Accuracy of genomic prediction using an evenly spaced, low-density single nucleotide polymorphism panel in broiler chickens.

One approach for cost-effective implementation of genomic selection is to genotype training individuals with a high-density (HD) panel and selection candidates with an evenly spaced, low-density (ELD) panel. The purpose of this study was to evaluate the extent to which the ELD approach reduces the accuracy of genomic estimated breeding values (GEBV) in a broiler line, in which 1,091 breeders from 3 generations were used for training and 160 progeny of the third generation for validation. All birds were genotyped with an Illumina Infinium platform HD panel that included 20,541 segregating markers. Two subsets of HD markers, with 377 (ELD-1) or 766 (ELD-2) markers, were selected as ELD panels. The ELD-1 panel was genotyped using KBiosciences KASPar SNP genotyping chemistry, whereas the ELD-2 panel was simulated by adding markers from the HD panel to the ELD-1 panel. The training data set was used for 2 traits: BW at 35 d on both sexes and hen house production (HHP) between wk 28 and 54. Methods Bayes-A, -B, -C and genomic best linear unbiased prediction were used to estimate HD-marker effects. Two scenarios were used: (1) the 160 progeny were ELD-genotyped, and (2) the 160 progeny and their dams (117 birds) were ELD-genotyped. The missing HD genotypes in ELD-genotyped birds were imputed by a Gibbs sampler, capitalizing on linkage within families. In scenario (1), the correlation of GEBV for BW (HHP) of the 160 progeny based on observed HD versus imputed genotypes was greater than 0.94 (0.98) with the ELD-1 panel and greater than 0.97 (0.99) with the ELD-2 panel. In scenario (2), the correlation of GEBV for BW (HHP) was greater than 0.92 (0.96) with the ELD-1 panel and greater than 0.95 (0.98) with the ELD-2 panel. Hence, in a pedigreed population, genomic selection can be implemented by genotyping selection candidates with about 400 ELD markers with less than 6% loss in accuracy. This leads to substantial savings in genotyping costs, with little sacrifice in accuracy.

Twenty-five years of selection for improved leg health in purebred broiler lines and underlying genetic parameters.

Leg health is an important component of broiler welfare and the economics of broiler production. This study presents the development of leg health in 3 purebred commercial broiler lines during 25 yr of selection and investigates the genetic background of leg health traits in current populations of these lines. The leg health traits were deformities of the long bones (LD) and crooked toes (CT), recorded since 1985, and tibial dyschondroplasia (TD) and hock burn (HB), recorded since 1990. The prevalence of CT and HB decreased mainly in the first decade (range among lines -1.2 to -2.3% and -1.3 to -1.5% per year, respectively), after which it stabilized at low levels. The prevalence of LD and TD decreased by -0.6 to -0.9% and -0.4 to -1.2% per year, respectively. Genetic parameters were estimated using data from 4 recent generations. The BW ranged from 2.0 to 2.4 kg at 5 wk of age; the prevalences of LD, CT, TD, and HB from 8.6 to 12.9%, 0.6 to 2.6%, 4.6 to 8.0%, and 4.0 to 12.2%, respectively. Estimates of heritability were 0.04 to 0.07 for LD, 0.01 to 0.10 for CT, 0.10 to 0.27 for TD, and 0.06 to 0.09 for HB (all SE ≤0.01). Estimates of the genetic correlations between LD and CT were 0.11 to 0.43 (all SE ≤0.09), between these traits and HB were negligible, and of TD with LD, CT, and HB were -0.26 to 0.16 (all SE ≤0.11). Estimates of genetic correlations between the leg health traits and BW were lowly to moderately unfavorable, ranging from 0.09 to 0.37 (all SE ≤0.06). The differences between the lines suggest that strategies for simultaneous improvement of all traits tailored for each line individually have been effective. This research demonstrates the long-term effectiveness of selection for improving leg health in broilers and highlights that, despite somewhat unfavorable genetic correlations with BW, these traits can be improved simultaneously in a balanced breeding program.

Genetic parameters of foot-pad dermatitis and body weight in purebred broiler lines in 2 contrasting environments.

