Raffaella Rebucci

Alpha-Tocopherol Counteracts the Cytotoxicity Induced by Ochratoxin A in Primary Porcine Fibroblasts

The aims of the current study were to determine the half-lethal concentration of ochratoxin A (OTA) as well as the levels of lactate dehydrogenase release and DNA fragmentation induced by OTA in primary porcine fibroblasts, and to examine the role of α-tocopherol in counteracting its toxicity. Cells showed a dose-, time- and origin-dependent (ear vs. embryo) sensitivity to ochratoxin A. Pre-incubation for 3 h with 1 nM α-tocopherol significantly (P < 0.01) reduced OTA cytotoxicity, lactate dehydrogenase release and DNA damage in both fibroblast cultures. These findings indicate that α-tocopherol supplementation may counteract short-term OTA toxicity, supporting its defensive role in the cell membrane.

Differential expression and secretion of alpha1-acid glycoprotein in bovine milk.

alpha1-Acid glycoprotein (AGP) is a lipocalin that is produced mainly by the liver and secreted into plasma in response to infections and injuries. In this study, we evaluated AGP isoforms that can be detected in bovine milk. We found that milk-AGP content is made up of at least two isoform groups, a low MW group (44 kDa) that is produced in the mammary gland (MG-AGP), and a higher MW group (55-70 kDa), that is produced by somatic cells (SC-AGP). Identical SC-AGP isoforms can be found both in milk and blood PMN cells. Analysis of the mammary tissue cDNA showed that the sequence of the MG-AGP isoform is identical to that of plasma AGP. Each group contains several proteins with different MWs and different isoelectric points, as shown by 2D-electrophoresis. The glycosylation patterns of these isoforms were analysed by means of specific lectin binding, to evaluate the degree of sialylation, fucosylation and branching. The MG-AGP glycan pattern was identical to plasma AGP produced by the liver. Several differences were detected, however, between plasma and SC-AGP isoforms, the most evident being the strong degree of fucosylation and the elevated number of di-antennary glycans in SC-AGP. Immunohistochemistry showed that AGP is found in all tissues that make up the mammary gland, but that it is most likely produced for the main part by the alveoli.

Effects of inclusion of selenium-enriched yeast in the diet of laying hens on performance, eggshell quality, and selenium tissue deposition

The aim of this study was to evaluate the bioavailability of ingested selenium (Se) yeast in laying hens and its effects on performance, eggshell quality, and tissue Se distribution. Forty-eight ISA brown laying hens were divided into 3 treatment groups: Group C, fed a basal diet containing 0.11 mg Se/kg of feed; Group SS, fed a basal diet plus 0.4 mg/kg of feed of Se from sodium selenite; and Group SY, fed a basal diet plus 0.4 mg/kg of feed of Se from selenium yeast. Feed intake, egg mass ratio, and production performance were not affected by Se supplementation, regardless of the Se source. Egg weight (+3.61% and +2.95%), eggshell weight (+4.26% and +5.38%), and eggshell surface (+2.43% and +1.96%) were higher (P<0.05) in SS and SY than C, whereas breaking strength was increased in SY (P<0.01). Breast muscle, liver and skin Se levels were higher in SY than in C, while kidney Se content was higher in SS hens. Eggs from SY had higher Se levels than SS. Blood metabolites were not affected in SS or SY groups than C. A higher Se level was detected in eggs and breast muscle of SY hens (P<0.05). Seleniumenriched eggs and edible tissues from organic Se sources in poultry diet could improve antioxidant status in humans and reduce possible Se deficiency-related diseases.

Rumen-protected choline and vitamin E supplementation in periparturient dairy goats: effects on milk production and folate, vitamin B 12 and vitamin E status

We investigated the effects of rumen-protected choline (RPC) and vitamin E (VITE) administration on milk production and status of folate, vitamin B12 and vitamin E during the periparturient period of dairy goats. Forty-eight Saanen multiparous goats were selected for the 72-day experiment, being moved to a maternity pen 30 days before expected parturition and assigned to one of the four experimental groups: control (CTR), no choline or vitamin E supplementation; choline (RPC), supplemented with 4 g/day choline chloride in rumen-protected form; vitamin E (VITE), supplemented with 200 IU/day vitamin E in rumen-protected form; and choline and vitamin E (RPCE), supplemented with 4 g/day RPC chloride and 200 IU/day vitamin E. Supplements were administered individually before the morning feed to ensure complete consumption, starting 30 days before kidding and continuing for 35 days after. During the experiment, milk yield and 4% fat-corrected milk (FCM) yield were, respectively, 210 and 350 g/day higher in RPC-supplemented goats than in non-supplemented goats. Milk fat concentration and fat yield were also increased by RPC treatment. Milk yield and composition were unaffected by vitamin E supplementation. There were no significant interactions between RPC and VITE for any of the variables measured. Plasma metabolites did not differ between treatments before and after kidding except that plasma folate at parturition was higher in RPC-supplemented goats. Neither choline nor vitamin E affected vitamin B12 plasma concentrations, while a time effect was evident after the second week of lactation, when B12 levels in each treatment group started to increase. Vitamin E administration resulted in plasma α-tocopherol levels that were 2 to 2.5 times higher than in non-supplemented goats. Overall, these results suggest that greater choline availability can improve milk production and methyl group metabolism in transition dairy goats.

