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BackgroundOptical spectral transmission (OST) is a modern diagnostic modality able to assess the blood-specific absorption of light transmitted through a tissue, promising quantification of inflammation in the finger and wrist joints of patients with rheumatoid arthritis (RA) (HandScan -Demcon/Hemics, The Netherlands). Early data on OST revealed both correlations with clinical and arthrosonographic activity markers of RA, as well as a high diagnostic value during disease follow-up [1-3]. However, in a previous work we could detect and describe several OST confounding factors that complicate the establishment of universal cut-off values [2].ObjectivesAim of this study was to examine and suggest OST-cut-off values after adjusting for the effect of statistically identified confounding factors. Moreover, we sought to describe a clinical model that would represent the probability of a positive RA diagnosis on the basis of OST values and presence of confounders in every individual patient.MethodsNewly diagnosed RA patients (ACR-EULAR Classification Criteria 2010) were examined by OST and compared with healthy controls. Moreover, RA patients underwent clinical (tender & swollen joint counts, DAS28) and laboratory (inflammation markers) examinations. To identify factors that could have a confounding effect on OST, we performed logistic multivariate regression analyses, taking into account patient-associated characteristics such as age, gender, BMI, hand size, nicotine use. After identifying confounders, we determined OST cut-offs for different patient categories. In addition, we calculated the probability of the presence of an RA-diagnosis, based on the measured OST value and the presence of the identified influencing factors. Finally, we examined correlations of OST with clinical and laboratory RA activity parameters.Results74 early RA patients [64%♀; median age 59 years (53-70, IQR); median DAS28 4.58 (3.5-5.3, IQR)] and 101 controls [80%♀; median age 45 years (35-56, IQR)] were recruited. Male gender and advanced age were statistically identified as OST influencing factors. Receiver operating characteristics (patients vs. controls) revealed an OST cut-off value of 11.2 (sensitivity 85%, specificity 72%) for female and an OST cut-off of 16.07 (sensitivity 75.7%, specificity 72.7%) for male patients.Two models were shown to be statistically appropriate for calculating the probability of the presence of RA based on the measured OST values and confounders: one model with included age for females and one model with included age and smoking status for males (Table 1).Table 1.Statistical models for calculating the probability of the presence of rheumatoid arthritis based on measured OST values and OST confounding factors.Statistical modelWomenMenOST: optical spectral transmission; P(RA): probability of RA diagnosisX1 for age, X2 for OST; X3 for nicotine useOST showed statistically significant moderate correlations with DAS28 (r=0.42, pConclusionThe establishment of OST cut-off values adjusted for confounding factors and the suggested statistical models could contribute to an increase of the diagnostic value of HandScan and assist clinicians during patient screening. To our knowledge, this is the first report in the literature on the specific topic. Further patient recruitment and research regarding additional confounders are currently taking place with the aim of improving the diagnostic capabilities of this new and promising diagnostic technology.References[1]van Onna M, et al. Ann Rheum Dis. 2016;75(3):511-8.[2]Triantafyllias K, et al. J Rheum. 2020;47(9):1314-22.[3]Triantafyllias K, et al. Ann Rheum Dis. 2020;79(Suppl 1):1244.1Disclosure of InterestsNone declared

Background:Cardiovascular (CV) and cerebrovascular (CVB) risk have not been adequately examined in the setting of patients with small vessel vasculitides. In particular, data on CV/CVB surogate markers are scarce and the diagnostic value of traditional CV risk scores, such as the Systematic COronary Risk Evaluation (SCORE) and ist EULAR modified version (mSCORE) is unknown.Objectives:Aims of this study were to examine gold standard surrogates of CV risk and subclinical atherosclerosis (1), as well as novel duplex-sonographic markers of CVB risk in a cohort of patients with small vessel vasculitides. Moreover, we sought to evaluate for the first time the diagnostic value of SCORE and mSCORE in identifying patients at high CV and CVB risk.Methods:SCORE/mSCORE and the gold standard marker of aortic stiffness (carotid-femoral pulse wave velocity; cfPWV) were examined in patients with small vessel vasculitides and healthy controls across 3 Rheumatology Centers. Moreover, sonography of the common- (CCA), internal- (ICA) and external- (ECA) carotid arteries was performed in subsets of both groups, evaluating carotid intima-media-thickness (cIMT), plaque presence and duplex-sonographic indices of CBV risk, such as the resistance- (RI) and pulsatility-index (PI). Disease characteristics, clinical and serological activity markers, as well as traditional CV risk factors were also documented.Results:We recruited 46 patients with small vessel vasculitides [granulomatosis with polyangiitis (n=33), eosinophilic granulomatosis with polyangiitis (n=6), microscopic polyangiitis (n=4), cryoglobulinemic vasculitis (n=1), urticaria vasculitis (n=2): 76.1% female, median age 61.5 years (52-72.25, IQR)] and 105 healthy controls [76.1% female, median age 61.5 years (52-72.25, IQR)]. cfPWV was significantly higher in the patient group compared to the control group, even after statistical corrections for confounding factors (i.e. age, gender: different in the two groups) (8.4 m/s vs. 6.8 m/s; padjAccording to SCORE and (EULAR)-mSCORE, 0.9% and 15.8% of the patients had high CV risk (defined as SCORE/mSCORE >5), respectivelly. However, cfPWV revealed an increased CV risk in 28.2% of the patients. Moreover, carotid sonography showed signs of subclinical carotid arteriosclerosis in 50% (plaques) and 71.7% (cIMT >0.9 mm) of the patients, respectively.Conclusion:Herein, we could show that patients with small vessel vasculitides had higher aortic stiffness and subclinical carotid atherosclerosis, compared to controls and could thus have higher CV and CVB risk. Moreover, we were able to demonstrate for the first time that SCORE/mSCORE performed poorly in identifying patients at high CV risk and carotid atherosclerosis compared to cfPWV and carotid sonography respectively. Thus, cfPWV and carotid sonography could improve CV and CBV screening in patients with small vessel vasculitides.Table 1.Descriptive characteristics.Controls(n=105)Patients(n=46)Significance(p)cfPWV† (m/s)6.80 (6.05-7.5)8.40 (7.26-10.66)Age† (years)50 (39.50-56.5)61.50 (52-72.25)Gender(female)95 (90.5%)35 (76.1%)0.019*Nicotin(smokers)21 (20%)5 (11.1%)0.190Antihypertensive drugs18 (17.1%)27 (65.9%)BMI†23.73 (21.1-27.1)25.14 (22.81-28.70)0.074Mean art. pressure¶93.45±10.6896.19±10.680.150ESR† (mm/h)-20.00 (11– 41.5)-ANCA-25 (54.3%)-Creatinine†0.77 (0.72-0.81)0.94 (0.8-1.31)Cholesterol¶ (mg/dL)-216.88±40.16 -Prednisolone† (mg)-5.63 (1.75-16.38)-Lung involvement17 (37%)-Kidney involvement17 (37%)-Ear-nose-throat (ENT)16 (34.8%)-†median (IQR): Mann Whitney test¶ mean±standard deviation: t test*pReferences:[1]Vlachopoulos C et al., Prediction of Cardiovascular Events and All-Cause Mortality With Arterial Stiffness: A Systematic Review and Meta-Analysis. J Am Coll Cardiol 2010 30;55(13):1318-27Disclosure of Interests:Konstantinos Triantafyllias Speakers bureau: Pfizer, Novartis, Janssen, Chugai, Leif-Erik Thiele: None declared, Michele De Blasi: None declared, Andreas Schwarting: None declared