The aims of this study were to investigate the genetic background of foot-pad dermatitis (FPD) in 4 different broiler lines reared in 2 contrasting environments (pedigree or sib-test) and to evaluate the performance of simultaneous genetic selection for improved FPD and BW. Data were available for 4 generations from 4 broiler lines, bred with varying intensities of selection for growth. The average BW ranged from 1.7 to 2.4 kg at 5 wk of age. In the pedigree environment, the prevalence of FPD ranged from 14 to 37%, with 3 to 9% being severely affected; in the sib-test environment, these values were correspondingly 45 to 79% and 35 to 70%. Both traits showed re-ranking of the 4 lines in terms of phenotype across the 2 environments, indicating the existence of a genotype-by-environment interaction. In both environments, females showed higher prevalences of FPD than males. In line with their higher prevalence, heritabilities of FPD in the sib-test environment ranged from 0.22 to 0.32, compared with 0.18 to 0.24 for FPD in the pedigree environment (all SE ≤0.02). Estimates of the genetic correlation between FPD in the pedigree and in the sib-test environments were high (0.78-0.82), which suggests that selection against FPD in a highly biosecure environment can improve the genetic merit for birds reared under commercial conditions. Estimates of the genetic associations between FPD and BW were small and varied in sign. Predicted responses to selection showed a yearly reduction in average score of -3.4 to -7.5% for FPD in the pedigree environment and -0.5 to -6.6% for FPD in the sib-test environment, while maintaining improvement of BW of 2.6 to 3.2% and 2.6 to 3.8% of the average BW per year, respectively. This research indicates that balanced genetic selection for both BW and FPD in contrasting environments is an effective strategy to reduce the genetic disposition to develop FPD in broilers.

Genetic parameters of feeding behavior traits and their relationship with live performance traits in modern broiler lines.

Current selection goals in broiler breeding focus on the improvement of live performance traits, such as feed intake, BW, and feed conversion ratio (FCR). The use of electronic feeders allows measurement of feed intake of individuals housed in groups as well as the identification of different feeding behaviors. Feed intake can thus be split into underlying feeding behavior traits, allowing the estimation of genetic correlations and assessment of the genetic consequences of selecting for performance traits on feeding behavior traits. To investigate the genetic relationships between performance traits and feeding behavior, data of visits to feeders by birds from 4 lines of broilers that differed in selection focus on growth and FCR were analyzed. Visits were recorded electronically and grouped into meals using an existing model for estimating meal criteria. Mean individual feeding behavior traits were then calculated across the entire test period (2 to 5 wk of age). Records were available for between 14,000 and 18,000 birds/line. Analyzed feeding behavior traits were meals per day, meal size, visits per meal, meal duration, nonfeeding time in meal, time feeding per day, proportion of meal spent feeding, feeding rate, and ADFI. Analyzed performance traits were 35-d BW, total feed intake over the entire test period, and FCR. All feeding behavior traits showed moderate to high heritabilities (0.24 to 0.57) but low genetic correlations with performance traits (-0.20 to 0.18), except for ADFI, which was moderately correlated with total intake on test (0.57) and highly correlated with FCR (0.91). The low genetic correlations indicate that the difference in selection intensity among lines for these performance traits has had limited effect on feeding behavior. Different feeding strategies that would result in favorable breeding values for FCR were identified, adding opportunities for further improvements in feed efficiency within and across environments.

Spectro-electrochemical characterization and quantification of Rutin in aqueous media.

From UV-Vis spectrophotometric measurements the acidity constants of Rutin in aqueous media, at 25 °C and 0.1 M ionic strength, were determined as: pK1 = 4.392 ±â€¯0.167, pK2 = 7.130 ±â€¯0.050, pK3 = 8.661 ±â€¯0.042 and pK4 = 12.354 ±â€¯0.020 and the molar absorptivity coefficients of all the Rutin pH-dependent species were reported as a function of wavelength. Furthermore, the electrochemical behavior of Rutin at neutral pH was investigated using a bare carbon paste electrode, CPE. It was found that both: Rutin electrochemical oxidation and reduction are reversible, adsorption-controlled processes, involving 2 electron transfers. Moreover, the bare CPE was used for the electrochemical quantification of Rutin in neutral aqueous media, displaying the following features: (1.078 ±â€¯0.440) µM, (3.594 ±â€¯0.400) µM and (0.308 ±â€¯0.014) µA µM-1 for the detection and quantification limits and sensitivity, respectively, within the 1-11 µM linear range. Meanwhile the spectrophotometric method displayed the following analytical features: (3.385 ±â€¯1.318) µM, (11.283 ±â€¯3.114) µM and (0.0120 ±â€¯0.0001) AU µM-1 for the detection and quantification limits and sensitivity, respectively within the 11-110 µM linear range. In like manner, the bare CPE is also shown as a robust electrochemical sensor that allows Rutin quantification even in the presence of ascorbic acid, commonly found in Rutin samples.

