bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of microwave leaky modes propagating through laser plasma filaments column waveguide
Authors: Alshershby Mostafa , Hao Zuoqiang , Lin Jingquan
Published: December 2012

A plasma column waveguide formed by a bundle of closely spaced plasma filaments induced by the propagation of ultrafast laser pulses in air and revived by a longer infrared laser pulse is shown to support microwave radiation. We consider values of both the plasma electron density and microwave frequency for which the refractive index of plasma is lower than the refractive index of air; therefore, a leaky plasma waveguide can be realized in extremely high frequency band. The guiding mechanism does not require high conductance of the plasma and can be easily excited by using commercial femtosecond laser sources. A theoretical study of leaky mode characteristics of isotropic and homogeneous plasma column waveguides is investigated with several values of plasma and waveguide structure parameters. The microwave transmission loss was found to be mainly caused by the microwave leakage through the air-plasma interface and is weakly dependent on the plasma absorption. In spite of losses of microwaves caused by leakage and plasma absorption, it is shown to be much lower than both that accompanying to surface waves attaching to single conducting plasma wire and the free space propagation over distances in the order of the filament length, which opens exciting perspectives for short distance point to point wireless transmission of pulsed-modulated microwaves.
Phase locking of an S-band wide-gap klystron amplifier with high power injection driven by a relativistic backward wave oscillator
Authors: Bai Xianchen , Zhang Jiande , Yang Jianhua , Jin Zhenxing
Published: December 2012

Theoretical analyses and preliminary experiments on the phase-locking characteristics of an inductively loaded 2-cavity wide-gap klystron amplifier (WKA) with high power injection driven by a GW-class relativistic backward wave oscillator (RBWO) are presented. Electric power of the amplifier and oscillator is supplied by a single accelerator being capable of producing dual electron beams. The well phase-locking effect of the RBWO-WKA system requires the oscillator have good frequency reproducibility and stability from pulse to pulse. Thus, the main switch of the accelerator is externally triggered to stabilize the diode voltage and then the working frequency. In the experiment, frequency of the WKA is linearly locked by the RBWO. With a diode voltage of 530 kV and an input power of ∼22 MW, an output power of ∼230 MW with the power gain of ∼10.2 dB is obtained from the WKA. As the main switch is triggered, the relative phase difference between the RBWO and the WKA is less than ±15° in a single shot, and phase jitter of ±11° is obtained within a series of shots with duration of about 40 ns.
Mode splitting of surface plasmon resonance in super-period metal nanohole array gratings
Authors: Guo Junpeng , Leong Haisheng
Published: December 2012

We experimentally observed surface plasmon resonance mode splitting in a super-period metal nanohole array grating under the transverse magnetic polarization excitation. The mode splitting was observed in the zeroth order transmission and also in the first order transmission. However, the mode splitting phenomenon is more evident in the first order transmission than in the zeroth order transmission. It is explained that the mode splitting is due to the coupling between the surface plasmon resonance mode in the subwavelength period metal nanohole arrays and the resonance mode of the super-period metal grating.
Thick CoFeB with perpendicular magnetic anisotropy in CoFeB-MgO based magnetic tunnel junction
Authors: Naik V. B. , Meng H. , Sbiaa R.
Published: December 2012

We have investigated the effect of an ultra-thin Ta insertion in the CoFeB (CoFeB/Ta/CoFeB) free layer (FL) on magnetic and tunneling magnetoresistance (TMR) properties of a CoFeB-MgO system with perpendicular magnetic anisotropy (PMA). It is found that the critical thickness (tc) to sustain PMA is doubled (tc = 2.6 nm) in Ta-inserted CoFeB FL as compared to single CoFeB layer (tc = 1.3 nm). While the effective magnetic anisotropy is found to increase with Ta insertion, the saturation magnetization showed a slight reduction. As the CoFeB thickness increasing, the thermal stability of Ta inserted structure is significantly increased by a factor of 2.5 for total CoFeB thickness less than 2 nm. We have observed a reasonable value of TMR for a much thicker CoFeB FL (thickness = 2-2.6 nm) with Ta insertion, and without significant increment in resistance-area product. Our results reveal that an ultra-thin Ta insertion in CoFeB might pay the way towards developing the high-density memory devices with enhanced thermal stability.
Sum-frequency generation in sol-gel material doped with binaphthol
Authors: Taupier Grégory , Saad May , Romeo Michelangelo , Crégut Olivier , Boeglin Alex , Mager Loic , Barsella Alberto , Arhach Hicham , Rehspringer Jean-Luc , (Honorat) Dorkenoo Kokou D.
Published: December 2012

A sum-frequency imaging system is used to investigate optical activity in a sol-gel material doped with the chiral molecule binaphthol. We show that this material can be optically structured to embed information that can be retrieved by sum-frequency.
Assisted reproduction techniques in the horse
Authors: Hinrichs Katrin
Published: December 2012

