts followed by somatic cell nuclear transfer. The second was incubation of IVP hatched ovine blastocysts in culture medium containing infectious recombinant lentiviral particles. Recipients were then sacrificed and analyzed for the presence of replication competent lentivirus (RCL). Tissues collected from each recipient included blood, lung, lymph node, kidney, liver, mammary gland, ovary, skeletal muscle, spleen, and uterus. In addition, when available, fetal and placental samples were collected. Analyses for RCL included a serum ELISA test for presence of the p24 HIV antigen as well as real-time quantitative PCR (qRT-PCR) on genomic DNA for the presence of VSV-G. To date, a total of 13 recipients including both sheep and cattle have been analyzed. All animals had p24 titers below the level of detection for the assay (<12.5 pg mL-1). Additionally, the tissues mentioned above have been analyzed by qRT-PCR for 6 of the 13 recipients so far, and all have been negative for VSV-G as determined by comparison with positive and negative control samples. Additional collections and analysis are ongoing. A lack of detection of RCL in these animals will build confidence in the use of lentiviral vectors in transgenic animal production and will lend support for their safety in both animal and human therapies.
439 OVARY ULTRASOUND EVALUATION OF Santa Inês EWES SUBMITTED TO SHORT DURATION PROTOCOLS WITH PROGESTOGENS ASSOCIATED WITH LECIRELIN AND/OR ESTRADIOL BENZOATE
Authors: Biscarde C. E. A. , Bicudo S. D. , Magalhães L. C. O. , Crocomo L. F. , Bittencourt R. F. , Ferreira D. O. L. , Monteiro C. D. , Oba E.
Published: August 2010

In order to enhance the use of biotechnology in females it is necessary to have good knowledge of the ovarian events, and thus enable the manipulation of the estrus cycle to concentrate ovulations in an established period of time. In this experiment, we evaluated the ovary response and corpus luteum (CL) parameters in a short-duration protocol associated or not to lecirelin (L)(GnRH) and/or estradiol benzoate (EB). Twenty-four cyclic Santa InÊs ewes were divided into 4 groups: Control animals (G1; n = 6) received 45 μg of d-cloprostenol (Prolise®, Tecnopec, Sao Paulo, Brazil) on Day 0. Intravaginal sponges containing medroxyprogesterone acetate (MAP60®, Tecnopec) were inserted on Day 3. Then on Day 7 sponges were removed and 400 IU of eCG and 45 μg of d-cloprostenol were administered. G2 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1; G3 (n = 6) animals underwent the same protocol as G1 but with administration of 25 μg of L (Gestran plus®, Tecnopec) 30 h after sponge withdrawal; G4 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1 and 25 μg of L 30 h after sponge withdrawal. Ewe ovaries were assessed via transrectal ultrasound (US), and CL were measured 4 days after ovulation. On the same day blood samples were taken to measure plasma progesterone levels via radioimmunoassay. Twenty-three out of 24 animals ovulated. One ewe from G4 that did not ovulate was taken out of the experiment. The average size of the largest pre-ovulatory follicle was 7.3 mm (G1), 7.0 mm (G2), 6.8 mm (G3), and 7.4 mm (G4) (P > 0.05). There were 1.8, 1.2, 2.0, and 1.4 ovulated follicles per animal in G1, G2, G3, and G4, respectively. The results for estrus start and duration after sponge withdrawal were G1: 32/38.6; G2: 37/69.3; G3: 29.3/37.3; G4: 44/68 h, respectively. Ovulation time span was G1: 65.3; G2: 88; G3: 53.3; G4: 82.4 h after sponge withdrawal. There was a high correlation of ovary mensuration and progesterone level (r = 0.64; P < 0.0001) on the fourth day after ovulation. Progesterone levels after ovulation were 2.37 ng mL-1 (G1); 1.5 ng mL-1 (G2); 3.22 ng mL-1 (G3) and 1.99 ng mL-1 (G4), being higher in G3 than in G2 (P < 0.05). It was seen that the EB is prejudicial to the CL function. Despite the fact that there was no significant difference of progesterone levels within G1, G3, and G4, animals in G3 displayed higher levels of progesterone; hence, there is a need for further studies using a larger number of animals and fertility test.

