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The action of glucocorticoids on target tissues is regulated by the glucocorticoid and mineralocorticoid receptors (codified by the NR3C1 and NR3C2 gene, respectively). Moreover, the prereceptor system, represented by the hydroxysteroid 11-beta dehydrogenases (HSD11Bs), catalyzes the interconversion from active glucocorticoids into inactive compounds. This study aimed to determine whether the expression of the prereceptor system, the corticosteroid receptors, and the molecules regulating their intracellular trafficking (FKBP prolyl isomerase 4 and FKBP prolyl isomerase 5) could be regulated in the hypothalamic-pituitary-adrenal axis and in different type of adipose tissue of calves by the administration of dexamethasone in combination with estradiol or prednisolone. Research about the glucocorticoid effects on bovine target tissues may allow development of new diagnostic methods that use potential molecular biomarkers of glucocorticoid treatment. The administration of dexamethasone in combination with estradiol increased the gene expression of HSD11B1 (P0.01), HSD11B2 (P0.05), NR3C1 (P0.01), and NR3C2 (P0.01) in the adrenal glands; NR3C2 in the intramuscular adipose tissue (P0.01), and HSD11B1 in the subcutaneous adipose tissue (P0.01). Prednisolone administration increased the gene expression of HSD11B1 (P0.01), NR3C1 (P0.05), and NR3C2 (P0.05) in the adrenal glands and HSD11B1 (P0.01) in the subcutaneous adipose tissue. Interestingly, most of the examined tissues/organs showed a significant variation of FKBP5 gene expression after the administration of dexamethasone in combination with estradiol. So, these changes suggest that the FKBP5 gene expression could be a possible biomarker of the illegal dexamethasone administration in calves.

The illegal administration of glucocorticoids in livestock is problematic and identification of pathways in which these hormones are involved is critically important, and new direct or indirect biomarkers should be identified. In this work, glucocorticoid transcriptional effects on some genes involved in the glucose metabolism were studied in the bovine liver. This study was conducted on adult Charolais male cattle treated with long-term low dose dexamethasone or prednisolone. Gene expression analysis was conducted in the liver by qPCR, and the geNorm algorithm was applied to select optimal reference genes. In line with the literature, a significant overexpression of genes involved in the gluconeogenic pathway and glycogen synthesis was detected in the liver of dexamethasone-treated animals, but histological and biochemical examination showed hepatocyte glycogen depletion particularly in dexamethasone-treated animals. It possible to hypothesize that glucocorticoids or adrenal insufficiency due to glucocorticoids withdrawal inhibit the enzymatic activity of glycogen synthase and/or induce glycogen autophagy in bovine liver. In fact, markers of glycophagy as starch-binding domain-containing protein 1 and γ-aminobutyric acid receptor-associated protein-like 1 mRNAs were upregulated in the liver by glucocorticoids treatment. Furthermore, glycogen synthase kinase-3 beta gene was significantly overexpressed in dexamethasone-treated animals, and this protein is also implicated in liver autophagy modulation and glycogen synthesis inhibition. These results showed that glucocorticoids likley have dual roles in hepatic glycogen metabolism of cattle, and investigation of these pathways could help find treatment biomarkers.

Antimicrobial (AM) resistance is largely acknowledged as one of the biggest global health and food safety challenges and the overuse of AMs is known to generate resistance in bacteria that may affect both animals and humans. Poultry meat is the second most-produced meat in the European Union and in recent years consumers are becoming more concerned about food safety, traceability, and animal welfare in poultry rearing system, increasingly requiring meats from broilers reared without AMs. In the present study, we performed RNA sequencing to analyze 64 liver and 54 muscle transcriptomic profiles in broilers reared without treatment or treated with different classes of AMs. Moreover, we validated the most differentially expressed genes among the treated groups to detect putative novel biomarkers able to discriminate meats of broilers reared without AMs. The PDK4, IGFBP1, and RHOB genes were identified as putative novel hepatic biomarkers, discriminating broilers treated with AMs compared to broilers reared without treatments. The whole transcriptome changes revealed the liver as a valuable target organ for AM administration screening. In addition, our results suggest a leading effect of the coccidiostat when associated with AMs, influencing several biological processes. Our study showed that RNA sequencing is a powerful and valuable method to detect aberrant regulated genes and to identify biomarker candidates for AM misuse detection in farm animals. Further validation on larger sample size and a wider spectrum of AMs are needed to confirm the viability of the aforementioned biomarkers in poultry population.

