bluuebunny/crossref_metadata_2025 huggingface dataset:
Bidimensional Particle-In-Cell simulations for laser-driven proton acceleration using ultra-short, ultra-high contrast laser
Authors: Scisciò M. , D'Humières E. , Fourmaux S. , Kieffer J. C. , Palumbo L. , Antici P.
Published: December 2014

In this paper, we report on bi-dimensional Particle-In-Cell simulations performed in order to reproduce the laser-driven proton acceleration obtained when a commercial 200 TW Ti:Sa Laser hits a solid target. The laser-to prepulse contrast was enhanced using plasma mirrors yielding to a main-to-prepulse contrast of ∼1012. We varied the pulse duration from 30 fs to 500 fs and the target thickness from 30 nm to several tens of μm. The on-target laser energy was up to 1.8 J leading to an intensity in excess of 1020 W cm−2. A comparison between numerical and existing experimental data [S. Fourmaux et al., Phys. Plasmas 20, 013110 (2013)] is performed, showing a good agreement between experimental results and simulations which confirms that for ultra-thin targets there is an optimum expansion regime. This regime depends on the target thickness and on the laser intensity: if the target is too expanded, the laser travels through the target without being able to deposit its energy within the target. If the target is not sufficiently expanded, the laser energy is reflected by the target. It is important to note that maximum proton energies are reached at longer pulse durations (in the 100 fs regime) than what is currently the best compression pulse length for this type of lasers (typically 20–30 fs). This duration, around 50–100 fs, can be considered a minimum energy transfer time between hot electrons to ions during the considered acceleration process.
Electrostatic turbulence intermittence driven by biasing in Texas Helimak
Authors: Toufen D. L. , Pereira F. A. C. , Guimarães-Filho Z. O. , Caldas I. L. , Gentle K. W.
Published: December 2014

We investigate changes in the intermittent sequence of bursts in the electrostatic turbulence due to imposed positive bias voltage applied to control the plasma radial electric field in Texas Helimak [K. W. Gentle and H. He, Plasma Sci. Technol. 10, 284 (2008)]—a toroidal plasma device with a one-dimensional equilibrium, magnetic curvature, and shear. We identify the burst characteristics by analyzing ion saturation current fluctuations collected in a large set of Langmuir probes. The number of bursts increase with positive biasing, giving rise to a long tailed skewed turbulence probability distribution function. The burst shape does not change much with the applied bias voltage, while their vertical velocity increases monotonically. For high values of bias voltage, the bursts propagate mainly in the vertical direction which is perpendicular to the radial density gradient and the toroidal magnetic field. Moreover, in contrast with the bursts in tokamaks, the burst velocity agrees with the phase velocity of the overall turbulence in both vertical and radial directions. For a fixed bias voltage, the time interval between bursts and their amplitudes follows exponential distributions. Altogether, these burst characteristics indicate that their production can be modelled by a stochastic process.
Evolution of chirped laser pulses in a magnetized plasma channel
Authors: Jha Pallavi , Hemlata  , Mishra Rohit Kumar
Published: December 2014

The propagation of intense, short, sinusoidal laser pulses in a magnetized plasma channel has been studied. The wave equation governing the evolution of the radiation field is set up and a variational technique is used to obtain the equations describing the evolution of the laser spot size, pulse length and chirp parameter. Numerical methods are used to analyze the simultaneous evolution of these parameters. The effect of the external magnetic field on initially chirped as well as unchirped laser pulses on the spot size, pulse length and chirping has been analyzed.
The Deep Space Network at 50
Authors: Lazio Joseph , Deutsch Les
Published: December 2014

From rovers on the surface of Mars to Voyager 1 near the edge of the solar system, spacecraft regularly call home to Earth. For five decades, the Deep Space Network has been at the other end of the line.
Limited application of irrigation water does not affect the nutritive characteristics of lucerne
Authors: Rogers M. E. , Lawson A. R. , Chandra S. , Kelly K. B.
Published: December 2014

Lucerne (Medicago sativa) has not been a significant part of the feedbase of the Murray-Dairy Region of Australia. However, under future climate regimes, which are indicating an overall decline in rainfall as well as water available for irrigation of pastures, lucerne is emerging as a potential forage species because of its adaptability, productivity and resilience. A large-scale field experiment was conducted at Tatura, Victoria, Australia, over four years to determine the dry matter (DM) production and nutritive characteristics of lucerne under a range of limiting and non-limiting irrigation management practices. Nine irrigation treatments were imposed that ranged from full irrigation to no irrigation in either a single, or over consecutive, irrigation seasons. Plots were harvested using plant-based criteria over the irrigation season and DM production and nutritive characteristics were determined. DM production generally increased as the total water supply (irrigation plus rainfall plus changes in soil water) increased, with the fully irrigated treatments producing significantly more DM than the treatments where the irrigation water had been restricted. Mean annual DM digestibility concentration was 66% of DM and was not affected by treatment. Similarly, mean crude protein concentration, which was 22% of DM, was also not affected by treatment. There was no consistent effect of restricted irrigation on the neutral detergent fibre concentration of the lucerne plants. These results suggest that in seasons where irrigation is restricted because of limited water supply, the reduction in lucerne productivity is not associated with a penalty in forage nutritive characteristics, with lucerne able to provide sufficient nutrients to form a large proportion of the total diet for a high-producing dairy cow.
Effects of calcium salts of medium-chain fatty acids on plasma metabolite and hormone concentrations in early lactating dairy cows
Authors: Sugino T. , Tateno A. , Ueno G. , Kawashima K. , Okimura T. , Hirabayashi H. , Suzuki A. , Asakuma S. , Kobayashi H. , Isobe N. , Obitsu T. , Kushibiki S.
Published: December 2014