CNPq e Tecnopec.
445 RELATIONSHIPS OF LUTEAL PHASE VARIABLES (PRIOR TO AI) WITH FOLLICULAR WAVES IN WATER BUFFALOES
Authors: Kohram H. , Mohammadi G. , Dirandeh E.
Published: August 2010

This study was done to consider relationships of luteal phase variables (prior to AI) with follicular waves. The estrous cycles of 15 buffaloes were synchronized with 2 i.m. injections of prostaglandin F2α given 11 days apart. The buffaloes were randomly assigned to 1 of 3 treatments. Buffaloes in the control treatment received no treatment, whereas G6 buffalos received a GnRH injection between Day 5 and 7 and G16 buffalos received a GnRH injection between Day 15 and 17 of the estrous cycle (estrus = Day 0). Daily, from estrus Day 0 to the next estrus Day 23, buffaloes had their ovaries scanned by ultrasound. Blood samples were collected by tail following each ultrasound examination from estrus until next estrus (estrus = 0). Concentrations of plasma progesterone were determined by radioimmunoassay kit. The limit of detection of the assay was 0.1 45 ng mL-1 and the intra- and interassay coeffients of variation were 7.4% and 9.2%, respectively. Data were analyzed by using PROC GLM of SAS (SAS Institute, Cary, NC, USA). For comparisons between groups, the 2-sample t-test was used for continuous traits, such as size of CL or hormone concentrations. Prospective comparisons of indices of progesterone indicated that the length of luteal lifespan was longer in 3-wave than in 2-wave buffaloes (P < 0.01). Plasma progesterone concentrations were similar at peak and measured as area under the curve on Day 5 through 17 preceding insemination in 2-wave (5.30 ± 0.40 ng mL-1) and 3-wave buffaloes (5.10 ± 0.20 ng mL-1). Length of the luteal phase (defined as from the day of estrus until the last day on which plasma progesterone remained >2 ng mL-1) was <2 days shorter in 2-wave buffaloes than in 3-wave buffaloes (15.20 ± 0.40 v. 17.10 ± 0.50 d; P < 0.05). In addition, the day of peak progesterone occurred earlier in 2-wave buffaloes (13.50 ± 0.30 v. 15.30 ± 0.70 d; P < 0.05).
448 RELATION BETWEEN GESTATIONAL AGE AND ULTRASONOGRAPHIC MEASUREMENTS OF CONCEPTUS PARAMETERS IN ANGLO-NUBIAN GOATS RAISED IN THE BRAZILIAN SEMIARID REGION
Authors: Teixeira D. I. A. , Pinheiro E. S. P. , Sousa F. C. , Pereira A. F. , Freitas V. J. F. , Rondina D.
Published: August 2010

The aim of this study was to determine the relation of gestational age with embryonic/fetal measurements in goats through the real-time ultrasound method. Ultrasonographic examinations began on the 25th day after first mating in eight Anglo-Nubian nulliparous goats, using a Falco 100 ultrasound scanner fitted to 6/8 MHz linear-array and 3.5 MHz convex probe. On recorded scanning images, the embryonic vesicle diameter (EVD) and crown-rump length (CRL), head length (HL), bi-parietal diameter (BPD), thoracic diameter (TD), umbilical cord diameter (UCD), long axis of heart (LAH), and short axis of heart (SAH) and placentomes of embryo/fetus were measured using computer software. The relationships between gestational age and each parameter of the ultrasonic measurements were expressed as straight-line equations and plotted as linear regression using Statistica 7.0 software, being statistically significant at a level of P < 0.05. Mean values were correlated to fetal age, and inverse regression equations were created for each parameter. Results obtained showed the following values: EVD (r = 0.94), CRL (r = 0.94), HL (r = 0.74), BPD (r = 0.70), TD (r = 0.88), UCD (r = 0.73), LAH (r = 0.77), SAH (r = 0.80), and placentome (r = 0.71). Additionally, embryonic size increased from the first detection of the embryo to the end of the study. In conclusion, the ultrasonographic technique is a reliable method to estimate gestational age through the embryonic/fetal measurement once its increase follows a linear regression standard.

CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico - Brasil).
63 ESTABLISHMENT OF GREEN FLUORESCENT PROTEIN EXPRESSED DOG CELL LINES CONTROLLED BY DOXYCYCLINE
Authors: Kim M. J. , Oh H. J. , Park J. E. , Hong S. G. , Kim J. E. , Jang G. , Kim T. A. , Kwon M. S. , Lee B. C.
Published: August 2010

An inducible gene expression system in transgenic animals has been widely used in biomedical science. The aim of this study was to establish green fluorescent protein (GFP) inducible dog cell line and evaluate the system in embryos using interspecies somatic cell nuclear transfer (iSCNT). Canine fetal fibroblasts were transfected with retroviral vector containing GFP, rtTA, and TRE and designated Gteton cell line. For iSCNT, bovine ovaries were collected from a local slaughterhouse and COCs were matured for 24 h. The denuded oocytes were enucleated, injected with Gteton cells, treated with 24 h of doxycycline (DOX), and electrically fused (NEPA GENE, 34 V, 15 μs, 2 pulses). The reconstructed oocytes were activated and then cultured in modified SOF medium. To verify the stability of the Gteton cells, 2 experiments were designed. Experiment 1 was designed to compare the cell size and viability of Gteton and nontransfected cells. Countness™ (Invitrogen, version 1.0, Carlsbad, CA, USA) was used for analysis. In experiment 2, the control of GFP gene expression was observed when the cells were cultured with 1 mg mL-1 of DOX. The cells were also cultured without DOX after 24 h of DOX treatment. Photographs were taken of cultured cells every 12 h. The intensity of GFP expression was analyzed by using Image J freeware (U.S. National Institutes of Health, version 1.42, NIH, Bethesda, MD, USA). To evaluate the reprogramming ability of the Gteton cells in embryos, another 2 experimental designs were planned. Experiment 3 estimated GFP expression in iSCNT embryos when they were cultured with and without DOX. Experiment 4 assessed the development of the iSCNT embryos under microscopy. Data were analyzed using statistical analysis system program (version 9.1, SAS Institute, Cary, NC, USA). In experiment 1, there was no significance (P < 0.05) in average viable cell size (13.7 v. 13.2 μm) or viability (97.0 v. 98.7%). In experiment 2, the GFP intensity increased steadily when cultured in medium containing DOX. The intensity was increased approximately two times after 24 h compared with 12 h of treatment. The intensity after 24 h of DOX treatment decreased to the basal level after 5 days. In experiment 3, the GFP intensity of iSCNT embryos cultured in mSOF containing DOX was increased approximately two times in 16-cell stage compared with 2-cell stage. In experiment 4, the cleavage rate was not significantly different between the 2 groups. In conclusion, we dtermined that the inducible system of Gteton cell line was established in a stable manner. Furthermore the results from iSCNT may indicate the possibility to produce GFP-expressed transgenic puppies controlled by doxycyline.

This study was supported by Korean MEST through KOSEF (grant # M10625030005-09N250300510) and BK21 program, RNL BIO, and Natural Balance Korea.
68 EFFECT OF XENOPUS EGG EXTRACT TREATMENT OF DONOR CELLS ON PORCINE SOMATIC CELL NUCLEAR TRANSFER
Authors: Liu Y. , Østrup O. , Li J. , Vajta G. , Lin L. , Kragh P. M. , Purup S. , Callesen H.
Published: August 2010

