bluuebunny/crossref_metadata_2025 huggingface dataset:
172 PREGNANCY RATES AND EARLY PREGNANCY LOSSES IN POSTPARTUM EQUINE EMBRYO RECIPIENTS
Authors: Demarchi M. E. , Tirone A. M. , Aguilar J. , Losinno L.
Published: August 2010

In commercial equine embryo transfer programs, the cost of recipient mares represents one of the major economical items that increases the final cost of the pregnancies (Douglas 1985; Losinno and Alvarenga 2006). Due to the risks to newborn foal, it is uncommon to use foaling recipients in the large equine embryo transfer programs in Argentina. The objective of this study was to evaluate pregnancy and early embryonic loss rates between foaling and non-foaling recipients in a commercial embryo transfer program. A total of 173 embryos were collected from Arabian donor mares during 3 consecutive breeding seasons, 2006-2009. Donor mare’s ages ranged from 2 to 22 years, and donors were inseminated with either fresh or frozen semen from 8 stallions 2-17 years old and of proved fertility. Uterine flushing was performed on Day 8 to 9 post-ovulation by standard techniques (Douglas 1985) and recovered embryos were transferred nonsurgically. Recipient mares 3-12 years old were selected clinically by rectal palpation and ultrasound. All embryos were transferred to synchronized recipient mares (-1 to +1 according to donor’s ovulation day) and randomly assigned to non-foaling (n = 84) or foaling (n = 89) recipient groups. For the analysis, we first considered pregnancy and early pregnancy loss rates in both groups, and second, the foaling mares were divided in 3 groups according to the days between foaling and transfer: 1) before 30 days postpartum (n = 10), 2) between 31 and 60 days postpartum (n = 30), and 3) after 61 days postpartum (n = 49). Pregnancy diagnosis was performed by ultrasonography on Days 14, 30, and 60 post-ovulation of the donor. Results were evaluated by chi-square test. No statistical differences (P > 0.05) were observed in pregnancy rates between non-foaling and foaling recipients (76.2% and 80.9%, respectively) and in post-foaling groups (80% for recipients transferred before 30 days postpartum, 73.3% for recipients transferred between 31 to 60 days postpartum, and 85.7% for recipients transferred after 61 days postpartum). No statistical differences (P > 0.05) were found in pregnancy loss rates between recipients transferred before 30 and after 61 days postpartum (25% v. 4.8%, respectively) and between recipients transferred before 30 days postpartum and the non-foaling group (25% v. 10.9%, respectively). Pregnancy loss rates for recipients transferred after 31 and 61 days postpartum (9.1% and 4.8%, respectively) were not significantly different with the non-foaling group, and both are within previously reported values. Our results show that postpartum recipients can be used 30 days after normal foaling in a commercial embryo transfer program with similar pregnancy and early pregnancy loss rates and can be a valuable tool maximizing the use of the recipient mares during consecutive breeding seasons.
182 MANAGING FACTORS ASSOCIATED WITH PREGNANCY RATE OF HEIFER RECIPIENTS IN LARGE IN VITRO PRODUCED EMBRYO PROGRAMS
Authors: Scanavez A. L. A. , Pupim F. P. V. , Destro G. R. , Nunes L. O. , Alves B. G. , Oliveira L. Z. , Santos R. M.
Published: August 2010

Large programs of in vitro embryo production have been developed in Brazil, but the results are very variable. The objective of this study was to evaluate managing factors that influence pregnancy rate of recipients in a large embryo transfer program. Results of 1104 embryo transfers performed from November 2008 to February 2009 by Transgen Desenvolvimento e Produção Agropecuária Ltda (Uberlˆndia, Minas Gerais, Brazil) were evaluated. Embryos produced in vitro were used: 1/2 Holstein/Gir embryos (n = 139) or 3/4 Holstein/Gir embryos (n = 961) produced from donors 1/2 Holstein/Gir and Gir with Holstein bull semen. Only excellent (grade 1) quality morulae (stage 4), early blastocysts (stage 5), mid- blastocysts (stage 6), or expanded blastocysts (stage 7) were assigned for fresh transfer using the International Embryo Transfer Society guideline for grading embryos. The heifer recipients were 1/2 crossbred Nelore/Simmental, in good health condition, 20 to 30 months of age, 330 to 400 kg of weight, and were maintained on pasture (Tifton) with mineral supplementation ad libitum. The recipients were synchronized as follows: Day 0-intravaginal device with 1.0 g of progesterone (PRIMER®, Tecnopec, São Paulo, SP, Brazil) +2 mg of estradiol benzoate (ESTROGIN®, Farmavet, São Paulo, SP, Brazil); Day 5-150 μg of D-Cloprostenol (PRELOBAN® Intervet, São Paulo, SP, Brazil) + 400IU of eCG (FOLLIGON®, Intervet); Day 8-progesterone device removed; Day 9-1 mg of estradiol benzoate. On Day 17, nonsurgical embryo transfers were performed by a trained technician and synchronization was confirmed by presence of a CL. The effects of the embryo breed, the number of transfers in each recipient (the heifers that failed to get pregnant were used in the next program), and the transfer sequence (i.e. 20 embryo transfers were performed per hour, and approximately 100 per day) on the pregnancy rate was analyzed by logistic regression with the LOGISTIC procedure of SAS (SAS Institute Inc., Cary, NC, USA). The breed of embryo (56.9% for 3/4 Holstein/Gir embryos v. 62.6% for 1/2 Holstein/Gir embryos), the number of transfers in each recipient (first: 56.5% v. second: 61.8% v. third: 55.5% v. ≥fourth: 55.0%), and the transfer sequence during the day of the program (first hour: 57.4% v. second hour: 60.0% v. third hour: 58.1% v. ≥fourth hour: 53.8%) did not influence the pregnancy rate. Within the confines of an extensive embryo transfer program with in vitro produced fresh embryos, a large number of embryo transfers can be executed per day without adversely affecting the pregnancy rate if high-quality embryos are used, the transfers are performed by a trained technician, and recipients are in good health condition and synchronized.
186 TRANSMISSION OF NONCYTOPATHIC BOVINE DIARRHEA VIRUS BY INFECTED SEMEN TO EMBRYO DONORS AND BY EMBRYOS TO EMBRYO TRANSFER RECIPIENTS AND OFFSPRING
Authors: Bielanski A. , Lalonde A. , Algire J.
Published: August 2010