Under European legislation, the use of growth promoters is forbidden in food-producing livestock. The application of unofficial protocols with diverse combinations of veterinary drugs, administered in very low concentrations, hinders reliable detection and subsequent operative prevention. It was observed that nandrolone (anabolic steroid) and ractopamine (β-adrenergic agonist) are occasionally administered to animals, but little is known about their synergic action when they are administered together. Two specific analytical methods based on liquid chromatography-tandem mass spectrometry have been developed, both of which include hydrolysis of the corresponding conjugates. For the nandrolone method, solid-phase extraction was necessary for the complete elimination of the interferences, while employment of the Quantitation Enhanced Data-Dependent scan mode during MS acquisition of ractopamine enabled the utilization of simple liquid-liquid extraction. The nandrolone method was linear in the range of 0.5-25 ng/mL, while the ractopamine calibration curve was constructed from 0.5 to 1000 ng/mL. The corresponding coefficients of correlations were >0.9907. The lower limit of quantification for both methods was 0.5 ng/mL, followed by overall recoveries >81%. Precisions expressed as relative standard deviations were

Under European legislation, the use of growth promoters is forbidden in food-producing livestock. The application of unofficial protocols with diverse combinations of veterinary drugs, administered in very low concentrations, hinders reliable detection and subsequent operative prevention. It was observed that nandrolone (anabolic steroid) and ractopamine (β-adrenergic agonist) are occasionally administered to animals, but little is known about their synergic action when they are administered together. Two specific analytical methods based on liquid chromatography-tandem mass spectrometry have been developed, both of which include hydrolysis of the corresponding conjugates. For the nandrolone method, solid-phase extraction was necessary for the complete elimination of the interferences, while employment of the Quantitation Enhanced Data-Dependent scan mode during MS acquisition of ractopamine enabled the utilization of simple liquid-liquid extraction. The nandrolone method was linear in the range of 0.5-25 ng/mL, while the ractopamine calibration curve was constructed from 0.5 to 1000 ng/mL. The corresponding coefficients of correlations were0.9907. The lower limit of quantification for both methods was 0.5 ng/mL, followed by overall recoveries81%. Precisions expressed as relative standard deviations were17%, while matrix effects were minimal. Urine samples taken at the slaughterhouse from veal calves enrolled in an experimental treatment consisting of intramuscular administration of β-nandrolone-phenylpropionate accompanied with a ractopamine-enriched diet were analysed. Those methods might be useful for studying the elimination patterns of the administered compounds along with characterization of the main metabolic pathways. Copyright © 2016 John WileySons, Ltd.

The administration of anabolic agents in farm animals to improve meat production has been prohibited in EU, due to the potential risks to human health. Meat quality was investigated to detect the effects of illegal administration of dexamethasone or prednisolone or 17β-estradiol on Charolais bulls. Three groups of 6 bulls were treated and 12 bulls were the control. Meat quality parameters were measured on live animals, carcasses and on samples of Longissimus thoracis and multivariate statistical data analysis was applied. In Charolais bulls, these parameters were affected by growth promoter administration and the multivariate canonical discriminant analysis was able to distinguish between treated and untreated animals mainly due to three electronic nose’s parameters, 24 h carcass temperature and drip loss. Therefore, meat quality control and the multivariate analysis could be useful as a first screening to address targeted controls on farms suspected of illicit use of growth promoters.

Growth promoter administration, in livestock, potentially poses a major threat to public health, due to the potential endocrine and carcinogenic activity of residues, accumulating in edible tissues, such as skeletal muscle. Therefore, development of new screening tests and methods for the detection of illicit treatments of food animals would be useful. In this study the serum concentrations of oxytocin peptide were measured in beef cattle receiving 17β oestradiol, dexamethasone or placebo over a period of 40 days. Changes in gene expression of oxytocin precursor in skeletal muscle were also examined in these animals. Serum analysis using an oxytocin EIA kit indicated a significant up-regulation of the biosynthesis of this nonapeptide only in cattle after 17β oestradiol, but not after dexamethasone or placebo treatment. Quantitative PCR (qPCR) analysis showed a significant overexpression of the oxytocin precursor gene by 33.5 and 13.3-fold in cattle treated with 17β oestradiol and dexamethasone, respectively, in comparison to placebo treated animals. Regulation of gene expression by some myogenic regulatory factors in skeletal muscle was also evaluated in these animal groups, confirming the activity of both growth promoters on this gene. To investigate the use of the oxytocin precursor gene as biomarker for 17β oestradiol and dexamethasone treatment in beef cattle, an absolute quantification of this gene by qPCR was developed. A standard curve was generated and developed with TaqMan® technology and optimal criterion value, sensitivity and specificity of this screening method were established through ROC analysis. This analysis suggested that the up-regulation of oxytocin precursor gene expression in skeletal muscle tissue is a valid marker for detection of illicit 17β oestradiol and/or dexamethasone use in beef cattle. This method may serve as a novel diagnostic tool in the screening phase, and, if introduced in routine testing, may significantly improve overall efficacy and success of the food screening process ordered by state authorities.

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17β-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17β-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CCα, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17β-oestradiol (5 mg week−1 per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17β-oestradiol (group C; 20 mg week−1 per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CCα on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CCα and they were classified as being suspected of 17β-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium.

Thymus atrophy and regeneration were studied in 13- to 22-month-old beef calves treated with dexamethasone (DMT), using anabolic dosages and implementing different withdrawal times. Two trials were conducted. In trial 1, group A (n=6) received 0.7 mg/day DMT orally for 40 days, group B (n=6) received 1.4 mg/day orally for 40 days and group C (n=6) was the control. In trial 2, group D (n=6) received 0.7 mg/day DMT orally for 40 days, group E (n=6) received 1.4 mg/day orally for 40 days and group K (n=6) was the control. DMT withdrawal times before slaughter were six days (groups A and B) and 26 days (groups D and E). At slaughter, thymus atrophy was severe and progressive in animals from groups A and B. In contrast, thymus weight and volume of the animals from groups D and E were almost normal. Slight atrophy was also detected in the calves in these groups. Histological changes and Ki67 immunostaining revealed a large number of positive lymphoid cells, mostly in the cortical area, associated with higher expression of apoptosis in the medulla compared with controls. This demonstrated that the thymus of beef cattle is still able to regenerate following DMT administration.

Public concern for animal welfare has progressively grown over the recent years. In this context, stress has a great economical impact on growth of animals and quality of animal products. The development and validation of methods to assess animal stress, particularly at the farm level, are desirable to evaluate animal production systems. Piemontese breed is traditionally tie-stall housed in the fattening period. Hence, the objective of this study was to characterise a profile of physiological and haematological changes of Piemontese beef cattle under different management conditions (tie-stall and loose housing). Our results suggest that the housing system is an important factor in animal welfare. Indeed, the values of the total protein, lysozyme, cortisol, serum and faecal corticosterone concentration and GR-α gene expression indicate that the tie-stall housing is more stressful than the loose system. All the alterations highlighted in this study considered together may be effective biomarkers of stress and disease susceptibility.

Glucocorticoids (GCs) are extensively used in livestock production, not only for their anti-inflammatory properties but also to improve the quality and quantity of meat in veal and beef production. In Italy, an increase in GC-positive cases has been observed in cattle since 2008, particularly prednisolone (PDN). Recent studies clearly demonstrate that both histopathological analysis and high-performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) were unable to detect PDN treatments. The aim of this study was to identify transcriptomic signatures of PDN administration in the thymus of experimentally treated animals by comparison with untreated controls, in order to identify gene expression changes or pathways alteration induced by the corticosteroid treatment. Microarray data analysis showed substantial modifications in thymus gene expression profiles after PDN treatment. Several of the 388 differentially expressed genes encoded pro-inflammatory and anti-inflammatory mediators or immune regulators which showed that PDN might have a role in the regulation of immunologic homeostasis, act on both innate and acquired components of the immunity and mainly induce the activation of immune tolerance and anti-inflammatory pathways. Thus, this study allowed to deepen the effects of PDN on the immune system and showed the potentiality of gene expression profiling by DNA-microarray as a powerful tool to complement the existing methods against the illegal use of growth promoting hormones, especially when working on samples collected after slaughtering.

E-cadherin and its associated cytoplasmic proteins, including β-catenin, have been examined as potential oncogenic markers due to the significant correlation between tumour dedifferentiation and the invasive capacity of epithelial tumours. The purpose of this study was to evaluate the expression of E-cadherin and β-catenin in canine colorectal cancer using immunohistochemistry and to examine the relationship between this expression and various clinicopathological variables. The expression pattern of E-cadherin and β-catenin was investigated in 44 colorectal canine carcinomas. In the intestinal mucosa of noncancerous areas, epithelial cells demonstrated equally strong membranous expression of E-cadherin and β-catenin localised to the cell-cell junctions. Reduced expression of E-cadherin and β-catenin was demonstrated in 75% and 81.8% of the colorectal carcinoma cases, respectively. The down-regulation of both E-cadherin and β-catenin was correlated with decreased differentiation and increased tumour grade. In addition, the expression of β-catenin was correlated with tumour size. These results suggest that dysfunction of the E-cadherin-catenin complex starts in the early stages of carcinogenesis and that the disruption of the tissue architecture is progressively associated with the invasion of the tumour.

17beta-Estradiol is one of the most powerful sex steroids illegally used in bovine production. The objective of this study was to evaluate the application and the specificity of the RIKILT yeast estrogen bioassay (REA) for the detection of molecules with estrogenic activities in the urine of calves experimentally treated with anabolics. Four groups of six calves each received an injection of 17beta-estradiol intramuscularly (group B), androsterone and gliburide (group A), and testosterone (group C) molecules at different dosage for 40 days. Group D was the control. The ability of the REA test to detect estrogenic activity in urine samples from all animals was assessed. All estrogen-treated animals (group B) showed as being positive up to 7 days after administration of the highest dosage of 17beta-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17beta-estradiol, 17alpha-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17beta-estradiol in the urine of calves treated with the hormone in vivo. The technique may offer an advantageous laboratory method for the veterinary surveillance of illegal steroid use.

Histopathology of male cattle previously found positive for beta-boldenone in urine in the Netherlands and in Italy was studied. The animals were derived from practice and several weeks had passed after the finding of beta-boldenone before the animals were examined. The animals consisted of 34 male veal calves and one finishing bull. In the prostate gland hypersecretion, cyst formation (45%) and hyperplasia of the urethral epithelium was observed, in the bulbo-urethral gland similar alterations were present. The testis showed reduced development and degeneration of the germinal epithelium (70%), leading to debris and syncytial cell formation in the lumina. Stromal proliferation was evident. In some animals the liver was sampled and showed periportal fibrosis, bile duct proliferation and sometimes necrosis. The bull also showed degeneration of the germinal epithelium of the testis and absence of sperm production, the prostate gland showed some secretion and had an atrophic appearance. It is concluded that beta-boldenone may lead to degeneration of the germinal epithelium of the testis and hypersecretion and cyst formation in the prostate and bulbo-urethral gland, which alterations may heal in time.

Receptor concentrations for adrenaline, steroid hormones, PGF2alpha, LH and FSH were measured in the hypophysis and ovary of dairy cows with ovarian cysts and the results were compared with those of healthy animals. Significant modifications were found in all receptor concentrations, either between follicular and luteal structures or between the hypophyseal and ovarian receptorial status. The correlations between catecholaminergic and steroidal systems have already been demonstrated, particularly those existing between beta-adrenoceptors and steroid hormone receptors. Particular attention has been given to the possibility that a derangement in neurogenic inputs may be at the basis of some ovarian pathologies. The results of the present study suggest that the modifications of the ovarian and hypophyseal receptorial status of healthy and affected cows could play an important role in the pathogenesis of ovarian cysts.

Forty-eight feline mammary carcinomas (FMC) were resected surgically from 48 cats to determine whether the Ki-67 index (Ki-67I) would provide an indication of the post-surgical survival time (PST). Twenty-four cats (50 per cent) were still alive (group A) one year after surgery, whilst 24 (50 per cent) (group B) had died. Formalin-fixed, paraffin wax-embedded histological sections were immunostained with a monoclonal antibody to Ki-67 (MIB-1) and at least 1000 nuclei in eight to 10 representative fields were counted. The Ki-67I was expressed as the percentage of positive nuclei. In FMC, the Ki-67I ranged from 7.5 to 49.7 (24.8+/-9.5). A statistically significant difference (P = 0.000006) in the Ki-67I was found between group A and group B cats. No other statistically significant differences were found between these groups. The Ki-67I did not correlate with age or different histological type, according to the WHO classification. A Ki-67I cut-off of 25.2 represents a useful tool for identifying FMC with a more aggressive course.

The hearts of 235 white sturgeons originating from an intensive farm system were examined in detail. Skin, muscle and abdominal viscera did not show any pathological features, except for the presence of liver and epicardial fat. All sturgeons had high levels of epicardial fat and 70% showed important variations in colouration, which was from grey/brownish to blackish. Histopathological examination revealed lymphocyte infiltrates, around the blood vessels as small focal lesions near the intercellular spaces, or as thin strips along the borders of fat tissue. In severe cases, heavy lymphoid infiltrates and proliferative lesions were evident and fatty tissue was replaced by inflammatory and proliferative tissue. The underlying myocardium was rarely affected. A diagnosis of infiltrative and proliferative steatitis is postulated. Moreover, a high percentage of the livers showed steatosis. Aetiology is still unknown, but the authors envisage a nutritional imbalance linked with oxidised fat intake.

A murine monoclonal antibody prepared against an ovine abortion isolate of Chlamydia psittaci (A22/Teramo) revealed specific binding to a 57 kDa chlamydial antigen in immunoblotting studies. The monoclonal antibody was able to detect intracytoplasmic chlamydial inclusions and scattered elementary bodies in infected McCoy cell culture, and on formalin-fixed paraffin-embedded tissue sections both from experimentally infected mice and from fetal membranes of cases of ovine enzootic abortion.

The histopathology of the uterine and salpingeal epithelium of five cows affected with genital tuberculosis was studied. Both optical and scanning electron microscopy were used and the results were compared. Various forms and degrees of cellular lesions are described, including hypertrophy, flattening, detachment, nuclear protrusions and alterations of the cilia. In the different sites considered (uterus: intercaruncular and caruncular areas; salpinges: isthmus and ampulla) various characteristic lesions were observed.

Pathologic findings, lectin histochemistry, and nuclear estrogen receptors were studied in the reproductive organs of gilts treated with clenbuterol. A ration containing 1 ppm of clenbuterol was fed for 40 days to four Landrace x Large white, 9-month-old gilts, weighing 134 to 172 kg at slaughter (gilt Nos. 5–8). Four gilts (Nos. 1–4) served as controls. Treated animals had macroscopic lesions characterized by microcystic ovaries and uterine atrophy. Histopathologic lesions included atretic degeneration of many ovarian follicles, complete absence of functional corpora lutea, a reduction in the number of endometrial glands, and a decrease in cytoplasmic volume of endometrial and glandular epithelial cells. In ovaries, uterus, and vagina lectin histochemistry, performed with thirteen different biotinylated lectins, revealed a different staining distribution between control and treated gilts. The binding pattern of Ricinus communis agglutinin-1 (RCA-I) and -II (RCA-II) in the ovaries of control gilts, displayed labeling of cytoplasm in theca interna cells of Graafian follicles. There was no labeling of the same cells in treated gilts. Labeling patterns with Griffonia simplicifolia agglutinin-I (GS-I), Phaseolus vulgaris agglutinin (PHA), RCA-I and RCA-II documented a difference in the vascularis of the theca interna between Graafian follicles of control and treated gilts. The GS-1 and Ulex europaeus agglutinin-I (UEA-I) binding patterns in uterus and vagina of treated gilts when compared to control gilts suggested that there was a block of the cycling activity in the proliferative stage. Immunohistochemical staining for estrogen receptors in the endometrium was positive in all but one treated gilts, and negative to weakly positive in control gilts. Serum progesterone concentrations were decreased in treated animals when compared to control: estradiol concentrations were similar in both group of gilts. Cystic ovaries, uterine atrophy, and reduction in progesterone concentrations suggested that clenbuterol changed ovarian hormonal activity in treated animals.

