Lieta Marinelli

Accuracy in naturally occurring anabolic steroid assays in cattle and first approach to quality control in Italy

This study assessed the accuracy of 16 commercial and three self-produced kits and drew the basis for using an external quality control (EQC) system. The commercial kits were mainly developed for blood sex steroid determination in humans but also have been used in cattle. Parallelism, recovery and precision tests were performed for progesterone (P4), testosterone (T) and oestradiol (E2) assays. Moreover, anonymous QC samples were sent to be analysed to some Italian laboratories. All kits showed a fair degree of parallelism (P < 0.01), even though 2/7 kits for T and 1/6 kits for E2 determination showed a regression coefficient (r2) lower than 0.98. For P4, an acceptable range of accuracy was achieved in the recovery test only by 1/6 kits; two kits showed fair or great overestimation and two kits considerable underestimation. For T, an acceptable range of accuracy was achieved only by 1/7 kits. For E2, 4/6 kits presented a variable degree of underestimation and two kits showed great overestimation. In the intra-assay precision test quite good repeatability was achieved only using samples with high hormone concentrations. While assaying samples with low concentrations we found a number of RSD > 10%. Moreover, the laboratories participating in the EQC produced statistically different (P < 0.05) results, particularly for high and medium concentrations. In conclusion, the use of commercial kits for screening naturally occurring sex steroid concentrations in cattle blood, in the case of suspected illegal treatments, requires preventive validation procedures and the development of an opportune EQC system.

Dehydroepiandrosterone secretion in dairy cattle is episodic and unaffected by ACTH stimulation

This paper describes the episodic release and response to adrenal stimulation of cortisol and dehydroepiandrosterone (DHEA) in cows. Observations made in samples taken every 10 min for 8 h (experiment 1) showed that plasma DHEA was significantly greater (P < 0.001) than DHEA-S, and release of these steroids was episodic and variable between animals (P < 0.01). No relationship was found between DHEA and cortisol. Significant (P < 0.001) DHEA-sulphate (DHEA-S) versus cortisol (R = -0.264) and DHEA-S versus DHEA (R = 0.200) correlations were found. DHEA and DHEA-S were not affected by a single ACTH challenge (experiment 2). In experiment 3, cortisol and DHEA secretions in response to prolonged ACTH administration (every 12 h for 6 days) were studied. On day 7, the episodic cortisol and DHEA release and response to the opioid antagonist naloxone were studied in blood samples taken every 10 min for 8 h. Animals were injected with naloxone after 4 h. A significant increase (P < 0.05) in mean circulating DHEA and DHEA pulse amplitude was observed during frequent sampling following ACTH treatment. DHEA and DHEA-S plasma concentrations were not affected following luteal regression (experiment 4). The effect of milk secretion around parturition on DHEA secretion was studied in dry and continuously milked cows (experiment 5). Plasma DHEA was significantly lower (P < 0.05) in milked cows. In the cow, ACTH is not an important DHEA secretagogue. Adrenal contribution to plasma DHEA is scarce. Likely, the placenta is the most important source of DHEA, and the lactating mammary gland can affect circulating DHEA levels. Investigation about the DHEA biological role in cows should be focused around parturition.

Oxidative stress indicators and metabolic adaptations in response to the omission of the dry period in dairy cows

The effects of dry period omission on oxidative stress and metabolic indicators around calving were studied. Seventeen Italian Friesian cows were randomly assigned to two groups, homogeneous for milk yield and parity, and managed either with a traditional 55-d dry off period (n=8) or continuously milked till parturition (n=9). Between 60 d before expected calving and 90 d after calving, body condition (BCS) was recorded and blood samples were collected to measure cortisol, urea, cholesterol, glucose, NEFA, triglycerides, insulin, malondialdehyde (MDA), total glutathione (GSH) and glutathione peroxidase (GPx) activity. BCS changes after calving were not different between the two groups. The normally dried group showed lower (P<0.05) glucose concentrations on day 7 before calving, greater (P<0.01) non-esterified fatty acid concentrations at 7 d and 15 d after calving, and greater (P<0.01) triglyceride concentrations for all the period before calving. On the other hand, plasma MDA was not different between groups. On average, plasma GSH concentrations were greater in continuously milked cows after calving (P<0.05), while plasma GPx was greater with continuous milking up to parturition (P<0.01). The results confirmed that omitting the dry period leads to an improved energy balance. The degree of oxidative stress was not detrimental for animal health, and the slight modifications of GPx observed prepartum were possibly related to continuous milk secretion. The differences in plasma GSH observed after calving may depend upon sulphur amino acid sparing in continuously milked cows.

Plasma steroid variations in bull calves repeatedly treated with testosterone, nortestosterone and oestradiol administered alone or in combination.

The aim of this work was to investigate the secretion of dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT) and oestradiol (E) as biological markers in response to illegal administration of testosterone, 19-nortestosterone (N) and oestradiol, either alone or in combination. Twenty male Friesian calves (age 13–14 months) were allotted to a control group (n = 5), and five experimental groups (n = 3) each. Each experimental animal was repeatedly injected with one of the following hormonal treatments: E, T, N, T+E and N+E. Circulating DHEA, T, DHT and E were determined by radioimmunoassay. The administration of T alone did not induce any variation in plasma DHEA, T, DHT and E, which were similar to those in the control group. In contrast, DHEA, T and DHT were on average significantly lower in the T+E and N-treated groups (χ<0.01), whereas the administration of N+E resulted in the reduction of plasma T and DHT without any modification of plasma DHEA. The administration of E alone or in combination increased circulating levels of E but did not affect androgen plasma profiles. The results indicate that plasma levels of T do not permit detection of illegal treatments because plasma androgens always remained within the physiological range. Illegal E treatment could be detected in blood samples when they were collected at least every 20 days.

Advanced oxidation protein products are generated by bovine neutrophils and inhibit free radical production in vitro.

Despite the recognised importance of oxidative stress in the health and immune function of dairy cows, protein oxidation markers have been poorly studied in this species. The current study aimed to characterise markers of protein oxidation generated by activated bovine neutrophils and investigate the biological effects of advanced oxidation protein products (AOPP) on bovine neutrophils. Markers of protein oxidation (AOPP, dityrosines and carbonyls) were measured in culture medium containing bovine serum albumin (BSA) exposed to neutrophils. The effect of AOPP-BSA on generation of reactive oxygen species (ROS) was assessed by chemiluminescence. Activation of caspases-3, -8 and -9 and the presence of DNA laddering were used as apoptosis markers. Greater amounts of AOPP were generated by phorbol myristate acetate (PMA)-activated than non-activated neutrophils (1.46 ± 0.13 vs. 0.75 ± 0.13 nmol/mg protein, respectively; P<0.05). Activated neutrophils and hypochlorous acid generated slightly different patterns of oxidized protein markers. Exposure to AOPP-BSA did not stimulate ROS production. Activated neutrophils generated a lesser amount of ROS when incubated with AOPP-BSA (P<0.001). Activation with PMA induced a loss of viable neutrophils after 3h, which was greater with AOPP-BSA incubation (P<0.05). Detectable amounts of active caspases-3, -8 and -9 were found in nearly all samples but differences in caspase activation or DNA laddering were not observed comparing treatment groups. Apoptosis was unlikely to be responsible for the greater loss of PMA-activated neutrophils cultured in AOPP-BSA and it is possible that primary necrosis occurred. The results suggest that accumulation of oxidized proteins at an inflammatory site might result in a progressive reduction of neutrophil viability.

Hierarchical stimulus processing by dogs (Canis familiaris)

AbstractThe purpose of this study was to assess the visual processing of global and local levels of hierarchical stimuli in domestic dogs. Fourteen dogs were trained to recognise a compound stimulus in a simultaneous conditioned discrimination procedure and were then tested for their local/global preference in a discrimination test. As a group, dogs showed a non-significant trend for global precedence, although large inter-individual variability was observed. Choices in the test were not affected by either dogs’ sex or the type of stimulus used for training. However, the less time a dog took to complete the discrimination training phase, the higher the probability that it chose the global level of test stimulus. Moreover, dogs that showed a clear preference for the global level in the test were significantly less likely to show positional responses during discrimination training. These differences in the speed of acquisition and response patterns may reflect individual differences in the cognitive requirements during discrimination training. The individual variability in global/local precedence suggests that experience in using visual information may be more important than predisposition in determining global/local processing in dogs.

The increase in plasma C19Δ5 steroids in subcutaneous abdominal and jugular veins of dairy cattle during pregnancy is unrelated to estrogenic activity

Plasma concentrations of dehydroepiandrosterone (DHEA), 5-androstene-3beta,17beta-diol (AED), and 17beta-estradiol (E2) in dairy cows and heifers and AED binding to uterine cytosolic estrogen receptor (ER) were studied. Plasma samples were collected from the subcutaneous abdominal (SA) and jugular (J) veins of heifers and cows in the non-pregnant state and at 15-45, 90-120, 180-210, and 250-280 days of pregnancy (N = 5-12). Plasma DHEA, AED, and E2 were determined by RIA. DHEA and AED significantly increased (P < 0.001) in heifers and cows throughout pregnancy. The stage of pregnancy significantly (P < 0.001) affected the three steroids in heifers and cows. Plasma DHEA increased throughout pregnancy in both heifers and cows, and in heifers it was significantly greater in SA than in J veins at 90-120 days (P < 0.01). Plasma AED was greater in heifers than in cows in J veins at 90-120 days (P < 0.01) and 180-210 days (P < 0.05), and in SA veins, at 15-45 days (P < 0.01) and 90-120 days (P < 0.05). In heifers, circulating AED showed concentration values significantly greater than those in non-pregnant animals from 90 to 120 days (P < 0.05) and was significantly greater in SA than in J veins at 90-120 days (P < 0.05). In cows, plasma AED was significantly greater than in non-pregnant animals at 250-280 days (P < 0.01). In heifers, plasma E2 was significantly greater in the SA than in the J veins from 180-210 to 250-280 days (P < 0.01). In cows, differences between E2 plasma concentrations in J and SA veins were observed only at 250-280 days of pregnancy. At 250-280 days, in both animal types plasma E2 was significantly greater than in non-pregnant animals (P < 0.001). We suggest that AED originates primarily from the feto-placental unit, while mammary E2 synthesis near term can affect plasma concentrations. Binding data showed that AED is a weak competitor for cytosolic ER (IC50 range: 1.44 x 10(-5) to 3.71 x 10(-5) M). These results suggest that a direct estrogenic activity for AED is unlikely in dairy cattle, and the physiological role of AED needs to be elucidated.

Omission of dry period and effects on the subsequent lactation curve and on milk quality around calving in Italian Holstein cows

The aim of this study was to investigate the effect of dry period omission on subsequent milk production over a whole lactation and the effect on milk quality around calving. Seventeen Italian Friesian cows, homogeneous for milk yield and parity, were managed either with a traditional dry-off period of 55 d (CTR group; n=8) or continuously milked up to parturition (CON group; n=9). Milk yield was recorded daily from 75±7 d before expected calving date to the end of subsequent lactation to fit the lactation curve. Before parturition milk samples were collected at −70 d, −60 d, and −55 d for CTR and CON groups and at −40 d, −30 d, and −7 d for CON cows. After calving, six milk samples were taken from 1 d to 90 d from both groups and analyzed for fat, protein, lactose, MUN and somatic cells. Body weight (BW) and body condition (BCS) were obtained 7 times from −55 d before expected calving to 90 d after calving. The omission of the dry period modified the shape of lactation in CON cows that reached the peak 10 d earlier and producing −5.5 kg/d than CTR. Over a 305 d period, the milk yield reduction was of −2241 kg in CON group. The higher amount of milk produced before calving, i.e., 560 kg of milk in 52 d of mean pre-partum period, did not compensate the milk yield reduction after a continuous lactation. Milk quality was unaffected by the omission of dry period after calving, and animals in both lactation groups showed a similar decreasing trends over time for fat and protein, and increasing trends for lactose and MUN. Somatic cell score remained greater (4.54 vs 3.40) in CON than in CTR cows after calving. No different BW or BCS changes were observed for both groups after calving. We conclude that despite the absence of great differences in milk quality, the complete omission of the dry period in cows reduce significantly the milk yield, suggesting no economical benefit from this management strategy.

Mammary steroid metabolizing enzymes in relation to hyperplasia and tumorigenesis in the dog

Progesterone and estradiol play a crucial role in the control of mammary gland proliferation and tumour formation in the dog. However, little is known whether steroid metabolizing enzymes are present within the canine mammary gland that may play a modulating role in the bioavailability of progesterone and estrogen. In this study we investigated the expression of the steroid metabolizing enzymes 5alpha-reductase (type I and type II) and aromatase in relation to hyperplasia or tumorigenesis in the canine mammary tissue. The relative mRNA concentrations were examined by a semi-quantitative reverse-transcriptase PCR analysis (RT-PCR). In addition the affinity of dihydroprogesterone (5alpha-reduced metabolite of progesterone) for canine progesterone receptors was investigated. Quantification of the RT-PCR products revealed that in mammary tumours a significantly higher expression of aromatase is present in comparison to normal mammary tissue. Furthermore, significant decrease in expression of both aromatase and 5alpha-reductase type II enzymes was found in hyperplasic mammary tissue compared to tumours. The changes in expression of type II 5alpha-reductase and aromatase were highly correlated. 5alpha-Reduction of progesterone to dihydroprogesterone resulted in a six-fold less affinity for the canine progesterone receptor. It is concluded that hyperplasia is associated with a decreased expression of type II 5alpha-reductase and aromatase enzymes, whereas in tumours the opposite situation is found.

Dog assisted interventions in a specialized centre and potential concerns for animal welfare

Dog assisted interventions in a specialized centre and potential concerns for animal welfare L. Marinelli & S. Normando & C. Siliprandi & M. Salvadoretti & P. Mongillo Published online: 4 July 2009 # Springer Science + Business Media B.V. 2009