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This study compared the nutrient-energy retention, digestive function, growth performance, and welfare of rainbow trout (ibw 54 g) fed isoproteic (42%), isolipidic (24%), fishmeal-free diets (CV) over 13 weeks. The diets consisted of plant-protein replacement with graded levels (10, 30, 60%) of protein from poultry by-product (PBM) and black soldier fly

The production of insects on an industrial scale has attracted the attention of the research and agricultural industry as novel protein sources. To detect the presence of Gryllodes sigillatus (GS) in feed and food, a real-time PCR method based on the mitochondrial cytochrome b (CYB) gene is proposed by this study. Forty DNA samples of animal and plant origin were used to confirm the specificity of the qPCR system. The detection method’s performance was evaluated on different processed GS matrices including native GS (UnGS) and different commercial products: crunchy roasted samples (RoGS), insect meal mixtures (ACGS) and energetic snacks containing GS (GSS). Data on sequencing were aligned with the reference gene to confirm the PCR products. The regression curve (y = −3.394 x + 42.521; R2 = 0.994, d.f. 14) between Ct values and Log DNA concentrations of Gryllodes sigillatus resulted in an efficiency of 96.4%. The severity of the technological processing treatments and the matrix structure affected the intensity of the PCR signal with the same amount of insect DNA as observed by different y-intercepts of the three-regression lines for RoGS, ACGS, and GSS. The real-time PCR method resulted in robust and sensitive outcomes able to detect low amounts of GS DNA (5 g/100 g) in a complex matrix, making it suitable for detecting the presence or absence of labeled Gryllodes sigillatus material both in feed and food.

This study compared the nutrient-energy retention, digestive function, growth performance, and welfare of rainbow trout (ibw 54 g) fed isoproteic (42%), isolipidic (24%), fishmeal-free diets (CV) over 13 weeks. The diets consisted of plant-protein replacement with graded levels (10, 30, 60%) of protein from poultry by-product (PBM) and black soldier fly H. illucens pupae (BSFM) meals, either singly or in combination. A fishmeal-based diet was also tested (CF). Nitrogen retention improved with moderate or high levels of dietary PBM and BSFM relative to CV (p < 0.05). Gut brush border enzyme activity was poorly affected by the diets. Gastric chitinase was up-regulated after high BSFM feeding (p < 0.05). The gut peptide and amino acid transport genes were differently regulated by protein source and level. Serum cortisol was unaffected, and the changes in metabolites stayed within the physiological range. High PBM and high BSFM lowered the leukocyte respiratory burst activity and increased the lysozyme activity compared to CV (p < 0.05). The BSFM and PBM both significantly changed the relative percentage of lymphocytes and monocytes (p < 0.05). In conclusion, moderate to high PBM and BSFM inclusions in fishmeal-free diets, either singly or in combination, improved gut function and nutrient retention, resulting in better growth performance and the good welfare of the rainbow trout.

Carpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. The aim of the present study was to test four different grossly isolipidic, isoproteic, and isoenergetic diets in which the main ingredients had a different origin. Specifically, a diet currently used by local farmers for carpione culture, largely based on marine ingredients, was used as control (CTRL), while the other three diets were formulated by partially replacing marine ingredients with plant ones (VEG) or with different percentages of processed animal proteins (PAP1 and PAP2). The feeding trial was run in triplicate, over a three-month period. No significant differences in growth performance among the experimental groups were observed. However, remarkable histological alterations and inflammatory markers upregulation were observed in VEG group, while PAP inclusion played a role in attenuating inflammation and improving nutrient uptake. Fillet analyses highlighted significant differences in marketable traits and flesh fatty acid composition among the experimental groups, including the reduction of polyunsaturated fatty acids related to PAPs inclusion. In conclusion, PAPs used in the present study promoted S. carpio gut health and absorption capacity, while further studies are required to maintain proper quality traits of the final product.

