Short communication: Reference limits for blood analytes in Holstein late-pregnant heifers and dry cows: Effects of parity, days relative to calving, and season.

Reference limits for metabolic profiles in Holstein late-pregnant heifers and dry cows were determined considering the effects of parity, days relative to calving, and season. Blood samples were collected from 104 pregnant heifers and 186 dry cows (68 primiparous and 118 pluriparous) from 60 to 10 d before the expected calving date in 31 dairy farms in northeastern Italy. Sampling was performed during summer (182 samples) and the following winter (108 samples). All the animals were judged as clinically healthy at a veterinary visit before sampling. Outliers were removed from data of each blood analyte, and variables that were not normally distributed were log transformed. A mixed model was used to test the fixed effects of parity (late-pregnant heifers, primiparous or pluriparous dry cows), class of days relative to calving (60-41 d, 40-21 d, 20-10 d), season (summer or winter), and the interactions between parity and class of days relative to calving and between parity and season, with farm as random effect. Single general reference limits and 95% confidence intervals were generated for analytes that did not vary according to fixed effects. Whenever a fixed effect included in the model significantly affected a given analyte, specific reference limits and 95% confidence intervals were generated for each of its levels. Albumin, urea, triglycerides, alanine aminotransferase, aspartate aminotransferase, creatinine kinase, conjugated bilirubin, calcium, phosphorus, magnesium, potassium, chloride, zinc, copper, and iron concentrations were not influenced by any of the fixed effects. Total protein, globulins, creatinine, glucose, alkaline phosphatase, gamma glutamyltransferase, lactate dehydrogenase, and sodium plasma concentrations were affected by parity. The class of days relative to calving had a significant effect on the concentrations of total protein, globulins, fatty acids, cholesterol, total bilirubin, and sodium. Season affected plasma concentrations of creatinine, glucose, fatty acids, lactate dehydrogenase, and sodium. Interactions between parity and class of days relative to calving and between parity and season did not significantly affect any of the blood analytes tested. The reference limits and the 95% confidence intervals for blood analytes determined in the study could help dairy practitioners to improve the accuracy of metabolic profile interpretation in Holstein late-pregnant cattle.

Refinement of a macaque transplantation model: application of a subcutaneous port as a means for long-term enteral drug administration and nutritional supplementation.

A new application of a device enabling the long-term enteral administration of drugs or nutritional supplementation was developed for implementing in research entailing the use of macaques (Macaca fascicularis). After implanting a subcutaneous port, a surgically-placed gastrostomy (SPG) was completed to afford access to the gastric lumen and enable the administration of substances. In this study, the device was left in place for a period ranging between two and 12 months in macaques (n= 16). In five cases, the SPG was used successfully for 8-12 months, until the experimental endpoint was reached. In six cases, the SPG had to be removed earlier due to local infection at the implant site, which promptly regressed after the SPG was removed and antibiotic treatment was administered. One SPG-implanted macaque was euthanized for reasons unrelated to the SPG or the xenotransplantation procedure. In four cases, the SPG was implanted without any complications but has yet to be used to administer substances to the animals. From an ethical standpoint, the SPG device described here minimizes the forced handling of macaques otherwise needed for the oral administration of viscous or unpalatable substances by gavage. The device thus represents an effective refinement that fully complies with the tenet of the '3 Rs' that should be considered by primate centres exposing non-human primates to the long-term daily administration of substances by oral gavage.

Target gene expression signatures in neutrophils and lymphocytes from cattle administered with dexamethasone at growth promoting purposes.

The glucocorticoid dexamethasone (DEX), when used as a growth promoter, cause morphological and functional alterations in cattle lymphoid organs and cells. In the present experiment, the transcriptional effects of an illicit DEX protocol upon six target genes were investigated in cattle neutrophils (NEU) and lymphocytes (LFC). Blood samples were taken before (T(0)) and 2, 3, 10, 19, 31 and 43 days from the beginning of DEX administration (T(1)-T(6)). Leukocytes were counted and cells isolated by gradient centrifugation; then, glutathione peroxidase 1 and 3 (GPX1 and GPX3), glucocorticoid receptor alpha (GRα), l-selectin, nuclear factor κB, subunit p65 (NFκB) and tumor necrosis factor alpha (TNFα) mRNA amounts were measured through a quantitative Real Time RT-PCR approach. A significant change vs controls in NEU/LFC ratio was noticed from T(3) forward. Compared to T(0), DEX significantly increased to a variable extent all candidate gene mRNAs abundances in NEU; in contrast, only l-selectin, GRα and GPX1 were significantly up-regulated in LFC. Present results suggest that illicit DEX affects transcription in cattle immune cells, that might be considered as a promising surrogate tissue for the screening of DEX abuse in cattle farming.

Short communication: reference values for blood parameters in Holstein dairy cows: effects of parity, stage of lactation, and season of production.

Confidence intervals for blood parameters used for nutritional and metabolic profile testing in cattle were calculated for clinically normal lactating Holstein cows, taking into account the effects of parity, stage of lactation, and season. Blood samples were collected from 740 cows in 33 Italian dairy herds according to a predefined protocol. Herds were visited during summer and the following winter, sampling 12 lactating cows at each visit (4 primiparous and 8 multiparous). Six cows were selected from the early-lactation group (days in milk: 10 to 89) and the other 6 were selected from the mid-lactation group (days in milk: 90 to 215). Cow selection criteria excluded animals clinically exposed to periparturient diseases as well as animals not considered in good health by a veterinary clinical examination. For each blood variable, outliers were identified and discarded. Data were then analyzed for their Gaussian distribution and variables with not normal distribution were log-transformed to adjust for lack of normality. Herd mean values were calculated for each blood parameter according to 3 main classification factors: parity (primiparous vs. multiparous), stage of lactation (early vs. mid) and season of production (summer vs. winter). The resulting data set was statistically analyzed using a mixed model with the fixed effects of these factors, their interactions, and the random effect of herd. General 95% confidence intervals were calculated for blood variables that showed a relevant herd variance component such as albumin, triglycerides, aspartate, urea, glucose, alanine aminotransferase, lactate dehydrogenase, direct and total bilirubin, calcium, magnesium, and potassium. For the remaining parameters, specific confidence intervals were calculated for each level of the significant main factors. Parity affected blood concentration of total protein, globulin, creatinine, alkaline phosphatase, gamma glutamyl transferase, creatinine kinase, and phosphorus. Blood nonesterified fatty acids, aspartate aminotransferase, gamma glutamyl transferase, creatinine kinase and cholesterol were influenced by stage of lactation. The season of production had a significant effect on total protein, globulin, creatinine, alkaline phosphatase, phosphorus, sodium, and chlorine. The outcomes of this work will improve the accuracy of the biochemical profile as a tool for dairy practitioners to assess the metabolic status of lactating Holstein cows.

Evaluation of indirect biomarkers for detecting corticosteroids used as illegal growth promoters in beef cattle.

The histological status of the thymus, blood cortisol concentration and circulating neutrophil:lymphocyte ratio were evaluated in 349 slaughtered beef cattle, to assess the potential of these parameters as indirect biomarkers of the illegal use of corticosteroids in meat production. The livers of 20 of the animals were analysed chemically for residues of corticosteroids. The morphology of the thymus was examined for adipose tissue infiltration, cortical atrophy and 'starry sky' appearance, and on the basis of these characteristics, the animals were considered to be negative, suspected or positive for illegal corticosteroid treatment. The animals considered to be negative had a mean cortisol concentration that was significantly higher (29 ng/ml) than that of the animals suspected for corticosteroid treatment (22 ng/ml). Using the chemical analysis as the gold standard for identifying illegally treated animals, the histological examination of the thymus had a sensitivity of 100 per cent and a specificity of 85 per cent. The samples that were positive by chemical analysis had cortisol concentrations of less than 2.0 ng/ml, whereas the mean cortisol concentration of the negative samples was 10.3 ng/ml.

Administration of dexamethasone per os in finishing bulls. II. Effects on blood parameters used as indicators of animal welfare.

A set of hormonal, haematological and biochemical parameters was used to evaluate the physiological response and welfare status of 14 finishing Marchigiana bulls treated for 49 days with a low daily dosage (0.75 mg/head per day) of dexamethasone per os. Compared to the Control group, dexamethasone decreased cortisol concentrations (42.3 v. 5.7 nmol/l; s.e.d. = 4.17; P < 0.001), and led to the reversal of the leukocyte formula in the animals treated (P < 0.05). Total serum proteins (70.2 v. 73.9 g/l; s.e.d. = 1.55; P < 0.05), in particular ß1 globulins (7.5 v. 9.1 g/l; s.e.d. = 0.24; P < 0.01) and fibrinogen (199 v. 258 mg/dl; s.e.d. = 32.70; P < 0.05), increased as a consequence of treatment. Prolonged dexamethasone administration led the bulls to an apparently chronic stress condition. Moreover, the study indicated various blood parameters that might be used by health officials as effective tools in identifying beef cattle suspected of being illegally treated with dexamethasone.

Refinement of a transplantation project in the non-human primate by the use of a humane endpoint.