Pretreatment of somatic cells to promote subsequent reprogramming during somatic cell nuclear transfer (SCNT) may significantly improve efficiency of the technique. The aim of this study was to evaluate the effect of Xenopus laevis egg extract pretreatment of porcine fetal fibroblast cells using different permeabilization agents prior to SCNT. Fibroblasts were permeabilized using streptolysin O (SLO; 300 ng mL-1, 30 min, 37°C) or digitonin (7 μg mL-1, 2 min, 4°C), and exposed to egg extract for 1 h or 0.5 h, respectively. Cell membranes were resealed in DMEM supplemented with 2 mM CaCl2 for 2 h. After culture for 1, 3, and 5 days (for SLO) or 3 and 5 days (for digitonin), the SLO extract-treated cells (SETC) and digitonin extract-treated cells (DETC) were used as donor karyoplasts for handmade cloning. Controls were SCNT with nontreated cells. Embryos were evaluated for cleavage rate (Day 2), blastocyst rate (Day 6), and total cell numbers of blastocysts. Statistical differences were analyzed by ANOVA. Results are summarized in Table 1. When SETC were used as donors, blastocyst rates were significantly lower compared with the controls, except when the donor cells were cultured for 3 days after treatment. Blastocysts of the latter group also had higher total cell number. With DETC as donors, blastocyst rates and total cell number of embryos at Day 6 reconstructed with cells cultured for 5 days were higher than those in other groups. Results indicate that extract treatment of the donor cells after SLO-permeabilization can give higher number of cells in cloned blastocysts but not improve overall embryo development. However, digitonin treatment for donor cell permeabilization improved both embryo development and cell number of blastocyst. The latter effect was detected only 5 days after the treatment. In conclusion, qualitative efficiency of porcine SCNT could be improved with a combined donor cell permeabilization and extract treatment.

Table 1.Effect of different permeabilization agents prior to SCNT
72 PREGNANCY OUTCOME AFTER OVIDUCTAL TRANSFER OF ZONA-FREE 1-CELL-STAGE PORCINE EMBRYOS PRODUCED BY HANDMADE CLONING
Authors: Ohlweiler L. U. , Mezzalira J. C. , Monaco E. , Mezzalira A. , Bertolini M. , Wilson S. M. , Ringwelski J. , Krisher R. L. , Rund L. A. , Wheeler M. B.
Published: August 2010

The pig is an important animal model for the study of human diseases. An important step for better use of this species in biomedical research is to obtain genetically identical individuals by procedures such as somatic cell nuclear transfer (SCNT). As the in vitro culture environment is usually sub-optimal for embryo development, the oviductal transfer of cloned embryos at the 1-cell stage may be more efficient for the establishment of pregnancies. However, the transfer at such an early stage usually requires the presence of zona pellucida or agar embedding to protect embryos from the recipient’s immune system (Loi et al. 1999 Livest. Prod. Sci. 60, 281-294). This study aimed to evaluate the developmental viability of 1-cell-stage porcine handmade cloned embryos directly transferred to the oviduct of female recipients without the zona pellucida or agar embedding. After 40 h of IVM in TCM-199 +10% follicular fluid, COCs obtained from sows were denuded, selected for the presence of a polar body (459/689), and submitted to a 0.2% pronase solution in 25% fetal bovine serum (FBS) for partial zona removal, followed by rinses in manipulation medium and pure FBS. Subsequent to oocyte splitting by manual bisection in a 5 μg mL-1 cytochalasin B solution (CCB), hemi-oocytes (87.1%) were screened under fluorescent microscopy using Hoechst 33 342 stain, resulting in 57.6% enucleated halves (461/800). A somatic cell culture established from a fetal clone pig biopsy (Adam et al. 2007 Oncogene 26, 1038-1045) at passage 4 was used for embryo reconstruction, which was done in a 0.05% phytohemagglutinin (PHA) solution, by sticking 2 cytoplasts and a somatic cell in a linear orientation. Reconstructed couplets, rinsed in calcium- and magnesium-free fusion medium, were electrofused in a fusion chamber after exposure to a 30-V AC pulse for 20 s for alignment, followed by two 24-μs-long DC fusion pulses of 1.3 kV cm-1. Fused couplets (154/214) were exposed for 10 min to a solution containing 5 μg mL-1 CCB and 10 μg mL-1 cycloheximide, followed by electrical activation (two 24-μs-long DC pulses of 0.9 kV cm-1) in fusion medium containing calcium and magnesium. Activated embryos were cultured in vitro for 12 h in 500 μL of PZM-3 medium in the well of the well (WOW) system, in a plastic bag filled with gas mixture (90% N2, 5% O2, 5% CO2), at 38.5°C. Then, a total of 70 and 80 non-agar-embedded, zona-free 1-cell-stage cloned porcine embryos were transferred directly to the oviducts of a sow and a gilt, respectively, both synchronous at approximately 12 h before ovulation. The recipient sow was diagnosed pregnant by ultrasonography on Day 66 of gestation. Although the sow was diagnosed open on Day 72, this study demonstrates that the transfer of 1-cell-stage zona-free embryos directly to the oviduct of a synchronous sow can result in pregnancy.
75 CHANGES IN GENE EXPRESSION PATTERN DURING PRE- AND PERI-IMPLANTATION STAGES IN BOVINE CLONED EMBRYOS
Authors: Rodriguez-Alvarez L. l. , Cox J. F. , Einspanier R. , Castro F. O.
Published: August 2010