This paper reviews current equine assisted reproduction techniques. Embryo transfer is the most common equine ART, but is still limited by the inability to superovulate mares effectively. Immature oocytes may be recovered by transvaginal ultrasound-guided aspiration of immature follicles, or from ovaries postmortem, and can be effectively matured in vitro. Notably, the in vivo-matured oocyte may be easily recovered from the stimulated preovulatory follicle. Standard IVF is still not repeatable in the horse; however, embryos and foals can be produced by surgical transfer of mature oocytes to the oviducts of inseminated recipient mares or via intracytoplasmic sperm injection (ICSI). Currently, ICSI and in vitro embryo culture are routinely performed by only a few laboratories, but reported blastocyst development rates approach those found after bovine IVF (i.e. 25%–35%). Nuclear transfer can be relatively efficient (up to 26% live foal rate per transferred embryo), but few laboratories are working in this area. Equine blastocysts may be biopsied via micromanipulation, with normal pregnancy rates after biopsy, and accurate genetic analysis. Equine expanded blastocysts may be vitrified after collapsing them via micromanipulation, with normal pregnancy rates after warming and transfer. Many of these recently developed techniques are now in clinical use.
10 SPERM BINDING TO PORCINE UTERINE EPITHELIAL CELLS MIGHT BE LECTIN MEDIATED
Authors: Bergmann A. , Taylor U. , Rath D.
Published: December 2012

Evidence suggests that porcine spermatozoa bind to the endometrium lining after entering the uterus. In previous studies, it was verified that this binding is specific to porcine uterine epithelial cells (UEC). Here, we present trials aimed at identifying the engaged binding mechanism. Several interactions of spermatozoa with the female reproductive tract are known to be lectin mediated. The respective sugar ligands are species specific for the oviductal as well as zona pellucida surface membrane. Therefore, we hypothesised that sperm-endometrium interactions are mediated by lectins, too. To block possibly existing sugar moieties on the sperm surface, we pre-incubated spermatozoa with lectins selected for their strong binding to viable spermatozoa [wheat germ agglutinin WGA, succinylated WGA (sWGA), concanavalin A (ConA)] and subsequently undertook co-incubation studies using an established cell culture model from primary porcine uterine epithelial cells. All trials were carried out in modified Dulbecco’s Modified Eagle’s Medium containing 20% serum (D20) because the UEC monolayer proved to react sensitive to PBS as well as to semen extender. Pre-trials confirmed that in D20 sperm vitality remained unaffected and lectin binding efficiency did not differ compared with PBS. The sperm-rich fraction of the ejaculate from 3 German Landrace boars was collected in D20 and washed by centrifugation. The pellet was resuspended in D20 and the concentration adjusted to 100 × 106 spermatozoa mL–1. Then, the samples were incubated with 1 of the 3 lectins (10 µg mL–1) for 15 min at 37°C. As a control, 1 aliquot of the sperm suspension was incubated without lectins. After another washing step, the pellet was resuspended in D20. For co-incubation with UEC, 500 µL of lectin-incubated sperm was released onto a UEC monolayer and the binding activity observed under a phase contrast microscope. The binding density was quantified by area under view and compared to results from the control incubation with untreated sperm. Images (2 repeats/boar and lectin) were divided into fields of 61.6 µm2 and the fields with and without sperm were counted. For statistical analysis, a Tukey Test was carried out. Sperm treated with WGA and sWGA, both binding to glucosamine molecules, showed significantly reduced binding activity to the UEC (P < 0.05), whereas ConA-treated and untreated sperm bound in the dense pattern as shown in previous trials. The results indicate that the sperm-UEC binding mechanism is mediated by glucosamine moieties on the sperm surface, which interact with matching lectins placed on the UEC membrane surface. Investigations for further confirmation of the suggested binding mechanism between porcine sperm and the uterine epithelial layer are ongoing.
Supported by IMV Technologies, L’Aigle, France.
104 3-D VISUALIZATION OF THE FIRST CLEAVAGE AND ABERRATIONS OF BOVINE ZYGOTES BY CONFOCAL LASER SCANNING MICROSCOPY
Authors: Gratao A. A. , Beck A. , Reichenbach M. , Reichenbach H. D. , Wolf E. , Habermann F. A.
Published: December 2012

A high proportion of bovine oocytes fertilized in vitro fail to develop beyond the first 4 cleavage cycles. The first mitotic division of the zygote and proper segregation of chromosomes and cytoplasmic components seems to be a particularly delicate task. Notably, zygotes cleaving with a delay of only a few hours seem to have a very low chance of developing to the blastocyst stage. But what exactly goes wrong, how often, and why? To answer such questions we have to visualize in greater detail basic structures and processes such as the sperm aster, DNA replication, migration and apposition of the 2 pronuclei, synchronous chromosome condensation and breakdown of the nuclear envelopes, assembly of the first mitotic spindle and chromosome congression, anaphase, and cytokinesis. Oocytes fertilized in vitro were fixed at different time points around the first cleavage and stained for DNA, Ser10-phosphorylated histone H3, microtubules, and microfilaments. Zygotes were imaged in toto by recording confocal serial sections at 1-µm intervals using a 40× objective (NA = 1.3). Details were recorded with high spatial sampling densities (pixel size 50 × 50 nm, z-step size of 200 nm) close to the Nyquist criterion and restored by maximum likelihood estimation deconvolution using the real point spread function. We present a series of 3-D confocal images captured at different stages of the first cleavage. The images reveal new insights into the formation, structure, and function of the first mitotic spindle and the occurrence of spindle aberrations, irregular chromosome segregation, and abnormal cytokinesis. The microscopic findings guide us to candidate proteins for localization analyses and functional studies based on 3-D fluorescence live-cell imaging of zygotes and early embryos.
This work is supported by the Deutsche Forschungsgemeinschaft (DFG FOR 1041).
105 COMPARATIVE ULTRASTRUCTURE OF IN VITRO-PRODUCED BOVINE BLASTOCYSTS (BOS INDICUS) DERIVED FROM IN VITRO FERTILIZATION, SNC, AND PARTHENOGENESIS
Authors: Oliveira F. , Perecin F. , Meireles F. , Sangalli J. , Watanabe Y. , Miglino A. , Assis Neto A.
Published: December 2012