Background:Treatment with Janus Kinase inhibitors (JAK-i) (Tofacitinib, Baricitinib) can cause an increase of serum lipids such as total cholesterol, low- (LDL) and high- (HDL) density lipoproteins in patients with arthritis (1). On the other hand, JAK-i can reduce systemic inflammation and have therefore a beneficial effect on the cardiovascular system of treated patients. However, the effects of JAK-i on the CV system have not been adequately examined. In particular, we are not aware of any ’’real world’’ data concerning CV risk of patients receiving JAK-i treatment.Stiffness of the aortic vasculature is a modifiable, valid and independent surrogate predictor of CV risk and can be measured by carotid femoral pulse wave velocity (cfPWV). Its predicted value has been shown in a series of epidemiological studies and cfPWV is characterized as the gold standard marker for the assessment of aortic stiffness (2)Objectives:Aim of this study was to evaluate for the first time changes of cfPWV, lipid profile and traditional CV risk factors in patients receiving JAK-i therapy.Methods:Measurements of cfPWV, total cholesterol, LDL, HDL and inflammation markers were performed directly before and 5-12 months (median 6.5 months; 5-7, IQR) after initiation of JAK-i therapy. Additionally, traditional CV risk factors such as nicotine, obesity (Body-Mass-Index), diabetes and arterial hypertension were documented for both time points next to clinical activity markers, such as the Disease Activity Score 28 (DAS28). Differences in lipids, DAS28 and inflammation markers between the two time points were examined by paired t-Test. Given the fact that cfPWV can be confounded by mean arterial pressure (MAP), a mixed linear model, with MAP as a covariate, was used in order to test for differences in adjusted cfPWV values between two measurements.Results:29 patients with rheumatoid arthritis (RA) (72.4%,female) with a median age of61.5(51-75, IQR) years and a median DAS28-CRP of5.27(3.62-6.21, IQR) were recruited before (planed) initiation of JAK-i therapy.30.7%of the patients were smokers,38.5%had arterial hypertension and0.4%diabetes. Median BMI was24kg/m2(22-29, IQR).Mean total cholesterol and LDL values increased significantly under treatment with JAK-i (196.76±38.70vs.220.28±40.41mg/dl;p=0.010and119.12±31.88vs.138.72±37.43mg/dl;p=0.032, respectively). Moreover, MAP increased significantly during the same time period (105± 9.82vs.109.91±11.22mmHg; p=0.005). On the other hand, C-reactive protein (CRP) had decreased significantly between the two measurements [15.27(3.82-38.9, IQR) vs.3.82(1.4-15.9) mg/dl;p=0.05]. No statistical significant difference of cfPWV values could be observed under JAK-i treatment[-0,035 95% CI (-0,615 - 0,545); p=0.903].Conclusion:Our results reveal that JAK-i induced hyperlipidaemia did not associate with an increase of a surrogate marker of CV risk, such as aortic stiffness. More data are needed to conclude whether JAK-i could have a (positive or negative) effect on the CV system.An additional examination of the current patients in 12-18 months from treatment initiation and the recruitment of new RA and psoriatic arthritis patients are currently taking place.References:[1]Charles-Schoeman C, et al. Cardiovascular safety findings in patients with rheumatoid arthritis treated with tofacitinib, an oral Januskinase inhibitor.Semin Arthritis Rheum. 2016;46(3):261-271[2]Laurent S, et al (2006) Expert consensus document on arterial stiffness: methodological issues and clinical applications. EurHear J 27:2588–2605.Disclosure of Interests:None declared