The structure of feeding behavior in commercial broiler lines selected for different growth rates.

Selection for increased growth rate in livestock is accompanied by increased requirements for food resources. It has been suggested that more intensively selected birds, such as broilers, have altered food intake control mechanisms and may be constantly hungry, due to the high demands of fast growth rates. If this is the case, it would be a major welfare issue. We investigated the hypothesis that more intensive selection for growth in some lines of broilers has altered feeding behavior by analyzing short-term feeding behavior bouts in relation to the roles of hunger and satiety mechanisms in the control of food intake. Using 4 genetic lines, resulting from different levels of selection for growth rate, meal pattern analysis was performed and the bouting of short-term feeding behavior was estimated. All lines showed bouted feeding behavior, although differences in meal size, number of meals, and meal duration were evident across lines. In all lines, the probability of birds starting a new meal was low immediately after finishing the previous meal and increased with time, as expected for feeding behavior governed by hunger and satiety mechanisms. Normal feeding behavior was, therefore, not affected by the intensity of selection. Feeding rate increased with growth rate, suggesting that this may be a consequence of selection. However the other characteristics of feeding behavior, such as meal duration, did not change consistently with higher growth rate. Due to differences between lines in bird size, the number and weight of birds per pen also differed between the lines. The differences in feeding behavior between lines were greatly diminished when weight of birds per square meter was taken into account but were still statistically significant. Overall, it is apparent that even when growth rate and body size have been substantially altered by genetic selection, the underlying normal controls of feeding behavior are conserved in broiler birds.

Genetic variability in residual variation of body weight and conformation scores in broiler chickens.

The aim of the study was to estimate genetic variance in residual variation of BW and body conformation scores of 34-d-old male and female broiler chickens. The data set included performance records of 52,349 female and 49,001 male birds. Two models were used: an iterative mean-variance restricted maximum likelihood sire plus dam model and a Bayesian sire plus dam model, in each case with heterogeneity in residual variances across sire families. They gave similar results. Estimates of heritability for BW in females and males were, respectively, 0.25 and 0.21, and for conformation score were 0.24 and 0.15, and genetic correlations for the same trait between sexes were high. Estimates of genetic correlations between means and variances were positive for both sexes and both traits. Estimates of the heritability of residual variation were low, between 0.023 and 0.038 for both traits, but genetic CV were substantial (between 0.25 and 0.40), indicating the possibility of improving uniformity of BW and conformation by means of selection.

Machine learning classification procedure for selecting SNPs in genomic selection: application to early mortality in broilers.

In genome-wide association studies using single nucleotide polymorphisms (SNPs), typically thousands of SNPs are genotyped, whereas the number of phenotypes for which there is genomic information may be smaller. Atwo-step SNP (feature) selection method was developed, which consisted of filtering (using information gain), and wrapping (using naïve Bayesian classification). This was based on discretization of the continuous phenotypic values. The method was applied to chick early mortality rates (0-14 days of age) on progeny from 201 sires in a commercial broiler line, with the goal of identifying SNPs (over 5000) related to progeny mortality. Sires were clustered into two groups, low and high, according to two arbitrarily chosen mortality rate thresholds. By varying these thresholds, 11 different "case-control" samples were formed, and the SNP selection procedure was applied to each sample. To compare the 11 sets of chosen SNPs, predicted residual sum of squares (PRESS)from a linear model was used. Naive Bayesian classification accuracy was improved over the case without feature selection (from 50% to 90%). Seventeen SNPs in the best case-control group (with smallest PRESS) accounted for 31% of the variance among sire family mortality rates.

New insights on the spectrophotometric determination of melatonin pKa values and melatonin-ßCD inclusion complex formation constant.

Using UV-Vis spectrophotometry a stability study of melatonin at different pH values was done in aqueous media, finding that at acidic pH melatonin is unstable when interacting with the environment, however it becomes stable protecting it from light and oxygen. From the UV-Vis spectra and SQUAD software, melatonin pKa values, in a completely protected aqueous medium, were estimated as 5.777±0.011 and 10.201±0.024. Using the same techniques, the melatonin and ß-cyclodextrin inclusion complex formation constants were assessed at pH3, 7 and 11.5, giving the values of log ß=(3.07±0.06), (2.94±0.01) and (3.07±0.06) M-1, respectively. From the global acidity formation constants and the complexes' formation constants, the molar fractions were determined for each species of MT and MT-ßCD, to build the molar fraction-[ßCD]-pH 3D diagram and the molar fraction-pH 2D diagrams, where it was possible to observe the predominance of the MT species with and without ßCD. A voltammetric study at pH3, allowed obtaining a value of log ß=(3.15±0.01) M-1, which corroborates that obtained through UV-Vis spectrophotometry, supporting strongly the rationale behind using simple, straightforward techniques.

Genetic basis of leg health and its relationship with body weight in purebred turkey lines.

The aims of this study were to estimate the genetic parameters for leg and foot health and mobility in purebred turkey lines and their genetic correlations with BW. Traits were gait score (GS) as an overall measure of leg health, footpad dermatitis (FPD), and 2 skeletal leg health traits, namely, valgus and varus deformities (VVD) and tibial dyschondroplasia (TD). Data from 4 different lines, comprising 3 yr of phenotypic records and 4 yr of pedigree information per line, were used. The sex average BW for the lines at 18 wk ranged from 19.1 kg (line A) to 12.4 kg (line D). The prevalence of VVD ranged from 5.2 to 14.6% and for TD from 4.1 to 23.2%. The average score for FPD on a scale of 0 to 100 ranged from 48.5 to 61.1. Gait Score was scored on a scale of 1 to 5, standardized to a mean of 3 and SD of 1. Heritabilities were estimated at 0.08 to 0.13 for GS, 0.01 to 0.07 for VVD, 0.06 to 0.12 for TD, and 0.10 to 0.15 for FPD (all SE ≤ 0.02). Estimates of the genetic correlations between VVD and TD ranged from 0.03 to 0.21 (all SE ≤ 0.08), and estimates of these with GS ranged from 0.07 to 0.87 (all SE ≤ 0.09). The genetic correlations of FPD with GS ranged from 0.00 to 0.34 (all SE ≤ 0.04), and with the skeletal leg health traits from -0.06 to 0.33 (all SE ≤ 0.06). Body weight showed estimated genetic correlations ranging from 0.28 to 0.51 (all SE ≤ 0.06) with GS, -0.06 to 0.50 (all SE ≤ 0.13) with VVD/TD and 0.05 to 0.34 (all SE ≤ 0.05) with FPD. The results suggest that selection for improved leg health can be incorporated effectively in a commercial turkey breeding program using balanced breeding goals, in which production traits and leg health traits are considered simultaneously.

Association of twelve immune-related genes with performance of three broiler lines in two different hygiene environments.

Elite populations of farm animals under genetic selection are often maintained in high hygiene conditions, yet the commercial populations may be raised in, and are expected to perform well in, environments of varied hygiene levels. This presents special challenges to genetically improve those traits for which genotype by environment interactions are important. Twelve immune-related genes were studied for associations with general mortality and other performance traits in 3 elite commercial broiler chicken lines raised in high and low hygiene environments. The genes were toll-like receptor 4, MD-2 (accessory' protein of TLR4), interferon-gamma, transforming growth factor-beta3, inducible nitric oxide synthase, macrophage migration inhibitory factor, interleukin-2, caspase-1, inhibitor of apoptosis protein-1, tumor necrosis factor-related apoptosis-inducing ligand, chicken B-cell marker, and bone morphogenetic protein-7. From a total of 56 identified single-nucleotide polymorphisms (SNP) in 12 genes, 14 SNP that had moderate allelic frequencies in at least 2 of the 3 lines were typed in about 100 progeny-tested sires from each of 3 elite commercial broiler chicken lines using restriction fragment length polymorphism techniques and then used in association analysis. The traits measured on the progeny (total progeny = 145,467) were: mortality from hatching to 14 d and from 14 to 40 d of age, BW at 7 and 40 d of age, feed conversion, ultrasound breast depth, percentage of breast, eviscerated carcass weight, twisted legs or evident tibial dyschondroplasia, x-ray-inspection-based subclinical or incipient development of tibial dyschondroplasia, curly or crooked toes or bowed legs, oxygen content of blood, and female's antibody titer to infectious bursal disease virus at 27 wk. Association analyses were conducted with allele and haplotype substitution effect models using progeny mean data adjusted for fixed and mate effects as sire trait records. Ten of the 12 genes had SNP associations with at least 1 trait. Most detected effects were with mortality and growth traits. Most gene-SNP trait associations varied by genetic line or with environment. These results indicate that associations of candidate genes with important broiler traits can be identified in multiple environments, and they offer a potential for the implementation of marker-assisted selection for traits expressed in the environment in which the commercial broiler needs to perform. The effects of these immune-related candidate genes, however, are complex and affected by genetic background and environment.

