Ingested versus inhaled limonene in sheep: A pilot study to explore potential different transfer to the mammary gland and effects on milk and Caciotta cheese aroma.

Concentration is a key determinant in the overall positive impact of terpenes on milk and cheese aroma; additionally, route of intake may affect the achievable concentrations of dietary terpenes in milk and cheese. In this study, we explored the possibility that the amount of the monoterpene limonene transferred to sheep milk and its corresponding cheese could differ depending on the route of intake and that the aroma profile of these products could also differ. To this aim, 12 lactating dairy ewes were repeatedly exposed to limonene by the oral or respiratory route during a 48-h test period, according to a 3 × 3 Latin square experimental design. Limonene content was measured in individual and bulk milk samples, in 1-d-old and 15-d-old Caciotta cheese obtained from that milk, in the related whey and curd, and in the air inhaled by the ewes in the respiratory treatment group (to obtain an estimate of the dose actually supplied by this route). Bulk milk and fresh (1-d-old) cheese underwent sensory analysis by ortho-olfactory evaluation. Both intake routes demonstrated transfer of limonene to milk, but the respiratory route transferred limonene with greater efficiency than the oral route. Moreover, according to the protocol used in this study, a short period of respiratory exposure induced a slightly higher limonene content in milk compared with oral exposure. As to the fate of limonene during cheesemaking, an important part of it was lost into the whey, perhaps through volatilization. The differences between milk and cheese tended to dissipate in curd and fresh cheese and disappeared completely after 15 d of ripening. Finally, it was possible to distinguish between the 2 routes of limonene intake using sensory analysis, even though no direct relationship was identified between the different aroma profiles of milks and cheeses from the oral and respiratory groups and their respective limonene contents. Overall, our results expand current knowledge on the biological pathways of terpene transfer from feed to sheep milk and cheese, as well as on the role played by terpenes in the formation of aroma in these products. Our observations may contribute to future development of strategies for external control and better standardization of the presence of odor compounds in milk and cheese from dairy ruminants.

A comparative study between responses of isolated bovine and equine digital arteries to vasoactive mediators.

Hemodynamic perturbations, partly resulting from abnormal vasoconstriction of digital vessels, have been implicated in the pathogenesis of bovine and equine laminitis. This study compared the responsiveness of isolated bovine (BDA) and equine (EDA) digital arteries to pharmacological agents that stimulate receptor systems involved in the regulation of normal vessel tone. The role of the endothelium and the short- and longer-term effects of an experimentally induced endothelial damage were also evaluated. Species-related differences were found in the vessel reactivity to all of the receptor agonists tested. In intact BDA, as compared to intact EDA, norepinephrine was a more effective vasoconstrictor, 5-hydroxytryptamine a more effective but less potent vasoconstrictor, isoproterenol a less effective vasodilator and carbamylcholine a less potent vasodilator. In BDA, but not in EDA, the contractile responses to norepinephrine and 5-hydroxytryptamine were enhanced immediately after endothelium removal. However, the contractile reactivity of denuded BDA returned to basal values following overnight incubation. The differences suggest species specificity for the pathophysiology of digital vasomotor tone and function in horses and cattle.

Effects of endotoxin and influence of cyclooxygenase-2 on ß-adrenergic mediated relaxation in isolated equine digital artery.

The effects of endotoxin on ß-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA ß-adrenoceptor functionality was also evaluated. Endothelium-intact (e(+)) and/or -denuded (e(-)) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS+NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e(-)) rings acutely exposed to NS398. Fresh and incubated (e(-)) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of ß-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e(-)) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA ß-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

A clonal cell line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1.

Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial cell line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1--1.0-8.0 microM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased linearly with time for 36-48 h and the percent biotransformation of AFB1 (2-4 microM) at 48 h was about 12-14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with cellular metabolism. MTT assay (from 24 h of cell exposure) and NRU assay (from 16 h of cell exposure) showed time-dependent and time/concentration-dependent decrease of cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC50 at 48 h = 12.00 +/- 2.66 microM; MTT: CC50 at 72 h = 20.42 +/- 7.30 microM). The results suggest that BME-UV1 cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.

Oral and intravenous administration of nimesulide in the horse: rational dosage regimen from pharmacokinetic and pharmacodynamic data.