Simple Summary Sustainability and fish welfare have been receiving increasing attention in the aquaculture sector, with an emphasis on the search for new, sustainable, and healthy aquafeed ingredients. For many years, plant ingredients have been widely used in aquafeed formulation; however, negative side effects on gut welfare have often been reported in several carnivorous fish species. From this perspective, alternative ingredients such as poultry by-products and insect meal are receiving attention due to their low ecological footprint and high nutritional value. In the present study, these two ingredients were used, singly or in combination, to formulate practical diets for gilthead seabream (Sparus aurata). After a twelve-week feeding trial, a multidisciplinary laboratory approach including histological, molecular, and spectroscopic techniques was adopted in order to investigate fish physiological responses to the new test diets. The results obtained showed excellent zootechnical performances and ameliorated gut health in fish fed dietary inclusions of poultry by-products and insect meal compared to those fed a vegetable-based diet. In addition, the modulation of nutrient absorption in relation to the ingredients used was highlighted by means of spectroscopic tools. The results obtained demonstrated that poultry by-products and insect meal can be successfully used to replace plant-derived ingredients in diets for gilthead seabream without negatively affecting fish welfare. Abstract The attempt to replace marine-derived ingredients for aquafeed formulation with plant-derived ones has met some limitations due to their negative side effects on many fish species. In this context, finding new, sustainable ingredients able to promote fish welfare is currently under exploration. In the present study, poultry by-products and Hermetia illucens meal were used to replace the vegetable protein fraction in diets totally deprived of fish meal intended for gilthead seabream (Sparus aurata). After a 12-week feeding trial, a multidisciplinary approach including histological, molecular, and spectroscopic techniques was adopted to investigate intestine and liver responses to the different dietary formulations. Regardless of the alternative ingredient used, the reduction in dietary vegetable proteins resulted in a lower incidence of intestine histological alterations and inflammatory responses. In addition, the dietary inclusion of insect meal positively affected the reduction in the molecular inflammatory markers analyzed. Spectroscopic analyses showed that poultry by-product meal improved lipid absorption in the intestine, while insect meal induced increased liver lipid deposition in fish. The results obtained demonstrated that both poultry by-products and H. illucens meal can successfully be used to replace plant-derived ingredients in diets for gilthead seabream, promoting healthy aquaculture.

By answering the need for increasing sustainability in aquaculture, the present study aimed to compare growth, gene expression involved in appetite regulation, physical characteristics, and chemical composition of Sparus aurata fed alternative protein sources. Fish were fed ten iso-proteic, iso-lipidic, and isoenergetic diets: a vegetable-based (CV) and a marine ingredient-rich (CF) diet were set as control diets. The others were prepared by replacing graded levels (10, 20 or 40%) of the vegetable proteins in the CV with proteins from a commercial defatted Hermetia illucens pupae meal (H), poultry by-product meal (PBM) singly (H10, H20, H40, P20, P40) or in combination (H10P30), red swamp crayfish meal (RC10) and from a blend (2:1, w:w) of Tisochrysis lutea and Tetraselmis suecica (MA10) dried biomasses. The increase in ghre gene expression observed in MA10 fed fish matched with increased feed intake and increased feed conversion ratio. Besides, the MA10 diet conferred a lighter aspect to the fish skin (p &lt, 0.05) than the others. Overall, no detrimental effects of H, PBM, and RC meal included in the diets were observed, and fish fatty acid profile resulted as comparable among these groups and CV, thus demonstrating the possibility to introduce H, PBM, and RC in partial replacement of vegetable proteins in the diet for Sparus aurata.

In the last decades, processed animal proteins, such as poultry by-product meal (PBM) and insect meals have received great attention as sustainable and nutritious aquafeed ingredients. The aim of the present study was to evaluate growth performances, liver and gut histology, macromolecular composition and inflammatory response in rainbow trout (Oncorhynchus mykiss) fed diets deprived of fish meal, where graded levels of vegetable protein-rich ingredients, were replaced by defatted Hermetia illucens pupae meal (HM) or PBM, singly or in combination. To this end eight grossly iso‐proteic (45% DM), isolipidic (26% DM) and isoenergetic (23.5 MJ/kg DM) were offered each to triplicated groups of juveniles' fish in a 91 days feeding trial. A diet rich in vegetable protein derivatives high in soybean meal (CV) was prepared to have a 10:90 and 20:80 fish to vegetable protein and lipid ratios respectively. By contrast, a fish-based diet (CF) was formulated with opposite fish to vegetable protein and lipid ratios. Six more diets, were obtained by replacing graded levels of protein (10, 30 and 60%) of diet CV, by protein from a defatted Hermetia illucens pupae meal and/or poultry by-product meal, singly or combined, while maintaining the same vegetable to fish lipid ratio as in the CV diet. Relative to diets CV and CF, a medium to high substitution (30 and 60%) of dietary vegetable protein-rich ingredients, with HM and/or PBM resulted in improved growth performance as well as in a minor incidence of distal intestine morphological alterations. The diet including both the test animal proteins led to nearly the best overall response in terms of growth and gut/liver health. Both HM and PBM when included at moderate or high levels in the diet, resulted in a downregulation of the expression of inflammatory-related genes relative to diet CV. This effect was greater with HM than that observed with PBM and goes beyond the parallel reduction of vegetable protein and SBM levels in the same diets, suggesting a beneficial role of insect meal that warrant further investigation. The results obtained so far, provide support to a reliable use of alternative/underexploited protein and lipid sources [(HM) or (PBM)] in developing a new generation of sustainable and healthy trout diets that meet the circular economy principles.

At the National Institute of Metrological Research (INRIM), an automated precision DC voltage fixed-ratios divider allowing the division ratios 10:1 and 100:1 has been built. It is made utilizing one hundred selected bulk metal foils 10 kΩ resistors with low temperature coefficient. Being the instrument inter-connectable to a multi-meter (DMM) characterized in linearity on the 10 V range and to a DC voltage generator, it can be quickly and automatically calibrated when necessary. This calibration method has been validated by comparing it with another method involving the INRIM DC voltage ratio standard. Other achievement is that the characterization of the linearity of the DMM on the 10 V range allow considering in the uncertainties budget of the calibration of the divider, the uncertainties of the linearity characterization of the DMM instead of its much larger accuracy specifications. The expanded uncertainties of the calibration of the 10:1 and 100:1 ratios are respectively 4.6 × 10−7 and 6.6 × 10−7, suitable to employ the divider in an automated measurement setup with a DMM and a DC voltage standard to calibrate DC voltage generators as top-level calibrators, widespread in secondary electrical laboratories.

At National Institute of Metrological Research (INRIM), an automated setup to calibrate dc voltage generators, mainly top-level calibrators, from 1 mV to 1 kV has been developed. The heart of the setup is an INRIM-built automated precision fixed ratios dc voltage divider. It can be interconnected to a DMM characterized in linearity, to a dc voltage standard and to a dc voltage generator under calibration to manage automatically the calibration process. Novelty of the system is the employment of the divider being such an instrument not commercially available. Main results are the improvement of the reliability and the increasing of the accuracy of the calibration process saving a lot of time. In addition, it is possible to avoid several standards (still manually operating) carrying out the whole process without changing the setup configuration and without the presence of operators. The expanded uncertainties of the system span from 6.0 × 10−7 to 1.3 × 10−4 improving the previous capabilities of the INRIM laboratory for calibration of multifunction instruments. The setup concept can be transferred to secondary high-level electrical laboratories to improve and expedite their calibrations.

Despite a growing interest in microalgae and cyanobacteria as potential sources of nutrients in aquafeeds, little information is presently available on their nutritive value for carnivorous fish species. The aim of this study was to evaluate chemical composition and nutrient digestibility of a panel of microalgae and cyanobacteria dried biomasses (MACB), using rainbow trout (Oncorhynchus mykiss W.) as a fish model. Nine test diets were obtained by mixing 80 parts of a reference diet, added with 20 g/kg of acid insoluble ash as an indigestible marker, to 20 parts of each of the following dried whole-cell biomass: Arthrospira platensis, Nostoc sphaeroides, two strains of Chlorella sorokiniana, Nannochloropsis oceanica, Tisochrysis lutea, Phaeodactylum tricornutum, Porphyridium purpureum and Tetraselmis suecica. The digestibility measurements were conducted with rainbow trout (52.4 ± 1.5 g) kept in six tank units each including three 60-L vessels singularly stocked with 12 fish and fitted with a settling column for faecal recovery. Per each diet, faeces were collected over three independent 10-day periods. Apparent digestibility coefficients (ADCs) of dry matter, crude protein (CP), organic matter and gross energy (GE) of single MACB were calculated by difference relative to those of the reference diet. The MACBs had heterogeneous chemical composition (CP, from 20 to 69%; Lipid, 5-27%; GE, 12.5--22.6 MJ/kg dry matter basis) reflecting their overall biodiversity. Most of them can be considered as virtually good sources of minerals and trace elements and exhibit an essential amino acid profile comparable or even better than that of soybean meal commonly used in fish feeds with P. purpureum showing the best protein profile. The digestibility results obtained with rainbow trout allowed ranking the MACBs into two major groups. A first one, including C. sorokiniana, N. oceanica and T. suecica, resulted in markedly lower (P < 0.05) crude protein and energy ADC (64-73%; 51-59%, respectively) compared to a second group including P. purpureum, T. lutea and cyanobacteria (CP-ADC, 83-88%; GE-ADC, 74-90%) while P. tricornutum resulted in intermediate values. Overall, the present study confirms the consistently reported role of cell-wall structure/composition in affecting accessibility of nutrients to digestive enzyme. Based on the overall outcomes, only T. lutea and cyanobacteria actually meet the requirements for being used as protein sources in aquafeeds provided their mass production becomes more feasible and cost-effective, hence attractive for the feed-mill industry in the near future.

At INRIM a DC Voltage precision resistive divider performing decade ratios from 10:1 to 107:1 was built. It can be automatically calibrated with a top-class calibrator and a precision multimeter calibrated in terms of deviation from linearity. It is made up of 90 kΩ, 9 kΩ, 900 Ω, 90 Ω, 9 Ω, 0.9 Ω, 90 mΩ and 10 mΩ bulk metal foil resistors connected in series, in four-terminal configuration. Peculiarities of the calibration method of the divider are: the evaluation of the DMM input impedance to correct its readings minimizing the load error and a solution to reduce the emfs effect of the relays. These operations are made during the calibration of the divider. The calibration and use uncertainties of the divider span respectively from 6.1 × 10−7 to 5.9 × 10−4 and from 6.7 × 10−7 to 6.5 × 10−4. The project is transferable to secondary laboratories in the framework of the INRIM knowledge transfer task.

At National Institute of Metrological Research (INRIM), a selectable-value transportable high dc voltage standard (THVS) operating in the range from 10 V to 100 V in steps of 10 V, was developed. This standard was built to cover the lack of high level dc voltage standards at values higher than 10 V to employ mainly as transportable standard for inter-laboratory comparisons (ILCs) or eventually as laboratory (local) high level dc voltage standard. An electronic technique was used to improve the THVS accuracy of the high values. The THVS shows lower noise, better short- term stability and accuracy than top level dc voltage and multifunction calibrators (MFCs) and satisfactory suitability to be transported. The project is extensible to 1000 V.

At the National Institute of Metrological Research (INRIM) a measuring setup able to vary, accurately and in a selective way, the temperature, humidity and pressure to detect eventual hysteretic phenomena and to evaluate their effects on electrical standards as high precision standard resistors and Zener-diode based DC Voltage standards. Main element of the setup is a pressure-tight case housing the standards and acting the stresses due to the desired parameters. By means of pressure-tight-connectors, the standards under test are connected to the measuring instruments, while the climatic conditions are acquired through inner and external sensors. Humidity is controlled by means of two conditioning containers filled respectively with water and silica-gel, which regulate the air-moisture percentage the case in a range of 10-90%. A pumping system establishes the pressure in the case from 600 hPa to 1400 hPa. The characterization vs. humidity and pressure is made at constant temperature by means of a commercial air-bath acting from 15 and 45 ° C. Preliminary results have been obtained for pressure dependence of commercial 100 Ω resistors and of an INRIM-developed 10 V Zener-diode based DC Voltage standard.

At the National Institute of Metrological Research (INRIM) an automatable precision DC voltage fixed-ratios divider allowing the division ratios of DC Voltages from 10:1 to 107:1. It can be quickly calibrated when necessary and involved in traceability transfer. Its resistors are selected bulk metal foils ones connected in series in four terminal configuration whose values are 90 kΩ, 9 kΩ, 900 Ω, 90 Ω, 9 Ω, 0.9 Ω, 90 mΩ and 10 mΩ. The main advantage of this divider is that it can be automatically calibrated with a calibrator and a digital multimeter. Its calibration starts from a10 V value. It takes advantage of the DMM linearity, in particular in the 10 V range that allows improving its calibration uncertainty. After calibration, it can be used to divide DC Voltages lower than 10 V. Preliminary evaluation of its calibration uncertainties and mid-term stabilities (a week) span respectively from 1.4×10-6 to 6.0×10-4 and from 2.4×10-7 to 4.5×10-4 for ratios from 10:1 to 107:1. This divider could be involved in the calibration of nanovoltmeters in a typical range from 10 V till down to 100 nV.

A portable temperature controlled DC Voltage and DC Resistance Reference with Switching Unit (RSU) has been developed at National Institute of Metrological Research (INRIM) to calibrate multifunction electrical instruments involving 10 V, a $\pmb{1 \Omega}$ and a $\pmb{10 \mathrm{k}\Omega}$ Standards. The resistors are made with two resistors nets while the 10 V is a low noise-drift INRIM-projected circuit. Preliminary measurement over one-month period shows standards uncertainties span from $\pmb{3.0\times 10^{-7}}$ to $\pmb{6.4\times 10^{-7},}$ suitable for artifact calibration. With a real clock calendar, the RSU can show both the RSU Standards calibration values and updated ones between two calibrations according to an internal algorithm.

Inter-laboratory comparisons (ILCs) are an effective mean to establish the compatibility of the measurements among laboratories. As consequence they are a mean to assure confidence in the competence and in the correctness of the dissemination processes from national Standards in different countries. This paper refers on a ILC between the National Institute of Metrological Research (INRIM-Italy) and the European space research and technology Centre (ESA- ESTEC), belonging to two different countries, on DC Voltage, DC Resistance and Electrical Capacitance carried out in 2016. The comparison showed a satisfactory agreement between the results of the two Laboratories.

A temperature controlled 10 V, 1Ω and 10 kΩ transportable setup has been developed at National Institute of Metrological Research (INRIM) for the calibration and adjustment of multifunction electrical instruments as digital multimeters (DMMs) and multifunction calibrators (MFCs) and for high level inter-comparisons. The Resistance Standards are made of two 10 Ω and 100 kΩ resistors nets connected in parallel while the 10 V Standard is an ultra-low noise-drift based reference INRIM developed circuit. The project of the setup started from a previous realization revisiting it and adding a 10 V Standard. The resistors net of the 1 Ω is inserted in oil-bath along with the internal side of its connectors lowering the thermo-electromotive forces (emfs). The three Standards are further enclosed in a thermo-regulated copper enclosure and in an external aluminium case. The setup is also equipped with a high insulation switch to select the desired Standard. Preliminary results (3 h relative stabilities ranging from 5×10−8 to 7×10−8 ) and temperature coefficients are satisfactory to the scope of the realization.

A l’Institut national de recherche metrologique (INRIM) il a ete developpe un etalon de haute tension continue avec des valeurs selectionnables de 10 V a 100 V pour compenser le manque d'etalons de haut niveau de tension continue d'une valeur superieure a 10 V pour les comparaisons interlaboratoires de haut niveau. Il a ete utilise une nouvelle technique electronique de terre mobile. L'etalon developpe a un bruit inferieur et il a la stabilite egal a celle des calibrateurs de tension continue ou multifonction de haut niveau largement utilise dans les laboratoires d'etalonnage electriques; il a aussi une meilleure attitude a etre transporte pour les comparaisons interlaboratoires. Le projet est extensible jusqu'a 1000 V.

The traditional scaling method used at the Istituto Elettrotecnico Nazionale (IEN) for the calibration of the 10-k/spl Omega/ standard is compared with more straightforward techniques: one based on the linearity of a DVM and the other on a commercial current comparator bridge. For the three methods, the measurement results and the uncertainty budgets are reported. The agreement is better than 1/spl times/10/sup -7/.

The high linearity and stability of modern top-level digital multimeters (DMM) can be exploited to measure voltage ratios with very low uncertainty by making two separate absolute voltage measurements. We developed an integrated approach to apply commercial multimeters as voltage ratio standards for the dissemination of the volt over a wide range of values, based on a JAVS as primary ratio standard and DMMs as secondary standards. The DMM characterized in linearity can find application in top level calibration laboratories, where it can replace the Kelvin-Varley divider in calibration of meters. Moreover, we developed a GPIB interfaced voltage divider that allows the fully automatic calibration in dc voltage of generators

Circulation of the travelling standard for international comparison CCEM-K8 on DC voltage ratio was completed in June 2001. Awaiting the conclusion of the process leading to the agreed final report, the final evaluation of the behavior of the standard is given.

Key comparisons CCEM-K8 and EUROMET.EM-K8 on DC voltage ratio [1, 2] were carried out in parallel, both having IEN as the pilot laboratory. Two Datron 4902S voltage dividers were used as travelling standards, each divider being dedicated to one comparison. On these dividers the measurements of the ratios 1000 V / 10 V and 100 V / 10 V were mandatory and were used to evaluate the degrees of equivalence of the participants with respect to the reference value of their own comparison. The mandatory measurements were performed by all the 15 participants in CCEM-K8 and by 19 out of the 20 participants in EUROMET.EM-K8.

Comparison CCEM-K8.1 is a follow-up to comparison CCEM-K8 and was organized for those participants in CCEM-K8 willing to improve their results. The same travelling standard as in CCEM-K8, a Datron 4902S DC resistive divider, was used, but the trimmers of the divider were adjusted in order to change the deviation of the divider's ratios from nominal value. The comparison protocol was the same as for CCEM-K8 and requested the measurement of voltage ratios 1000?V/10?V and 100?V/10?V, the measurement of other ratios being optional. Two laboratories participated, but one of them withdrew before the draft?A report was released. In the comparison's final report the results of the other laboratory, CEM from Spain, are linked to those of comparison CCEM-K8 through the measurements of IEN, which was the pilot laboratory of both comparisons. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCEM, according to the provisions of the Mutual Recognition Arrangement (MRA).

The purpose of the regional comparison EUROMET.EM-K8 was to compare the scaling capabilities up to 1000 V DC of the European national metrology institutes, thus extending to Europe the evaluation of equivalence of voltage ratio measurements obtained by the comparison CCEM-K8. Twenty laboratories participated, with the Istituto Elettrotecnico Nazionale Galileo Ferraris (Torino, Italy) acting as pilot laboratory. The circulation of the travelling standard, a resistive voltage divider, started in July 1998 and ended in January 2002. The technical protocol requested the measurement of voltage ratios 1000?V/10?V and 100?V/10?V, the measurement of other ratios being optional. Nineteen laboratories reported measurements of the mandatory ratios. For these ratios, the measurement methods used are described and the process to evaluate the comparison reference value and the degrees of equivalence is reported in detail. The relative uncertainty of the comparison, given by the combination of the uncertainty of the reference value and the instability of the transfer standard, was about 0.26?10-6 for ratio 1000?V/10?V and about 0.16?10-6 for ratio 100?V/10?V, at 95% level of confidence. The results for the optional ratios are reported in an appendix. With some exceptions, the agreement between the results is satisfactory. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCEM, according to the provisions of the Mutual Recognition Arrangement (MRA).

The purpose of CIPM comparison CCEM-K8 was to compare the scaling capabilities up to 1000 V dc of national metrology institutes. Fifteen institutes participated, with the Istituto Elettrotecnico Nazionale Galileo Ferraris (Turin, Italy) acting as pilot laboratory. Circulation of the travelling standard, a resistive voltage divider, started in October 1998 and ended in June 2001. The technical protocol requested the measurement of voltage ratios 1000 V/10 V and 100 V/10 V, the measurement of other ratios being optional. For the mandatory ratios, the measurement methods used by the participants are described and the evaluation of the key comparison reference value and the degrees of equivalence is reported in detail. For these ratios, the relative uncertainty of the comparison, given by the combination of the uncertainty of the reference value and the instability of the transfer standard, was about 0.23?10-6 at 95% level of confidence. Results for the supplementary ratios are reported in an appendix. With certain exceptions, agreement between the results is satisfactory; in some cases the measurement uncertainty was underestimated.