In ruminants after blastocyst formation and before implantation the embryo elongates drastically. This peri-implantation stage is characterized by active proliferation of trophoblastic cells and by changes in gene expression. In cloned embryos inappropriate expression of genes during early stages could be the cause of embryonic losses. The aim of this study was to compare the expression pattern of selected developmentally important genes in Day 7 embryos produced by somatic cell nuclear transfer with gene expression at Day 17 during elongation. IVF embryos at each stage were used for comparison. For analysis we used an RT-PCR approach (conventional + quantitative real-time PCR; qPCR). For nuclear transfer we used handmade cloning with an adult fibroblast cell line that previously yielded live cloned calves. Cells were confluent, non-serum starved in passage 4. Cloned embryos were cultured in SOF medium for 7 days in the well of the well (WOW) system. Semen from a proven bull was used for IVF and the presumptive zygotes were cultured under the same conditions as cloned embryos. At Day 7, embryos (cloned or IVF) were either transferred to recipients or pooled in tens for RNA extraction using NucleoSpin RNA XS Kit (Macherey-Nagel, Düren, Germany), converted to complementary (c)DNA and amplified with specific primers in PCR reactions. Transferred embryos were recovered at elongation stage (Day 17); their RNA was extracted and treated as above. The genes studied were NANOG, FGF4, OCT4, EOMES, IFNtau, and ACTB (internal standard for quantification). Data were analyzed by Kruskal Wallis test using the InfoStat program (Buenos Aires, Argentina). Differences were considered significant at P < 0.05. Development to blastocysts was 65% for clones and 32% for IVF embryos; 19 and 6 elongated embryos were flushed back, respectively. For gene expression analysis we used 5 pools of 10 blastocyts each and 12 (6 IVF; 6 cloned) elongated embryos with embryonic disc. At pre-implantation, we found OCT4, FGF4, IFNtau, and NANOG expressed and their relative levels were quantified (except for NANOG). Expression of OCT4 was higher in the cloned embryos; expression of EOMES was not detected at this stage. At Day 17, all 5 genes were expressed and quantified via qPCR, and interesting differences were found: relative levels of expression of FGF4 decreased in both types of embryos from Days 7 to 17, whereas those for IFNtau, NANOG, and EOMES increased significantly. Expression of OCT4 remained constant in IVF embryos but not in the cloned embryos (higher at Day 7). Our results provide an initial approach to the dynamic changes occurring in the expression of developmentally important genes in the transition from expansion to elongation. The decrease in FGF4 expression as well as the increase in EOMES and NANOG expression is a new finding that could shed light on the mechanisms of trophoblastic differentiation in bovine elongation.
80 USE OF THE SHORT DISPOSABLE NEEDLE SYSTEM FOR FOLLICLE ASPIRATION IN MARES
Authors: Song K. , Lee W. , Chun Y. , Lee I. , Yeon S. , Vanderwall D. K.
Published: August 2010

To achieve success in equine somatic cell nuclear transfer (SCNT), it is important to obtain recipient oocytes of good quality. Transvaginal ultrasoundguided follicle aspiration (TVUFA) is one of the methods to obtain recipient oocytes in equine SCNT, but the commercial long double-lumen needle for TVUFA in large animals is not currently purchasable. The aims of the present study were (1) to compare the recovery rate of short disposable needle system (14G) with that of the long double-lumen needle (12G) and (2) to investigate the developmental competency of recovered oocytes after SCNT and embryo transfer (ET). A real-time ultrasound scanner (Mylab30 vet, Esaote, Italy) equipped with a 7.5-MHz convex array transducer (model EC123) housed in a plastic vaginal device with stainless steel needle guidance was used for TVUFA from pre-ovulatory follicles 25 to 40 mm in diameter on synchronized thoroughbred mares between 6 and 9 years of age. Two types of needles were used: (1) a 12G double-lumen long needle (V-EOAD-1260L; Cook, Brisbane, Australia); (2) a 14G single-lumen disposable needle (2.1 × 80mm; Bovi-vet, Kruuse, Denmark) inserted with 18G inner needle (0.8 × 600 mm) using stainless steel connector and tube. Recovered oocytes were matured in vitro in TCM-199 with 5 mU mL-1 FSH (Folltropin-V, Bioniche, Belleville, ON, Canada) and 10% fetal bovine serum (Sigma, St. Louis, MO, USA) for 12 to 16 h. Matured oocytes were enucleated and electrically fused with equine skin fibroblasts (2.25 kV cm-1, 20μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 1 mM 6-DMAP. Immediately following SCNT procedures, cloned embryos were surgically transferred to the oviducts of recipient mares (n = 2 to 5 embryos per recipient) that had ovulated within 24 hours before the transfer. An initial pregnancy examination was performed using transrectal ultrasonography between Days 14 and 16 (Day 0 = surgery). The recovery rate of the short disposable needle (n = 89, 44.1%) was slightly increased compared with that of the long needle (n = 34, 29.8%), but the difference was not significant. Nineteen SCNT embryos were transferred to 8 mares, and 1 mare is maintaining pregnancy for 60 days. The results of this study demonstrated that our short disposable needle system could be used instead of the commercial long needle and in vivo development of oocyte recovered with the 14G needle could be maintained after nuclear transfer and embryo transfer.
91 MOTILITY (CASA) AND VIABILITY OF FROZEN SPERM AFTER DIFFERENT TIMES OF EXPOSURE AT 15°C
Authors: Garcia Guerra A. , Lüssenhoff M. G. , Brogliatti G. M.
Published: August 2010

One of the key factors for successful long-term cryopreservation in liquid nitrogen is maintaining the samples at -130°C or lower at all times to avoid cell damage (Barth A 1991 Proc. 10th Annu. Conv. Am. ET Assoc., 20-26). Previous data reported that exposure of semen straw to ambient temperature for more than 15 s can raise the temperature above -130°C and could reduce sperm motility by subjective evaluation (Berndston et al. 1976 Proc. 6th NAAB Tech. Conf. Artif. Insem. Reprod., 51-60). The CASA system provides an opportunity to assess multiple motility characteristics on a semen sample objectively and with high repeatability. Two experiments were designed to evaluate the effect of exposing frozen semen in 0.5-mL straws for 15, 30, or 60 s to room temperature on motility characteristics assessed by CASA and viability parameters by vital stain, HOS test, and acrosome integrity. Thirty-three ejaculates from different bulls (88% British breeds) were used for CASA evaluation, and 12 ejaculates were from other bulls (7 Bos indicus and 4 Bos taurus) were used for viability evaluation. All ejaculates were diluted using a chemically semi-defined media (Andromed, Minitub, Germany) and frozen in an automatic freezer (Digicool, IMV, France). Five frozen straws per bull were used, one for each time of exposure and one as control (0 s = 0 time). Straws were exposed to room temperature (15°C ± 1.78) for different times and then placed back into liquid nitrogen. Semen thawing was conduced in a water bath at 37°C during 1 min. Motility characteristics were evaluated by the IVOS Sperm Analyzer (Hamilton Thorne Research). Two chambers of 20 μm depth and 5 fields per chamber were analyzed (30 frames/0.5 s for each field). Six motility parameters were evaluated: % of motile sperm; % of progressive sperm; VAP (path velocity, μm s-1); ALH (lateral amplitude, μm); BCF (beat frequency, Hz); and LIN (linearity, %). Viability characteristics were evaluated by % of live sperm (eosin-nigrosin); % positive to HOS test, and % of intact acrosome (Giemsa stain). A nonparametric AOV (Kruskal Wallis) test was used to compare variables among groups, and results are shown in Table 1. There was a reduction (P < 0.05) in the percentage of motile and progressive sperm when exposure to 15°C was longer than 30 s. The alive cells have similar motile characteristics as VAP, VCL, ALH, BCF, and LIN. The viability of spermatozoa was reduced (P < 0.05) when they were exposed to room temperature beyond 30 s. Also, a lower proportion of positive spermatozoa for HOS test was detected for exposures beyond 15 s. In conclusion, these results suggest sperm motility and viability would not be affected if straws are exposed up to 30 s to 15°C. Further study should be done regarding viability tests.

Table 1.Motility and viability parameters of exposed frozen semen
92 CRYOTIPS: A SIMPLE VITRIFICATION TECHNIQUE FOR SHEEP AND GOAT EMBRYOS
Authors: Gibbons A. E. , Pereyra-Bonnet F. , Cueto M. I.
Published: August 2010

Vitrification procedure for embryos has not been used massively because there is not a standard protocol according to species. We evaluated reproductive efficiency of vitrified embryos in plastic tips of micropipettes. Estrous cycles of 36 Criolla goats (donors, n = 10; recipients, n = 26) and 48 Merino sheep (donors, n = 10; recipients, n = 38) were synchronized by sponges containing 60mgof MAP (Progespon®). At sponge removal (goats, Day 18; sheep, Day 14), recipients received 200 UI of eCG (Novormon®, Syntex, Argentina). Sheep and goat donors were superovulated with a total of 80 (igpFSH (Folltropin V®, Bioniche, Canada) every 12 h in 6 decreasing doses (18, 18, 14, 14, 8, 8) during the last 3 days of progestagen treatment. A dose of eCG (200 UI, Novormon®) was applied at progestagen removal. Donors of both species were inseminated by laparoscopy with frozen/thawed semen (200 × 106 spz) 12 to 14h after the onset of estrus. Embryo recovery was done by surgical prepubic laparotomy, and uterine horns were flushed with PBS + 10% SFB at Day 8 after sponge withdrawal. Before vitrification, embryos were kept in commercial embryo holding medium (Syngro, Bioniche, USA) at 25°C. Vitrification solutions were prepared using holding solution (HS) contents, PBS + 20% FBS. Embryos were maintained at 25°C and processed according to the following procedures: (1) HS + 10% glycerol (G) for 5 min, (2) HS +10% G + 20% ethylene glycol (EG) for 5 min, and (3) HS + 25% G + 25% EG for 30 s. Embryos were aspirated in 1 μL of solution 3, into the center of a plastic micropipette tip (0.36 mm inner diameter), and plunged into a cryotube filled with liquid nitrogen. Before embryo transfer, tips were warmed with fingers for 10 s and immersed in 4 dilution steps during 5 min each with 2.5 mL of HS containing (1) 12.5% G+ 12.5% EG+ 0.5 M of sucrose (S), (2) 0.5 M S, (3) 0.25 M S, and (4) HS alone at 25°C. In both species, at Day 8 after sponge withdrawal, 2 embryos of similar stage were transferred. Under laparoscopy view, the tip of the uterine horn was exteriorized by a nontraumatic clamp, through a 1-cm-long midline incision. The embryos were placed into the uterine lumen after punction and the uterine horn was allowed to return to the abdomen (semi-laparoscopic technique). Pregnancy was diagnosed by ultrasonography (Aloka 500) at Day 28 after being transferred. Reproduction efficiency in both species is shown in Table 1. This technique proves to be simple and efficient for both ovine embryo stages but only for goat blastocysts and can be used by veterinarians in field conditions at a very low cost. Differences of morulae survival between species require further study.