Background:Valid assessment of disease activity leads to outcome improvement in patients with rheumatoid arthritis (RA) (1). Optical spectral transmission (OST) is a modern diagnostic tool able to assess the blood-specific absorption of light transmitted through a tissue, promising quantification of inflammation in the finger and wrist joints of RA patients (commercial device: HandScan - Hemics, The Netherlands) (2).Objectives:To our knowledge, there are no data regarding the diagnostic value of OST in the evaluation of inflammatory activity changes during arthitis follow up.Thus, aims of this study were to examine the ability of OST to detect response to anti-inflammatory therapy in patients with arthritis and to explore OST associations with clinical, laboratory and ultrasonographic (US) activity markers.Methods:OST measurements were performed in patients with active arthritides of the wrist and finger joints before and after administration of glucocorticoids (GC), during a disease flare. For the same points in time (a and b) patients and healthy controls underwent clinical, laboratory and joint US [Grey Scale (GSUS) - Power Doppler (PDUS)] examinations. OST-values before and after therapy were subsequently compared with their corresponding DAS28- and US-values. The distributions of Delta-PDUS und OST-values between the two time points were compared by Bayesian statistics. Moreover, OST diagnostic performance was tested by Receiver Operating Characteristics (ROC).Results:We recruited 54 patients with active inflammatory arthritis: 39 RA, 4 gout, 7 peripheral spondylarthritides and 4 other miscellaneous arthritides (66.7% females) and 114 controls.Previous to therapy with GC, median OST was [OST(a):8.75(5.38-16.25, IQR)] and after therapy [OST(b):4.75(2.38-8.63, IQR)] (p). Similarly, DAS28 dropped significantly after GC therapy [DAS28(a):5.12(4.33-6.10, IQR) vs. DAS28(b):3.85(3.40-4.82),p)]. OST correlated moderately with PDUS at both time points: (a)rho=0.449and (b)rho=0.414, respectively (both;p). Moreover, OST correlated significantly with swollen joint count at both time points (a)rho=0.379and (b)rho=0.382,prespectively.OST and US performed similarly in the assessment of inflammatory changes caused by the administration of GC (same tendency in the change of OST values in83.2%of the cases). Furthermore, Bayesian statistic revealed no significant differences between OST and US for all 3 examined joint categories (MCP:p=0.81; PIP:p=0.74; wrists:p=0.60).In addition, ROC revealed that OST is a very good tool to distinguish patients with arthritis from healthy controls at both examination points [AUC(a):0.883(95% CI=0.83-0.94) and AUC(b):0.811(95% CI=0.74-0.881)].Conclusion:OST was able to assess response to therapy in arthritis patients comparable to US. Moreover, OST correlated with disease activity markers and could effectively differentiate between arthritis patients and controls. Therefore, OST could prove to be a valuable non-interventional time- and resource-saving diagnostic tool to assist arthritis monitoring.References:[1]Katchamart W, et al. Systematic monitoring of disease activity using an outcome measure improves outcomes in rheumatoid arthritis. J Rheumatol 2010;37:1411–1415.[2]Onna M Van, et al. Assessment of disease activity in patients with rheumatoid arthritis using optical spectral transmission measurements, a non-invasive imaging technique. Ann Rheum Dis 2016;75:511–518.Figure 1.OST-, PDUS- and DAS28- values before and after GC therapy.Figure 2.OST- (group 1) und Ultrasound- (group 2) Bayesian distributions, means- and standard deviation-differences for MCP (A), PIP (B) and wrists (C).Disclosure of Interests:None declared

Background:Antisynthetase Syndrom (ASyS) is an autoimmune overlap disease characterized by antiaminoacyl-tRNA-synthetase (anti-ARS) antibodies and the classic triad of arthritis, myositis and interstitial lung disease (ILD) (1). Markers of cardiovascular (CV) or cerebrovascular (CVB) risk have never been examined in ASyS.Objectives:Aim of this study (RIsk of CARdiovascular Disease in ASyS: RI.CAR.D.A.) was to test the ability of an established traditional CV risk prediction score (Systematic Coronary Risk Evaluation-SCORE) and its EULAR modified version (mSCORE) to identify ASyS patients at high CV risk. Moreover, we sought to examine for the first time associations of CV surrogate markers with clinical and immunological ASyS parameters.Methods:SCORE/mSCORE and the gold standard marker of aortic stiffness (carotid-femoral pulse wave velocity-cfPWV) were examined in patients with ASyS and healthy controls in a multicenter setting (6 Rheumatology Centers). Moreover, sonography of the common- (CCA), internal- (ICA) and external- (ECA) carotid arteries was performed in subsets of both groups, evaluating carotid intima-media-thickness (cIMT), plaques and duplex-sonographic indices of CBV risk such as the resistance- (RI) and pulsatility-index (PI).Figure 1.Carotid Doppler surrogate markers of cardiovascular and cerebrovascular risk in controls and ASyS (case).cIMTCarotid intima media thickness;CAA(common-),ICA(internal),ECA(external) carotid artery;RIresistance index;PIpulsatility index. (all;p< 0.05)Results:We recruited 66 ASyS patients with different anti-ARS and 88 controls. According to mSCORE5/50 (10%) of the patients had high CV risk. However, cfPWV and carotid sonography (CS) revealed an increased CV risk in21.2%and subclinical carotid arteriosclerosis (plaques and/or cIMT>0.9 mm) (SCA) in85.7%of the patients respectively. ROC analyses showed similarly poor diagnostic performances of SCORE/mSCORE in comparison to cfPWV(>10 m/s) and SAC by areas under the curve (AUC) of0.56 (95%CI=0.39-0.73) and0.63 (95%CI=0.3-0.96),respectively. cfPWV and SCA were higher in ASyS patients compared to controls (padj=0.021andp=0.003, respectively). cfPWV and cIMT correlated in the patient group significantly with age (r=0.679; prespectively).Moreover, cfPWV correlated with BMI (padj=0.001) and diabetes(padj=0.043). ACC-RI and ACC-PI showed significant associations with a marker of myositis activity [creatine phosphokinase (CPK):r=0.629;p=0.012andr=0.574;p=0.032, respectively]. Finally, ACI-RI and ACI-PI values were higher in patients with ILD (both;p=0.039).Conclusion:This is the first report of higher aortic stiffness and SCA in ASyS patients compared to controls. Active myositis and presence of ILD were associated with higher CVB risk parameters. Furthermore, SCORE/mSCORE performed poorly in identifying patients at high CV risk and carotid arteriosclerosis compared to cfPWV and CS respectively. Thus, cfPWV and CS could improve CV and CBV screening in ASyS patients.References:[1]Cavagna L, et al. Clinical Spectrum Time Course in Anti Jo-1 Positive Antisynthetase Syndrome.Medicine2015;94:1144.Disclosure of Interests:None declared

Background Autonomic dysfunction, a basic element of fibromyalgia (FM), has been in some cases related to increased risk of cardiovascular (CV) disease. CV risk associates with aortic stiffness, which can be reliably assessed by carotid-femoral pulse wave velocity (cfPWV). Objectives Aims of this study were to test the hypothesis of increased cfPWV in a group of patients with FM and to examine its association with FM associated parameters and selected traditional CV risk factors. Methods We performed measurements of cfPWV in 99 FM patients and 102 healthy controls. The difference between cfPWV values in the two groups after controlling for possible confounding factors was evaluated through multiple regression analysis. The associations of cfPWV with FM related parameters such as pain severity on the EuroQol visual analogue scale (EQ-VAS) and FM tender points were also analysed. Finally, we explored the relationship of cfPWV with various laboratory parameters (patients’ group) and traditional CV risk factors (both groups). Results Adjusted statistical analyses for confounding factors showed significantly higher cfPWV values in FM patients in comparison to controls (padj=0.044). cfPWV associated significantly with age in both the patients and the control group (rho=0.614, p Conclusions Our data reveal that patients with FM have higher aortic stiffness than healthy controls, even after adjusting for confounding factors of cfPWV. Therefore, FM may be associated with an increased CV risk. To our knowledge, this is the largest study to examine the gold standard assessment method of aortic stiffness in patients with FM and the first one to find increased cfPWV-values in comparison to healthy subjects. Disclosure of Interest None declared