The effects of 17beta-oestradiol (E2) on gene expression in cultures of bovine primary prostate stromal cells (BPSCs) and prostate gland tissue were studied. In the first part of the study, BPSCs were grown in the presence of E2 from the first passage to the end of the experiment; a second group was treated in the same way but the treatment was suspended for 48 hours before the end of the experiment; a third group of BPSCs served as a control. In the second part of the study, five male veal calves, aged 130 days, were treated four times intramuscularly with 10 mg of E2 at intervals of two weeks and then euthanased two weeks after the last treatment. Quantitative PCR and immunohistochemistry were used to evaluate the expression of fibroblast growth factor (FGF) receptors (FGFRs), FGFs, progesterone receptor, androgen receptor and oestrogen receptor in BPSCs and prostate tissue. E2 induced a significant over-expression of progesterone receptor in both BPSCs and prostate tissue. There was also a marked up-regulation of FGFR types 1, 2 and 3 genes observed in the BPSCs.

TRANSPORTATION of cattle is a routine management practice. However, mixing groups of unfamiliar animals and loading, unloading and driving the cattle are associated with psychological stress, physical damage and injury. These stresses have adverse effects on animal welfare and lead to reduced meat quality and economic losses to the farmer (Knowles 1999, Eicher 2001). Biochemical parameters in the blood (for example, levels of cortisol, adrenaline, non-esteridied fatty acids, iron, urea and glucose) are useful in the assessment of transport stress (Grasso and others 1989, Agnes and others 1990, Warriss and others 1995, Steinhardt and others 1997). The most commonly measured indicator of shortterm stress is cortisol, but its levels are highly variable and comparisons should not be made between different studies (Grandin 1997). The evaluation of antioxidant capacity (that is, the ability of a compound to reduce pro-oxidants) is commonly used in human medicine, in cystic fibrosis (Lands and others 2000), diabetes (Ceriello and others 1997) and HIV infection (De Martino and others 2001). This short communication describes a study to determine whether antioxidant capacity is a reliable marker in the evaluation of transport stress in veal calves transported by road. Fifty calves aged between one and two months were purchased in Rumilly, France, and transported by road to Centallo, northern Italy, over a distance of approximately 330 km, a journey which took five hours. Transportation took place at the end of January 2000, at a mean atmospheric temperature of 5°C. There were 20 male and 30 female cattle, all Garonnaise crosses, from different facilities, weighing 60 to 65 kg. They were all transported in one two-floor trailer at a stocking density of 0·65 m2 per calf, and they were not given food or water during the journey; this has been shown to lead to weight loss and dehydration, especially in young calves (Knowles 1995). Blood samples were collected immediately after the journey by jugular venipuncture, and the sera obtained were immediately frozen. The calves were boxed singly and fed a milk replacer. They were in good health and two months after their arrival, when a second blood sampling was performed, they weighed approximately 110 to 120 kg. Each animal acted as its own control in the study; as suggested by Palme and others (2000), this reduces the influence of variation between individuals. The sera were tested by means of the PAO kit (MED.DIA) in order to determine their antioxidant capacity, according to the manufacturer’s instructions. This test is based on the evaluation of the concentration of Cu+ ion obtained from the reduction of a known amount of Cu2+ ion by antioxidant substances in the sample. The antioxidant capacity of the sample can be quantified by comparing the obtained value with a standard curve. Briefly, serum samples were diluted in the buffer supplied with the kit and placed in 96-well plates, and their absorbance at 490 nm was determined in a microplate reader (Bio-Rad) to obtain blank values. After the addition of the Cu+-containing solution and incubation for three minutes at room temperature, stopping solution was added and a second absorbance value at 490 nm was determined. The antioxidant capacity value for each sample, expressed as reducing equivalents in μmol/litre, was obtained by subtracting the first measure from the second one, comparing the result with the standard curve and multiplying by a correction factor. The tests were performed in duplicate. The results were analysed using Student’s t test for paired values and InStat software (GraphPad Software). The antioxidant capacity values were normalised by applying the formula