Evaluation of the damage induced by ochratoxin A and the protective role of α-tocopherol in cultured bovine mammary epithelial cells

Evaluation of the damage induced by ochratoxin A and the protective role of α-tocopherol in cultured bovine mammary epithelial cells E. Fusi & R. Rebucci & C. Pecorini & L. Rossi & F. D’Ambrosio & A. Baldi Published online: 7 August 2008 # Springer Science + Business Media B.V. 2008

Effects of putrescine, cadaverine, spermine, spermidine and β-phenylethylamine on cultured bovine mammary epithelial cells

Abstract A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermidine and β-phenylethyla-mine on mammary epithelial cells. Each biogenic amine was diluted in several concentrations (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures. In BME-UV1, spermine significantly inhibited cell proliferation (P<0.001), while the other amines inhibited at higher concentrations (50mM). In primary bovine organoids, β−phenylethylamine significantly (P<0.001) inhibited cell proliferation at 4 mM. Organoids cultured in the presence of all amines, except β-phenylethylamine, had stellate projections indicating intense cell proliferation. Proliferation of mammary epithelial cells was stimulated at low concentrations, while at high concentrations it was inhibited. Our results suggested that the effects of each compound on mammary epithelial cells could be related to the compound itself and not to mediating by the bovine amino oxidase, responsible of the formation of toxic metabolites.

Effect of growth factors and lactogenic hormones on expression of plasminogen activator-related genes and cell proliferation in a bovine mammary epithelial cell line.

There is conflicting evidence in the literature as to whether up-regulation of urokinase plasminogen activator (u-PA) expression is related to bovine mammary epithelial cell growth. The role of u-PA receptor (u-PAR) and that of the plasminogen activator inhibitors type 1 and type 2 (PAI-1 and PAI-2) in bovine mammary epithelial cell proliferation is not known. The effect of growth factors and various hormones known to affect mammary function on expression of u-PA, u-PAR, PAI-1, PAI-2 and cell proliferation using the BME-UV1 bovine mammary epithelial cell line was examined. Cell proliferation was measured using the MTT assay and direct cell enumeration. Results showed that both IGF-1 and EGF increased cell proliferation but EGF was a more potent mitogen than IGF-1. Furthermore, IGF-1 increased by 2-fold expression of both u-PA and u-PAR while EGF increased by 3·8-fold the expression of only u-PAR. Both growth factors had no effect on expression of PAI-1 and PAI-2. In a manner consistent with changes in gene expression, EGF and to a lesser extent IGF-1 up-regulated total cell associated, membrane-bound and secreted u-PA activity. Thus, a strong correlation exists between u-PAR gene expression along with the activity of u-PA present on cell membranes and cell proliferation. Dexamethasone, prolactin and surprisingly insulin had no effect on cell proliferation. Dexamethasone alone and when combined with insulin or prolactin up-regulated gene expression of both PAI- and PAI-2 but not that of u-PA and u-PAR. Decreased total cell-associated, membrane-bound and secreted u-PA activity was detected in cells cultured in the presence of dexamethasone when combined with insulin or prolactin. However no such effect was observed in the presence of dexamethasone alone. Thus, dexamethasone acting synergistically with prolactin or insulin inhibits the activation of the plasmin-plasminogen system but this inhibition is not correlated with any changes in cell proliferation.

Effects of Rumen-protected Choline Supplementation in Periparturient Dairy Goats

D’Ambrosio, F., Campagnoli, A., Susca, F., Fusi, E., Rebucci, R., Agazzi, A, Pinotti, L. and Baldi, A., 2007. Effects of rumen-protected choline supplementation in periparturient dairy goats. Veterinary Research Communications, 31(Suppl. 1), 393–396

Milk Choline, α-Tocopherol and Neutrophil Chemotaxis in the Periparturient Dairy Cow

L. Pinotti*, R. Rebucci, E. Fusi, L. Rossi and A. Baldi Department of Veterinary Sciences and T echnology for Food Safety, Faculty of Veterinary Medicine, University of Milan, Milan, Italy *Correspondence: Dipartimento di Scienze e T ecnologie Veterinarie per la Sicurezza Alimentare, Facolta di Medicina Veterinaria, Universita degli Studi di Milano, V ia T rentacoste 2, 20134 Milano, Italy E-mail: luciano.pinotti@unimi.it

Effect of Escherichia coli lipopolysaccharide on u-PA activity and u-PA and u-PAR RNA expression in a bovine mammary epithelial cell line.

It is well known that the plasminogen-activating (PA) system plays a key role in the bovine mammary gland during tissue remodelling. However, the modulation of the PA cascade after bacterial infections needs to be elucidated. This study examined the effects of Escherichia coli lipopolysaccharide (LPS) on cell viability, the modulation of cell-associated u-PA activity, and the regulation of u-PA and u-PA receptor (u-PAR) RNA expression using the BME-UV1 bovine mammary epithelial cell line. LPS did not affect cell viability, but induced an increase in u-PA activity, with the maximum response after 6 h of incubation. Moreover, u-PA and u-PAR mRNA expression were both up-regulated in BME-UV1 cells after 3 h of incubation with LPS. These data indicated that E. coli LPS led to an increase in u-PA activity and RNA expression of u-PA and u-PAR in BME-UV1 cells, thus strengthening the role of the PA system during pathological processes.