Objective: To evaluate the ability of the BioFire FilmArray Blood Culture Identification (BCID) panel to rapidly detect pathogens producing late-onset ventilator-associated pneumonia (VAP), a severe infection often produced by Gram-negative bacteria. These microorganisms are frequently multidrug resistant and typically require broad-spectrum empiric treatment. Methods: In the context of an international multicentre clinical trial (MagicBullet), respiratory samples were collected at the time of suspicion of VAP from 165 patients in 32 participating hospitals in Spain, Greece and Italy. Microorganisms were identified using the BCID panel and compared with results obtained by conventional microbiologic techniques. Results: Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae were the most commonly identified species, representing 54.7% (70/128) of microorganisms. The BCID panel showed high global specificity (98.1%; 95% confidence interval, 96–100) and negative predictive values (96.6%) and a global sensitivity and positive predictive value of 78.6% (95% confidence interval, 70–88) and 87.3%, respectively, for these microorganisms. Importantly, the BCID panel provided results in only 1 hour directly from respiratory samples with minimal sample processing times. Conclusions: The BCID panel may have clinical utility in rapidly ruling out microorganisms causing VAP, specifically multidrug-resistant Gram-negative species. This could facilitate the optimization of empiric treatment. © 2018 European Society of Clinical Microbiology and Infectious Diseases

This paper presents the design and the experimental results of a CMOS Automatic Control System (ACS) for the biasing of High-Electron-Mobility-Transistors (HEMT). The ACS is the first low-power mixed-signal Application-Specified-Integrated-Circuit (ASIC) able to automatically set and regulate the operating point of an off-chip 6 HEMT Low-Noise-Amplifiers (LNAs), hence it composes a two-chip system (the ACS+LNAs) to be used in the Large Scale Polarization Explorer (LSPE) stratospheric balloon for Cosmic Microwave Background (CMB) signal observation. The hereby presented ACS ASIC provides a reliable instrumentation for gradual and very stable LNAs characterization, switching-on, and operating point (

A 90 nm-CMOS power-optimized analog baseband chain for ultra-low-power impulse-radio ultra-wideband (IR-UWB) receivers is presented. The proposed device merges the functions of a programmable gain amplifier (PGA) and a low-pass filter (LPF). It consists of the cascade of three biquadratic cells made up by opamps in a series-shunt configuration, which features high input impedance, low load effects in the cascade blocks, and better frequency response. The opamp parameters are included in the overall biquad transfer function. This allows getting very low power performance, since the opamp bandwidth is not required to be much larger than the filter cutoff frequency. Moreover, the current consumption is optimized according to the selected gain level (1.3 mA at 0 dB-gain up to 1.9 mA at 40 dB-gain). The PGA features a 0-40 dB programmable gain range with a 5 dB gain-step. The LPF performs a sixth-order 255 MHz low-pass frequency response. For the overall chain the IIP3 is 14 dBm at 0 dB gain, while the input referred noise is 12.5 nV/√Hz at 40 dB gain. © 2012 IEEE.

In vitro-produced embryos are less viable than their in vivo counterparts. It is known that the developmental speed is a reliable marker of embryo viability. One of the major factors impairing in vitro embryo development is oxidative stress. The aim of the study was to evaluate oxidative stress and lipid peroxidation in bovine in vitro-produced embryos that reached different developmental stages at the end of culture. Abattoir-derived oocytes were matured in vitro in TCM-199 with 15% bovine serum, 0.5mugmL(-1) of FSH, 5mugmL(-1) of LH, 0.8mM L-glutamine and 50mgmL(-1) of gentamicin. Mature cumulus-oocyte complexes (COC) were fertilized in Tyrode's modified medium, supplemented by 5.3 SImL(-1) of heparin, 30muM penicillamine, 15muM hypotaurine, 1muM epinephrine and 1% of bovine serum. Both in vitro maturation and IVF were carried out at 39degreesC and 5% CO(2) in air. After 20 to 22h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF for 7 days at 39degreesC under humidified air with 5% CO(2), 7% O(2) and 88% N(2) in air. At the end of culture, embryos were assessed according to the stage of development as tight morulae (TM), early blastocysts (eBl), blastocysts (Bl), expanded blastocysts (XBl) and hatched blastocysts (HBl). For each stage of development, an average of 20 embryos were used to determine manganese superoxide dismutase (MnSOD) activity and levels of nitric oxide (NO(2)(-)) and thiobarbituric acid-reactive substances (TBARS). The SOD activity was determined by a colourimetric method (Caraglia M et al. 2011 Cell Death Dis. 2, 150, doi:10.1038/cddis.2011.34) whereas NO(2)(-) and TBARS were measured by a spectrophotometric method (Balestrieri et al. 2011 J. Cell. Physiol. doi:10.1002/jcp.22874). Data were analysed by t-test. Greater (P

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183???1192; Takahashi and First 1992 Theriogenology 37, 963???978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 ??g mL???1 FSH, 5 ??g mL???1 LH, 0.8 mM L-glutamine, and 50 mg mL???1 gentamicin. Mature COC were fertilized in Tyrode???s modified medium, with 30 mg mL???1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose???phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P < 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P < 0.05) and 1.5 mM (P < 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P < 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (>4 cells) was higher (P < 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative control; n = 513), in the presence of 0.01 mM heparin (positive control; n = 775), 10 μM melatonin (n = 684), 100 μM melatonin (n = 751), and 1 mM melatonin (n = 650), for 2 h. Sperm were then exposed for 15 min to 60 μg mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction (AR) only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The percentage of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. The experiment was repeated 4 times. Differences among groups were analyzed by chi-square test. Acrosomal loss was observed only in 2.1% of the sperm population at time 0. Interestingly, sperm treatment with both heparin and the different concentrations of melatonin resulted in a significantly higher incidence of AR compared to the negative control (24.4, 20.5, 20.0, 23.6 v. 8.0% for the positive control, 10 μM melatonin, 100 μM melatonin, 1 mM melatonin, and the negative control, respectively; P < 0.01). These results demonstrated that melatonin determines capacitation of buffalo spermatozoa in vitro. Furthermore, the effect of melatonin was comparable to that of heparin, that is, the capacitating agent currently used in the IVF system. The capacitating effect was observed at all the tested concentrations, and viability was not affected. This suggests to extend the range of concentrations to test in future studies, in order to identify the optimal dose. Moreover, considering the seasonality of the species and the great differences in fertility attitude of buffalo bulls, it would be interesting to investigate the capacitation effect of melatonin in relation to both the season and the bull.