Study on the stability of the serotonin and on the determination of its acidity constants.

The present work aimed at describing the spectral behaviour of the serotonin and to evaluate its acidity constants using three different methods, using two spectrophotometry titrations and a third method that involved point-by-point analysis, which permitted to monitor closely and determine the evolution of the serotonin species in solution as a function of time. The three methods allowed estimation of three acidity constants associated to the same number of functional groups that form part of the molecule. The results given by the point-by-point analysis were: log(beta1) = 24.95 +/- 0.12; log(beta2) = 20.20 +/- 0.10; log(beta3) = 10.89 +/- 0.018.

Study on the stability of adrenaline and on the determination of its acidity constants.

In this work, the results are presented concerning the influence of time on the spectral behaviour of adrenaline (C(9)H(13)NO(3)) (AD) and of the determination of its acidity constants by means of spectrophotometry titrations and point-by-point analysis, using for the latter freshly prepared samples for each analysis at every single pH. As the catecholamines are sensitive to light, all samples were protected against it during the course of the experiments. Each method rendered four acidity constants corresponding each to the four acid protons belonging to the functional groups present in the molecule; for the point-by-point analysis the values found were: log beta(1) = 38.25 +/- 0.21, log beta(2) = 29.65 +/- 0.17, log beta (3) = 21.01 +/- 0.14, log beta(4) = 11.34 +/- 0.071.

Evaluation of the acidity constants of the 4-hidroxy-5-[salicylideneamino]-2-7-naphthalenedisulfonic acid (Azomethine-H) using UV-vis spectrophotometry.

The time stability of the azomethine-H species was determined not to be better than 10 min in the absence of oxygen and light, however under phosphate buffered conditions the azomethine-H species remained stable for longer periods, as indicated by the spectrophotometric behaviour. Nevertheless, the analysis time still exceeded the stability allowance. Therefore, the determination of the acidity constants of the Azomethine-H species was studied by means of UV-vis spectrophotometry in buffered media by means of the point-by-point analysis and data processing with SQUAD to refine the resulting constants, which were: pK(a1) = 3.39, pK(a2) 7.36 and pK(a3) 8.73. The latter were associated to the corresponding acid-base equilibria of the amine and hydroxy groups constituting the molecule.

Study on the stability of noradrenaline and on the determination of its acidity constants.

Noradrenaline is a catecholamine which has been largely recognised to play a very important role in biological systems. In view of the neurotransmitter's alleged importance, this work aimed at showing the influence of time on its spectral behaviour using different analytical methods and determining its acidity constants through spectrophotometric titration and by the so-called point-by-point analysis, where the samples are freshly prepared for each pH value investigated at the instant required. Because the catecholamines are light-sensitive and likely to react with the oxygen in the surrounding air, both methods used preclude its incidence onto the samples being analysed under the presence of a nitrogen atmosphere maintained over the solutions. The constants obtained through point-by-point analysis were log beta1 = 30.71+/-0.16, log beta2 = 22.00+/-0.15 and log beta3 = 11.69+/-0.16.

Effect of gamma irradiation on the microbiological quality of minimally processed vegetables.

