bluuebunny/crossref_metadata_2025 huggingface dataset:
159 USE OF PHYTOHEMAGGLUTININ-L, AS AN AGGLUTINATOR AGENT, TO PRODUCE CHIMERAS OF IVF BOVINE EMBRYOS
Authors: Emanuelli I. P. , Agostinho B. F. , Mancini M. P. M. , Barros C. M. , Nogueira M. F. G.
Published: August 2010

Embryonic chimeras have been used as a tool to understand embryogenesis and organogenesis, as well as to prove, in vivo, the pluripotency of the embryonic stem cells. One of the techniques used to obtain embryonic chimeras is aggregation, which can be performed with intact or half-embryos and in different stages of the development, produced by in vivo or in vitro systems and in different wells. However, its efficiency tends to reduce when advanced stages, such as morulae and blastocysts, are used. The aim of this work was to evaluate the effect of the treatment with an agglutinating agent (phytohemagglutinin-L; PHA) in the percentage of chimeras produced with IVF bovine embryos. Bovine ovaries (from abattoir) were used to obtain 270 COC that were matured in drops (90 μL) of TCM-199 bicarbonate medium, supplemented with 10% of FCS, and incubated in vitro for 22 to 24 h. The fertilization occurred in TALP-IVF medium, and the COC were maintained in the incubator for 18 h. After fertilization, the presumptive zygotes were transferred to SOF culture medium to in vitro culture. In vitro maturation, fertilization, and culture were performed under 38.5°C, 5% CO2 in air and saturated humidity. The chimerism by aggregation was tested between 2 intact (zona-free) 8- to 16-cell stage embryos in the presence (G1, n = 16) or absence of PHA (G2, n = 14) and between one half-morula and one half-blastocyst with (G3, n = 15) or without PHA (G4, n = 12). The embryos in groups G1 and G3 were treated with PHA in a concentration of 500 μLg mL-1 for 3 min. After PHA treatment, the pairs of embryos were allocated in wells, under previously described culture conditions, until expanded blastocyst stage could be observed (Day 7 of culture). At 24 h of culture, embryonic aggregation pairs were first evaluated to detect only cohesive masses of cells. The results (chimerism rate) were 62.5%, 42.9%, 40.0%, and 25.0%, respectively, for groups G1, G2, G3, and G4. There were no significant differences neither among groups (chi-square, P = 0.252) nor between G1 and G2 (P = 0.464), G3, and G4 (P = 0.683; Fisher’s exact test). Main effects as use of PHA (G1 + G3 v. G2 + G4, P = 0.284) and stage of embryos (G1 + G2 v. G3 + G4, P = 0.183; Fisher’s exact test) were not statistically significant. However, when all groups were compared, the power of the performed test (0.354) was below the desired power of 0.800 (i.e. one must be cautious in over-interpreting the lack of difference among them). In the conditions of this study, it was concluded that the treatment with PHA did not increase the rate of aggregation in the embryonic chimera production, even for half-embryos in advanced stage of development (morulae and blastocysts).

Granted by FAPESP, Brazil: 06/06491-2 and 07/07705-9 (MFGN) and 07/04291-9 (MPMM).
202 EFFECTS OF ASCORBIC ACID ON IN VITRO CULTURE OF CATTLE PREANTRAL FOLLICLES
Authors: Andrade E. R. , van den Hurk R. , Lisboa L. A. , Hertel M. F. , Melo-Sterza F. A. , Moreno K. , Bracarense A. P. F. R. L. , Landim-Alvarenga F. C. , Seneda M. M. , Alfieri A. A.
Published: August 2010

The mechanisms that regulate the gradual exit of ovarian follicles from the nongrowing, primordial pool are poorly understood. The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine the effects of this addition on the growth activation and viability of preantral follicles. The ovarian cortex was divided into small fragments; 1 fragment was immediately fixed in Bouin’s solution (control). The other fragments were cultured for 2, 4, 6, or 8 days on culture plates in minimum essential medium (MEM) supplemented with insulin-transferrin-selenium (ITS), pyruvate, glutamine, hypoxantine, BSA, and antibiotics (MEM+) or in MEM+ plus ascorbic acid (5, 25, 50, 100, or 200 μg mL-1). Ovarian tissue was processed for classical histology, TEM, and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Preantral follicles were classified according to their development stage (primordial, intermediate, primary, and secondary) and on the basis of morphological features (normal or degenerated). Pair-wise comparisons were done using Tukey’s procedure. Chi-square test was used to compare percentages of follicles with PCNA-positive granulosa cells. All analyses were done with Statistical Analysis System (SAS Institute, Cary, NC, USA); P ≤ 0.05 was considered significant. Compared with control fragments, the percentage of primordial follicles was reduced (P ≤ 0.05) and the percentage of growing follicles was increased (P ≤ 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (P ≤0.05), but not when cultures were supplemented with 25, 50, and 100 μg mL-1 of ascorbic acid. Ultrastructural and immunohistochemical analysis of ovarian cortical fragments cultured for 8 days, however, showed the integrity and viability of follicles only when fragments were cultured in the presence of 50 μg mL-1 of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 μg mL-1 not only stimulates the activation and subsequent growth of cattle primordial follicles that are cultured in vitro for 8 days but also safeguards the viability of these preantral follicles.

E. R. Andrade and A. A. Alfieri are recipients of the PRODOC/CAPES fellowship.
217 EFFECT OF FREQUENCY OF MILKING ON POSTPARTUM EVENTS IN SISTANI CATTLE
Authors: Niasari-Naslaji A. , Afsharian A. , Naghzali E.
Published: August 2010

Prolonged postpartum anestrus is a major concern in Bos indicus breeds. Sistani cattle is an Iranian Bos indicus beef breed with more than 5000 years of history in the northwest part of Iran, Sistan. This breed is well adapted to the harsh, arid environment, which includes 120 days of hot winds. Small holders in the Sistan region use Sistani cattle for both meat and milk. The purpose of this study was to investigate the effect of frequency of milking on the postpartum events in this breed. Healthy Sistani cattle (n = 41) with 3.3 ± 0.3 parity and normal parturition were randomly assigned to 3 groups after parturition and were monitored for 3 months. Cows in Groups 1 and 2 were milked once (n = 15) or twice (n = 14) per day, respectively. The control (n = 12) cows were not milked. All cows were allowed to be suckled by their calves once per day. Throughout the 3 months of monitoring after calving, blood samples were collected weekly and assayed for progesterone using RIA. Standing estrus was detected every day at 0700, 1400, and 2100 h. The first rise in progesterone after calving was considered when it was above 1.5 ng/mL. Data were analyzed using GLM procedure in SAS (SAS Institute, Cary, NC). There was no significant difference in the variables measured among groups (P > 0.05); therefore, the data were pooled (Table 1). The number of estrus, the day of the first estrus, and the first rise in progesterone after calving were 2.9 ± 1.42, 31.2 ± 1.77, and 38.8 ± 2.20, respectively (Table 1). Average number of service per conception (1.6 ± 0.12) and days open (94.4 ± 7.63) in Sistani cattle may be considered to be superior reproductive indices to those reported in the majority of Bos indicus breeds. In conclusion, once-daily or twice-daily milking did not influence postpartum reproductive indices of Sistani cattle, indicating that twice-daily milking may be advised to enhance the profitability of small holders. In Sistani cattle, ovarian activity and estrous behavior resumed fairly soon after partition, allowing for earlier conception in this breeds.

Table 1.Postpartum reproductive indices of Sistani cattle with varying frequencies of milking

This research was funded by the Research Deputy of the University of Tehran and Animal Science Research Institute.
258 CLASSIFICATION AND IDENTIFICATION OF THE CLONED HANWOO CALVES DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER USING MS GENOTYPES AND mtDNA POLYMORPHISMS
Authors: Yang B. C. , Hwang S. S. , Oh K. B. , Park C. H. , Park E. W. , Kim D. H. , Lee S. S. , Han S. H. , Park S. B.
Published: August 2010

The calves produced by somatic cell nuclear transfer (SCNT) had identical genetic background except for cytoplasmic transmission molecules, which originated from recipient oocytes. To identify the SCNT calves generated from 3 nucleus donors, genotypes for 16 microsatellite (MS) markers and mitochondrial DNA (mtDNA) polymorphisms were analyzed. Using MS genotypes, the parentage test results were not only classified as the single donor-derived calves but mtDNA sequence variations might also discriminated all SCNT calves within the donor-derived families. Comparing MS genotypes, AI-derived progenies were easily discriminated from each other. However, those genotypes could not supply the useful information for identifying the SCNT calves produced from each donor. Informative sequence variations were detected in several regions including D-loop, 12 S rDNA, and ND5 genes. About 19 nucleotide substitutions found within D-loop allowed individual identification for most SCNT-derived progeny except for 5 individuals. However, further investigation on 12 S rDNA and ND5 genes provided the useful polymorphic information for those 5 individuals. Although the experiment had been carried out to produce SCNT calves without previous investigation of mtDNA polymorphism, polymorphic mtDNA sequences provided interesting information that discriminated individuals, even those from the same donor cells. In addition, we could distinguish the 2nd generations produced by AI combinations using SCNT donors and SCNT progeny as the dams and/or the sires when combined molecular data obtained from MS genotypes and mtDNA polymorphisms were derived. These results suggested that mtDNA polymorphisms might supply the critical information for identification and traceability for SCNT-derived calves when combined with MS data.

This work received grant support from the Agenda Program (No. 200901FHT010305191), Rural Development Administration, Republic of Korea.
265 MATRIX-ASSISTED LASER DESORPTION IONIZATION MASS SPECTROMETRY (MALDI-MS) CHARACTERIZATION OF SPERM LIPID PROFILES OF BULLS WITH DIFFERENT CAPACITIES OF EMBRYO IN VITRO PRODUCTION
Authors: Ferreira C. R , Borges J. C. , Santos L. F. A. , Gozzo F. C. , Franscechini P. H. , Sanvido G. B. , Eberlin M. N.
Published: August 2010

Matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) has been applied to study sperm lipid profiles. Lipids are known to play a crucial role in sperm membrane physico-chemical behavior during cryopreservation. In this work, we show the results of characterization of sperm lipid profiles from 2 bulls with different capacities of in vitro embryo production by MALDI-MS direct analysis. The bull capacities judged by the rate of blastocyst formation after IVP with semen from seven different ejaculates per each animal were 19.1 and 35.3% for bulls 1 and 2, respectively (P < 0.05). For MALDI-MS analysis, frozen semen from each ejaculate was thawed in water at 25°C for 40 s. Sperm was washed 3 times by centrifugatin in 1 mL of PBS at 3000 × g for 10 min. Samples were stored at -20°C in 200 μL of methanol:PBS (vol/vol) solution until analysis. A Synapt HDMS mass spectrometer (Waters Corp., Milford, MA, USA) equipped with a MALDI was used. All spectra were collected for 45 s in the positive ion mode at the mass range of m/z 450 to 1200. The volume of 1 μL of the semen pellet was spotted in the target plate and allowed to dry. Afterward, 1 μL of 2,5-dihydroxybenzoic acid (DHB) was added as matrix. The 50 most intense monoisotopic ions were considered for principal component analysis (PCA). Values of m/z and relative ion intensities were processed using the software Pirouette v.3.11 (Infometrix, Woodinville, WA, USA). Direct MALDI-MS analysis of bulls 1 and 2 spermatozoa with no extraction provided informative spectra containing either [M + Na]+ or [M + H]+ ions characteristic of sphingomyelins, such as m/z 753.6 for SM 18:0, phosphocholines (m/z 780.6 for PC 34 : 2; 782.6 for PC 34 : 1; 806.6 for PC 36 : 6; 808.6 for 36 :2; 828.6 for PC38 : 6; and 830.6 for PC38 : 5), plasmalogens (m/z 790.6 for 1-palmitenyl-2-docosahexanoyl-GPC and 814.6 for 1-palmityl-2-docosaheaenoyl-GPC); and triacylglycerols (m/z 881.7 for sn-glycerol-palmitoleate-oleate-oleate). PCA showed clear separation between bulls 1 and 2 ejaculates, indicating that each bull presented a characteristic and reproducible (from different ejaculates) profiles. Differences in the relative intensities of the ions mentioned above contributed for bulls 1 and 2 differentiation by PCA. PC1 and PC2 explained 86.5% of the data variance. In conclusion, a fast sample preparation protocol followed by MALDI-MS appears to provide characteristic lipid fingerprints for crude spermatozoa (ejaculates) of bulls with different capacities of embryo in vitro production. Experiments involving a larger and more statically relevant set of samples are underway.

We thank the Brazilian research foundations FAPESP (2008/10756-7) and CNPq.
281 INFLUENCE OF DEFINED AND COMPLEX CULTURE MEDIA ON FERTILIZATION AND EMBRYONIC DEVELOPMENT OF IN VITRO-MATURED CAPRINE OOCYTES
Authors: Kharche S. D. , Yadav P. , Goel A. K. , Jindal S. K. , Sharma M. C.
Published: August 2010

Complex media containing serum and/or co-culture with somatic cells result in satisfactory development, although the undefined conditions make it impossible to examine requirements of the embryos. Our objective was to compare defined and complex culture systems for their ability to support normal caprine embryonic development. In total, 3844 selected cumulus oocyte complexes (COC) were used for maturation in TCM-199 containing 10% newborn calf serum (NCS), 3 mg mL-1 BSA, 5 μg mL-1 FSH, 5 μg mL-1 LH, and 1 μg mL-1 estradiol-17β and 10 ng mL-1 epidermal growth factor. After 27 h of maturation, oocytes were separated from cumulus and corona cells by treatment with 0.1% hyaluronidase and by passing through a fine-bore pipette. They were then washed in sperm TALP and fertilized in a drop of fertilization TALP (20% estrous goat serum and 10 μg mL-1 heparin) containing 1 to 2 × 106 spermatozoa per mL. Oocytes-sperm after 18 h of co-incubation were washed in embryo development medium 15 to 20 times and randomly divided into 3 groups. Fertilized oocytes were cultured for 10 days in TCM-199 containing 10% NCS and 4 mg mL-1 BSA (group 1; n = 1511), synthetic oviductal fluid (SOF) containing 10% NCS and 4 mg mL-1 BSA (group 2; n = 1333) or potassium simplex optimization medium (KSOM) containing 10% NCS and 4 mg mL-1 BSA (group 3; n = 1000). The cleavage rate for groups 1, 2, and 3 were 13.6, 11.7, and 31.4%, respectively. All data were analyzed by one-way ANOVA; developmental data were arc sin transformed. The cleavage rate was significantly higher (P < 0.01) for group 3 than for groups 1 and 2. Similarly, embryo development up to morula stage was higher (P < 0.05) in KSOM compared with TCM-199 and SOF. This study shows that good development of embryos can be obtained in a completely defined medium and was better in KSOM than in SOF.
290 CYTOKINE PROFILE IN THE AMNIOTIC FLUID OF NELORE CALVES CONCEIVED BY IN VITRO PRODUCTION AND EMBRYO TRANSFER
Authors: Moya-Araujo C. F. , Preste N. C. , Piagentini M. , Araujo G. H. M. , Silva M. G.
Published: August 2010

Cytokines have several functions in many cells and their appropriate balance in the maternal-fetal connections is necessary to maintain a successful pregnancy. The incidence of fetal problems reported at the delivery moment in cows with conceptus conceived by in vitro production (IVP) and lack of literature in the immunology gestation area in bovine prompted this study. The aim of the present study was to determine the levels of cytokines such as tumor necrosis factor a (TNF-α), interferon-y (IFN-γ), interleukin-6 (IL-6), and IL-8 in amniotic fluid of calves from IVP and conventional embryo transfer at the moment of delivery. Forty animals were used and divided into 2 groups: 1 - Twenty Nelore cross-breed cows pregnant with Nelore calves conceived by conventional embryos transfer method (ET group); 2 - Twenty Nelore cross-breed cows pregnant with Nelore calves obtained by IVP after follicular aspiration (IVP group). Around labor, cows were transferred to a maternal paddock to permit observation of delivery. During the expulsion phase the amnion was punctured and 15 mL of fluid was collected in a plastic tube and stored in a freezer for later analysis. The cytokine levels were measured by immunoenzymatic assay (ELISA). The commercial kits used were ESS0011 (Bovine TNF Alpha), ESS0026 (Bovine IFN-gamma), and ESS0029 (Bovine IL-6) from Pierce Biotechnology™ (Rockford, IL, USA) and kit D8000C (Human IL-8) from R&D Systems™ (Minneapolis, MN, USA). The protocol was performed according to the manufacturer’s recommendations. The densities were evaluated by using an automatic optical reader ELISA. The well plates were read at 450 nm. The ANOVA on ranks was used for statistical analysis because of nonparametric distribution of data, with 5% of significance. No calves in this experiment showed signs of sepsis or death during the peripartum period. There was no significant difference in the concentration of TNF-α between the animals of the ET (250.40 pg mL-1) and IVP (398.70 pg mL-1) groups (P > 0.05). There was no difference statistically for IFN-γ; the medians of this cytokine were 35.7 pg mL-1 for the ET group and 48.22 pg mL-1 for the IVP group (P > 0.05). Only samples 15 and 18 of the IVP group had detectable concentrations of IL-6 (2227.47 and 2686.30 pg mL-1, respectively) and for IL-8 only 15 of the samples in the same group (577.6 pg mL-1). The results for TNF-α and INF-γ in this experiment can be adopted as standards for normal pregnancy in cattle, because there are no reports of such data in the literature. However, additional research in the area of gestation immunology area is necessary to describe the role of cytokines in the maintenance of pregnancy and at the moment of delivery in cattle.
Supported by FAPESP, Brazil.
313 ASSESSMENT OF SPERM DNA FRAGMENTATION IN CANINE EJACULATES USING THE Sperm-Halomax® KIT: PRELIMINARY RESULTS
Authors: Hidalgo M. , Murabito M. R. , Gálvez M. J. , Demyda S. , De Luca L. J. , Moreno M. , Dorado J.
Published: August 2010

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for humans and different mammalian species, using a commercial kit based on the sperm chromatin dispersion (SCD) test; however, a descriptive study in canine semen has not been performed. The aim of this work was to assess the sperm DNA fragmentation in canine ejaculates using the SCD test and 2 different staining techniques. For this purpose, ejaculates were collectedby digital manipulation from4 healthy dogs of different breeds (1 German Pointer, 2 Spanish Greyhounds, and 1 Crossbreed). After collection, the sperm-rich fraction of the ejaculates from 3 dogs were pooled each time (n = 4) and then extended in Dulbecco’s phosphate buffered saline. All the pooled semen samples presented physiological values concerning routine semen parameters (motility, morphology, and sperm concentration). The sperm DNA fragmentation was assessed using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain). Two semen aliquots of the diluted pooled semen samples were processed on each pre-treated slide provided in the kit following the manufacturer’s instructions. The last step was the staining technique. We stained each slide with 2 different staining procedures. The first half of the slide was stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) mixed in a proportion 1 : 1 with an antifading solution. The second half of the slide was stained for 15 min in Wright solution (1.01383.0500, Merck, Whitehouse Station, NJ, USA) 1 :1 in Phosphate Buffer pH 6.88 (1.07294.1000, Merck). The stained slides were observed using fluorescence and light microscopy, respectively. Five hundred sperm per slide were counted. Spermatozoa with fragmented DNA showed a large and spotty halo of chromatin dispersion. Unfragmented sperm only showed a small and compact halo. Statistical analyses were performed using the Statistical Package for Social Science version 12.0 (SPSS Inc., Chicago, IL, USA). The sperm DNA fragmentation index was compared between Wright and fluorescence staining methods by ANOVA. Results were expressed as mean ± standard error of the mean. The first report of the sperm DNA fragmentation index in canine ejaculates was 2.26 ± 0.53% for Wright staining and 1.99 ± 0.10% for fluorescence technique. No differences were found between staining procedures. In conclusion, it was possible to assess the sperm DNA fragmentation of canine ejaculates using 2 different staining procedures, expecting that continuous research could be useful in defining the role of DNA fragmentation SCD test in canine semen evaluation and cryopreservation.
34 OVINE SPERM IS HIGHLY SUSCEPTIBLE TO ATTACK BY HYDROGEN PEROXIDE
Authors: Perez E. G. A. , Nichi M. , Oliveira Neto F. A. , Silva R. O. C. , Dalmazzo A. , Viana C. H. C. , Pesce D. M. C. , Góes P. A. A. , Ferreira G. H. , Barnabe V. H. , Barnabe R. C.
Published: August 2010

Ram sperm membrane displays a particular lipid composition, especially regarding the high quantity of polyunsaturated cholesterol. This trait improves membrane fluidity; however, the spermatozoa become more susceptible to the attack of reactive oxygen species (ROS), which may lead to structural and functional damage, impairment or even impeded fecundity. The aim of the present experiment was to study the resistance of ovine spermatozoa to different ROS. Sperm samples from 4 rams were collected using an artificial vagina. Sperm samples were then incubated (1 h, 37°C) with four ROS inducer mechanisms: xanthine/xanthine oxidase (produces superoxide anion), hydrogen peroxide (4 mM), ascorbate/ferrous sulfate (4 mM; produces hydroxyl radical), and malondialdehyde (MDA, lipid peroxidation product). Samples were analysed using the 3-3′ diamino benzidine (DAB) stain as an index of mitochondrial activity, the eosin nigrosin stain as an index of membrane integrity; the simple stain (fast green/Bengal rose) as an index of acrosome integrity; and the measurement of thiobarbituric acid reactive substances (TBARS) as an index of lipid peroxidation. Results showed that acrosome and membrane integrity as well as mitochondrial potential were highly impaired by hydrogen peroxide, which was not the case for the other ROS (Table 1). Surprisingly, TBARS production was higher in samples incubated with ascorbate and ferrous sulfate (hydroxyl radical). Furthermore, sperm showing impaired mitochondrial potential were negatively correlated with membrane and acrosome integrities (r = -0.83, P < 0.0001 and r = -0.62, P = 0.01, respectively). Results of the present experiment suggest that semen of rams is extremely susceptible to attack by hydrogen peroxide. However, the mechanism by which this substance impairs sperm quality apparently does not involve oxidative stress, because no increase in TBARS was observed. Despite the necessity of further studies to investigate how hydrogen peroxide negatively influences sperm function, the use of catalase and glutathione peroxidase, important hydrogen peroxide scavengers, appears to be an alternative to improve the quality of ram sperm.

Table 1.Effect of different reactive oxygen species in semen of rams

The authors thank Nutricell for the media used in the experiment and CAPES for financial support.
343 PHOSPHATIDYLINOSITOL 3-KINASE IS INVOLVED IN THE MAINTENANCE OF METAPHASE II ARREST IN PORCINE OOCYTES MATURED IN VITRO
Authors: Kashiwazaki N. , Shimada M. , Ito J.
Published: August 2010

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study, the effect of a PI3K inhibitor, LY294002, on the mitogen-activated protein kinase (MAPK) and p34cdc2 kinase activities of matured porcine oocytes was examined. Immature oocytes were collected from ovaries and cultured in modified NCSU37 up to 48 hr. After culture, cumulus cells were removed and oocytes were cultured up to 24 h in medium supplemented with 25 or 50 μM LY294002. Groups of 10 or 20 oocytes were collected at each culture period for in vitro kinase assay of p34cdc2 kinase and MAPK, respectively. Groups of 40 oocytes were also used for detection of PKB phosphorylation by Western blotting. After maturation culture, both the p34cdc2 kinase and MAPK activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 μM LY294002 did not affect either the p34cdc2 kinase or MAPK activities, 50 μM LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34cdc2 kinase activity. Next, the effect of 10 μM Ca2+ ionophore which was reported as inducing a transient decrease of p342+ kinase but not MAPK activities, was examined in LY294002-treated oocytes. Pronuclear formation of the oocytes was also evaluated by the aceto-orcein staining. By additional treatment with LY294002 after Ca2+ ionophore, both the MAPK and p34cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is possibly involved in the maintenance of MAPK activity in matured porcine oocytes.
The work was supported in part by Grant-in-Aid for Scientific Research from JSPS (KAKENHI) (21789253) to J.I. This work was also supported in part by the Promotion and Mutual Aid Corporation for Private Schools of Japan through a Grant-in-Aid for Matching Fund Subsidy for Private Universities to J.I. and N.K.
356 MODULATION OF PORCINE NUCLEAR MATURATION STATUS USING A SPECIFIC PHOSPHODIESTERASE INHIBITOR
Authors: Sutton-McDowall M. L. , Gilchrist R. B. , Thompson J. G.
Published: August 2010

Compared to other livestock species, IVM porcine COCs have poor developmental competence.Thisismost likely due to poor cytoplasmic maturation and asynchronous nuclear maturation. The resumption of nuclear maturation is largely regulated by cyclic adenosine monophosphate(cAMP), with increasing intra-oocyte levels prolonging cumulus-oocyte gap junction communication and delaying meiotic resumption. Modulation of cAMP levels using phosphodiesterase (PDE) inhibitors during bovine IVM significantly improves developmental competence (Thomas et al. 2004 Biol. Reprod. 71, 1142-1149). Hence, the aim of this study was to determine the effect of a type 3 PDE inhibitor (cilostamide, CIL) supplementation during porcine IVM on nuclear maturation, using a defined culture system. COCs derived from follicles of prepubertal gilts were cultured in groups of 10 in 100μL IVM medium (VitroMat, IVF Vet Solutions, Adelaide, Australia) +100 m IU mL-1 rhFSH +4 mg mL-1 BSA and nuclear maturation was assessed using orcein dye. Exp. 1: IVM medium was supplemented with 0, 5, 10, and 20 μM CIL or 0, 0.01, 0.1, 1, and 10 μM CIL and nuclear maturation was determined at 24 h and 44 h. Exp. 2: COCs were cultured in ± 0.1 μM CIL and nuclear maturation was assessed at 24 h, 44 h, 48 h, 52 h, and 56 h. Proportions of COCs at each stage of nuclear maturation were arcsine transformed and differences determined using a general linear model and Bonferroni post hoc test. Four replicates per experiment were performed with 20 COCs used for each treatment and/or time point. Results for Exp. 1 revealed no differences in the rate of nuclear maturation after 24 h of culture. After 44 h, 77% of COCs incubated in the absence of CIL were at metaphase II (MII) compared to 35-45% MII when cultured in the presence of 1, 5, 10, or 20 μM CIL (P < 0.001). Furthermore, CIL supplementation resulted in approximately half the COCs arresting at germinal vesicle stage (GV) after 44 h of culture. While there were no significant differences in the MII rates of COCs cultured in 0, 0.01, and 0.1 μM CIL, significantly more COCs were at metaphase I (MI) at 44 h compared to control COCs (0 μM CIL, P < 0.05). The time course experiment (Exp 2) demonstrated that nuclear maturation was delayed by 12 h with 0.1 μM CIL, compared to the absence of CIL, with comparable MII rates being achieved at 56 h for 0.1 μM CIL (72%) and 44 h for controls (0 μM CIL = 73%).
These results demonstrate that porcine oocyte maturation can be induced in vitro by FSH in the presence of a low dose of type 3 PDE inhibitor resulting in a delay in the resumption and completion of nuclear maturation. Further investigations are underway to determine if CIL treatment prolongs gap junction communication and improves the developmental competence of porcine oocytes.

This work was supported by the National Institutes of Health (USA) and National Health and Medical Research Council (Australia).
388 OPTIMIZATION OF INDUCED PLURIPOTENT STEM CELL GENERATION FROM BOVINE FIBROBLASTS
Authors: Lim M. L. , Sumer H. , Liu J. , Verma P. J.
Published: August 2010

Difficulties associated with isolation and culturing bovine embryonic stem (ES) cells has led to the exploration of alternative methods for generating pluripotent stem cells. The viral delivery of reprogramming factors Oct4, Sox2, cMyc, and Klf4 has resulted in generation of induced pluripotent stem cells (iPS) in rodent, human, rhesus monkey, and pig somatic cells. In the current study, we improved the efficiency of retroviral transduction of bovine adult fibroblasts (BAF) for the generation of bovine iPS cells. Bovine adult fibroblasts were transduced with 4 human factors: Oct 4, Sox 2, cMyc, and Klf4. To determine transfection efficiency, pMXs-GFP was used as a reporter. The effect of change in cell density of the Platinum A retroviral packaging cell line (Plat A), cell densities of the target BAF, and infection regimes on the transfection rates was examined. A reduction in Plat A cell density from 8 × 106 to 2 × 106 did not alter transfection rates. Reduced target cell density from 4 × 105 to 4 × 104 (10-fold) improved the transfection rates from 0.31 to 7.06%, P < 0.001 (n = 3). Subjecting the BAF to 2 sequential rounds of viral transduction further improved the transfection rates to 13.88%, P < 0.001 (n = 3). These preliminary results suggest that optimizing the density of target cells can greatly improve transduction outcomes. Following viral induction with the 4 reprogramming factors, putative bovine iPS colonies were observed when the transfection rate was >1%. The putative bovine iPS cells were cultured in alpha-minimal essential medium supplemented with 20% FCS and 10 ng mL-1 human leukemia inhibitory factor. These putative bovine iPS colonies had mouse ES-like morphology, were multilayered, and had high nucleus-to-cytoplasmic ratio. They stained positive for alkaline phosphatase activity. The colonies were manually passaged onto mitomycin C-inactivated mouse embryonic fibroblasts every 5 to 7 days but could only be expanded for a limited number of passages. Other strategies are currently being explored to improve stable reprogramming of BAF such as epigenetic modification of cells, lentivirus-mediated transduction, and investigation of media suitable to maintain putative bovine iPS colonies for further characterization including RT-PCR or immunohistochemical detection for pluripotent markers and in vivo differentiation ability.

Acknowledgments are given to Dairy Australia.
402 SUPEROVULATION OF TROPICAL-ADAPTED BOS TAURUS AND BOS INDICUS COWS IN A TROPICAL ENVIRONMENT
Authors: Alvarez R. H. , Martinez A. C. , Pires R. M. L.
Published: August 2010

Breed differences in ovarian function were found among beef Bos indicus and Bos taurus cows maintained in a subtropical environment (Alvarez P et al. 2000 J. Anim. Sci. 78, 1291-1302). The aim of this study was to compare ovarian response to superovulation and embryo production of tropical-adapted Bos taurus and Bos indicus cows. The experiment was carried out in a tropical wet climate at the experimental station of Instituto de Zootecnia (latitude 22°46′S, longitude 47°17′W) from November to February (average maximum temperature = 30.0 ± 0.8°C and average absolute precipitation = 153.1 ± 78.8 mm3). Forty Caracu (a local Bos taurus breed) and 50 Nelore (Bos indicus breed) lactating cows were treated with an intravaginal device containing progesterone (1.38 mg; CIDR-B®, Pfizer Animal Health, Montreal, Québec, Canada) and 2.5 mg i.m. of estradiol benzoate (Estrogin®, Farmavet, São Paulo, Brazil). Four days later, the animals were superovulated with multiple i.m. injections of 400 IU of FSH (Pluset®, Calier, Spain) in decreasing doses (75-75, 75-50, 50-25, and 25-25 IU) at 12-h intervals over 4 days. The CIDR-B® device was removed 3 days after the first superovulatory injection and cows received i.m. 150 μg of cloprostenol (Veteglan®, Calier, Spain). Cows were inseminated 48 and 62 h after the cloprostenol injection and embryos were recovered nonsurgically 7 days after insemination. Differences in the number of CL (assessed by ultrasound scanning), total number of ova/embryos, and number of transferable embryos were analyzed by ANOVA. Differences in the number of animals with low response (<3 CL) to superovulation were analyzed by chi-square test. All donors (with the exception of 1 Caracu and 2 Nelore) with ovarian response >3 CL showed estrus at insemination. Three (8.9%) Caracu and 5 (10.0%) Nelore cows had <3 CL following the superovulation treatment (P = 0.68). There was no difference (P > 0.05) in the mean (± SEM) CL counts of Caracu (11.4 ± 3.3) and Nelore (12.0 ± 4.1) cows. Similarly, there were no differences (P > 0.05) between Caracu and Nelore cows for total number of ova/embryos collected (8.6 ± 2.6 v. 9.0 ± 4.3) or transferable embryos (6.0 ± 2.4 v. 5.1 ± 2.9). In conclusion, the superovulation of Caracu and Nelore cows carried out in a tropical climate resulted in similar ovarian responses and embryo production.
Supported by FAPESP.
Implementation of quantitative bushfire risk analysis in a GIS environment
Authors: Atkinson Dale , Chladil Mark , Janssen Volker , Lucieer Arko
Published: August 2010

Bushfires pose a significant threat to lives and property. Fire management authorities aim to minimise this threat by employing risk-management procedures. This paper proposes a process of implementing, in a Geographic Information System environment, contemporary integrated approaches to bushfire risk analysis that incorporate the dynamic effects of bushfires. The system is illustrated with a case study combining ignition, fire behaviour and fire propagation models with climate, fuel, terrain, historical ignition and asset data from Hobart, Tasmania, and its surroundings. Many of the implementation issues involved with dynamic risk modelling are resolved, such as increasing processing efficiency and quantifying probabilities using historical data. A raster-based, risk-specific bushfire simulation system is created, using a new, efficient approach to model fire spread and a spatiotemporal algorithm to estimate spread probabilities. We define a method for modelling ignition probabilities using representative conditions in order to manage large fire weather datasets. Validation of the case study shows that the system can be used efficiently to produce a realistic output in order to assess the risk posed by bushfire. The model has the potential to be used as a reliable near-real-time tool for assisting fire management decision making.
Local prolactin is a target to prevent expansion of basal/stem cells in prostate tumors
Authors: Rouet Vincent , Bogorad Roman L. , Kayser Christine , Kessal Karima , Genestie Catherine , Bardier Armelle , Grattan David R. , Kelder Bruce , Kopchick John J. , Kelly Paul A. , Goffin Vincent
Published: August 2010

Androgen-independent recurrence is the major limit of androgen ablation therapy for prostate cancer. Identification of alternative pathways promoting prostate tumor growth is thus needed. Stat5 has been recently shown to promote human prostate cancer cell survival/proliferation and to be associated with early prostate cancer recurrence. Stat5 is the main signaling pathway triggered by prolactin (PRL), a growth factor whose local production is also increased in high-grade prostate cancers. The first aim of this study was to use prostate-specific PRL transgenic mice to address the mechanisms by which local PRL induces prostate tumorogenesis. We report that (
i
) Stat5 is the major signaling cascade triggered by local PRL in the mouse dorsal prostate, (
ii
) this model recapitulates prostate tumorogenesis from precancer lesions to invasive carcinoma, and (
iii
) tumorogenesis involves dramatic accumulation and abnormal spreading of p63-positive basal cells, and of stem cell antigen-1–positive cells identified as a stem/progenitor-like subpopulation. Because basal epithelial stem cells are proposed to serve as tumor-initiating cells, we challenged the relevance of local PRL as a previously unexplored therapeutic target. Using a double-transgenic approach, we show that Δ1–9-G129R-hPRL, a competitive PRL-receptor antagonist, prevented early stages of prostate tumorogenesis by reducing or inhibiting Stat5 activation, cell proliferation, abnormal basal-cell pattern, and frequency or grade of intraepithelial neoplasia. This study identifies PRL receptor/Stat5 as a unique pathway, initiating prostate tumorogenesis by altering basal-/stem-like cell subpopulations, and strongly supports the importance of further developing strategies to target locally overexpressed PRL in human prostate cancer.
Sleep homeostasis in the rat is preserved during chronic sleep restriction
Authors: Leemburg Susan , Vyazovskiy Vladyslav V. , Olcese Umberto , Bassetti Claudio L. , Tononi Giulio , Cirelli Chiara
Published: August 2010

Sleep is homeostatically regulated in all animal species that have been carefully studied so far. The best characterized marker of sleep homeostasis is slow wave activity (SWA), the EEG power between 0.5 and 4 Hz during nonrapid eye movement (NREM) sleep. SWA reflects the accumulation of sleep pressure as a function of duration and/or intensity of prior wake: it increases after spontaneous wake and short-term (3–24 h) sleep deprivation and decreases during sleep. However, recent evidence suggests that during chronic sleep restriction (SR) sleep may be regulated by both allostatic and homeostatic mechanisms. Here, we performed continuous, almost completely artifact-free EEG recordings from frontal, parietal, and occipital cortex in freely moving rats (
n
= 11) during and after 5 d of SR. During SR, rats were allowed to sleep during the first 4 h of the light period (4S
+
) but not during the following 20 h (20S
−
). During the daily 20S
−
most sleep was prevented, whereas the number of short (<20 s) sleep attempts increased. Low-frequency EEG power (1–6 Hz) in both sleep and wake also increased during 20S
−
, most notably in the occipital cortex. In all animals NREM SWA increased above baseline levels during the 4S
+
periods and in post-SR recovery. The SWA increase was more pronounced in frontal cortex, and its magnitude was determined by the efficiency of SR. Analysis of cumulative slow wave energy demonstrated that the loss of SWA during SR was compensated by the end of the second recovery day. Thus, the homeostatic regulation of sleep is preserved under conditions of chronic SR.
Filtration of submicrometer particles by pelagic tunicates
Authors: Sutherland Kelly R. , Madin Laurence P. , Stocker Roman
Published: August 2010

Salps are common in oceanic waters and have higher per-individual filtration rates than any other zooplankton filter feeder. Although salps are centimeters in length, feeding via particle capture occurs on a fine, mucous mesh (fiber diameterd∼0.1 μm) at low velocity (U= 1.6 ± 0.6 cm·s−1, mean ± SD) and is thus a low Reynolds-number (Re ∼10−3) process. In contrast to the current view that particle encounter is dictated by simple sieving of particles larger than the mesh spacing, a low-Re mathematical model of encounter rates by the salp feeding apparatus for realistic oceanic particle-size distributions shows that submicron particles, due to their higher abundances, are encountered at higher rates (particles per time) than larger particles. Data from feeding experiments with 0.5-, 1-, and 3-μm diameter polystyrene spheres corroborate these findings. Although particles larger than 1 μm (e.g., flagellates, small diatoms) represent a larger carbon pool, smaller particles in the 0.1- to 1-μm range (e.g., bacteria,Prochlorococcus) may be more quickly digestible because they present more surface area, and we find that particles smaller than the mesh size (1.4 μm) can fully satisfy salp energetic needs. Furthermore, by packaging submicrometer particles into rapidly sinking fecal pellets, pelagic tunicates can substantially change particle-size spectra and increase downward fluxes in the ocean.
Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H
2
O
2
and superoxide dismutase activity
Authors: Mesquita Ana , Weinberger Martin , Silva Alexandra , Sampaio-Marques Belém , Almeida Bruno , Leão Cecília , Costa Vítor , Rodrigues Fernando , Burhans William C. , Ludovico Paula
Published: August 2010

The free radical theory of aging posits oxidative damage to macromolecules as a primary determinant of lifespan. Recent studies challenge this theory by demonstrating that in some cases, longevity is enhanced by inactivation of oxidative stress defenses or is correlated with increased, rather than decreased reactive oxygen species and oxidative damage. Here we show that, in
Saccharomyces cerevisiae
, caloric restriction or inactivation of catalases extends chronological lifespan by inducing elevated levels of the reactive oxygen species hydrogen peroxide, which activate superoxide dismutases that inhibit the accumulation of superoxide anions. Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases.
Structural basis for high-affinity HER2 receptor binding by an engineered protein
Authors: Eigenbrot Charles , Ultsch Mark , Dubnovitsky Anatoly , Abrahmsén Lars , Härd Torleif
Published: August 2010

The human epidermal growth factor receptor 2 (HER2) is specifically overexpressed in tumors of several cancers, including an aggressive form of breast cancer. It is therefore a target for both cancer diagnostics and therapy. The 58 amino acid residue Z
her
2 affibody molecule was previously engineered as a high-affinity binder of HER2. Here we determined the structure of Z
her
2 in solution and the crystal structure of Z
her
2 in complex with the HER2 extracellular domain. Z
her
2 binds to a conformational epitope on HER2 that is distant from those recognized by the therapeutic antibodies trastuzumab and pertuzumab. Its small size and lack of interference may provide Z
her
2 with advantages for diagnostic use or even for delivery of therapeutic agents to HER2-expressing tumors when trastuzumab or pertuzumab are already employed. Biophysical characterization shows that Z
her
2 is thermodynamically stable in the folded state yet undergoing conformational interconversion on a submillisecond time scale. The data suggest that it is the HER2-binding conformation that is formed transiently prior to binding. Still, binding is very strong with a dissociation constant
K

D

 = 22 pM, and perfect conformational homogeneity is therefore not necessarily required in engineered binding proteins. A comparison of the original Z domain scaffold to free and bound Z
her
2 structures reveals how high-affinity binding has evolved during selection and affinity maturation and suggests how a compromise between binding surface optimization and stability and dynamics of the unbound state has been reached.
Entrainment of disrupted circadian behavior through inhibition of casein kinase 1 (CK1) enzymes
Authors: Meng Qing-Jun , Maywood Elizabeth S. , Bechtold David A. , Lu Wei-Qun , Li Jian , Gibbs Julie E. , Dupré Sandrine M. , Chesham Johanna E. , Rajamohan Francis , Knafels John , Sneed Blossom , Zawadzke Laura E. , Ohren Jeffrey F. , Walton Kevin M. , Wager Travis T. , Hastings Michael H. , Loudon Andrew S. I.
Published: August 2010

Circadian pacemaking requires the orderly synthesis, posttranslational modification, and degradation of clock proteins. In mammals, mutations in casein kinase 1 (CK1) ε or δ can alter the circadian period, but the particular functions of the WT isoforms within the pacemaker remain unclear. We selectively targeted WT CK1ε and CK1δ using pharmacological inhibitors (PF-4800567 and PF-670462, respectively) alongside genetic knockout and knockdown to reveal that CK1 activity is essential to molecular pacemaking. Moreover, CK1δ is the principal regulator of the clock period: pharmacological inhibition of CK1δ, but not CK1ε, significantly lengthened circadian rhythms in locomotor activity in vivo and molecular oscillations in the suprachiasmatic nucleus (SCN) and peripheral tissue slices in vitro. Period lengthening mediated by CK1δ inhibition was accompanied by nuclear retention of PER2 protein both in vitro and in vivo. Furthermore, phase mapping of the molecular clockwork in vitro showed that PF-670462 treatment lengthened the period in a phase-specific manner, selectively extending the duration of PER2-mediated transcriptional feedback. These findings suggested that CK1δ inhibition might be effective in increasing the amplitude and synchronization of disrupted circadian oscillators. This was tested using arrhythmic SCN slices derived from

Vipr2
−/−

mice, in which PF-670462 treatment transiently restored robust circadian rhythms of PER2::Luc bioluminescence. Moreover, in mice rendered behaviorally arrhythmic by the

Vipr2
−/−

mutation or by constant light, daily treatment with PF-670462 elicited robust 24-h activity cycles that persisted throughout treatment. Accordingly, selective pharmacological targeting of the endogenous circadian regulator CK1δ offers an avenue for therapeutic modulation of perturbed circadian behavior.
Carbohydrate-binding modules promote the enzymatic deconstruction of intact plant cell walls by targeting and proximity effects
Authors: Hervé Cécile , Rogowski Artur , Blake Anthony W. , Marcus Susan E. , Gilbert Harry J. , Knox J. Paul
Published: August 2010

Cell wall degrading enzymes have a complex molecular architecture consisting of catalytic modules and noncatalytic carbohydrate-binding modules (CBMs). The function of CBMs in cell wall degrading processes is poorly understood. Here, we have evaluated the potential enzyme-targeting function of CBMs in the context of intact primary and secondary cell wall deconstruction. The capacity of a pectate lyase to degrade pectic homogalacturonan in primary cell walls was potentiated by cellulose-directed CBMs but not by xylan-directed CBMs. Conversely, the arabinofuranosidase-mediated removal of side chains from arabinoxylan in xylan-rich and cellulose-poor wheat grain endosperm cell walls was enhanced by a xylan-binding CBM but less so by a crystalline cellulose-specific module. The capacity of xylanases to degrade xylan in secondary cell walls was potentiated by both xylan- and cellulose-directed CBMs. These studies demonstrate that CBMs can potentiate the action of a cognate catalytic module toward polysaccharides in intact cell walls through the recognition of nonsubstrate polysaccharides. The targeting actions of CBMs therefore have strong proximity effects within cell wall structures, explaining why cellulose-directed CBMs are appended to many noncellulase cell wall hydrolases.