An assessment scheme was developed to establish a humane endpoint in a pig-to-primate renal xenotransplantation project, with a view to minimizing and controlling any pain or suffering conditions in the animals involved while still achieving the scientific objective. In particular, the assessment criteria for identifying the earliest endpoint are described, bearing in mind both the researcher's need to obtain top-quality data and the ethical need to safeguard the animals. The scheme should also be applicable to other experiments involving non-human primates (e.g. allotransplantation, survival after major surgery, pharmacological safety tests) because it considers reproducible general parameters together with aspects specific to each experimental model.

Illicit treatments in cattle and urinary 6beta-hydroxycortisol/cortisol ratio.

Dexamethasone (DXM) is often illegally used as a growth promoter. To identify indirect biomarkers of illicit treatments, the urinary ratio between 6beta-hydroxycortisol (6beta-OHF) and cortisol (F) was measured in urines obtained from bulls experimentally treated per os and intramuscularly (i.m.) with different DXM dosages. Dexamethasone, given per os at low doses elicited an early and lasting significant reduction of 6beta-OHF/F. No significant variations were seen in urines from bulls given DXM intramuscularly. These results suggest 6beta-OHF/F as a rapid, non-invasive, screening test for oral, low-dose, long-term corticosteroid treatment in cattle. Further studies are required to go deep inside the biochemical and molecular events underlying such an effect.

Efficacy of silymarin-phospholipid complex in reducing the toxicity of aflatoxin B1 in broiler chicks.

Silymarin, the standardized extract of Silybum marianum, is used as a hepatoprotector in man, and is a potent antihepatotoxic agent. This study focused on the effects of a silymarin-phospholipid complex in reducing the toxic effects of aflatoxin B1 (AFB1) in broiler chickens. Twenty-one 14-d-old male commercial broilers were randomly allotted to 3 groups and treated as follows: basal diet alone [Group C (Control)]; AFB1 at 0.8 mg/kg of feed [Group B1]; AFB1 at 0.8 mg/kg of feed plus silymarin phytosome, a silymarin complexed form with phospholipids from soy, at 600 mg/kg of BW [Group B1+Sil]. Considering the whole growth cycle, BW gain and feed intake were lower in AFB1-treated birds with respect to controls (P < 0.05). In the B1+Sil group, BW gain and feed intake were higher with respect to birds receiving AFB1 alone (P < 0.05), and not different from the control birds. Serum biochemistry showed no difference among groups, except for a decrease of alanine amino transferase (ALT) in chicks treated only with AFB1. Alanine amino transferase activity in AFB1 plus silymarin phytosome treated birds was not different from the controls. No treatment differences were noted on liver weight. In conclusion, our results suggest that silymarin phytosome can provide protection against the negative effects of AFB1 on performance of broiler chicks.

The provision of drinking water to veal calves for welfare purposes.

Growth performance, behavior, physiology, forestomach development, abomasal lesions, and meat quality of veal calves fed a milk-replacer diet (No Water) were compared to those obtained from calves fed the same diet and provided with increasing amounts of drinking water (Water). Two groups of 69 Polish Friesian calves, balanced according to initial BW, were assigned to two water treatments in a 3 x 2 x 2 factorial arrangement that provided solid feed in addition to the milk-replacer diet (No solid feed, 250 g x calf(-1) x d(-1) of wheat straw or the same amount of beet pulp), and the adoption of two housing systems (individual stall vs group pen). The fattening trial lasted 160 d, and calves received drinking water starting from the 2nd wk of the study. The amount of drinking water was progressively increased from 3 to 8 L x cal(-1) x d(-1). Although not dehydrated, as shown by hematocrit and Na, K, and total protein hemoconcentration, calves consumed almost all the offered amount of water throughout the fattening period. Therefore, the water provided by the milk replacer alone, which ranged between 6 to 16 L calf d(-1), was not sufficient to satisfy the need of the animal. Drinking water did not affect the calves' growth performance but it reduced nonnutritive oral behavior throughout the fattening period. Based on these results, drinking water did not cover a shortage in the calves' water requirement but it played a role in environmental enrichment. Health status was similar between treatments, although water provision reduced the episodes of feed refusal. The measurement of chronic stress by ACTH challenge showed that the administration of drinking water would be advisable when calves are fed with small amounts of solid feed for well-being. Feces consistency and animal cleanliness were not affected by drinking water. At slaughter, forestomach development was similar between treatments, and drinking water did not affect the number of calves showing rumen hair-balls and abomasal lesions. No differences in color and other meat quality traits were observed between Water and No Water calves. Despite the lack of direct effects on productive traits, when water was available, the calves drank it, and positive effects were noticed on their nonnutritive oral behaviors and chronic stress indicators.