The initial microflora of minimally processed celery and cabbage packaged under a modified atmosphere was determined. The samples came from the same producer and were sold in a supermarket chain of Santiago, Chile. Although neither E. coli nor Salmonella spp. were detected, initial total plate and Enterobacteriaceae counts were high (= 10(5) cfu/g), not meeting in most cases the specifications of the Chilean legislation. The D10 value for two strains of E. coli (ATCC 8739 and a wild type) inoculated as indicator microorganisms was determined. After irradiating with 5 D10 doses (1kGy), the variation of the microbial population and sensory quality during a 7 days storage period at 5 degrees C was studied. In irradiated celery, a reduction of 4.7 and 3.8 logs for total plate and Enterobacteriaceae counts respectively was observed. There was a decrease of 3.8 and 3.6 logs in cabbage for total plate and Enterobacteriaceae counts respectively. In both irradiated and non-irradiated vegetables, neither E. coli nor Salmonella spp. were detected. An increase of 1.6 - 1.7 logs in both microbiological parameters in non-irradiated samples was observed during storage. In irradiated products, only celery showed an increase of 1.2 log in total plate count. Both Enterobacteriaceae count in the two vegetables and Total plate count in cabbage presented essentially no variation in time. No significant differences (p = 0.05) were detected in sensory total quality between the control and the irradiated vegetable and between days of storage.

Spectrophotometric quantification of the thermodynamic constants of the complexes formed by dopamine and Cu(II) in aqueous media.

The thermodynamic constants of the complex Cu(II)-dopamine in aqueous solution were evaluated from spectrophotometric data using the software SQUAD. It was found that there exist Cu(II):DA complexes with 1:1 and 1:2 stoichiometries and that their predominance depends on both the solution pH and the [Cu(II)]/[DA] ratio. Moreover, it is shown that the solubility of Cu(OH)2(s) increases drastically when these complexes are thermodynamically stable.

A seven-cell human egg recovered from the oviduct.

A seven-cell human egg recovered from the proximal middle quarter of the oviduct is described. Whether or not it is a normal representative of this stage of human development cannot be established at the present time. The specimen was recovered 83 hours after intercourse and 77 hours after the first significant elevation of the luteinizing hormone level in the urine. According to these data and the results of the endometrial and corpus luteum biopsies, the age of the egg was estimated to be approximately 72 hours. An analysis of size and the reaction of the blastomeres to toluidine blue suggests that they already show some differentiation at this early stage of development. The addition of these findings to previous reports of eggs recovered from human oviducts and uteri gives support to the concept that human eggs are delivered to the endometrial cavity when they contain between 7 and 12 blastomeres.

Expected increases in genetic merit from using optimized contributions in two livestock populations of beef cattle and sheep.

The expected benefits from optimized selection in real livestock populations were evaluated by applying dynamic selection algorithms to two livestock populations of sheep (Meatlinc) and beef cattle (Aberdeen Angus). In addition, the effects of introducing BLUP evaluations on the population structure, genetic gain, and inbreeding were investigated. The use of BLUP-EBV accelerated the rates of gain in the Meatlinc, but the effects of BLUP evaluations on Aberdeen Angus are not as evident. Although steady increases in the average coefficient of inbreeding (F) were observed, the inbreeding rates (deltaF) before and after the introduction of BLUP evaluations were not significantly different. The observed deltaF in the last generation was 1.0% for Meatlinc and 0.2% for Aberdeen Angus. The application of the dynamic selection algorithms for maximizing genetic gain at a fixed deltaF led to important expected increases in the rate of genetic gain (deltaG). When deltaF was restricted to the value observed in both populations, increments per year in deltaG of 4.6 (i.e., 17%) index units for Meatlinc and 3.5 (i.e., 30%) index units for Aberdeen Angus were found in comparison to the deltaG expected from conventional truncation BLUP selection. More relaxed constraints on deltaF allowed even higher expected increases in deltaG in both populations. This study demonstrates that the optimization tools constitute a potentially highly effective way of managing gain and inbreeding under a broad range of schemes in terms of scale and inbreeding level. No losses in genetic gain were associated with the use of dynamic optimization selection when schemes were compared at the same deltaF.

Spectrophotometric study on the stability of dopamine and the determination of its acidity constants.

The interest in determining the acidity constants of the catecholamines stems from the fact that they play rather an important biological role. The present work reveals the effect of different parameters such as oxygen, light, analysis time and pH on the dopamine oxidation process, where oxygen has an effect on the dopamine oxidation of 40% and up to 20% is attributed to exposure to light as a function of the pH. The application of adequate control on the said parameters (which ensured stability of the dopamine) facilitated the determination of the corresponding three acidity constants, 9.046+/-0.147, 10.579+/-0.148 and 12.071+/-0.069.