It is known that embryos produced in vitro may have structural alterations that often compromise the normal embryo development, generating a high rate of pregnancy loss. The study of these changes is of great importance because it may elucidate the cause of embryonic loss during the first trimester of pregnancy. Thus, the objective of this study was to characterize and compare ultrastructurally bovine blastocysts in the 7th day of development produced by IVF, cloning by somatic cell nuclear transfer (SCNT), and parthenogenesis. In vitro-produced embryos were derived from in vitro-matured oocytes. The somatic cell used to make cloning was fibroblasts of adult cows, and the protocol for parthenogenetic activation of the embryos was done with ionomycin-DMAP. The blastocysts derived from the different experimental groups were fixed in 2.5% glutaraldehyde and processed for transmission electron microscopy evaluation. The results showed that blastocysts derived by SNC and parthenogenesis exhibited a significantly reduced size; the inner cell mass and the blastocoel were not well defined compared with IVF embryos, indicating a less-advanced state of development. Furthermore, organelles of blastocysts derived from SCNT and parthenogenesis were fewer in number and had changes in form, when compared with IVF blastocysts. In parthenogenetic embryos there was the presence of phagosomes, suggesting a high degradation activity of cellular. Mitochondria showed the most significant changes. Although they occur in large quantities in all blastocysts, the morphology of them was impaired in SNC and parthenogenetic embryos (vacuolization, abnormal shape). Such modifications could suggest changes in mitochondria functionality, which may decrease cellular metabolic activity. Thus, we find that the D7 blastocysts derived from SCNT and parthenogenesis showed several ultrastructural differences compared with IVF embryos, with particular reference to a reduced number and morphology of embryo organelles.
109 EFFECT OF SHORT TERM PROGESTERONE SUPPLEMENTATION ON CIRCULATING PROGESTERONE CONCENTRATION, CORPUS LUTEUM SIZE, AND EARLY EMBRYO DEVELOPMENT IN CATTLE
Authors: O'Hara L. , Forde N. , Rizos D. , Maillo V. , Ealy A. D. , Kelly A. K. , Rodriguez P. , Evans A. C. O. , Lonergan P.
Published: December 2012

The aim of this study was to investigate the effect of short term progesterone (P4) supplementation on circulating P4 concentrations, corpus luteum (CL) size, and conceptus development in cattle. The oestrous cycles of crossbred beef heifers were synchronised using a 7-day PRID® Delta (1.55 g P4) treatment with administration of a PGF2α analog (Enzaprost®) the day before PRID® Delta removal. Only those recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to 1 of 5 groups: (1) control: no treatment, (2) placebo: insertion of a blank device (no P4) from Day 3 to 7, (3) insertion of a PRID® Delta from Day 3 to 7, (4) insertion of a PRID® Delta from Day 3 to 5, or (v5) insertion of a PRID® Delta from Day 5 to 7. In vitro produced blastocysts were transferred to each heifer on Day 7 (10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. In Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to 1 of 3 treatment groups (1) placebo, (2) PRID® Delta from Day 3 to 5, or (3) PRID® Delta from Day 3 to 7. All heifers were slaughtered on Day 16, and recovered conceptuses were incubated in synthetic oviduct fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-tau (IFNT). In both experiments, daily blood samples were taken to measure serum P4 concentration. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA). Insertion of a PRID® Delta resulted in an increase (P < 0.05) in serum P4, which declined following removal. In Experiment 1, serum P4 concentration was significantly lower from Day 9 to 14 (P < 0.05) and Day 14 CL weight was lower in the PRID® Delta Day 3 to 7 group than the placebo or control groups. P4 supplementation from Day 3 to 5 (17.0 ± 1.4 mm) or Day 3 to 7 (11.3 ± 2.3 mm) increased conceptus length compared to the placebo (2.1 ± 1.8 mm). In Experiment 2, serum P4 was significantly lower in the two supplemented groups following PRID® Delta removal compared with the placebo (P < 0.05) and was associated with a lower CL weight in the Day 3 to 7 group. Supplementation from Day 3 to 5 (94.0 ± 18.8 mm) or Day 3 to 7 (143.6 ± 20.6 mm) increased conceptus length on Day 16 compared to the placebo (50.3 ± 17.4 mm). Conceptus length was strongly correlated with the concentration of IFNT in the uterine flush (r = 0.58; P = 0.011) and spent culture medium (r = 0.68; P < 0.002). These findings highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrus period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.
Supported by CEVA Sante Animale and Science Foundation Ireland (07/SRC/B1156).
115 EFFECT OF FEED SUPLEMENTATION ON PREGNANCY RATE AND EMBRYONIC SURVIVAL IN ALPACAS (VICUGNA PACOS)
Authors: Huanca W. , Cordero A. , Huaman H. , Ampuero A.
Published: December 2012