Bovine diarrhea virus (BVDV) causes a variety of economically important enteric and infertility problems. For that reason, several countries have eradicated the disease and some others have schemes in progress to achieve freedom from it. Although there is a considerable amount of information about the risk of BVDV transmission through contaminated semen used for AI, there is no available evidence to indicate whether the resulting embryos, when used for embryo transfer (ET), can lead to the transmission of BVDV to recipients and offspring. For this experiment, semen from a bull persistently infected with BVDV (105 TCID50/mL of NY strain) was used for insemination (2 times at estrus) of BVDV-seronegative, superovulated heifers (n = 27).All heifers seroconverted to BVDV within 10 days post-insemination. Embryos and unfertilized oocytes were collected nonsurgically (n = 92) or postmortem (n = 52) 7 days post-insemination and were either washed according to IETS recommendations (without trypsin treatment) or left unwashed. In total, out of 144 unfertilized oocytes and embryos collected, 23 (16%) were of ET quality. Most of the embryos were degenerated or unfertilized. On 17 occasions, 1 or 2 washed embryos were transferred to BVDV-seronegative recipients. After ET, all pregnant and nonpregnant recipients remained free of BVDV and antibodies. In total, 6 heifers became pregnant and 5 calves free of BVDV and BVDV antibodies, including 2 sets of twins, have been born to date. Post-insemination, BVDV was detected in 29% (12/41) of unwashed and 10% (4/40) of washed embryos, 100% (4/4) of follicular fluid samples, oviductal epithelial cells, endometrium, and corpora lutea tissues as determined by the virus isolation test. Results herein suggest that BVDV can be transmitted by AI, resulting in the production of some proportion of contaminated embryos. However, it appears that such embryos, when washed according to the IETS guidelines, do not cause BVDV transmission to recipients or their offspring.
218 THE EFFECT OF OVULATING AGENTS ON MATURE FOLLICLES IN BACTRIAN CAMEL (CAMELUS BACTRIANUS)
Authors: Nikjou Darab , Niasari-Naslaji Amir
Published: August 2010

Mature follicle response and CL formation following induction of ovulation using GnRH analogues and LH were investigated in Bactrian camel. Bactriancamels (n = 58) withamature follicle (12-25 mm)received 1) alarelin (Vetaroline®, Aburaihan,Tehran, Iran; 25 μg, i.m. 37/58); 2) buserelin (Receptal®, Intervet, Boxmeer, the Netherlands; 20 μg i.v. 16/58); or 3) LH (Lutropin®-V, Bioniche, Belleville, Ontario, Canada; 25 mg i.v. 5/58; Day 0 of experiment = day of inducing ovulation). Ovarian follicles were considered mature according to previous definition in Bactrian camel (Nikjou D et al. 2008 Theriogenology 69 491-500). Daily ultrasonography was conducted between Days -5 and 15 of the experiment using a real-time ultrasound scanner (Aloka 500, Tokyo, Japan) equipped with a 5-MHz linear array transrectal transducer. The day after inducing ovulation, the size of the mature follicle increased in 89.7% (52/58; 0.72 mm ± 0.11) or decreased in 10.3% (6/58; 0.75 mm ± 0.13) of observations. All mature follicles ovulated by 48 h after treatment. Corpus luteum was detected on Day 4.9 ± 0.13 at the diameter of 14.0 mm ± 0.31 and reached the maximum diameter of 20.2 mm ± 0.58 on Day 9.0 ± 0.13 after treatment. Vacuolated CL was detected in 14% of observations. Corpuralutea started to regress 1 to 2 days after reaching the maximum diameter. In conclusion, mature follicles, at the diameter of 12 to 25 mm, ovulated and normal CL with the short life span (about 10 days) formed consistently in response to the use of ovulating agent in Bactrian camel.

Table 1.Changes in ovarian structures following injection of different GnRH analogues and LH

Research was funded by the Deputy of Research, University of Tehran. The authors thank the companies Bioniche (Canada), Aburaihan (Iran) and Intervet (the Netherlands) for kind provision of pharmaceuticals.
223 PREANTRAL FOLLICLES POPULATION IN BOS TAURUS INDICUS AND BOS TAURUS TAURUS FEMALES
Authors: Silva K. C. F. , Santos G. M. G. , Siloto L.S. , Hertel M. F. , Andrade E. R. , Rubin M. I. B. , Sturion L. , Sterza F. A. M. , Seneda M. M.
Published: August 2010

Bos taurus indicus females provide a greater in vivo oocyte recovery (2-4 times more) in comparision with Bos taurus taurus. This aspect has strongly contributed for the success of the embryo industry in Brazil. Little information is available to explain this difference. Our objective was to test the hypothesis that the difference in oocyte yield is a result of higher numbers of preantral follicles in the ovaries of B. indicus females. Ovaries (n = 44) of fetuses aging from 180 to 240 days and of heifers from 20 and 24 months were collected from Bos taurus indicus (Nelore) and Bos taurus taurus (Aberdeen Angus) females at a slaughterhouse. Fetal age was estimated from the crown-rump length (Evans HE and Sack WO 1973 Anat. Hist. Embryol. 2, 11-45). Ovaries from Nelore fetuses (n = 10) and heifers (n = 12) and Aberdeen Angus fetuses (n = 10) and heifers (n = 12) were cut longitudinally into two halves, fixed in Bouin’s solution, and processed for histological evaluation. The ovarian halves were dehydrated in alcohol, cleared with xylene, embbebed in paraffin, and serially sectioned at 7 μm. Every 120th section was mounted and stained with periodic acid Schiff and hematoxylin. The number of preantral follicles was estimated through the counting of follicles in each section using the nucleus of the oocyte as a marker and a correction factor (Gougeon A and Chainy GBN 1987 J. Reprod. Fertil. 81, 433-442). Only 1 ovary per female was analyzed. Preantral follicles were classified according to the developmental stage as primordial (1 layer of flattened granulosa cells surrounding the oocyte), primary (1 layer of cuboidal granulosa cells), or secondary (2 or more layers of cuboidal granulosa cells), and as normal or degenerated according to their morphological appearance. The number of preantral follicles was not normally distributed and was compared using the Mann-Whitney test. There was no difference (P > 0.05) between the average number of preantral follicles in the ovaries of Bos indicus and Bos taurus females. The average number of preantral follicles per female was 143 929 ± 253 (mean ± SD) and 285 155 ± 570 for Bos taurus and Bos indicus fetuses, respectively, and 76 851 ± 280 and 109 673 ± 293 for Bos taurus and Bos indicus heifers (P > 0.05), respectively. A large variation in numbers of preantral follicles was observed among individuals within the same category and between breeds. Our results suggest that there might be differences in mechanisms controlling follicle development after the preantral stage accounting for the greater oocyte yield from Bos indicus females.

This work was supported by grants from the Universidad Nacional del Litoral (CAI+D program), Universidad Nacional de Lomas de Zamora, the Argentine National Agency for the Promotion of Science and Technology (ANPCyT), and the Argentine National Council for Science and Technology (CONICET).
241 GENE EXPRESSION OF DNMT1 ISOFORMS IN PORCINE OOCYTES, EMBRYOS, AND SOMATIC CELLS
Authors: Giraldo A. M. , Mendicino M. V. , Vance A. M. , Ayares D. L.
Published: August 2010

In human, mouse, and some marsupials, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (DNMT1o) enzyme. The identification and characterization of a similar oocyte transcript variant in farm animals would greatly contribute to the understanding of the methylation processes that occur during nuclear remodeling of in vivo embryos as well as in cloned embryos. The objectives of this study were to identify the alternative splicing variants of DNMT1 in porcine oocytes and determine the gene expression pattern of the different DNMT1 isoforms during embryo development. Total RNA was isolated from a pool of 50 zona free mature porcine oocytes using the Trizol method. A RACE System (Invitrogen, Carlsbad, CA) was used to amplify the 5′ cDNA end of DNMT1. Polymerase chain reaction products were separated by electrophoresis, recovered from the agarose gel, subcloned into a cloning vector, and sequenced. The location of exons and introns of every transcript variant was determined by aligning the 5′RACE-derived sequences to the Sus scrofa DNMT1 genomic sequence located on chromosome 2 (CU462940). RNA levels of the DNMT1 isoforms were analyzed in porcine oocytes and in vivo embryos, as well and in cells from ovary, lung, spleen, liver, heart, and skin by quantitative PCR using the AACT method. DNMT1 protein levels of porcine oocytes and different somatic cells types were analyzed by Western blot. Two new DNMT1o RNA isoforms were identified (EU908730 and EU908731). The previously reported DNMT1s isoform (DQ060156) was expressed at low but constant levels in oocytes, as well as in embryos from the 2-cell to the blastocyst stage. Abundant RNA levels of EU908730 and EU908731 were detected in oocytes and embryos from the 2-cell to the 8- to 16-cell stage. Levels of these DNMT1o transcripts were low at the morula and blastocyst stage. Although DQ060156 was present in all the somatic cell types analyzed, EU908730 and EU908731 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by Western blot analysis, EU908730 and EU908731 RNAs translate one DNMT1o enzyme (ˆ170 KDa). Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, while somatic cells produce only DNMT1s protein. In conclusion, this study demonstrates that porcine oocytes express a DNMT1o RNA in addition to the DNMT1s somatic transcript. The 2 newly identified RNA isoforms are produced by alternative splicing of the DNMT1 gene and translate a DNMT1o protein that is unique to oocytes and early embryos. Analysis of the oocyte and somatic DNMT1 isoforms in pre-implantation embryos will determine the expression pattern of this transcript during genomic methylation and its involvement during nuclear reprogramming and cellular differentiation.
246 ASSOCIATION OF SINGLE NUCLEOTIDE POLYMORPHISMS WITHIN THE BOVINE HEAT SHOCK PROTEIN 70 GENE AND CALVING RATES
Authors: Rosenkrans Jr C. , Roe M. , Brown M. , Johnson Z. , Brown H. , Looper M.
Published: August 2010

Heat shock proteins (Hsp) are induced by various stressors such as heat, cold, toxins, and oxygen deprivation. Our objective was to determine the relationship among polymorphisms in the Hsp70 gene, forage system, and calving rates. Genomic DNA for 77 cows was purified from the buffy coats of EDTA-treated whole blood. The cows were Angus (n = 20), Brahman (n = 26), and reciprocal crosses (n = 31). Cows were assigned to and remained on their respective forage system for the duration of the experiment (8 years). Forage systems were endophyte-infected toxic tall fescue (E+) or common bermudagrass (CB). Specific primers for bovine Hsp70 (HSP1778F: CGCTGGAGTCGTACGCCTTC; HSP2326R: CTTGGAAGTAAACAGAAACGGG) were used for PCR amplification of a 523-base segment (based on GenBank accession number U09861). The PCR product was sequenced in both directions. Seven single nucleotide polymorphisms (SNP) were identified, and they were located at base positions 1851 (n = 6), 1902 (n = 4), 1917 (n = 4), 1926 (n =4), 2033 (n = 20), 2087 (n = 6), and 2098 (n =4). Concentrations of Hsp70, Julian date, and lifetime calving rate were analyzed by ANOVA, with each SNP represented as the main effect in the model. Two SNP resulted in altered peptide sequences, also known as mis-sense mutations (1926, aspartic acid to glutamic acid, and 2033, glycine to alanine). Five unique haplotypes were deduced based on the SNP profile (GCGCGCT, GCGCCCT, ACGCGCT, GCGCGGT, GTTGGCA, respectively, for haplotype 1, 2, 3, 4, and 5). Plasma concentrations of Hsp70 were affected by an interaction (P < 0.05) between Hsp70 haplotype and forage system. Cows with haplotypes 4 and 5 consuming fescue had higher plasma Hsp70 concentrations than other cows (5.4, 5.1, 3.8, 5.1, 5.2, 5.1, 5.7, 4.2, 22.4, and 9 MSE 1.5 ng mL-1, respectively, for 1-5 CB and 1-5 E+). That same interaction tended (P < 0.09) to be associated with lifetime calving percentage. Cows with haplotype 4 consuming bermudagrass had the lowest calving rate (58%). These results suggest that the Hsp70 gene in cattle is polymorphic, and those polymorphisms are related to cattle fertility.
29 SHORT-TERM v. LONG-TERM PROGESTERONE PROTOCOL USING CERVICAL OR INTRAUTERINE FIXED-TIME INSEMINATION IN SHEEP
Authors: Menchaca A. , Vilariño M. , Rubianes E.
Published: August 2010

The short-term protocol with progesterone, prostaglandin F2α (PGF2α), and eCG is used to control follicular dynamics and luteal activity synchronizing the ovulation for fixed-time AI in sheep. The objective of this experiment was to compare the pregnancy rate obtained with short-term protocol (6 d) and long-term protocol (14 d) using cervical or intrauterine fixed-time AI in sheep. Three hundred fifty-two Merino ewes with a body condition score of 2.9 ± 0.3 (mean ± SD; scale 0 to 5) were used during the breeding season (April, 33S, Uruguay). All the females received a CIDR-G (0.3 g of progesterone, InterAg, Hamilton, New Zealand) for 6 d (short-term protocol; n = 178) or 14 d (long-term protocol, n = 174). One imdose of eCG (300 IU, Novormon, Syntex, BA, Argentina) was given at the moment of device withdrawal for the both protocols, and one imdose of PGF2α (10 mg of dinoprost, Lutalyse, Pfizer, New York, NY, USA) was given at the end of the short-term protocol to ensure luteolysis. Cervical AI (short-term protocol, n = 85; long-term protocol, n = 104) or intrauterine AI (short-term protocol, n = 93; long-term protocol, n = 70) was performed 48 or 54 h after device withdrawal, using 200 × 106 or 100 × 106 spermatozoa per ewe, respectively. Fresh semen was extended in UHT skim milk (1000 × 106 spermatozoa mL-1) and used within 1 h of collection. Estrus was recorded twice a day for 4 days after device withdrawal using vasectomized males. Pregnancy diagnosis was performed by transrectal ultrasonography 40 d after AI (5.0 MHz, Aloka, Tokyo, Japan). Logistic regression was used to analyze the effect of the treatment (P < 0.05), the AI technique (P < 0.05), and their interaction (P = NS). Pregnancy rate was higher for the short-term than for the long-term protocol, and for intrauterine than for cervical AI (Table 1). The highest pregnancy rate was achieved with short-term protocol using intrauterine AI (54.8%, 51/93), and the lowest response was obtained with long-term protocol using cervical AI (33.7%, 35/104; P < 0.05). These data were not different from data of short-term protocol using cervical AI or long-term protocol using intrauterine AI (42.4%, 36/85; and 44.3% 31/70, respectively). Ewes in estrus/treated ewes was not different among short-term and long-term protocols (83.7%, 149/178; and 82.8%, 144/174, respectively; P = NS). In summary, regardless of insemination technique, short-term protocol of 6 d enhances pregnancy rate in fixed-time AI programs in sheep.

Table 1.Main effects of short-term (6 d) v. long-term (14 d) protocol using cervical or intrauterine fixed-time AI on pregnancy rate in sheep

Financially supported by Pfizer, SP, Brazil.
357 MOLECULAR AND SUBCELLULAR CHARACTERIZATION OF BOVINE OOCYTES AND THEIR SURROUNDING FOLLICULAR CELLS IN SUBJECT TO THEIR DEVELOPMENTAL COMPETENCE
Authors: Torner H. , Janowski D. , Ghanem N. , Salilew-Wondim D. , Alm H. , Tomek W. , Viergutz T. , Tesfaye D.
Published: August 2010

Though many factors have been shown to affect the oocyte developmental potential, it remains difficult to draw clear and reliable criteria for oocyte selection. With the urgent need for establishing non-invasive means for oocyte selection, the brilliant cresyl blue (BCB) staining test based on glucose- 6-phosphate dehydrogenase (G6PDH) activity has been successfully used to differentiate competent and non-competent bovine oocytes (Alm et al. 2005 Theriogenology 63, 2194-2205). Also it has been hypothesized that there is a correlation between the appearance of light atretic granulosa cells (GC) in the follicle and an increased developmental competence of the oocyte. Here we aim to investigate whether different developmental competent oocytes show differences concerning the degree of apoptosis or in the gene expression pattern of their follicular environment [GC and cumulus cells (CC)]. After follicular aspiration, the immature COCs were separately stained with 26 μM BCB for 90 minutes. Based on their colouration, oocytes were grouped into BCB- (colourless cytoplasm, low developmental competence) and BCB+ (coloured cytoplasm, high developmental competence). The corresponding CC and GC were also grouped according to the colouration of the enclosed oocytes. BCB+ oocytes were found to result in a higher blastocyst rate at Day 8 of in vitro culture (34.1%) compared to BCB- ones (3.9%) (n = 601 COCs). Apoptosis in GC was determined either by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL) or by Annexin-V-staining followed by flow cytometric measurement. The degree of apoptosis in GC of BCB+ oocytes was slightly increased in contrast to the BCB- group (17.0 v. 11.0%; P < 0.05). The abundance and activity of protein kinases Akt, MAP kinase, and Caspase-3 were estimated by western blot analysis. CC, GC, and oocytes from the BCB+ group showed a higher ratio of cleaved Caspase-3/Caspase-3 in contrast to all compartments of the BCB- group (P < 0.05). Moreover, a bovine Affymetrix microarray plate form (Affymetrix Inc., Santa Clara, CA, USA) was used to analyze the gene expression profiles in oocytes, CC, and GC. The BCB+ oocytes were found to be enriched with genes regulating the oocyte maturation (EIF3F, PRKCSH) and the transition from maternal to embryonic genome activation (HMG2L1). Also BCB+ derived follicular compartments showed elevated expression of genes related to steroidgenesis, cumulus expansion and gonadotropins. In conclusion, the results demonstrate that the developmental competence of oocytes is associated with the apoptotic level and altered expression of genes in cells of their follicular environment.

This work was supported financially by Deutsche Forschungsgemeinschaft (To 138/5-1).
44 MUSCLE FATTY ACID COMPOSITION AND LIPOGENIC GENE EXPRESSION IN ADULT BOVINE CLONES AND CONTROL CATTLE
Authors: Berthelot V. , Bernard L. , Richard C. , Chavatte-Palmer P. , Heyman Y.
Published: August 2010

Previous evaluation of milk and meat from clone cattle compared with AI control cows indicated that these products were in the normal range of data, but slight differences were observed in their fatty acid (FA) composition and muscle Δ9-desaturase indexes (Heyman et al. 2007 Animal 1, 936-972). It was therefore hypothesized that epigenic modifications induced by the nuclear transfer technology may affect the expression of the 2 genes [stearoyl-coenzyme A desaturase (SCD)-1, SCD5] responsible for Δ9-desaturation in bovines. The aim of the present experiment was to analyze the differences between clones and controls on FA composition and on SCD1 and SCD5 gene expression of the semitendinosus (ST) muscle. Biopsies of ST were taken from 5 clones from 2 different Holstein genotypes and 5 Holstein AI controls at 26 months of age. Each sample was immediately split into 3 aliquots, frozen in liquid nitrogen, and stored at -80°C until analysis. Fatty acid composition was analyzed by gas chromatography after lipid extraction and methylation according to Bas et al. (2005 Meat Sci. 71, 317-326). Total RNA was isolated from 300 mg of muscle tissue and abundance of SCD1 and SCD5 genes transcripts was determined by RT-PCRas described by Bernard et al. (2005 J. Dairy Sci. 88, 1478-1489) and Lengi and Corl (2007 Lipids 42, 499-508). Results are expressed as percentage of total FA for the FA composition and mRNA abundance of SCD1 and SCD5 determined as relative to the abundance of cyclophilin A mRNA. Statistical analyses were performed using the GLM procedure of SAS (SAS Institute, Cary, NC). Single degree of freedom orthogonal contrasts were used to compare effects of cloning (AI controls v. clones) as well as effects of clone genotypes (genotype 1 v. genotype 2). The C14:0, C14:1 cis-9, C16:0, C16:1 cis-9, and C18:0 proportions in ST were not different between clones and controls. However, clones tended to have a lower proportion of C18:1 n - 9 (-3.1% of total FA; P < 0.07) and higher proportions of C18:2 n - 6 (+1.2% of total FA; P < 0.01), C18:3 n - 3 (+0.7% of total FA; P < 0.05) and n - 3 polyunsaturated FA (+1.17% of total FA; P < 0.05) than controls. Ratios of C14 and C16 Δ9-desaturation in ST were not different between clones and controls but a lower C18 Δ9-desaturation ratio for the clones compared with controls was observed (0.76 v. 0.79; P < 0.05). The mRNA abundance of SCD1 was lower in clone compared with control cows (3.8 v. 8.5; P < 0.05), which could be explained by the higher proportion of n - 3 polyunsaturated FA observed in clones because of the negative effects of these polyunsaturated FA on SCD gene expression. The only difference observed between genotypes was for the C18:0 proportion in muscle (P < 0.05). In conclusion, in our set of animals, cloning decreased the ST muscle gene expression of SCD1 but not of SCD5, which is related to a slight decrease in C18 Δ9-desaturation ratio.
96 RECOVERY RATES OF BOVINE IVP EMBRYOS CRYOPRESERVED BY SLOW FREEZING AND VITRIFICATION (OPS AND SSV) METHODS
Authors: Landim Junior L. P. , Yamazaki L. T. S. , Watanabe O. , Benzi E. C. D. , Corneglian D. P. , Romano M. , Guidorizzi F. J. , Santos G. L. , Yamazaki W.
Published: August 2010

In current commercial bovine in vitro embryo production (IVP) systems, the majority of costs are related to acquisition and preparation of recipient cows, which must be proportional to the produced embryos. Nevertheless, most of the time this relation is not obtained, and one option is the cryopreservation of extra embryos. Unfortunately, there is a large difference in the pregnancy rates of fresh and cryopreserved IVP embryos or the rates of recovery of frozen-thawed embryos according to the technique used for cryopreservation. The aim of this work was to compare recovery taxes (re-expansion and eclosion) of bovine IVP embryos produced following conventional systems, without any specific media or supply for cryopreservation, simulating one condition of extra produced embryos. The COC obtained from abattoir ovaries were matured (TCM-199, supplemented with FCS, LH, FSH, E2, pyruvate, and antibiotic) for 24 h and fertilized (Fert-TALP supplemented with BSA, PHE, and heparin) for 18 to 22 h (Day 0) in vitro. At Day 1, presumptive zygotes were transferred to development media (SOFaa supplemented with BSA and FCS), and at Day 7, grade I embryos were submitted to 3 different cryopreservation methods: slow freezing (ethylene glycol 1.5 M from 6°C to -35°C, 1°C min-1) and vitrification (DMSO, ethylene glycol, and sucrose) by OPS or SSV After the cryopreservation process, all embryos were specifically thawed according to their method and re-cultured in SOFaa for 48 h when the re-expansion and eclosion taxes were evaluated (number of viable embryos after the cryopreservation process). According to results, the taxes of viable embryos cryopreserved by vitrification methods were better than slow freezing, except expanded blastocysts cryopreserved by slow freezing, disproving literature data that show more ability for ice crystal formation in this embryo stage than others due to liquid storage. When the total of embryos is considered in the different methods (n = 1464), the vitrification method was superior to the others, but when new methodologies could be applied aimed at less lipid storage in structures (oocytes and embryos) by gas atmosphere control or total-defined cultured media, better rates can most likely be obtained with the OPS method as SSV or slow freezing.

Table 1.Viable embryo rates after different cryopreservation methods

WTA-Watanabe Applied Technology.
Dysregulation of the mevalonate pathway promotes transformation
Authors: Clendening James W. , Pandyra Aleks , Boutros Paul C. , Ghamrasni Samah El , Khosravi Fereshteh , Trentin Grace A. , Martirosyan Anna , Hakem Anne , Hakem Razqallah , Jurisica Igor , Penn Linda Z.
Published: August 2010

The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents.
Rice yields in tropical/subtropical Asia exhibit large but opposing sensitivities to minimum and maximum temperatures
Authors: Welch Jarrod R. , Vincent Jeffrey R. , Auffhammer Maximilian , Moya Piedad F. , Dobermann Achim , Dawe David
Published: August 2010

Data from farmer-managed fields have not been used previously to disentangle the impacts of daily minimum and maximum temperatures and solar radiation on rice yields in tropical/subtropical Asia. We used a multiple regression model to analyze data from 227 intensively managed irrigated rice farms in six important rice-producing countries. The farm-level detail, observed over multiple growing seasons, enabled us to construct farm-specific weather variables, control for unobserved factors that either were unique to each farm but did not vary over time or were common to all farms at a given site but varied by season and year, and obtain more precise estimates by including farm- and site-specific economic variables. Temperature and radiation had statistically significant impacts during both the vegetative and ripening phases of the rice plant. Higher minimum temperature reduced yield, whereas higher maximum temperature raised it; radiation impact varied by growth phase. Combined, these effects imply that yield at most sites would have grown more rapidly during the high-yielding season but less rapidly during the low-yielding season if observed temperature and radiation trends at the end of the 20th century had not occurred, with temperature trends being more influential. Looking ahead, they imply a net negative impact on yield from moderate warming in coming decades. Beyond that, the impact would likely become more negative, because prior research indicates that the impact of maximum temperature becomes negative at higher levels. Diurnal temperature variation must be considered when investigating the impacts of climate change on irrigated rice in Asia.
Cortical depth-specific microvascular dilation underlies laminar differences in blood oxygenation level-dependent functional MRI signal
Authors: Tian Peifang , Teng Ivan C. , May Larry D. , Kurz Ronald , Lu Kun , Scadeng Miriam , Hillman Elizabeth M. C. , De Crespigny Alex J. , D’Arceuil Helen E. , Mandeville Joseph B. , Marota John J. A. , Rosen Bruce R. , Liu Thomas T. , Boas David A. , Buxton Richard B. , Dale Anders M. , Devor Anna
Published: August 2010

Changes in neuronal activity are accompanied by the release of vasoactive mediators that cause microscopic dilation and constriction of the cerebral microvasculature and are manifested in macroscopic blood oxygenation level-dependent (BOLD) functional MRI (fMRI) signals. We used two-photon microscopy to measure the diameters of single arterioles and capillaries at different depths within the rat primary somatosensory cortex. These measurements were compared with cortical depth-resolved fMRI signal changes. Our microscopic results demonstrate a spatial gradient of dilation onset and peak times consistent with “upstream” propagation of vasodilation toward the cortical surface along the diving arterioles and “downstream” propagation into local capillary beds. The observed BOLD response exhibited the fastest onset in deep layers, and the “initial dip” was most pronounced in layer I. The present results indicate that both the onset of the BOLD response and the initial dip depend on cortical depth and can be explained, at least in part, by the spatial gradient of delays in microvascular dilation, the fastest response being in the deep layers and the most delayed response in the capillary bed of layer I.
Accents in the workplace: Their effects during a job interview
Authors: Deprez‐Sims Anne‐Sophie , Morris Scott B.
Published: August 2010

As the workplace becomes increasingly global, organizations are more likely to employ persons from other countries whose accents clearly identify them as different from the local workforce. Understanding the impact of accents in the workplace is important because accents can be salient in the same way as ethnicity, age, gender, and skin color and may be a source of employment discrimination. The present study looked at the influence of accents on the evaluation of job applicants during an interview for a human resource manager position. Participants from the US were asked to evaluate an applicant with one of three accents (Midwestern US, French, Colombian) by listening to an audiofile. The results showed that the applicant with the Midwestern US accent was evaluated more positively than the applicant with the French accent; however, the applicant with the Colombian accent did not receive an evaluation that differed significantly from those given to the applicants with either the French or the Midwestern US accent. Analyses of process variables indicated that the bias against the French‐accented applicant was mediated by perceived lower similarity. These results are consistent with the similarity‐attraction hypothesis, which states that demographic variables will impact judgments to the extent to which they make the decision‐maker view the applicant as similar or dissimilar. The ability of accent to trigger bias highlights the importance of considering the full array of characteristics that can lead to discrimination in employment settings. Research on employment discrimination has traditionally focused on visual cues such as gender and ethnicity, but in an interview situation, the way the applicant speaks is also important.
Simultaneity and temporal order perception: Different sides of the same coin? Evidence from a visual prior-entry study
Authors: Weiß Katharina , Scharlau Ingrid
Published: August 2010

Attended stimuli are perceived as occurring earlier than unattended stimuli. This phenomenon of prior entry is usually identified by a shift in the point of subjective simultaneity (PSS) in temporal order judgements (TOJs). According to its traditional psychophysical interpretation, the PSS coincides with the perception of simultaneity. This assumption is, however, questionable. Technically, the PSS represents the temporal interval between two stimuli at which the two alternative TOJs are equally likely. Thus it also seems possible that observers perceive not simultaneity, but uncertainty of temporal order. This possibility is supported by prior-entry studies, which find that perception of simultaneity is not very likely at the PSS. The present study tested the percept at the PSS in prior entry, using peripheral cues to orient attention. We found that manipulating attention caused varying temporal perceptions around the PSS. On some occasions observers perceived the two stimuli as simultaneous, but on others they were simply uncertain about the order in which they had been presented. This finding contradicts the implicit assumption of most models of temporal order perception, that perception of simultaneity inevitably results if temporal order cannot be discriminated.
Follow you, Follow me: Continuous Mutual Prediction and Adaptation in Joint Tapping
Authors: Konvalinka Ivana , Vuust Peter , Roepstorff Andreas , Frith Chris D.
Published: August 2010

To study the mechanisms of coordination that are fundamental to successful interactions we carried out a joint finger tapping experiment in which pairs of participants were asked to maintain a given beat while synchronizing to an auditory signal coming from the other person or the computer. When both were hearing each other, the pair became a coupled, mutually and continuously adaptive unit of two “hyper-followers”, with their intertap intervals (ITIs) oscillating in opposite directions on a tap-to-tap basis. There was thus no evidence for the emergence of a leader–follower strategy. We also found that dyads were equally good at synchronizing with the irregular, but responsive other as with the predictable, unresponsive computer. However, they performed worse when the “other” was both irregular and unresponsive. We thus propose that interpersonal coordination is facilitated by the mutual abilities to (a) predict the other's subsequent action and (b) adapt accordingly on a millisecond timescale.
Terrorists brought down the plane!—No, actually it was a technical fault: Processing corrections of emotive information
Authors: Ecker Ullrich K. H. , Lewandowsky Stephan , Apai Joe
Published: August 2010

It is well known that people often continue to rely on initial misinformation even if this information is later corrected and even if the correction itself is remembered. This article investigated the impact of emotionality of the material on people's ability to discount corrected misinformation. The focus was on moderate levels of emotionality comparable to those elicited by real-world news reports. Emotionality has frequently been shown to have an impact upon reasoning and memory, but the generality of this influence remains unclear. In three experiments, participants read a report of a fictitious plane crash that was initially associated with either an emotionally laden cause (terrorist attack) or an emotionally more neutral cause (bad weather). This initial attribution was followed by a retraction and presentation of an alternative cause (faulty fuel tank). The scenarios demonstrably affected participants’ self-reported feelings. However, all three experiments showed that emotionality does not affect the continued influence of misinformation.
Stimulation of ribosomal frameshifting by antisense LNA
Authors: Yu Chien-Hung , Noteborn Mathieu H. M. , Olsthoorn René C. L.
Published: August 2010

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12–18 nt. Antisense oligonucleotides bearing locked nucleid acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.
Sinorhizobium meliloti Requires a Cobalamin-Dependent Ribonucleotide Reductase for Symbiosis With Its Plant Host
Authors: Taga Michiko E. , Walker Graham C.
Published: August 2010

Vitamin B12 (cobalamin) is a critical cofactor for animals and protists, yet its biosynthesis is limited to prokaryotes. We previously showed that the symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti requires cobalamin to establish a symbiotic relationship with its plant host, Medicago sativa (alfalfa). Here, the specific requirement for cobalamin in the S. meliloti–alfalfa symbiosis was investigated. Of the three known cobalamin-dependent enzymes in S. meliloti, the methylmalonyl CoA mutase (BhbA) does not affect symbiosis, whereas disruption of the metH gene encoding the cobalamin-dependent methionine synthase causes a significant defect in symbiosis. Expression of the cobalamin-independent methionine synthase MetE alleviates this symbiotic defect, indicating that the requirement for methionine synthesis does not reflect a need for the cobalamin-dependent enzyme. To investigate the function of the cobalamin-dependent ribonucleotide reductase (RNR) encoded by nrdJ, S. meliloti was engineered to express an Escherichia coli cobalamin-independent (class Ia) RNR instead of nrdJ. This strain is severely defective in symbiosis. Electron micrographs show that these cells can penetrate alfalfa nodules but are unable to differentiate into nitrogen-fixing bacteroids and, instead, are lysed in the plant cytoplasm. Flow cytometry analysis indicates that these bacteria are largely unable to undergo endoreduplication. These phenotypes may be due either to the inactivation of the class Ia RNR by reactive oxygen species, inadequate oxygen availability in the nodule, or both. These results show that the critical role of the cobalamin-dependent RNR for survival of S. meliloti in its plant host can account for the considerable resources that S. meliloti dedicates to cobalamin biosynthesis.
An Experimental Host Range for Triticum mosaic virus
Authors: Seifers Dallas L. , Martin T. J. , Fellers J. P.
Published: August 2010

Triticum mosaic virus (TriMV) is a newly discovered virus isolated from wheat (Triticum aestivum). This study was conducted to determine an experimental host range for TriMV and identify species that could serve as differential hosts for isolating TriMV from Wheat streak mosaic virus (WSMV). Plants tested were mechanically inoculated with the 06-123 isolate of TriMV or the Sidney 81 isolate of WSMV. Some plants were analyzed by enzyme-linked immunosorbent assay (ELISA) using antibodies of TriMV and WSMV. Plants infected with TriMV always produced mosaic symptoms and only extracts of symptomatic plants reacted with antibodies of TriMV. Maize is not a host for TriMV but barley, oat, rye, and triticale are hosts of TriMV. Certain barley and triticale accessions are hosts for TriMV but not WSMV. These plants can be used in combination with maize to separate WSMV and TriMV in plants infected by both viruses. We also showed that 8 wild grass species were susceptible to TriMV and 25 were not. All of the grasses susceptible to infection with TriMV have been reported as susceptible to infection with WSMV. Because of their growth habits, these plant species would be less desirable for use as differential hosts than maize, barley, and triticale.