F.T. Cannizzo

Effects of anabolic and therapeutic doses of dexamethasone on thymus morphology and apoptosis in veal calves

Three groups of 10 veal calves were treated, respectively, with 5 mg of dexamethasone-21-isonicotinate administered intramuscularly on days 0 and 7 (group A); 0·4 mg/day of dexamethasone-21-phosphate administered orally for 20 days (group B); or left untreated as controls (group C). Two animals from each group were slaughtered on day 3, 7, 14, 32 and 52. The size and weight of the thymus decreased progressively in both treated groups until day 32. On day 14, in comparison with the controls, there was a mean reduction of 76 per cent in the thymus weight of group A and 35 per cent in group B. On day 32, the reductions were 13 per cent in group A and 50 per cent in group B, but the thymus weight of both groups had recovered completely by day 52. Dexamethasone-induced changes in thymus weight associated with lymphoid depletion and fat replacement, and there were clear correlations between these changes and apoptosis of thymocytes.

Antioxidant capacity as a reliable marker of stress in dairy calves transported by road

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:

Progesterone receptor gene expression in the accessory sex glands of veal calves

This study investigated progesterone receptor (PR) cDNA expression in the testes, prostate and bulbourethral glands of prepubertal calves treated experimentally with high and low doses of 17β-oestradiol and with testosterone. Tissue samples were examined histologically and immunohistochemically for PR. Western blot analysis and quantitative PCR against PR was performed on cDNA and protein extracted from the same tissues. Bulbourethral glands from animals treated with low and high dosages of 17β-oestradiol had 39- and 429-fold increases of PR transcript, respectively, compared with controls. In the prostate there were 7.5- and 16-fold increases, respectively. Animals treated with testosterone showed no increases in PR transcript. The results demonstrate that 17β-oestradiol specifically induces marked overexpression of the PR gene and protein, particularly in the bulbourethral gland.

Thymus atrophy and regeneration following dexamethasone administration to beef cattle

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.

Gene expression profiling of thymus in beef cattle treated with prednisolone

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.

17β-oestradiol-induced gene expression in cattle prostate: biomarkers to detect illegal use of growth promoters

The effects of 17β-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.

Progesterone receptor up-regulation: a diagnostic tool for the illicit use of oestrogens in adult beef cattle

The monitoring of gene regulation via mRNA levels to detect anabolic sex steroid administration in cattle is a novel approach to detecting the illicit treatment of livestock in meat production. A previous study revealed that progesterone receptor (PR) gene expression levels were increased in the bulbourethral glands and prostates of 17β-oestradiol-treated prepubertal calves, suggesting that the PR can be used as a specific molecular biomarker for oestrogen treatment. The aim of this study was to verify the specificity and applicability of the PR to detect the illegal use of 17β-oestradiol in sexually mature beef cattle. Accessory sex glands were sampled from 42 male beef cattle that were divided into six experimental groups, including two control groups, K1 and K2. Group A cattle were treated with 17β-oestradiol (five weekly intramuscular doses of 20 mg), and group B cattle were treated with dexamethasone (40 daily doses of 0.7 mg per os). Group C cattle received an implant of Revalor-200 (200 mg of trenbolone acetate and 20 mg of 17β-oestradiol), and group D cattle received Revalor-200 plus dexamethasone (0.7 mg daily per os). 17β-Oestradiol, either alone or in combination with other steroids, up-regulated the PR gene and protein expression, even in the absence of detectable histological changes in the accessory sex glands, confirming the high sensitivity of PR gene expression as an indirect diagnostic screening tool to detect illicit oestrogen treatment in sexually mature male bovine.

A RIKILT yeast estrogen bioassay (REA) for estrogen residue detection in urine of calves experimentally treated with 17β-estradiol

17β-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 17β-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 17β-estradiol, while the other three groups showed as being negative. The identity of estrogenic molecules in the urine of group B (17β-estradiol, 17α-estradiol) was confirmed by gas chromatography-mass spectrometry (GC/MS). This is the first time the REA test has been applied to detect 17β-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.

Oxytocin precursor gene expression in bovine skeletal muscle is regulated by 17β-oestradiol and dexamethasone.

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.

Determination of prednisolone metabolites in beef cattle

Prednisolone is a synthetic corticosteroid acting on both hydrosaline balance and metabolism that is liable to fraudulent administration to meat-producing animals for growth-promoting purposes. Its use outside strict therapeutic control and prescription is banned by the European legislation, but official controls are hampered by its negligible direct excretion into the urinary matrix. Recent studies reported on a potential endogenous origin of prednisolone in animals subjected to stressful conditions, accounting for its occasional detection in control urines. The objective of the present study was the identification and quantification of prednisolone urinary metabolites to be used as illicit treatment biomarkers in place of the parent drug. An LC-MS/MS screening was conducted on urine samples collected from a bullock intramuscularly administered with prednisolone acetate by using a therapeutic protocol (2 × 0.52 mg kg−1 at 48-hour interval). Four prednisolone metabolites were identified: 20β-dihydroprednisolone, 20α-dihydroprednisolone, 6β-hydroxyprednisolone and 20β-dihydroprednisone; the first was detected at relatively high concentrations. An existing quantitative LC-MS/MS method was expanded and revalidated to include these metabolites. The new analytical method proved sensitive (LODs: 0.35–0.42 ng mL−1) and specific and was applied to urine samples collected from eight beef cattle subjected to low-dosage oral administration of prednisolone acetate for a 35-day period, as in standard growth-promoting treatments. 20β-Dihydroprednisolone was detected in all urine samples collected during the treatment, at relatively high concentration (1.2−27 ng mL−1), whereas the prednisolone concentration was virtually negligible (<0.7 ng mL−1). 20β-Dihydroprednisolone was no longer present in almost all samples collected 6 days after the end of the treatment, but trace amounts of this metabolite were found in two urine samples from control animals. 20β-Dihydroprednisolone is proposed as an effective biomarker to test illegal growth-promoting treatments with prednisolone in meat cattle, alternatively to the parent drug.