REASONS FOR PERFORMING STUDY: The selective COX-2-inhibitor nimesulide is used extra-label in equine veterinary practice as an anti-inflammatory agent. However, there are no data on which to base the rational use of the drug in this species. OBJECTIVES: To determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. METHODS: The pharmacokinetics of nimesulide in the horse after oral administration (1 mg/kg bwt), and oral and i.v. administration (1.5 mg/kg bwt) were investigated, effects of feeding status on bioavailability determined, and plasma protein binding of the drug and its principal metabolites measured. Compartmental and noncompartmental pharmacokinetic analyses were performed. The plasma concentration-time profile was used, together with in vitro literature data on nimesulide inhibition of COX isoforms, to determine the effective COX selectivity of nimesulide in the horse, and suggest a suitable dosing schedule. RESULTS AND CONCLUSIONS: The findings suggest that 1.5 mg/kg bwt may produce adequate clinical effects, and the dosing interval should be 12-24 h depending on condition severity. However, at that dose, the concentration in the animal exceeds the in vitro IC50 for both isoforms, so that COX-1/COX-2 selectivity is lost and side-effects due to COX-1 inhibition are a possibility. Nimesulide should therefore be used with caution in equine clinical practice.

Pharmacokinetics and mammary elimination of imidocarb in sheep and goats.

The pharmacokinetics and mammary excretion of imidocarb dipropionate, a therapeutic/prophylactic agent against a variety of tick-borne hemoparasitic diseases in domestic animals, have been investigated in sheep and goats. A commercial formulation of imidocarb di-propionate was injected i.m. at a single dose of 3 mg/kg of body weight in 7 mature lactating ewes and 8 lactating does in good health. Blood samples were collected for 48 h after administration and milk samples were collected every 12 h for 10 d. A weak cation-exchange solid-phase procedure was used to remove imidocarb from plasma. A hexane/isoamyl alcohol liquid-liquid procedure was adopted to extract the drug from the milk of sheep. The same method was used for goat milk after exposing the matrices to enzymatic digestion. The extracted samples were analyzed by HPLC. The i.m. disposition kinetics of imidocarb in the 2 species showed significant differences in the rate of elimination (0.0075 +/- 0.002 and 0.025 +/- 0.004 L/h in sheep and goats, respectively), being faster in ewes than in does. Nevertheless, a smaller area under the concentration-time curve (12.21 +/- 0.76 and 9.49 +/- 0.54 microg/mL per h in sheep and goats, respectively), a larger volume of distribution (4.18 +/- 0.44 and 7.68 +/- 0.57 L/kg in sheep and goats, respectively), and a longer mean residence time (9.07 +/- 0.77 and 14.75 +/- 2.20 h in sheep and goats, respectively) were found in goats, suggesting a more rapid and effective drug storage in tissues during the first 48 h after the injection. The concentrations of imidocarb in milk of both species were higher than in plasma. However, a fast passage through the blood-milk barrier and a high storage of imidocarb were observed in the milk of ewes, whereas the drug concentrations were not as high nor was the extent of drug penetration from blood to milk as great in the milk of goats (AUC(milk 0-48)/AUC(plasma 0-48) = 2.5 +/- 0.45 and 1.26 +/- 0.27 in sheep and goat, respectively). Despite the differences in pharmacokinetic behavior, and considering the sensitivity of pathogens to imidocarb, the same dosage regimen can be used for clinical efficacy against Babesia spp. infection in both species. In contrast, the differences in depletion of imidocarb residue in milk and the large variability in mammary drug elimination found in goats suggests that great care should be taken in defining the withdrawal time in small ruminant dairy species.

Identification of functional alpha-adrenoceptor subtypes in the bovine female genital tract during different phases of the oestrous cycle.

The concentration and functionality of the alpha-adrenoceptor (alpha-AR) subtypes in the genital tract of cyclic heifers were investigated. In each tissue sample, a single class of alpha1-ARs was observed, whereas two distinct classes of alpha2-ARs were discriminated: low-affinity (LA) and high-affinity (HA) alpha2-ARs. Statistical analysis showed the presence of significantly (p < 0.05) higher concentrations of all alpha-AR subtypes in the follicle than in the corpus luteum. No significant differences were found in the ovary or myometrium between the luteal and follicular phases. In the ovary, the density of alpha1-ARs was significantly (p < 0.05) higher than that of alpha2-ARs. By contrast, there were significantly (p < 0.05) more alpha2-ARs than alpha1-ARs in the myometrium. As far as alpha2-ARs are concerned, LA alpha2-ARs were significantly (p<0.05) higher than HA alpha2-ARs in all tested tissues. Competition studies suggested that the rank order of potency of antagonists for alpha1-ARs was prazosin > phentolamine > yohimbine, whereas for alpha2-ARs the order of potency was yohimbine > or = phentolamine>prazosin. Functional assays performed on myometrium showed that noradrenaline, phenylephrine and clonidine elicited concentration-dependent contractions only in dioestrus and pro-oestrus preparations and that clonidine was more effective than phenylephrine as a contractile agent. It appeared that there were no significant modifications in alpha-AR affinity or concentration during the different stages of bovine oestrous cycle, whereas the uterine spontaneous activity and the responsiveness to alpha-adrenergic stimulation was strongly influenced by hormonal levels. The modifications of uterine contractility observed during the oestrous cycle may be related to modifications induced in the transductional mechanisms of alpha-ARs.