To elucidate the effects of medium-chain fatty acids (MCFA) on milk production and plasma metabolite and hormone concentrations in early lactating dairy cows, 10 multiparous Holstein dairy cows were randomly assigned to two dietary treatment groups after parturition. One group was fed a diet supplemented with calcium salts of MCFA (MCFA-Ca) for 8 weeks after parturition, while the other group was fed the same diet without the supplement (control). MCFA-Ca, containing 60% caprylic acid and 40% capric acid, was added to a total mixed ration (TMR) at 1.5% of the dietary dry matter (DM). Cows were offered the TMR ad libitum. DM intake, daily gain in bodyweight, milk yield, milk fat content and milk protein content did not differ between the two treatment groups. The MCFA-Ca diet decreased plasma glucose and triglyceride concentrations (P < 0.05), while plasma concentrations of total and free cholesterols tended to increase (P < 0.10). Plasma ghrelin was maintained at a higher concentration (P < 0.05) in cows fed the MCFA-Ca diet than in the control group. Relative to the control diet, the MCFA-Ca diet decreased plasma insulin concentration (P < 0.05) and numerically increased plasma glucagon concentration, resulting in a lower insulin : glucagon ratio (P < 0.05). In conclusion, plasma metabolite and hormone concentrations were affected by the MCFA-Ca diet, suggesting that MCFA-Ca supplementation may change endocrine functions and nutrient metabolism in early lactating cows, ultimately resulting in an enhanced catabolic state.
The effect of maternal arginine supplementation on the development of the thermogenic program in the ovine fetus
Authors: McCoard S. , Wards N. , Koolaard J. , Salerno M. Senna
Published: December 2014

Brown adipose tissue (BAT) is a specialised fat store that is metabolised by the newborn lamb to ensure effective adaptation to the cold challenge of the extra-uterine environment. Increasing BAT reserves therefore has the potential to increase neonatal thermogenesis and survival. It is established that arginine supplementation can increase fetal BAT stores but the biological mechanisms involved are unclear. The objective of this study was to test the hypothesis that increased fetal BAT stores resulting from maternal arginine supplementation is mediated by activation of the thermogenic program. Brown adipose tissue was collected from fetuses of ewes supplemented with arginine from 100 to 140 days of gestation. Increased peri-renal fat stores in fetuses from arginine-supplemented ewes was associated with an increase in uncoupling protein 1 (UCP-1) and PRD1-BF-1-RIZ1 homologous domain containing protein-16 expression, but not proliferator-activated receptor gamma or proliferator-activated receptor gamma-co-activator-1α in BAT. The activity of UCP-1 is regulated by hormones including cortisol and thyroid hormones. Cortisol level in fetuses from supplemented sheep was 68% greater than those from control ewes, indicating that cortisol may control upregulation of UCP-1 expression in the ovine neonate. The DNA and RNA concentration in BAT of both groups suggest that increased peri-renal fat stores is not associated with an increase in cell number or number of ribosomes, but rather an increase in the size of individual fat cells. Collectively, these results indicate that maternal arginine supplementation during mid to late gestation improved the thermoregulatory ability of lambs and this could potentially increase their survival in early life.
Effects of site of assessment and variation in wool quality traits on the tactile assessment of textural greasy wool handle
Authors: Preston J. W. V. , Hatcher S. , McGregor B. A.
Published: December 2014

Variation in textural greasy wool handle across the fleece of Merino sheep was explored. Results demonstrate that there were significant differences across nine sites on the fleece. The inclusion of the covariates fibre diameter, fibre diameter co-efficient of variation, staple length, dust penetration, staple structure, greasy wool colour and resistance to compression, helped to explain some of the variation in textural greasy wool handle between sites such that site of assessment was no longer a significant factor (P = 0.065). However, in practice, those involved with clip preparation or sheep selection based on textural greasy wool handle assessment are unlikely to have these measurements available. The results highlight the importance of nominating a site of assessment when recording textural greasy wool handle.
119 A SHORT PERICONCEPTIONAL MATERNAL HYPERGLYCEMIA IS SUFFICIENT TO DISRUPT THE FETO-PLACENTAL PHENOTYPE IN A RABBIT MODEL
Authors: Rousseau-Ralliard D. , Tarrade A. , Thieme R. , Brat R. , Aubrière MC , Dahirel M. , Rolland A. , Daniel N. , Fournier N. , Boileau P. , Duranthon V. , Navarrette-Santos A. , Fischer B. , Chavatte-Palmer P.
Published: December 2014

Pre-gestational type 1-diabetes (T1D) increases the risk of miscarriage and congenital malformations and programs the offspring to develop metabolic syndrome in adulthood. Management of maternal diabetes is essential during gestation but could be also highly important around the time of conception. Using a rabbit model, the effects of maternal T1D during the periconceptional period on pre-implantation blastocysts have been well documented, but the effects on feto-placental phenotype at 28 dpc (term = 31 days) has not been explored. Diabetes was induced by Alloxan in dams 7 days before mating. Glycemia was maintained at 15 to 20 mmol L–1 with exogenous insulin injections. At 4 dpc, embryos were collected and transferred into nondiabetic recipients. At 28 dpc, control (C) and diabetic (D) fetuses were collected for biometric records, placental analyses including stereology and gene expression, and lipid profiles of feto-placental tissues by gas chromatography. Lipid data were analysed by principal component analysis. D-fetuses were growth retarded, hyperglycemic, and dyslipidemic compared with C fetuses. Moreover, placental efficiency was much higher in D- than in C-fetuses. The volume density of fetal vessels was significantly decreased in D-placentas compared to C-placentas, whereas the volume density of trophoblast tended to increase (P = 0.051). This morphometric disruption was associated with a deregulation of the expression of genes related to nutrient supply and lipid metabolism. In fetal plasma, a specific fatty acid signature was observed in D- and C-groups. Moreover, the composition of placental and fetal liver membranes differed according to maternal status and fetal sex. Tissues from D-fetuses contained significantly more n-6 polyunsaturated fatty acids compared with C. Docosahexaenoic acid decreased whereas linoleic acid increased in the cardiac membranes of D-fetuses, indicating a higher risk of ischemia. This study demonstrates that exposure to high plasma glucose during the short periconceptional period is sufficient to adversely program fetal phenotype by reducing fetal growth, altering placental function and lipid profiles in all fetal tissues.
148 EXPRESSION OF ARTEMIN IN THE BOVINE ENDOMETRIUM, UTERINE FLUID, AND EARLY EMBRYOS
Authors: Muñoz M. , Martín D. , Caamaño J. N. , Carrocera S. , Gómez E.
Published: December 2014