This paper describes a chopper stabilized amplifier, obtained using an input chopper for modulation, an AC coupling for offset rejection and a correlated double sampling structure for demodulation, avoiding ripple spurs. The operational amplifier has a rail-to-rail input stage, with a supply voltage range from 1.8V to 5V. It is characterized by a multipath nested Miller compensation network with double pole-zero cancellation. The circuit achieves a simulated 130dB-dc-gain with a current consumption of 20μA, suitable for low-power application. Equivalent input offset has been evaluated by Montecarlo simulations demonstrating a maximum standard deviation of about 4μV.

In this paper a dedicated integrated front-end for the Triple-GEM (Gas Electron Multiplier) detector is presented. The design has been realized in 0.13 μm CMOS technology. This system aims to improve performance with respect to the state-of-the-art on these types of detectors, regarding adaptability, portability, power consumption and on-chip data processing. The front-end is composed by 8-input-channels. Each channel performs the charge-vs-time conversion, and then the signal is definitively converted into digital domain. For this aim a Charge-Sensitive Preamplifier (CSP), and a Charge-to-Time Converter (CTC) are implemented. An automatic on-chip calibration circuit is also included, in order to compensate CMOS technological process/temperature variations. The system is able to manage a 15 pF detector capacitance. The maximum count rate is 4·106 counts-per-second (cps) and the power consumption is 3.8 mW/ch. The Equivalent Noise Charge (ENC) is 418 e-. The front-end compares favorably with the state-of-the-art.

The design of an automatic biasing system for High-Electron-Mobility-Transistors is here presented (HEMT-Biasing-System, HBS). The HBS automatically regulates the operating point of an off-chip multistage HEMT block. A proper automatic algorithm is implemented in order to maximize the HEMT transconductance (gm) efficiency (defined as gm/IDS ratio). This is an important feature in several HEMT-based systems, since they are used in LNA stages, where noise/power trade-off is a key aspect. Interfacing CMOS and HEMT technologies features several design issues, like negative gate-source voltage (to be managed by a CMOS circuits for HEMT switch-off operation) and closed-loop stability (due to the presence of very large off-chip capacitance - 100nF). The System here presented manages all these issues with tailored circuit solutions. A prototype has been designed in CMOS 0.35μm technology. It consumes 6.2mW (excluding the current consumption for HEMT driving).

In this paper a 5th order low-pass continuous time analog filter in CMOS 45nm technology is presented. The filter design is based on a single compact cell used to synthesize five poles. The circuit performs the low-pass filtering of the in-band signal, while the in-band thermal noise is high-pass filtered, improving Signal-To-Noise-Ratio. 1-GHz -3dB-bandwidth is obtained by a prototype of the filter simulated in 45nm CMOS technology node (also Process-Voltage-Temperature simulations are available). The overall power consumption is 9mW from a single 1.2V supply voltage. 54dB@THD>40dBc is the SNR at 0dB gain.

Background The significance of early diagnosis and early therapy initiation in patients with RA is tremendous. ADAPTHERA is a state wide rheumatology network based in Rhineland-Palatinate, Germany. ADAPTHERA aims to improve RA prognosis and quality of patients` life by reducing the time-to-RA-diagnosis. While there have been a lot of studies regarding quality of life in patients with established RA 1,2 , adequate data concerning very early rheumatoid arthritis (VERA) (disease duration Objectives To study quality of life in VERA patients and to investigate its associations with various RA associated clinical and laboratory parameters. Methods In a two center subgroup analysis from the ADAPTHERA cohort, we studied quality of life indexes such as Hannover Functional Ability Questionnaire (HFAQ), Health Assessment Questionnaire (HAQ), EuroQol-5D-3L (EQ-5D-3L), Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and World Health Organisation-5 Well Being Index (WHO-5) in patients with VERA. Moreover, we examined the statistical relationship of these indexes with various clinical and laboratory RA associated parameters such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF)-/ cyclic citrullinated peptide antibodies (anti-CCP)-status and Disease Activity Score 28 (DAS28) as well as its components [counts of tender and swollen joints, Visual Analogue Scale (VAS)]. Results 74 Patients (50 females, 24 males) with a median age of 62 years (IQR 49, 71) were identified as VERA patients. 36 of them were anti-CCP positive and 34 RF positive. Median scores for HFAQ, HAQ, EQ-5D-3L, FACIT-Fatigue, and WHO-5 were 75.5 (IQR 61, 92.5), 0.75 (IQR 0.26, 1.25), 0.89 (IQR 0.79, 0.89), 40 (IQR 32, 46) and 60 (IQR 32, 76) accordingly. The results of the statistical associations between these scores and RA associated parameters are seen in Table 1. Conclusions The quality of life indexes HFAQ, HAQ, EQ-5D-3L and WHO-5 are associated with two or more markers of disease activity in patients with VERA. Therefore, improvement of patients9 quality of life is linked to the achievement of disease remission. In contrast, the FACIT-fatigue index seems to be solely associated with arthritis pain intensity. Thus, improvement of fatigue requires an effective pain management strategy. References Gossec L et al., RMD Open. 2015 Apr 2;1(1):e000019. Her M et al., Curr Opin Rheumatol, 2012 May;24(3):327–34. Disclosure of Interest None declared

In this paper, aiming to overcome the problems related to mobility in a Satellite Internet scenario, the data link reliable transmission over a mobile geo-stationary satellite link has been analysed. In such environment, the Selective Repeat (SR) classical version does not perform well due to high BER values and burst errors. In order to cope with these impairments, an Automatic Repeat Request (ARQ) protocol, which represents a SR version particularly suitable to mobile channel characteristics has been considered. A performance analysis of this SR version, through a simulations campaign aimed to the evaluation of throughput and delay measures, is presented in comparison with those of other SR approaches. The results of the analysis show how an accurate design of lower layers can determine an appreciable efficiency gain

In this paper a 8.2MHz-f @−3dB bandwidth Filter-&-Amplifier to be embedded in a DVB-T RX chain is presented. The filter has been integrated in 65nm CMOS node, working with a V DD /V TH ratio (supply/threshold voltage) as low as 2. Operating point issues due to low V DD /V TH ratio has been resolved by a proper bias circuit. Since such bias circuit is part of the filter circuit, its impact on filter performance have been studied and considered in the design. Power consumption is minimized by using a novel algorithm based on Matlab procedure, which guarantees the minimum power consumption for a given transfer function, noise and linearity requirements. The device consumes 1.3mW from a single 1.2V supply voltage, features −10dBm-IIP3 at 32dB pass-band Gain, and 1.6mV rms output integrated noise over the pass-band (300kHz÷8.2MHz).