Table 1.Reproductive efficiency after vitrified/warmed embryo transfer in sheep and goats according to the embryo stage
Behavioral dynamics and influence in networked coloring and consensus
Authors: Judd Stephen , Kearns Michael , Vorobeychik Yevgeniy
Published: August 2010

We report on human-subject experiments on the problems of
coloring
(a social differentiation task) and
consensus
(a social agreement task) in a networked setting. Both tasks can be viewed as coordination games, and despite their cognitive similarity, we find that within a parameterized family of social networks, network structure elicits
opposing
behavioral effects in the two problems, with increased long-distance connectivity making consensus easier for subjects and coloring harder. We investigate the influence that subjects have on their network neighbors and the collective outcome, and find that it varies considerably, beyond what can be explained by network position alone. We also find strong correlations between influence and other features of individual subject behavior. In contrast to much of the recent research in network science, which often emphasizes network topology out of the context of any specific problem and places primacy on network position, our findings highlight the potential importance of the details of tasks and individuals in social networks.
Economic choices can be made using only stimulus values
Authors: Wunderlich Klaus , Rangel Antonio , O'Doherty John P.
Published: August 2010

Decision-making often involves choices between different stimuli, each of which is associated with a different physical action. A growing consensus suggests that the brain makes such decisions by assigning a value to each available option and then comparing them to make a choice. An open question in decision neuroscience is whether the brain computes these choices by comparing the values of stimuli directly in goods space or instead by first assigning values to the associated actions and then making a choice over actions. We used a functional MRI paradigm in which human subjects made choices between different stimuli with and without knowledge of the actions required to obtain the different stimuli. We found neural correlates of the value of the chosen stimulus (a postdecision signal) in ventromedial prefrontal cortex before the actual stimulus–action pairing was revealed. These findings provide support for the hypothesis that the brain is capable of making choices in the space of goods without first transferring values into action space.
Polar front shift and atmospheric CO
2
during the glacial maximum of the Early Paleozoic Icehouse
Authors: Vandenbroucke Thijs R. A. , Armstrong Howard A. , Williams Mark , Paris Florentin , Zalasiewicz Jan A. , Sabbe Koen , Nõlvak Jaak , Challands Thomas J. , Verniers Jacques , Servais Thomas
Published: August 2010

Our new data address the paradox of Late Ordovician glaciation under supposedly high
p
CO
2
(8 to 22× PAL: preindustrial atmospheric level). The paleobiogeographical distribution of chitinozoan (“mixed layer”) marine zooplankton biotopes for the Hirnantian glacial maximum (440 Ma) are reconstructed and compared to those from the Sandbian (460 Ma): They demonstrate a steeper latitudinal temperature gradient and an equatorwards shift of the Polar Front through time from 55°–70° S to ∼40° S. These changes are comparable to those during Pleistocene interglacial-glacial cycles. In comparison with the Pleistocene, we hypothesize a significant decline in mean global temperature from the Sandbian to Hirnantian, proportional with a fall in
p
CO
2
from a modeled Sandbian level of ∼8× PAL to ∼5× PAL during the Hirnantian. Our data suggest that a compression of midlatitudinal biotopes and ecospace in response to the developing glaciation was a likely cause of the end-Ordovician mass extinction.