Artemin, a member of the glial cell line-derived neurotrophic factor (GDNF) family, is expressed in human and mice pre-implantation embryos and reproductive tract (Li et al. 2009 FEBS Lett. 583; Kawamura et al. 2012 PLoS One 7). In mice, artemin promotes in vitro embryo development and decreases apoptosis (Li et al. 2009 FEBS Lett. 583). The presence of artemin in cattle embryos and endometrium, however, is unknown. In this work we analysed artemin expression in bovine blastocysts and endometrium by immunohistochemistry and in uterine fluid (UF) by Western blot (WB). Briefly, Day-6 in vitro-produced (IVP) embryos (n = 50) were nonsurgically transferred to the uterus of heifers (n = 10, 50 IVP embryos per heifer) at nonconsecutive oestrus cycles. On Day 8, embryos and their corresponding diluted UF were flushed; blastocysts that developed entirely in vitro were also collected. In addition, endometrial samples were collected on Day 8 from slaughtered females that were embryo transferred (n = 6) and sham transferred (n = 6) on Day 5. Artemin localization was investigated in blastocysts and in endometrial samples, using immunohistochemical staining methods described elsewhere (Muñoz et al. 2012 J. Proteome Res. 11; Gómez et al. 2014 Reproduction pii: REP-14–0304). The signal-strength comparisons between uterus-exposed and IVP blastocysts were analysed using the software Confocal Uniovi Image-J. Quantification of WB protein bands was achieved by computer-assisted densitometry using Image-J software. Artemin was detected, with similar intensity, in the inner cell mass and trophectoderm from both uterus-developed and IVP blastocyst. All embryos analysed expressed artemin. The signal intensity and staining pattern observed did not differ between uterus-exposed and IVP blastocysts. In the endometrium, the most intense staining for artemin was localised to the apical sites in the luminal epithelium and in the glandular epithelium of superficial glands. There was also diffuse staining in the stroma and deep uterine glands. The uterine region and pregnant or cyclic status did not affect the artemin staining pattern. Artemin was detected by WB in all UF samples analysed (embryo transferred N = 10, sham transferred N = 10). However reliable quantitation of artemin by WB was unfeasible due to the broad dynamic range of artemin expression through samples. In conclusion, our results demonstrate the presence of artemin in bovine uterine endometrium and UF, and embryos during early development. As shown in mice, it is feasible that artemin might exert an autocrine/paracrine role during early embryo development in the cow.
The study received grant support: Spanish Ministry of Science and Innovation (MINECO, project AGL2012–37772 and FEDER). M. M. was supported by grant MICINN-RYC08–03454. The authors are members of the COST Action FA1201 Epiconcept.
151 REPRODUCTIVE PARAMETERS OF MINI-HORSE MARES
Authors: Uliani R. C. , Ramires-Neto C. , Alvarenga M. A.
Published: December 2014

The Mini-horse is a new equine breed that is on the rise in Brazil. These are animals with a maximum adult height of 98 cm for females and 93 cm for males, with a well-proportioned body. Those animals are easily handled by children and require small spaces with low maintenance costs. The Mini-horse is under constant genetic selection that is aimed mainly at decreasing its height. For this reason, mares of low stature are required, justifying their use in assisted reproduction programs. Reproductive data of these animals are not available in the literature. Only data of full-size mares are referenced, not taking into account the particularities of this breed. Therefore, the aim of this study was to describe one technique of embryo transfer (ET) for Mini-horse mares. Reproductive rates obtained during the 2012/2013 Brazilian breeding season are presented. Thirteen Mini-horse donor mares were monitored daily by transrectal ultrasonography and ovulation induction was performed with injection of deslorelin acetate or hCG, along with natural breeding using the same stallion for all mares. Eight days after detection of ovulation, flushing for embryo collection was performed. The recipient mares were also monitored daily to detect ovulation and those selected ovulated in the range of up to four days after the donor mare. In order to prevent cervical injuries, the large catheter traditionally employed in uterine washing of large mares was replaced by a Foley catheter (24 Fr in diameter). A range of 50 to 250 mL of Ringer's lactate solution was used to fill the uterus up to 5 times or until the embryo was observed in the collector filter. After the embryo collection, the donor mare received a dose of luteinizing drug (sodic cloprostenol – Sincrocio®, Ouro Fino, Brazil). The recipient mare was examined for a corpus luteum, absence of uterine fluid or endometrial oedema, and good cervical and uterine tone. Recipients were also selected by height (similar to donors) so that the newborn foal could reach the recipient mare's udder to nurse. For non-surgical transfer, the embryos were aspirated by an artificial insemination pipette with ~0.5 mL of holding medium interspersed with small columns of air and then inserted into the uterine body of the recipient mares. Daily follicular growth was 2.2 ± 1.0 mm and the size of follicles at ovulation induction was 31.6 ± 2.8 mm. In 57 embryo collections performed, 46 embryos were obtained and transferred into the uteri of recipient mares, resulting in 35 pregnancies. The embryo recovery and pregnancy rate were 80.7% and 76.1% respectively. The relationship between pregnancy and number of embryo flushes performed was 61.4%. The time between two consecutive flushes was 20 ± 4.5 days. The rate of embryonic loss between 20 and 60 days was 20%. The easy handling of the animals and the high rates of ET program success were promising. However, information regarding full-size mares should be used with caution, respecting the specific characteristics of this breed.
16 BIOCHEMICAL ANALYSIS OF COMPONENT IN SEMINAL GEL SECRETED WITH BOAR SEMEN
Authors: Takahashi G. , Maeda M. , Kimura Y. , Funahashi H.
Published: December 2014