In this paper a 115μW Programmable Gain Amplifier (PGA) in 90nm CMOS technology is presented. The PGA is embedded in the baseband chain of a low-data-rate UWB PAN receiver. Since a vibration-based energy scavenger powers the receiver, low power consumption is required, while maintaining large dynamic range and wide-band frequency response. Moreover, high input impedance is required from system level constraints and this is achieved with MOS input devices, exploiting dual differential input Opamp. The PGA gain is programmable in the 15dB-to-50dB range with 1dB step. For the maximum gain level (50dB), the Input-Referred-Noise power spectral density is lower than 20nV/√Hz. From a single 1V supply, for the minimum gain level (15dB), a 0.8Vzero-peak output signal is processed with THD=−40dBc.

In this work a new concept of hybrid lamps burner is presented. The High Intensity Discharge (HID) lamps and the new generation Light Emitting Diodes (LED) lamps can be driven by the same electronic ballast using this hybrid ASIC in 0.35m CMOS technology. In the state-of-art the electronic ballasts are managed by powerful general-purpose devices (usually FPGA or microcontroller). These devices are not customized for the specific application. Thus they have a larger area occupation and power consumption compared to ASIC solution. The designed ASIC power consumption is 35 mW and the package area is about 6.5 cm2 against 550 mW and 6.5 cm2 for commonly used PIC 18FXX2. © 2011 IEEE.

In this paper the design of a 3rd-order 16 bit 20 kHz-bandwidth discrete-time σδ modulator with voltage- controlled oscillator (VCO)-based quantizer is presented. This design is motivated by the trends towards die size and power consumption reduction together with performance robustness. In this direction, the flash converter embedded in a multi-bit σδM is here replaced by a VCO and digital counter, resulting in a compact fully-digital quantizer implementation. This solution would greatly exploit the technology scaling. Moreover in the proposed feed-forward architecture all feed-forward paths are summed within the last integrator of the σδ loop filter, thereby eliminating the need for an analog summation amplifier at the quantizer input The presented 3rd-order σδM features 110 dB-SNR, 104 dB-SNDR (i.e. 16 bit-ENOB). © Springer Science+Business Media, LLC 2011.

Radio frequency identification (RFID) technology is becoming more and more important for tracing and tracking systems. Furthermore, the application of standards, such as EPCglobal, is contributing substantially to diffusing the important concept of Internet of Things. Although, these elements promise enormous benefits, there remain barriers that are limiting a large-scale deployment of these innovative technologies in supply chain management systems. An analysis of the main effects of the use of these technologies on the main processes of a supply chain is useful. This paper attempts to identify key performance indicators (KPIs) to trace the impacts of the combined use of different standards, such as RFID, EPCglobal, and ebXML, on a pharmaceutical supply chain. The current and possible future visions of the pharmaceutical supply chain are described through the AS-IS and TO-BE models. These models are used to perform a first KPI analysis; even if the reported results are still only partial they sketch out the main guidelines identifying the advantages obtainable by adopting an integrated system with B2B messaging and tracing and tracking capabilities in the pharmaceutical supply chain.

The item-level tagging is one of the main challenges of many application scenarios. Among these, the pharmaceutical supply chain represents a very interesting case where the new auto-identification technologies, based on RFID and EPCglobal, will play a very import role. The authors present practical experiences gained from a recent research project on the item-level traceability in the pharmaceutical supply chain. Furthermore, a discussion on several areas of improvement opportunities for future large-scale deployments of these technologies is reported.

The item-level tagging is one of the main challenges in the pharmaceutical distribution in order to improve track and trace systems. The RFID technology will play a very important role in development of auto-identification solutions. In this chapter, we focus on the use of passive UHF tags, in order to analyze a performance comparison between near field and far field UHF RFID systems. Some different passive UHF tags, suitably chosen, have been tested at item-level, i.e., on each single medicine package, simulating each stage of the pharmaceutical supply chain in a controlled laboratory environment. The obtained experimental results, in terms of successful read rates, have shown that the use of passive far field UHF tags represents an advantage solution to guarantee high performance in an item-level tracing system in the whole supply chain.

Implementation of a computer architecture simulator prototype written in Turbo Prolog, which is suitable for educational purposes, is described. Besides adapting to teaching needs for conventional use, the simulator design incorporates features for an easy alteration of computer architectural structures appropriate to the trainee's learning level. Thus, this simulator is not limited to a particular architecture but permits the student to vary dynamically the architectural characteristics of the computer under study in order to verify the consequences of his decisions and to experiment with families of computers and alternatives of design. >

Recently, there has been much effort to integrate heterogeneous wireless technologies in order to make overlay networks able to provide mobile users with ubiquitous Internet access. Handover management is still one of the most challenging issues to be solved for seamless integration of wireless networks. In this paper, an algorithm for vertical handover with QoS guarantees between IEEE 802.16e and IEEE 802.11e networks, without computational complexity added on mobile nodes, has been proposed. It uses a decision mechanism based on the following metrics: the available bandwidth, the signal-to-noise ratio (SNR) and the access delay. Its characteristics are perfectly compatible with the guidelines specified in the IEEE 802.21 standard. The effectiveness of the defined algorithm has been evaluated, using the ns-2 tool, in terms of packet loss rate (PLR), satisfaction of minimum applications requirements and number of useless handovers. The performance of the defined algorithm has been compared with that obtained by using the algorithm suggested by IEEE 802.21 standard that exploits only the SNR in the decision procedure.