Alpacas, like all camelids are induced ovulators which means mating is required to induce ovulation. Studies over the last decades have clearly established the link between nutrition and ruminant fertility. These animals live above 4000 m with a deficient quality and quantity of pastures and with a rate of birth less than 50%. The study was carried out with the objective to evaluate feed supplementation before service on the pregnancy rate and embryonic survival in alpacas. One hundred ninety-four alpaca adults of 4–6 years were assigned to 4 treatments: T1 (n = 49): alpacas lactating with supplementation; T2 (n = 48): alpacas lactating without supplementation; T3 (n = 49): alpacas non-lactating with supplementation and T4 (n = 48): slpacas non-lactating without supplementation. Body weight and body condition score were measured before the initiation of the experiment and every two weeks. Animals in T1 and T3 were supplemented with 2 kg of alfalfa hay and 500.00 UI mL–1 of vitamin A, 75 000 UI mL–1 vitamin D3 and 500 mg mL–1 vitamin E four weeks before the initiation of the experiment. Animals were maintained on Festuca dolichophylla and Calamagrostis vicunarum grasses. Ultrasound examinations with a rectal probe of 7.5 MHz were performed on mating day (Day 0) and Days 2, 9, 20, 30, and 60 after mating. Pregnancy rate was evaluated with a Fisher test. Follicular size before mating was 9.1 ± 0.7, 8.3 ± 0.5, 8.2 ± 0.6 and 7.9 ± 0.8 mm for T1, T2, T3, and T4; differences between T1 and T4 were significant (P < 0.05); corpus luteum size: 13.6 ± 1.1; 11.2 ± 0.9; 11.6 ± 1.3 and 10.8 ± 1.9 mm for T1, T2, T3, and T4; different T1 and T3 and T4 (P < 0.05); conception rate on Day 20: 65.6; 51.7; 52.9 and 51.1% for T1, T2, T3, and T4 different between T1 with respect to T2, T3, and T4 (P < 0.05). Day 30: 62.5; 37.9; 47.1 and 33.3% for T1, T2, T3, and T4 different T1 with respect to T2, T3, and T4 (P < 0.05); D60: 62.5; 34.5; 47.1 and 30.3% different for T1 with respect to T2, T3, and T4 (P < 0.05). Feed supplementation before mating was important to conception rate on Day 20 but appeared to be more important to embryo and fetal survival.
117 TRANSLATION INITIATION IS ESSENTIALLY MODIFIED IN THE PORCINE ENDOMETRIAL TISSUE DURING EARLY PREGNANCY
Authors: Wollenhaupt K. , Brüssow K. P. , Tomek W.
Published: December 2012

Protein synthesis is a major determinate in cell growth and differentiation. Here, we investigated the porcine uterine tissue during early pregnancy. The objective was the analysis of the initiation of translation, which is believed to be the rate-limiting process of protein synthesis. Performing Western blot screening during implantation and placentation, in vitro protease assays and mass spectrometry, we revealed that essential components of the cap-binding complex eIF4F and its regulators are targets of specific proteolytically processing. This proteolytical activity shifts from the implantation site to the peri-placental area later in development. The results show that the cap-binding protein eIF4E is N-terminally truncated which results in lower binding to the repressors 4E-BP1 and 4E-BP2. On the other hand, 4E-BP1 is mainly degraded, whereas 4E-BP2 emerges as N-terminally truncated stable fragments. It is likely that these fragments have lost an essential sequence motif that enables the phosphorylation and inactivation of the repressor. Furthermore, the Akt/mTor signaling cascade (which phosphorylates 4E-BP1 and 2) but not the MAPK pathway (which phosphorylated eIF4E and together with Akt/mTor hierarchically 4E-BP1 and 2) was inactivated. This results in long-term translation repression of eIF4E-sensitive and 4E-BP2-dependent mRNAs. However, other translation initiation factors like eIF4A, eIF2α, and the poly(A)-binding protein PABP are not modified. In conclusion, we suggest that the observed specific modification of the mRNA-cap-binding complex in the porcine endometrium during early pregnancy is essential to regulate gene expression to establish the noninvasive implantation and to sustain the ongoing gravidity.
124 THE REGULATION OF CALCIUM TRANSPORT GENE EXPRESSIONS DURING PREGNANCY AFTER EXPOSURE TO OCTYLPHENOL AND BISPHENOL A
Authors: Kim S. , Jeung E. B.
Published: December 2012

Octylphenol (OP) is a degradation product of alkylphenol ethoxylates that are widely used to in rubber, pesticides, and paints. Bisphenol A (BPA) is an organic compound with two functional phenol groups and used to make polycarbonate plastic and epoxy resins, along with other applications. OP and BPA are known as endocrine disruptors that can induce inappropriate estrogenic action, and may disturb natural calcium metabolism. In the present study, the effects of OP and BPA on the calcium levels of serum and urine, and calcium transport genes were investigated in the duodenum and kidney of the pregnant mice. From 6.5 to 16.5 days post-coitus (dpc), 5 pregnant mice for each group were orally given with ethylestradiol (EE, 0.2 mg kg–1 day –1), OP (15, 45, or 135 mg kg–1 day–1) or BPA (5 or 50 mg kg–1 day –1) dissolved in corn oil. The duodenum, kidney, blood, and urine were obtained from the mice at day 18.5 of pregnancy. As a result, serum and urinary calcium levels were decreased by OP and BPA in a dose-dependent manner. The mRNA expression levels of calcium transport genes TRPV6 and CaBP-9k were decreased in the kidney after treatment with OP and BPA, while duodenal expression of TRPV6 was reduced by a high dose of BPA. The protein levels of these genes showed similar pattern with those of mRNA in the kidney and duodenum. The data were analyzed using a one-way ANOVA. P < 0.05 was considered statistically significant. Taken together, OP and BPA altered gene expressions associated with calcium transport in the pregnant mice, which may cause reduced serum and urine calcium levels. These results suggest that estrogenic actions of OP and BPA may lead to influence the calcium levels during pregnancy in the mice.
136 EFFECTS OF INSULIN-LIKE GROWTH FACTOR I (IGF-1) ON THE DEVELOPMENT AND APOPTOSIS OF PREIMPLANTATION BUFFALO (BUBALUS BUBALIS) EMBRYOS
Authors: Lu F. , Luo T. , Sun H. , Li N. , Liu X. , Meng L. , Jiang J. , Shi D.
Published: December 2012

The aim of this study was to explore the effects of insulin-like growth factor-I (IGF-1) on the development and apoptosis of preimplantation buffalo (Bubalus bubalis) embryos derived from IVF or somatic cell nuclear transfer (SCNT) in order to improve the quality of in vitro embryo culture (IVC). Buffalo oocytes collected from ovaries at slaughter were cultured in the maturation medium (TCM-199 + 26.2 mmol L–1 NaHCO3 + 5 mmol L–1 HEPES + 5% FBS) for 22–24 h, and fertilized in vitro, or enucleated and reconstructed for SCNT. Embryos were then cultured in the culture medium (CM: TCM-199 + 3% FBS) supplemented with different concentrations of IGF-1. Blastocyst development was evaluated after 7 days of culture. A total of 1566 oocytes were used in this study. The experimental data were analyzed using two-way ANOVA, P < 0.05 was considered to be statistically significant. The results showed that the cleavage rates of IVF or SCNT embryos cultured with 0, 10, 50, or 100 ng mL–1 IGF-I, were not significantly different (P > 0.05). However, the blastocyst rate of IVF embryos cultured with 50 ng mL–1 IGF-1 was significantly increased compared to the 0 ng mL–1 group (35.1 v. 23.0%; P < 0.05), but not significantly different among the 0, 10, and 100 ng mL–1 groups (23.0 v. 28.2 and 26.5%; P > 0.05). In the same line, more SCNT embryos could develop to the blastocyst stage when cultured in the CM supplemented with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (32.3 v. 20.2%; P < 0.05), but the blastocyst development decreased with 100 ng mL–1 (32.3 v. 21.4%; P < 0.05). Apoptosis and total cell number (TCN) of IVF/SCNT blastocysts were respectively detected by TUNEL or Hoechst 33342 staining. By comparison with the 0 ng mL–1 group, the TCN of IVF/SCNT blastocysts was significantly increased (IVF: 91.7 ± 6.9 v. 108.7 ± 3.9, SCNT: 76.3 ± 5.6 v. 92.8 ± 3.9; P < 0.05) and the apoptotic index was obviously decreased (IVF: 3.9 ± 0.7 v. 2.5 ± 0.7; 7.2 ± 0.5 v. 2.9 ± 0.5; P < 0.05) when the embryos were cultured in the CM with 50 ng mL–1 IGF-I. The result of RT-qPCR analysis showed that the mRNA expression level of the anti-apoptotic bcl-2 gene was distinctly enhanced, while the mRNA expression level of the pro-apoptotic bax gene was remarkably reduced in IVF/SCNT embryos cultured with 50 ng mL–1 IGF-I by comparison with the 0 ng mL–1 group (P < 0.05). These results demonstrated that supplementing CM with 50 ng mL–1 IGF-1 could improve the developmental competence of buffalo embryos, increase the TCN of blastocysts and decrease their apoptotic index, probably by down-regulating the mRNA level of pro-apoptotic bax gene and up-regulating the mRNA level of anti-apoptotic bcl-2 gene.
This work was funded by the China High Technology Development Program (2011AA100607), China Natural Science Foundation (31072033), and Guangxi Science Foundation (2012GXNSFFA060004).
155 CHARACTERIZATION OF LIPID DROPLETS IN BOS INDICUS AND BOS TAURUS EMBRYOS
Authors: López-Damián E. P. , Fiordelisio T. , Lammoglia M. A. , Alarcón M. , Asprón M. , Galina C. S.
Published: December 2012

Accurate evaluation of bovine embryos for assessing developmental stage and quality is critical to the success of any embryo transfer program. However, this evaluation process has been reported to be highly subjective in Bos indicus (BI) and can vary as much as 23% compared with that of Bos taurus (BT). These differences in assessment may be related to the quantity of lipid droplets (LD) within the embryo, which has been shown to have a negative effect in cryopreserving embryos. The aim of the present study was to characterize the number and size of LD in different developmental stages of fresh embryos from BI and BT and to compare LD across the three different embryo quality grades (1 = excellent or good, 2 = fair, and 3 = poor). Nonsurgical embryo collection was performed 7 days post-insemination in 10 BI and 10 BT females. Forty-eight embryos were evaluated for stage and grade using stereoscopic microscopy, processed for transmission electron microscopy, and stained with Nile red. Digitalized images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD, USA), contour of lipid droplets were designed, and values of perimeter, area, and fluorescence intensity were assessed. Nonparametric statistical analysis (Mann–Whitney) was utilized. There was no difference in LD number for BT or BI for morulae and blastocyst; however, BI morulae presented larger LD compared with blastocyst stage embryos (286 µm2 v. 223 µm2; P < 0.05). Likewise, BI TF cells had more LD compared with inner cell mass (ICM) cells (48 v. 36; P < 0.05). BT TF cells exhibited larger LD compared with ICM cells (149 µm2 v. 128 µm2; P < 0.05), while BI embryos exhibited a larger area of LD in the ICM compared with the TF (591 µm2 v. 472 µm2; P < 0.05). In all embryos, BI contained more lipid droplets than BT (78 v. 49; P < 0.05). Across all quality grades (good, fair, and poor) there was no difference in the number of LD in BT embryos; however, BI grade-3 embryos presented more LD than grade-1 (36 v. 25). BT embryos LD were larger than BI LD (907 µm2 v. 625 µm2; P < 0.05). Fluorescence images showed higher arbitrary units of fluorescence (auf) for LD in BI. Compared with BT embryos (386 auf v. 280 auf; P < 0.05). These results suggest that BI embryos contain more and smaller LD than BT embryos and the LD described for BI embryo quality grade 1 are larger than those of quality grades 2 and 3, and even though the number of LD in morulae and blastocyst stage embryos are not different LD size is reduced as development occurs.
Research funding provided by UNAM-DGAPA-PAPIIT IN200810.
165 THE EFFECT OF shRNA TARGETING CLUSTER OF DIFFERENTIATION ANTIGEN 14 ON GENE EXPRESSION OF TNF-α, TLR4, AND IL-6 IN LIPOPOLYSACCHARIDE-INDUCED BUFFALO PERIPHERAL BLOOD MONOCYTE/MACROPHAGE
Authors: Li X. , Li M. , Huang S. , Qiao S. , Kang C. , Shi D.
Published: December 2012

Cluster of differentiation antigen 14 (CD14) plays a crucial role in the inflammatory response to lipopolysaccharide (LPS), which interacts with TLR4 and MD-2 to enable cell activation, leading to inflammation. Several studies have proved that upstream inhibition of bacterial LPS/toll-like receptor 4 (TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. In this study, to explore the effect of CD14 down-regulation on TLR4 signal conductive-related genes expression after stimulation by LPS, five CD14 shRNA (319/421/755/970/1041) sequences and a negative control sequence (NC-1864) were synthesised and used to construct lentiviral recombinant plasmid pSicoR-GFP-shRNA. Lentiviral recombinant plasmids of pSicoR-GFP-shRNA and fusion expression vector of pDsRed-N1-buffalo CD14 were co-transfected into HEK293 using liposome. At 72 h after transfection, the expression of exogenous buffalo CD14 mRNA was reduced at different level for all shRNA plasmids, in which shRNA-1041 had the highest interfering efficiency by RT-qPCR and fluorescence-activated cell sorting analysis. Then, buffalo peripheral blood monocyte/macrophage was purified and infected by the CD14 shRNA lentivirus. After 7 days of infection, the cells were stimulated by 1 µg mL–1 LPS for 3 h, then the mRNA expression level of CD14, TLR4, IL-6, and TNF-α transcripts in the cells were detected by the RT-qPCR method. After stimulation by LPS, the expression of endogenous CD14 was significantly reduced by CD14 shRNA-1041, the mRNA expression level of TLR4, IL-6, and TNF-α genes was also significantly down-regulated in comparison with control group (P ≤ 0.01). In conclusion, the selected CD14 shRNA-1041 cannot only inhibit the expression of endogenous CD14 mRNA in buffalo peripheral blood monocyte/macrophage, but also downregulate the mRNA expression of CD14, TLR4, IL-6, and TNF-α. The above results demonstrate that knockdown of endogenous CD14 has obvious coordination effects on the signal conductive function of TLR4 after stimulating by LPS, and shRNA technology will provide a new way to prevent endotoxin-related diseases in livestock.
This work was supported by the National Transgenic Project (2009ZX08007-009B), Guangxi natural science funding (2012GXNSFCB053002), and funding of State Key Laboratory of Subtropical Bioresource Conservation and Utilisation (KSL-CUSAb-2012-02).
166 CAN COXIELLA BURNETII BE TRANSMITTED BY GOAT EMBRYO TRANSFER?
Authors: Alsaleh A. , Pellerin J. L. , Roux C. , Larrat M. , Chatagnon G. , Fieni F.
Published: December 2012

Coxiella burnetii, an obligate intracellular bacterium of worldwide distribution, is responsible for Q fever. Detection of significant bacterial loads in flushing media and tissue samples (oviducts and uterine horns) from the genital tracts of nonpregnant goats is a risk factor for in utero infection and transmission during embryo transfer (Alsaleh et al. 2011 CIMID 34, 355–360). The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the washing protocol recommend by the IETS for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and PCR, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9–9, 11–11, and 4–4 in replicates 1, 2, and 3, respectively) were placed in 1 mL of MEM containing 107 C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37°C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3–3, 5–5, and 2–2 in replicates 1, 2, and 3, respectively) were submitted to the same procedures but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 h at 13 000g. The washed embryos and pellets were tested by PCR. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first 5 washing fluids for ZP-free embryos and in the first 8 washing fluids for ZP-intact embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly demonstrate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to cling to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.
167 BLACK TEA DOES NOT INTERFERE IN THE NUMBER OF CORPORA LUTEA IN SUPEROVULATED RATS
Authors: Kikuchi E. S. C. , Castilho C. , Papa P. C. , Laposy C. B. , Nogueira R. M. B. , Giometti J. , Machado L. G. , Giuffrida R. , Vieira D. P. , Giometti I. C.
Published: December 2012

The black tea originating from the Camellia sinensis plant is considered a healthy drink because it is associated at prevention of cancer, cardiovascular disease, and osteoporosis. It has been also stated that its catechins have antioxidative, antibacterial, and antitumoral activity. Despite many potential benefits of black tea consumption, the catechins present in black tea can significantly inhibit proliferation, steroidogenesis, and vascular endothelial growth factor production by swine granulosa cells. Based on these statements, the aim of this work was to verify the influence of black tea in the ovulation rate and the ovary weight in superovulated rats. This study was approved by the ethics committee of the university. Wistar rats were distributed in two groups: treated group (BT): 30 rats drinking only black tea (2.5%) ad libitum; control group (CG): 30 rats drinking water ad libitum. The experiment lasted for 3 months. Ten animals of each group were superovulated monthly with 150 UI kg–1 of eCG (Folligon®, Intervet Schering-Plough) and 150 UI kg–1 of hCG (Vetecor®, Hertape Calier) and sacrificed 48 h later. Ovaries were collected and weighted, and the corpora lutea were dissected and counted. The obtained data were submitted to the unpaired t-test with Welch’s correction, and the effect of time was assessed by ANOVA (P < 0.05). It was observed that the animal of both groups responded to the hormonal treatment because superovulation was noticed for the number of corpora lutea in both groups. The mean and standard deviation values for ovary weight (g) and corpora lutea number in both groups were as follows: first month (CG = 0.30 ± 0.10, 61.10 ± 10.94 and BT = 0.30 ± 0.07, 55.00 ± 23.03); second month (CG = 0.19 ± 0.06, 64.90 ± 27.77 and BT = 0.17 ± 0.05, 50.60 ± 19.35); and third month (CG = 0.21 ± 0.05, 62.30 ± 20.17 and BT = 0.19 ± 0.07, 48.6 ± 10.59), respectively. No difference was observed between the groups in the time points. The chronic consumption of black tea for 3 months does not interfere with ovulation aornd weight of the ovaries in superovulated rats.
Financial support was provided by FAPESP 2010/20583-2 and 2010/20274-0.
170 ASSESSMENT OF FRESH AND FROZEN - THAWED BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) SPERM DNA FRAGMENTATION USING THE SPERM CHROMATIN DISPERSION TEST
Authors: Sanchez-Calabuig M. J. , de la Fuente J. , Beltrán-Breña P. , Martinez-Nevado E. , Perez-Gutierrez J. F. , Blyde D. , Johnston S. , Harrison K. , Lopez-Fernandez C. , Gosalvez J.
Published: December 2012

There has been significant development over the last 20 years to improve genetic management of the captive bottlenose dolphin (Tursiops truncatus) by means of genome resource banking and assisted reproduction. Although standard semen parameters have been analysed in some detail, very little is known about sperm DNA fragmentation (SDF) in this species. The aim of this study was to develop a sperm chromatin dispersion test (SCDt) for the bottlenose dolphin to establish the baseline level of SDF immediately after ejaculation and cryopreservation and to determine the dynamic loss of sperm DNA quality after ex vivo handing and incubation in conditions that mimic the female reproductive tract. Semen from 8 bottlenose dolphins was collected by manual stimulation. Initial validation of the SCDt was conducted by means of in situ nick translation and neutral comet assay using a proven fertile male. To investigate the dynamic loss of sperm chromatin (rate of sDF loss), thawed sperm samples were incubated at 37.9°C for up to 48 h, and aliquots of spermatozoa were assessed after 1, 4, 8, 24, and 48 h. Dolphin sperm nuclei with fragmented DNA exhibited large halos of dispersed DNA fragments, whereas those with nonfragmented DNA displayed small halos of nondispersed DNA. A high correlation (r2 = 0.82; P ≤ 0.01) was found between the respective assessments of the SCDt and the neutral comet assay. All nucleoids resulting in a large halo of dispersed chromatin were intensely positive to in situ nick translation. The level of sDF fragmentation observed immediately after ejaculation in fresh and frozen samples was relatively low (1–5%). After comparing different ejaculates of the same individual, differences were found. Chromatin stability was high during the first 48 h of ejaculation or post-thawing and incubation. Evaluation of the sDF dynamics of fresh and frozen–thawed spermatozoa revealed no significant increase in the baseline level of sDF or in the relative increase of DNA damage after 48 h of incubation. Our data suggest that cryopreservation does not induce a dramatic increase in sperm chromatin damage. Interestingly, sperm samples derived from aged animals resulted in an increased rate of DNA loss, which was observed after 60 min post-incubation.
181 XENOTRANSPLANTATION OF BOVINE OVARIAN CORTEX IN SEVERE COMBINED IMMUNODEFICIENT MICE TO STUDY PRE-ANTRAL FOLLICULAR DEVELOPMENT: DETERMINATION OF THE OPTIMAL GRAFT SITE
Authors: Langbeen A. , Jorssen E. P. A. , Andries S. , Merckx E. , Leroy J. L. M. R. , Bols P. E. J.
Published: December 2012

Nowadays, a lot of attention is given to female reproductive preservation strategies. However, opportunities to study the dynamics of pre-antral folliculogenesis in vitro are not readily available because routine procedures for culture of (ruminant) isolated follicles are still lacking. One of the current techniques to study follicular dynamics and activation is xenografting bovine ovarian cortex into immune-deficient mice. Several transplantation sites have been described (Bols et al. 2010 Theriogenology 73, 740–747), such as subcutaneously (SC), underneath the kidney capsule (SK), intramuscularly (IM), and underneath the peritoneum (SP). In the present study, our objective was to determine the optimal host type and graft location in order to maximize the success of graft retrieval and follicular development. In total, 22 mice [12 conventional, 10 severe combined immunodeficient (SCID)] were used as graft recipients. All mice were anesthetized with an intraperitoneal injection of ketamine and xylazine and subsequently sterilized. Small pieces (maximum dimension of 9 mm3) of adult bovine ovarian cortex, retrieved from slaughterhouse ovaries, were then grafted at 4 different sites: SC grafts were localised at the left-hand side of the neck of the host, IM at the left hamstring (between the semimembranosus and semitendinosus muscle), SK under the left kidney capsule or SP, namely on the left and right-hand side, retroperitoneal. Blood vessels were macroscopically localised and stimulated by curettage before the cortex piece was grafted. Fourteen days later, mice were killed and the graft was localised and retrieved (if possible), after which the presence of follicles was assessed by visualisation following hematoxilin-eosin staining of histological slides. Data (Table 1) show that graft retrieval rates were highest when cortex fragments were grafted underneath the peritoneum (SP site). Although the extent of follicular presence and quality assessment of the detected follicles surely require additional experiments, our data do not support a difference between SCID or a conventional mouse strain as an optimal host type when it comes to graft retrieval rates and the determination of follicular activity when grafts are left in place for a limited period of 14 days.

Table 1.Graft retrieval and follicular activity of xenotransplanted, bovine adult cortex fragments: conventional strain compared with a SCID mouse strain
187 EXPRESSION AND REGULATION OF THE FIBROBLAST GROWTH FACTOR GENE FAMILY DURING MOUSE FOLLICULAR DEVELOPMENT
Authors: Furukawa S. , Naito K. , Sugiura K.
Published: December 2012

Recent studies have shown the critical roles of fibroblast growth factors (FGFs), including FGF8 produced by oocytes, in regulating follicular development. However, the expression and regulation of the FGF gene family, which consists of 22 ligands and 4 receptors, in the mouse ovary have not been well understood. The aim of the present study was to assess the expression and regulation of FGF ligands and receptors in the mouse ovary. Transcript levels of FGF ligands and receptors in immature (3-week-old) and adult (7- to 8-week-old) ovaries as well as other tissues of B6/DBA2F1 mice were analysed with RT-PCR. Furthermore, expression levels of FGF receptors in cumulus cells (CC) and mural granulosa cells (MG) before and after equine chorionic gonadotropin (eCG) treatment were determined with RT-quantitative PCR. Among 21 FGF ligands examined, 12 and 9 transcripts were detectable in immature and adult ovaries, respectively. More FGF ligands were detected in ovary, testis, heart, and brain compared to other tissues, including liver and spleen. Transcripts of all 4 FGF receptors (Fgfr1–4) were detectable in both immature and adult ovaries. Expression levels of Fgfr1 and Fgfr2 were significantly higher in MG compared with CC before and after the eCG treatment. Levels of Fgfr4 were comparable between MG and CC before the eCG treatment, but became significantly different with higher expression levels in MG after the eCG treatment. Fgfr3 transcripts were barely detectable in CC and MG. Overall levels of Fgfr1 in granulosa cells (CC and MG) were downregulated by eCG treatment, whereas those of Fgfr2 and Fgfr4 were upregulated. In summary, many FGF ligands are expressed, at least in mRNA levels, in mouse ovaries. Moreover, the expression levels of Fgfr transcripts in granulosa cells are dynamically regulated during follicular development.
190 JNK SIGNALLING IN FOLLICLE ACTIVATION
Authors: Bertoldo M. J. , Duffard N. , Calais L. , Frapsauce C. , Monniaux D. , Mermillod P. , Locatelli Y.
Published: December 2012

Primordial follicles are maintained in a quiescent state until they receive a signal to activate and join the growing pool. It is essential that the rate of follicle activation is well coordinated as it determines the reproductive lifespan of the female. This finely tuned process is under the control of a variety of positive and negative factors, and there is evidence that the JNK pathway is involved. The aim of this study was to assess the effect of a JNK inhibitor (SP600125) on follicle activation in vitro. Ovaries from 6 prepubertal sheep were dissected into 1-mm3 fragments and cultured in the presence or absence of SP600125 (0, 5, or 25 µM; SP0, SP5, and SP25, respectively). After 0, 2, 5, or 9 days, fragments were fixed, sectioned, and analysed by histological morphometry to determine the number and type of follicles in addition to TUNEL analysis for apoptosis. In total, 21 584 follicles were assessed. Follicles were classified as either primordial, intermediate, primary, secondary, or antral. Culture media were also assayed for steroid content. After multinomial regression analysis, there were no differences in the rate of follicle activation between groups on Day 2 of culture. However, after 5 days of culture there were significantly more primordial follicles in SP25 (69 ± 9.15%; P < 0.01) and SP5 (52 ± 8.12%; P < 0.05) groups when compared with SP0 (34 ± 3.98%). At the end of culture (Day 9), these differences continued to be observed (SP25: 63 ± 8.47%; SP5: 51 ± 6.62%; SP0: 34 ± 5.61%; P < 0.05). On Day 9 there were more intermediate follicles following treatment with SP0 (52 ± 3.27%) compared with SP5 and SP25 (42 ± 4.72% and 31 ± 7.41% respectively; P < 0.05). There was also a tendency for more primary follicles on Day 9 for the control (11 ± 4.29%) when compared with the other treatments (4 to 7%; P = 0.06). The proportion of apoptotic primordial follicles was greatest in the SP25 group when compared with SP5 and SP0 at all timepoints (P < 0.05). After 2 days of culture, progesterone concentration was highest in the SP25 group (4.1 ± 0.45 ng mL–1) compared with the SP5 and SP0 groups (2.9 ± 0.39 ng mL–1 and 1.6 ± 0.79 ng mL–1, respectively; P < 0.05). However, this dose effect was lost by the end of culture. There was no effect of the inhibitor on oestradiol synthesis. By using SP600125, we have observed a dose-dependent block on follicle activation. These results suggest that the JNK pathway is a novel regulator of follicle activation and development. However, further confirmation is required to determine its specific action. The mechanisms by which SP25 promotes progesterone synthesis and increases apoptosis are unknown and also require clarification.