Seminal gel (SG), a part of semen, of the boar originates from secretions from the Cowper's gland and has a high viscosity and water-holding capacity, preventing backflow of semen at natural mating. However, there are is little information available about biochemical and functional characteristics of boar SG. In this study, as a first step to elucidate the chemical features of the SG, we examined the structure of O-glycans and the primary structure of protein from the boar SG. Seminal gel was collected from ejaculated semen of a Berkshire boar with high fertility and freeze-dried. Samples were preserved in a refrigerator until experiments were conducted. For Exp. 1 the presence of O-glycans in SG was confirmed by detection of the amino sugar, galactosamine (GalNH2), from acid hydrolysis of GalNAc. The freeze-dried SG (1 mg) was hydrolyzed with 4N trifluoroacetic acid at 110°C for 2 h. The resulting amino sugar was labelled with phenyl isothiocyanate (PITC) and then analysed by RP-HPLC. The GalNAc was detected as a main amino sugar, suggesting that the SG contains O-glycosylated glycoprotein. For Exp. 2 the O-glycans were prepared from the freeze-dried SG (5 mg) by hydrazinolysis at 100°C for 2 h. After N-acetylation, the O-glycans were pyridylaminated. The structures were identified by anion-exchange HPLC, size-fractionation HPLC, glycosidase digestion, and ESI-MS and MS/MS analysis. Almost all glycans were digested by α2–3,6-sialidasae, indicating that these O-glycans are sialylated and give the glycoproteins viscosity. Furthermore, the MS analysis showed that the de-sialylated O-glycans consist of HexNAc-PA (m/z 300.0) and Hex-HexNAc-PA (m/z 462.0) and major glycans are di- or tri-saccharides. For Exp. 3 proteins in the SG were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition with 5% 2-mercaptoethanol. Proteins were stained with Coomassie Brilliant Blue R-250. Three bands (~160, 140, and 70 kDa) were found on 7.5% polyacrylamide gel, but two bands (160, 140 kDa) were converted to ~130 kDa after the sialidase digestion, indicating that native two proteins (160 and 140 kDa) may be highly sialylated. For Exp. 4 internal amino acid sequence was analysed using one of the peptic peptides. The freeze-dried SG (5 mg) was digested with porcine pepsin in 5% formic acid at 37°C for 3 h. The resulting peptides were separated by RP-HPLC. N-terminal sequence of one of the peptic peptides was WSEKYGIPGGKAH. The amino acid sequence showed a high homology with tyrosine-protein kinase ZAP-70. These results suggest that boar SG contains mucin-like glycoproteins carrying heavily sialylated O-glycans. Additionally, the current study suggests a possibility that some protein components of the boar SG derive from high concentration of the kinase in (dead) sperms.
165 EFFECT OF INJECTION OF SEMINAL PLASMA ON OVULATION RATE, CORPUS LUTEUM DEVELOPMENT, AND SENSITIVITY TO PROSTAGLANDIN IN ALPACAS (VICUGNA PACOS)
Authors: Huanca W. F. , Mamani C. , Huanca W.
Published: December 2014

The seminal plasma (SP) of camelids contains a protein identified as β Nerve Growth Factor with capacity of induced ovulation and develop a corpus luteum. A study was designed to evaluate the effect of application of SP on the interval of time from injection of stimulus to the ovulation and corpus luteum (CL) size (Experiment 1, n = 24) and on the sensitivity of CL to the injection of prostaglandin (196 µg of tiaprost) (PG) at different periods from ovulation (Experiment 2, n = 86). Exp. 1: Adult female alpacas with presence of a follicle ≥7 mm were assigned to the application of 1 mL of SP via IM (T: n = 12) or application of 50 µg of acetate of busereline IM (T2: n = 12). Exp. 2: Alpacas with presence of a follicle ≥7 mm were induced to ovulation with 50 µg of acetate of busereline or 1 mL IM of SP. Animals were evaluated by ultrasound to confirm the ovulation and were assigned to the following treatment: T1 (n = 8): SP + PG Day 4; T2 (n = 8): buserelin acetate + PG Day 4; T3 (n = 8): SP + PG Day 5; T4 (n = 8): buserelin acetate + PG Day 5; T5 (n = 8): SP + PG Day 6; T6 (n = 8): buserelin acetate + PG Day 6; T7 (n = 8): SP + PG Day 7; T8 (n = 8): buserelin acetate + PG Day 7; T9 (n = 8): SP + PG Day 8; T10 (n = 8): buserelin acetate + PG Day 8 and T11 (n = 6) (Control): Application of 1 mL of saline solution. The animals were evaluated by ultrasound with an Aloka SSD500 (Aloka, Tokyo, Japan) and 7.5-MHz linear transducer each 2 h (Exp. 1) and each 12 h (Exp. 2) after of application of PG. In Exp. 1, the ovulation rate was 95.7% to T1 and T2 and an interval of time between injection of stimulus and ovulation was 27.4 ± 2.5 h and 26.8 ± 1.8 to T1 and T2, respectively. In Exp. 2, luteolysis was 0.0%, 0.0%, 25.0%, 0.0%, 100.0%, 100.0%, 100.0%, 100.0%, 100.0%, 100.0%, and 0.0% for the treatments T1, T2, T3, T4, T5, T6, T7, T8, T9, T10, and T11 respectively. The results suggest that no differences exist between in the ovulation rate and interval to the ovulation between the application of buserelin acetate or SP and that the CL was sensible at Day 5 to the prostaglandin respect SP and with similar response to the sensibility of CL from Day 6 to Day 8.
202 ASSOCIATION BETWEEN BMP4 GENE POLYMORPHISM AND OVUM PICK-UP IN VITRO PRODUCTION TRAITS IN BRAZILIAN Gyr COWS
Authors: Quirino C. R. , Vega W. H. O. , Pacheco A. , Azevedo A. S. , Dias A. J. B.
Published: December 2014

In Bos taurus taurus, previous studies have reported an association between single nucleotide polymorphism (SNP) in BMP4 gene and the blastocyst rate in cows evaluated for in vitro embryo production efficacy. The aim of this study was to evaluate the degree of association between a polymorphism in the BMP4 gene and characteristics related to in vitro embryo production as well as pregnancies thereof in Bos taurus indicus cattle (Gyr breed). Data from 212 ovum pick-up in vitro (OPU-IPV) sessions were collected of 50 Gyr cows in the Rio de Janeiro state, Brazil. The OPU-IVP procedures were performed and associated with marker SNP. The OPU-IVP traits were number and proportion of viable cumulus-oocyte complexes, number and proportion of cleaved embryos at Day 4 of culture, number and ratio of transferable embryos at Day 7 of culture, number and proportion of pregnancies at Day 30 after transfer, and number and proportion of pregnancies at Day 60 after transfer. DNA was extracted from hair follicles. BMP4 polymorphism, the segment of the BMP4 gene (exon 2, SNP rs109778173) was amplified by PCR with a specific primer. PCR-restriction fragment length polymorphism (RFLP) was used for genotyping. HinfI restriction enzyme that recognises and cleaves the sequence 5′ G ? ANTC 3′ was used. The samples were submitted to capillary electrophoresis Fragment Analyzer™ (Advanced Analytical Technologies Inc., Ames, IA) for allelic discrimination by size and polymorphism identification. Genotypic frequency, allele frequency, Hardy–Weinberg equilibrium probability, gene homozygosity, gene heterozygosity, effective allele number, and polymorphism information content were statistically analysed in the software PowerMarker v.3.25 (North Carolina State University, Raleigh, NC, USA). The association between DNA marker and the OPU-IVP traits and pregnancy rates was make by analysis of variance of repeated data PROC GLIMMIX of SAS 2009 (SAS Institute Inc., Cary, NC, USA). The domain of the protein coding of BMP4 gene is located in exon 2. A fragment of the sequence of DNA in exon 2 was amplified, where the mutation SNP rs109778173 was identified (G > T) in Gyr cows using PCR-RFLP and capillary electrophoresis methods. Three genotypes were identified (GG, TT, and GT) with genotypic frequencies of 0.64, 0.32, and 0.04 respectively; allelic frequency and Hardy–Weinberg equilibrium. The polymorphism was significantly associated with the number of viable oocyte complexes, ratio of viable oocyte complex, and ratio of pregnancies at 30 days (P > 0.01). The GT genotype affects negatively the number and proportion of viable cumulus-oocyte complexes and the proportion of pregnancies at Day 30 after transfer. This finding is an indication of a genetic effect of these characteristics.
205 SITE OF OVULATION ALTERS GENE PROFILE IN THE OVIDUCT FROM NELORE (BOS TAURUS INDICUS) AND ANGUS HEIFERS (BOS TAURUS TAURUS)
Authors: Fontes P. K. , Castilho A. C. S. , Pinto R. F. P. , Trinca L. A. , Carvalho R. F. , Ereno R. L. , Barros C. M.
Published: December 2014

The oviduct plays a key role promoting a favourable microenvironment to gametes transport, fertilization and early embryo development. Numerous differences in reproductive physiology are known among animals of Zebu and European breeds. Reports indicate that female Zebu cattle have a higher number of follicles per wave than female European cattle and individual distinctions in the number of follicles recruited are present in both breeds, namely animals with high follicular count (HFC) and low follicular count (LFC). Furthermore, the follicular count is related to animal fertility and is greatly influenced by the activity of FSH, oestradiol, and androgens. However, little is known about the effects of follicular count differences between Zebu and European cattle, and between breeds in the oviduct molecular profile. Based in these information, we hypothesised that differences in bovine breed (Nelore and Aberdeen Angus), differences in the follicular count (FC), and differences in the antimere related to ovulation (ipsilateral and contralateral) alter the molecular profile of genes involved in oviducal functions during the initial period after ovulation. To do so, oviducts from Nelore heifers (HFC, n = 4; LFC, n = 4) and oviducts from Aberdeen Angus heifers (HFC, n = 4; LFC, n = 4) were isolated and oviducal segments were divided (infundibulum, ampulla, isthmus) from ipsilateral and contralateral antimere. Total RNA was extracted using Illustra TriplePrep Kit (GE Healthcare, Waukesha, WI, USA) and then reverse transcription was performed using a high-capacity cDNA kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocols. Relative RT-qPCR analysis was performed with TaqMan® Low Density Array (TLDA, Life Technologies). The mRNA abundance of the target was tested by ANOVA analysis, using PROC GLM procedure of SAS (SAS, 9.2, SAS Institute Inc., Cary, NC, USA). Individual differences were analysed through pair-wise comparisons (SAS). All the comparisons were performed in each segment (ampulla, infundibulum, and isthmus); no comparisons were performed between segments. The differences were considered significant when P < 0.05. In the ampulla, the mRNA abundance of COX2, OVGP1, GPR78, FUCA1, and ANXA4 showed higher levels in ipsilateral antimere compared to contralateral. Similarly, in the infundibulum the mRNA abundance of GRP78, PGTER4, FUCA2, and FUCA1 was higher in ipsilateral antimere. No difference was found in the isthmus. In conclusion, the breed and the follicular count have no effect on the molecular profile of bovine oviduct, suggesting the site of ovulation has the main effect in gene expression related to gametes transport, fertilization, and early embryo development.Research supported by FAPESP 2012/09498-9 and 2012/50514-8.
211 TRANSCRIPTS EXPRESSION RELATED TO MAPK/SMAD2 PATHWAY IN OVIDUCT CELL, CUMULUS CELL, AND OOCYTE DERIVED FROM DIESTRUS BITCHES
Authors: Lee S. H. , Kim M. J. , Oh H. J. , Kim G. A. , Setyawan E. M. N. , Kwang Jo Y. , Choi Y. B. , Lee B. C.
Published: December 2014

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are known to play an essential role in regulating ovarian follicular development and ovulation rate via SMAD pathway in several species and have different functions depending on the species. However, their expression and function have not yet been reported in dogs. Therefore, the aim of present study is to analyse expressions of transcripts related to the MAPK/SMAD2 pathway in oviduct cell, cumulus cell, and oocyte derived from diestrus bitches. Ovariohysterectomy was performed in 5 large, mixed breed dogs 3 days after serum progesterone reached 5–10 ng mL–1. Oviduct cells were obtained by scraping the superficial oviduct tissue and oocytes were obtained by mincing the ovary. Both cumulus cells derived from non-ovulated oocytes (IM-cumulus) and ovulated oocytes (M-cumulus) were obtained. Oviduct, IM-cumulus, and M-cumulus cells were cultured using RCMEP media and cryopreserved using FBS containing 10% DMSO. Total RNA was extracted from Oviduct, IM-cumulus, and M-cumulus cells and oocytes. After cDNA synthesis, transcript expression of β-actin, GDF9, MAPK1, BMP6, and SMAD2 genes were analysed. The data was analysed by one-way ANOVA using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). The threshold for statistical significance was set at P < 0.05. In results, mRNA expression of MAPK1 was not significantly different among the cells. However, expressions of BMP6, GDF9, and SMAD2 were significantly higher in oocytes compared to oviduct, IM-cumulus, and M-cumulus cells. Although oviduct cells showed increased GDF9 and SMAD2 transcripts similar to that of oocytes, there was no significant difference between them. In conclusion, MAPK/SMAD2 pathway may have important roles in growth of immature oocytes in dogs and oviduct cells may help the maturation of ovulated immature oocytes. Further studies will be conducted to analyse MAPK/SMAD2 pathway in anestrus, proestrus, and oestrus periods.This research was supported by RDA (#PJ008975022014), IPET (#311062-04-3SB010), Research Institute for Veterinary Science, the BK21 plus program, and global PhD Fellowship Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2014H1A2A1021187).
219 EFFECT OF THE ENDOCRINE DISRUPTING CHEMICALS ON PLACENTAL TRANSPORT IN BeWo CELLS AS AN IN VITRO MODEL
Authors: Lee J.-H. , Jeung E.-B.
Published: December 2014

The placenta exchanges vital factors, including oxygen, carbon dioxide, copper, iron, calcium cations, and glucose, which are essential to fetal growth. Each molecule is transferred by specific receptors that are located at the cell membrane or in the cytoplasm. Copper, iron, calcium cations, and glucose transfer genes are regulated by estrogens, vitamin D, and human placental lactogen. Regulations of these receptors depend on pregnancy time length and maternal and fetal nutrient environment with various pathways. Some synthetic plastics known as endocrine disrupting chemicals (EDC) have a similar structure to reproductive hormones such as estrogens. Thus, these substances have a potential effect on the expression of genes which are regulated by estrogens or progesterone by interfering their pathways. Having an estrogenic property, EDC interact with oestrogen receptors and elevate or decrease the expression of target genes which are responsible for transporting essential molecules such as copper, iron, and calcium. To examine the effects of EDC exposure during pregnancy, we conducted an in vitro model study using the BeWo human trophoblast cell line. The BeWo cell was treated with well-known EDC, octyl-phenol (OP), nonyl-phenol (NP), and bisphenol A (BPA) in a dose-dependent manner (10–7, 10–6, and 10–5 M) for 24 h. The expression of copper (CTR1, ATP7A), iron (IREG1, HEPH), and calcium transporting genes (PMCA1, TRPV6), were measured by real-time RT–PCR and Western blot. The expression of copper, iron, and calcium transporting genes were elevated in a dose-dependent manner by all well-known EDC, including OP, NP, and BPA, as well as E2. To unveil the mechanism of these elevations of ionic transporting genes, an ERE promoter study will be needed. Taken together, essential cation transporting genes in placenta are modulated by EDC.
22 SEMEN AND REPRODUCTIVE PROFILES OF CLONED ANATOLIAN GREY CATTLE
Authors: Arat S. , Pabuccuoglu S. , Sagirkaya H. , Demir K. , Arici R. , Ustuner B. , Alcay S. , Toker B. , Alkan S. , Nak Y. , Nak D. , Kilicaslan R.
Published: December 2014

Anatolian grey cattle (endangered native Anatolian cattle) as 1 male (clone 1) and 4 females (clones 2–5) were produced from cells of 1 male and 1 female cattle by somatic cell nuclear transfer (SCNT) in a previous study. In this study, we examined the reproductive potential of these cloned animals, which are now 4 and 5 years old. The parameters evaluated by phase contrast microscopy for motility, TUNEL for DNA fragmentation, eosin staining for viability, Hoechst 33258 staining and hypo-osmotic swelling test (HOST) for membrane integrity, and fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA) for acrosome integrity of frozen-thawed spermatozoa, as well as birth and survival of calves following insemination with frozen-thawed semen of cloned and nuclear donor bull and normal bull. Six ejaculates and 3 samples per ejaculate from each bull were tested, and the Mann-Whitney U test was used to analyse the data. The spermatological parameters of cloned bull semen – volume, concentration, and motility of fresh – were within accepted limits for artificial insemination (4.60 ± 0.47 mL, 1.55 ± 0.21 × 109 spermatozoa mL–1, 80.00 ± 1.07%, respectively). Frozen-thawed sperm motility and viability rate were higher in the cloned bull (56.6%, 56.7%) than in its nuclear donor (47%, 43%; P < 0.05). Intact membrane and DNA fragmentation rate of cloned bull and its nuclear donor bull sperm were similar (P > 0.05) but the intact acrosome rate of cloned bull was higher than that of its nuclear donor (P < 0.05). Low rates in frozen-thawed sperm of nuclear donor can be related to storage time of sperm which were frozen 5 years before. One (clone 4) of the cloned grey heifers was artificially inseminated with frozen semen from nuclear donor bull and the other (clone 5) was naturally mated with a Holstein bull. Two healthy calves were delivered naturally. When same cloned cows (clones 4–5) and 2 other cloned heifers (clones 2–3) were artificially inseminated with frozen semen of the cloned grey bull, clones 2 and 4 gave birth to 2 healthy female calves. One cloned cow (clone 3) aborted in the third month of gestation and other one (clone 5) is currently 8 months pregnant. Two calves of clone 4 and 5 are 17 months old and 2 other calves of clone 2 and 4 are now 6 and 1 months old. Except for clone 3, our results show that cloned Anatolian grey bull and cows produced from frozen cells in gene bank have normal fertility.
220 THE ESTROGEN-LIKE EFFECT OF 2-METHOXYESTRADIOL (2-ME) IN IN VITRO AND IN VIVO MODELS
Authors: Lee J.-S. , Jeung E.-B.
Published: December 2014

2-Methoxyestradiol (2-ME), an endogenous metabolite of 17β-oestradiol, interacts with oestrogen receptors and microtubules and has a low affinity for oestrogen receptors (ER). It has attracted considerable interest due to its potential anti-cancer therapeutic effects. 2-ME is also recognised for its unique and profound actions on various tumour cell lines and cancer independent of the hormone receptor status. Regardless of differences in function, 2-ME has an affinity for ER, however, the exact mechanisms of 2-ME action via the ER are not fully understood. In the current study, we examined the estrogenic effect of 2-ME on mRNA levels of CaBP-9k, ER, and progesterone receptor (PR) in the absence or presence of the 17β-oestradiol (E2) and progesterone (P4) in both in vivo and in vitro models by real-time RT–PCR. In vitro, cells (n = 3 per group) were exposed to a single dose of E2 (10–9 M), P4 (10–6 M), 2-ME (10–8 M, 10–7 M, 10–6 M). The mechanism of CaBP-9k induction by these chemicals pre-treated with 10–7 M ICI 182, 780 and 10–6 M RU 486 for 30 min before exposure to E2 and 2-ME were analysed. In vivo, 35 female ICR mice (PND 14 days) were divided into 7 groups (n = 5 per group), and each group was administered subcutaneously with 24% DMSO, 38% ethanol, and 38% sterile saline as a vehicle, E2 [40 μg kg–1 of body weight (BW)] a physiological dose level), 2-ME (4, 40, and 80 mg kg–1 of BW) for 3 days. The mice were killed 24 h after the final injection. To investigate the effect of antagonism, 10 mice were injected SC with ICI 182 780 (10 mg kg–1 of BW) and RU 486 (10 mg kg–1 of BW) at 30 min before injection with 2-ME (40 mg kg–1 of BW) for 3 days and killed 24 h after the final injection. Results are presented as mean ± s.e.m.; P-values were calculated using one-way ANOVA. In GH3 cells, the mRNA level of CaBP-9k was induced in the E2 (10–9 M) treatment group, and expression of CaBP-9k was also up-regulated in the 2-ME (10–7 M)-treated group. Uterine lactoferrin (Ltf) mRNA expression was also increased in the 2-ME (40 mg kg–1 of BW) group, similar to the response with E2 (40 μg kg–1 of BW) in mice. As a blocker for ER and PR activity, ICI 182 780 and RU 486 reversed the E2 or 2-ME mediated increase of CaBP-9k and Ltf mRNA expression. We found that 2-ME significantly increased the levels of ERa and PR transcripts. In parallel with in vitro results, the mRNA levels of ERa and PR were induced by treatment with E2 and 2-ME. Taken together, our findings demonstrated that expression of estrogenic markers, CaBP-9k and Ltf, was regulated by 2-ME in both in vitro and in vivo, which may increase their estrogenic activities in female during the cycle through ER and/or PR-mediated pathway.
243 THE SEX OF THE BOVINE EMBRYO AFFECTS APOPTOTIC RATES AT THE BLASTOCYST STAGE
Authors: Ghys E. , De Troy D. , Donnay I.
Published: December 2014

Male and female pre-implantation bovine embryos may differ in several aspects such as kinetics of development, metabolism, or gene expression. These differences vary between culture conditions and may lead to shifts in sex ratio. In a previous study, we showed that female Day 7 blastocysts display a higher apoptotic rate than male ones when cultured in the presence of 5% FCS (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The difference was less important in the presence of BSA. This previous study was performed on in vitro-produced embryos produced with sexed semen and analysed using confocal microscopy. The objective of the present work is to confirm these differences using a) first, the unsexed semen of the same bull (bull 1) as the one used in the previous study in order to exclude any potential bias induced by the use of sexed semen and b) second, the unsexed semen of another bull (bull 2) in order to generalise our findings to the Bos taurus species. Levels of apoptosis were assessed in Day 7 blastocysts using immunohistochemical staining of cleaved caspase-3 and detection of fragmented DNA by TUNEL reaction on Day-7 blastocysts cultured with 5% FCS. Quantification of the number of stained cells was achieved with a fluorescence microscope. After cell counting, embryos were recovered and sexed by PCR. In both experiments, a higher proportion of cells showing caspase staining was observed in female (n = 145) than in male (n = 215) embryos (Bull 1: male: 11.8 ± 0.6%; female: 17.6 ± 1.1%, 2-way ANOVA, P ≤ 0.0001; bull 2: male: 9.5 ± 0.4%; female: 13.3 ± 0.6%, P ≤ 0.0001), whereas the proportion of TUNEL-stained cells was only significantly higher in female embryos produced with the semen of bull 2 (bull 1: male: 10.8 ± 0.4%; female: 11.4 ± 0.6%, P = 0.12; bull 2: male: 7.2 ± 0.3%; female: 9.1 ± 0.4%, P = 0.0009). A significant difference in cell number was observed between male and female blastocysts produced with the semen of bull 2 (male: 172 ± 5; female: 154 ± 5, P = 0.02) and the same tendency was observed for embryos generated with the semen of bull 1 (male: 143 ± 4; female: 132 ± 4, P = 0.07). In conclusion, our study demonstrated that the use of sexed semen does not interfere with the pattern of caspase and TUNEL staining previously observed. Moreover, a similar pattern was observed with 2 different bulls. We can thus conclude that the level of apoptosis of bovine Day 7 blastocysts produced in the presence of FCS is higher in female than male embryos. This could be related to the tendency towards a lower cell number in female blastocysts and to the shift in sex ratio in favour of male embryos often observed in the presence of serum.
246 USE OF IONOPHORE A23187 ON BOVINE FROZEN SPERM INCREASES HYPERACTIVATED MOTILITY
Authors: Garaguso R. , Franco M. J. , Ortega N. M. , Fanti T. , Mutto A. A.
Published: December 2014

Sperm capacitation is critical for oocyte fertilization in mammals. The capacitated state is acquired by the spermatozoon during its passage through the female reproductive tract and can be induced in vitro. At a functional level, sperm capacitation is associated with changes in the motility pattern – the hyperactivated state (HA) – and terminates with the acrosome reaction (AR). These events are characteristically regulated by different Ca2+-signalling pathways. For this reason, Ca2+ ionophores, such as A23187, are commonly used in sperm capacitation studies. The induction of AR and IVF, as well as the assessment of protein phosporylation of tyrosine substrates are useful methods for the evaluation of the capacitation state of spermatozoa. Although the increase of protein tyrosine phosphorylation via PKA is associated with sperm capacitation, in the mouse, A23187 capacitates the spermatozoa, thus bypassing this pathway. The aim of the present work was to test the effect of the ionophore A23187 on acquisition of the capacitated state by evaluation of HA motility and protein phosphorylation pattern in frozen bovine spermatozoa. Motile bovine spermatozoa were isolated by gradient centrifugation (Percoll) from frozen/thawed samples and treated with different concentrations of A23187 (0.05, 0.1, 0.2, and 0.3 µM) in H-TYH medium without BSA. After 10 min of treatment, spermatozoa were washed in medium with BSA (11.2%) and incubated in H-TYH with BSA (6%) for 30 min. For control groups, sperm were incubated in H-TYH medium and DMSO. The motility pattern was visually identified and quantified using a computer-assisted sperm analysis system. The motile/immotile spermatozoa and the HA motility patterns of each group were statistically analysed by applying Fisher's tests. The protein tyrosine phosphorylation pattern was evaluated by a Western immunoblotting assay using heparin as a positive control of sperm capacitation. Our results showed that spermatozoa treated with A23187 had a significant increase in HA motility. The proportions of HA spermatozoa were 10.92, 31.27, and 75.4% for 0.1, 0.2, and 0.3 µM A23187, respectively (P < 0.05). On the other hand, the pattern of PKA-tyrosine phoshorylation characteristic of capacitated spermatozoa was absent after incubation with A23187, similar to the response seen in mouse spermatozoa. The percentage of motile spermatozoa in the control groups (H-TYH medium: 36% and DMSO: 27.95%; P < 0.05) was reduced as compared with that of the basal sperm suspension (65.4%, P < 0.05) after 30 to 40 min of incubation. Motility in the spermatozoa treated with 0.1 and 0.2 µM ionophore was similar – 65.9 and 68.1%, respectively, but it was reduced in the 0.3 group – 57.5% (P < 0.05). Thus, treatment with A23187 increased the percentage of spermatozoa with HA motility, probably suggesting an improvement in fertilizing capacity. Nevertheless, these promising results remain to be confirmed by IVF assay.