Numerous cases of acute myocardial infarction (AMI) have been reported in the literature following closed chest injuries, due to post-traumatic dissection or thrombosis of a coronary artery. In the follow-up of AMI, wall thickness during diastole and systole provides important information on heart viability. Multidetector computed tomography (MDCT) is currently the only noninvasive instrumental investigation which provides an appreciable assessment of the coronary arteries, as well as heart wall thickness measurements. We describe and discuss the clinical and imaging findings, especially of MDCT, in a case of post-traumatic regional myocardial necrosis with normal coronary arteries.

Although primary cardiac lymphoma is an extremely rare disease and is associated with high mortality, it is treatable when diagnosed appropriately. We describe the transthoracic echocardiography, 16-row multidetector computed tomography (16-MDCT) and magnetic resonance findings of primary cardiac lymphoma in an immunocompromised patient and review of the literature.

Despite the great improvement of in vitro embryo production (IVEP) efficiency recorded over the years in cattle, the in vitro produced (IVP) embryos are still less viable and resistant to cryopreservation than their in vivo counterparts. One of the major factor impairing in vitro embryo development is oxidative stress. Resveratrol is an important antioxidant polyphenolic compound found in several vegetal sources, that contributes to red wine’s beneficial effects on the prevention of human cardiovascular disease. Recently, the interest in resveratrol has increased exponentially following the major findings that this molecule has positive effects on cancer chemoprevention, cardioprotection, inflammatory processes, several aspects of metabolism, leading to increased lifespan of various organisms from yeasts to vertebrates (Pirola et al. 2008 IUBMB Life 60, 323–332). A positive effect of resveratrol on in vitro embryonic development was demonstrated in swine (Lee et al. 2010 J. Reprod. Dev. 56, 330–335). The aim of this study was to evaluate whether supplementation of culture medium with resveratrol improves in vitro blastocyst development and the embryo resistance to cryopreservation in cattle. A preliminary dose response trial indicated that the optimal concentration in the range tested (from 0.5 to 10 µM) was 0.5 µM, with evident toxic effects at concentration higher than 5 µM. Abattoir-derived oocytes (n = 581, over 5 replicates) were matured and fertilized in vitro according to our standard procedure (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Twenty hours after IVF, presumptive zygotes were cultured in SOF medium, supplemented with 5% bovine serum, in the absence (control, n = 271) or presence of 0.5 µM resveratrol (n = 310) at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7 (IVF = Day 0), embryo yields were assessed and the blastocysts (except the hatched blastocysts) were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5M sucrose (Rubessa et al. 2011). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, and hatching rate after 48 h culture. Data were analyzed by chi-square test. Resveratrol supplementation during culture did not affect either cleavage (69.1 v. 72.0%, in the control and resveratrol groups, respectively) or blastocyst yields (38.3 v. 36.3%, in the control and resveratrol groups, respectively). However, treatment with resveratrol increased the cryotolerance of IVP embryos, as indicated by higher survival rates (74.7 v. 88.4%, in the control and resveratrol groups, respectively; P

In buffalo, in vitro embryo production (IVEP) technology is the best tool to improve the genetic merit through the maternal lineage. A major limitation of IVEP technology in buffalo species is the poor cryotolerance of the embryos, likely due to their high lipid content (Gasparrini 2002 Theriogenology 57, 237–256). It was previously demonstrated that supplementing bovine culture media with L-carnitine, a cofactor of β-oxidation, improves in vitro embryo development (Sutton-McDowall et al. 2012 Theriogenology 77, 1632–1641). The aim of this work was to evaluate whether L-carnitine supplementation during in vitro culture (IVC) improves blastocyst development and cryotolerance of in vitro produced buffalo embryos. After a preliminary dose response trial, we selected the concentration of 0.25 mM for the experiment. Cumulus–oocytes complexes (n = 288, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006 Theriogenology 65, 275–287). On Day 1 (Day 0 = IVF), zygotes were cultured in SOF supplemented with 8 mg mL–1 BSA, in the absence (control, n = 143) or presence of 0.25 mM L-carnitine (n = 145). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 5, when the cleaved embryos were transferred into fresh medium for further 2 days. On Day 7 after IVF, embryo outcome was assessed and all the embryos were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide (DMSO), and 0.5 M sucrose (De Rosa et al. 2007 Ital. J. Anim. Sci. 6(Suppl 2), 747–750). The resistance to cryopreservation was evaluated by assessing the survival rate, on the basis of morphological criteria, after 24 h culture. Data were analyzed by chi-square test. No differences were found in cleavage rates between the control (81.5%) and the L-carnitine group (78.8%). The blastocyst yields (calculated in relation to the cleaved embryos) were not significantly influenced by the L-carnitine treatment (40.2 and 52.9%, in the control and the L-carnitine groups, respectively). However, buffalo embryos cultured in the presence of L-carnitine showed an increased resistance to cryopreservation, as indicated by the higher survival rates recorded after 24 h culture (78.7 and 96.4%, in the control and the L-carnitine groups, respectively; P

Most systems for producing bovine embryos in vitro use glucose as an energy source despite putative toxic effects. Glucose has a selective embryotoxicity towards female embryos, due to the higher expression of the X-linked glucose-6-phosphate dehydrogenase gene (Kimura et al. 2005 Mol. Reprod. Dev. 72, 201–207). Recently, the replacement of glucose with citrate and myo-inositol in SOF medium supplemented with 5% bovine serum (BS) increased the percentage of female embryos (Rubessa et al. 2011 Theriogenology 76, 1347–1355). Serum also affects the sex ratio of in vitro-produced (IVP) bovine embryos, favoring the male gender (Gutierrez-Adan et al. 2001 Theriogenology 55, 1117–1126). The aim of this work was to evaluate the effect of glucose replacement with myo-inositol during in vitro culture, in the presence of either BS or BSA, on bovine embryo sex ratio. Abattoir-derived oocytes (n = 1164, over 4 replicates) were matured and fertilized in vitro as previously described (Rubessa et al. 2011). After 20 to 22 h of gametes co-incubation, zygotes were denuded and cultured for 7 days in SOF with: group A) 0.34 mM trisodium citrate + 2.77 mM myo-inositol + 5% BS (n = 287); group B) 0.34 mM tri-sodium citrate + 2.77 mM myo-inositol + 8 mg mL–1 BSA(n = 290); group C) 1.5 mM glucose + 5% BS (n = 302) and group D) 1.5 mM glucose + 8 mg mL–1 BSA (n = 285). Representative samples of blastocysts produced in each group (n = 96, 58, 99, and 70, respectively in groups A, B, C, and D) were sexed by PCR as previously described (Rubessa et al. 2011). Differences among groups in blastocyst yields were analyzed by ANOVA. The percentages of female embryos were analyzed by chi-square test. Blastocyst rates in group C were lower (28.1%) than those recorded in groups A, B, and D (35.9, 41.0 and 36.1%, respectively; P

Although in vitro embryo production efficiency in buffalos has greatly improved over the years, the in vitro-produced embryos show lower viability and resistance to cryopreservation. Therefore, it is necessary to optimize the in vitro culture conditions to improve embryo quality. Hyaluronic acid, a glycosaminoglican present in oviducal and uterine fluids, has been shown to successfully support in vitro development of bovine embryos (Stojkovic et al. 2002 Reproduction 124, 141–153). The aim of this study was to evaluate the influence of high concentrations of hyaluronic acid (HA) during late in vitro culture on blastocyst development, as well as on their cryotolerance after cryotop vitrification in buffalos. In vitro matured and fertilized buffalo oocytes (n = 1007) from slaughterhouse ovaries were cultured for 4 days in SOFaa supplemented by 8 mg mL–1 of BSA in a controlled gas atmosphere consisting of 5% CO2, 7% O2 and 88% N2, in humidified air, at 38.5°C. On Day 4, cleavage rate was assessed (75.2%) and all of the cleaved elements were divided into 3 different late culture groups: 8 mg mL–1 of BSA (n = 244; group A), 8 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 251; group B) and 1 mg mL–1 of BSA supplemented by 6 mg mL–1 of HA (n = 262; group C). On Day 7 after IVF, embryo outcome was assessed and all of the embryos were vitrified by cryotop [De Rosa et al. 2007 Ital. J. Anim. Sci. 6 (Suppl 2), 747–750] and cultured for 24 h. The resistance to cryopreservation was evaluated by assessing the survival rate on the basis of morphological criteria and the percentage of embryos reaching a more advanced developmental stage after 24 h culture. Data were analysed by the chi-square test. No differences in blastocyst rate were recorded among groups (43.9, 44.3 and 40.0%, respectively in A, B and C groups). However, out of the total embryos, a higher percentage of Grade 1 hatched blastocysts (Robertson and Nelson 1998 Manual of the International Embryo Transfer Society 9, 103–16) was observed in group C (P

In vitro-produced embryos are less viable than their in vivo counterparts. It is known that the developmental speed is a reliable marker of embryo viability. One of the major factors impairing in vitro embryo development is oxidative stress. The aim of the study was to evaluate oxidative stress and lipid peroxidation in bovine in vitro-produced embryos that reached different developmental stages at the end of culture. Abattoir-derived oocytes were matured in vitro in TCM-199 with 15% bovine serum, 0.5 μg mL–1 of FSH, 5 μg mL–1 of LH, 0.8 mM L-glutamine and 50 mg mL–1 of gentamicin. Mature cumulus–oocyte complexes (COC) were fertilized in Tyrode's modified medium, supplemented by 5.3 SI mL–1 of heparin, 30 μM penicillamine, 15 μM hypotaurine, 1 μM epinephrine and 1% of bovine serum. Both in vitro maturation and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF for 7 days at 39°C under humidified air with 5% CO2, 7% O2 and 88% N2 in air. At the end of culture, embryos were assessed according to the stage of development as tight morulae (TM), early blastocysts (eBl), blastocysts (Bl), expanded blastocysts (XBl) and hatched blastocysts (HBl). For each stage of development, an average of 20 embryos were used to determine manganese superoxide dismutase (MnSOD) activity and levels of nitric oxide (NO2–) and thiobarbituric acid-reactive substances (TBARS). The SOD activity was determined by a colourimetric method (Caraglia M et al. 2011 Cell Death Dis. 2, 150, doi:10.1038/cddis.2011.34) whereas NO2– and TBARS were measured by a spectrophotometric method (Balestrieri et al. 2011 J. Cell. Physiol. doi:10.1002/jcp.22874). Data were analysed by t-test. Greater (P

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P

The current knowledge on metabolism and glucose utilisation of preimplantation bovine and ovine embryos suggest the reduction of glucose concentration during early culture. On the contrary, it has been demonstrated that glucose is absolutely required for in vitro culture of buffalo embryos, as indicated by the poor efficiency recorded in the absence of this substrate during early embryonic development (Monaco et al. 2006 Reprod. Domest. Anim. 41, 332). However, complete removal of glucose from culture medium throughout pre-elongation development is unlikely to benefit the embryo because glucose plays other roles including ribose and NADPH production through the pentose-phosphate pathway. Therefore, the aim of this study was to investigate the effect of reducing glucose concentration up to 0.15 mM (1/10 compared to the standard concentration in SOF) on embryo development in buffalo. In order to evaluate the role of this substrate during development, glucose was reduced at different stages of embryo culture. Cumulus–oocyte complexes (n = 573, over 4 replicates), recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287). On day 1 (Day 0 = IVF), zygotes were cultured in SOF with group A) 1.5 mM glucose (standard concentration in SOF) throughout culture (control); group B) 1.5 mM glucose for early culture (Day 1 to Day 4) and 0.15 mM glucose for late culture (Day 4 to Day 7); group C) 0.15 mM glucose throughout culture; and group D) 0.15 mM glucose for early culture and 1.5 mM glucose for subsequent culture. In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Cleavage rate was evaluated on Day 4, and blastocyst yield, in relation to cleaved embryos, was recorded on Day 7. Differences among groups in blastocyst rate were analysed by chi-square test. The reduction of glucose concentration did not affect cleavage rate (73.7 v. 65.1%, respectively, for Groups A-B and C-D). Nevertheless, blastocyst rates significantly decreased when glucose was reduced throughout culture (Group C: 10.1%; P

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183–1192; Takahashi and First 1992 Theriogenology 37, 963–978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 μg mL–1 FSH, 5 μg mL–1 LH, 0.8 mM L-glutamine, and 50 mg mL–1 gentamicin. Mature COC were fertilized in Tyrode’s modified medium, with 30 mg mL–1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose–phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P < 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (>4 cells) was higher (P < 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants