Pharmacokinetic assessment of the monepantel plus oxfendazole combined administration in dairy cows

Monepantel (MNP) is a novel anthelmintic compound launched into the veterinary pharmaceutical market. MNP is not licenced for use in dairy animals due to the prolonged elimination of its metabolite monepantel sulphone (MNPSO₂) into milk. The goal of this study was to evaluate the presence of potential in vivo drug‐drug interactions affecting the pattern of milk excretion after the coadministration of the anthelmintics MNP and oxfendazole (OFZ) to lactating dairy cows. The concentrations of both parent drugs and their metabolites were measured in plasma and milk samples by HPLC. MNPSO₂ was the main metabolite recovered from plasma and milk after oral administration of MNP. A high distribution of MNPSO₂ into milk was observed. The milk‐to‐plasma ratio (M/P ratio) for this metabolite was equal to 6.75. Conversely, the M/P ratio of OFZ was 1.26. Plasma concentration profiles of MNP and MNPSO₂ were not modified in the presence of OFZ. The pattern of MNPSO₂ excretion into milk was also unchanged in animals receiving MNP plus OFZ. The percentage of the total administered dose recovered from milk was 0.09 ± 0.04% (MNP) and 2.79 ± 1.54% (MNPSO₂) after the administration of MNP alone and 0.06 ± 0.04% (MNP) and 2.34 ± 1.38% (MNPSO₂) after the combined treatment. The presence of MNP did not alter the plasma and milk disposition kinetics of OFZ. The concentrations of the metabolite fenbendazole sulphone tended to be slightly higher in the coadministered group. Although from a pharmacodynamic point of view the coadministration of MNP and OFZ may be a useful tool, the presence of OFZ did not modify the in vivo pharmacokinetic behaviour of MNP and therefore did not result in reduced milk concentrations of MNPSO₂.     

Comparison of hepatic in vitro metabolism of the pyrrolizidine alkaloid senecionine in sheep and cattle

OBJECTIVE: To compare hepatic metabolism of pyrrolizidine alkaloids (PAs) between sheep and cattle and elucidate the protective mechanism of sheep. SAMPLE POPULATION: Liver microsomes and cytosol from 8 sheep and 8 cattle. PROCEDURE: The PA senecionine, senecionine N-oxide (nontoxic metabolite) and 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP; toxic metabolite) were measured in microsomal incubations. The kcat (turnover number) was determined for DHP and N-oxide formation. Chemical and immunochemical inhibitors were used to assess the role of cytochrome P450s, flavin-containing monooxygenases (FMOs), and carboxylesterases in senecionine metabolism. The CYP3A, CYP2B, and FMO concentrations and activities were determined, in addition to the role of glutathione (GSH) in senecionine metabolism. RESULTS: DHP concentration did not differ between species. Sheep formed more N-oxide, had higher N-oxide kcat, and metabolized senecionine faster than cattle. The P450 concentrations and isoforms had a large influence on DHP formation, whereas FMOs had a large influence on N-oxide formation. In cattle, CYP3A played a larger role in DHP formation than in sheep. FMO activity was greater in sheep than in cattle. Addition of GSH to in vitro microsomal incubations decreased DHP formation; addition of cytosol decreased N-oxide formation. CONCLUSIONS AND CLINICAL RELEVANCE: Hepatic metabolism differences alone do not account for the variation in susceptibility seen between these species. Rather, increased ruminal metabolism in sheep appears to be an important protective mechanism, with hepatic enzymes providing a secondary means to degrade any PAs that are absorbed from the rumen.     

Midazolam oxidation in cattle liver microsomes: The role of cytochrome P450 3A

In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1′- and 4-hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme- and species-specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC-UV method was used to measure 1′- and 4-hydroxy MDZ (1′- and 4-OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single-enzyme Michaelis–Menten kinetics. Values of V_max and K_m were 0.67 nmol/min/mg protein and 6.16 μM for 4-OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1′-OHMDZ. An anti-rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1′- and 4-OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1′-OHMDZ and 4-OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4-OHMDZ formation and the involvement of other CYPs in 1′-OHMDZ production, paving the way for further investigations.     

Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.     

Species differences in hepatic biotransformation of the anthelmintic drug flubendazole

Flubendazole (FLBZ) is a broad‐spectrum benzimidazole anthelmintic used in pigs, poultry, and humans. It has been proposed as a candidate for development for use in elimination programmes for lymphatic filariasis and onchocerciasis in humans. Moreover, FLBZ has shown promise in cancer chemotherapy, particularly for neuroblastoma. This work investigated the hepatic carbonyl‐reducing pathway of FLBZ in different species, including humans. Microsomal and cytosolic fractions were obtained from sheep, cattle, pig, hen, rat, and human liver. Both subcellular fractions of each species converted FLBZ into a reduced metabolite (red‐FLBZ). The rate of microsomal red‐FLBZ production was highest in sheep (1.92 ± 0.13 nmol/min.mg) and lowest in pigs (0.04 ± 0.02 nmol/min.mg); cytosolic red‐FLBZ production ranged from 0.02 ± 0.01 (pig) to 1.86 ± 0.61 nmol/min.mg (sheep). Only subcellular fractions from sheep liver oxidized red‐FLBZ to FLBZ in a NADP⁺‐dependent oxidative reaction. Liver microsomes from both pigs and humans transformed FLBZ to red‐FLBZ and a hydrolyzed metabolite. Very significant differences in the pattern of FLBZ metabolism were observed among the tested species and humans. These results reinforce the need for caution in extrapolating data on metabolism, efficacy, and safety of drugs derived from studies performed in different species.     

Benzydamine as a useful substrate of hepatic flavin‐containing monooxygenase activity in veterinary species

Capolongo, F., Santi, A., Anfossi, P., Montesissa, C. Benzydamine as a useful substrate of hepatic flavin‐containing monooxygenase activity in veterinary species. J. vet. Pharmacol. Therap. doi: 10.1111/j.1365‐2885.2009.01145.x. Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N‐oxide (BZ‐NO), is produced in the liver and brain by flavin‐containing monooxigenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, K_m and V_max, of BZ‐NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered.     

The effect of leucaena hay as a source of effective fibre and nutrients in diets with forage palm for finishing sheep in semi-arid regions

The present study was conducted to evaluate the intake, digestibility and nitrogen balance of diets for finishing sheep, containing leucaena hay as a source of effective fibre and forage palm. Twenty-one male sheep were used in a randomized block design with three treatments, represented by total diets, where the forage was leucaena hay and forage palm in natura in proportions 350:650, 450:550 and 550:450 g/kg respectively. For the pH and NH₃-N in the ruminal fluid, and urea in the blood serum, plots split in time were adopted. Dry matter and nutrient digestibility, and the nitrogen balance were not influenced (p > .05) by the diets. The neutral detergent fibre digestibility decreased (p < .05) as the proportions of leucaena hay in the diets increased. The pH and NH₃-N were suitable for ruminal fermentation, and blood urea was maintained under normal physiological conditions for sheep. Combining 350 to 550 g/kg of leucaena hay with 450 to 650 g/kg of forage palm in total diets with a forage:concentrate ratio of 60:40 in late-maturing sheep meets the nutritional requirements for daily gain 200 g, characterizing leucaena hay as important source of effective fibre and nutrients in diets.     

Effect of increasing levels of wasted date palm on digestion,rumen fermentation and microbial protein synthesis in sheep

This study was conducted to evaluate the effects of increasing levels of wasted date (WD) in sheep (initial body weight 50 ± 2.3 kg) diets on apparent diet digestibility, rumen fermentation and microbial protein synthesis. The experimental diets contained 0 (0 WD), 9% (9 WD), 18% (18 WD) or 27% (27 WD) of wasted dates. These diets were evaluated through a 4 × 4 Latin square design with four 28‐day periods. Sheep were individually housed in metabolic cages and were fed diets ad libitum. To keep the diets isoenergetic and isonitrogenous, increasing partial replacement of WD for barley grain was used with increasing levels of soya bean meal. Increasing the amount of WD in diets of sheep did not influence (p > 0.05) diet intake and digestibility of nutrients. Inclusion of WD in diets of sheep did not affect (p > 0.05) ruminal pH but decreased (p < 0.05) NH₃‐N concentration and blood urea nitrogen. Also there was no effect (p > 0.05) of the WD provision on N intake and faecal N loss of experimental animals, but urinary nitrogen excretion and retained nitrogen were affected (p < 0.05) by dietary treatments. Purine derivatives and microbial protein synthesis increased (p < 0.05) linearly with the level of WD in the diet. In conclusion, the increase in microbial protein synthesis and concomitant reduction in ruminal NH₃‐N concentration and blood urea nitrogen in response to wasted date supply suggest improved efficiency of ruminal nitrogen metabolism without adverse effects on fermentation characteristics and animal performance.     

Molecular cloning and expression of FGF2 gene in pre‐implantation developmental stages of in vitro‐produced sheep embryos

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO₂, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO₂ incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.     

Effect of breed and gender on bovine liver cytochrome P450 3A (CYP3A) expression and inter-species comparison with other domestic ruminants

The cytochrome P450 (P450) superfamily represents a group of relevant enzymes in the field of drug metabolism and several exogenous or constitutional factors contribute to regulate its expression. Cattle represent an important source of animal-derived food-products and studies concerning the P450 expression are needed for the extrapolation of pharmacotoxicological data from one species to another and for the evaluation of the consumer's risk associated with the consumption of harmful residues found in foodstuffs. In the present study, possible breed-, gender- and species-differences in P4503A (the P450 subfamily more expressed in the human liver) expression were studied in vitro in Piedmontese (PDM) and Limousin (LIM) meat cattle breeds of both sexes and in domestic Ruminants (cattle, sheep and goats). Cytochrome P450 and P4503A contents as well as CYP3A-dependent drug metabolising enzymes (DME) were measured in liver microsomes. Significant lower levels of P450 (P < 0.001) and P4503A (P < 0.05) contents were observed in PDM vs. LIM of both sexes; the P4503A-dependent DME activities were significantly (P values ranging from 0.05 up to 0.001) higher in PDM cattle, particularly in males. A gender-effect in DME activities was noticed (P < 0.05) only in PDM male cattle. With regards to the species, the expression of both P4503A apoprotein and some of the related DME activities were more pronounced in sheep (P < 0.01 vs. cattle) and in goats (P < 0.05 vs. sheep; P < 0.01 vs. cattle) than in cattle. The significant differences in P4503A expression observed in LIM and PDM cattle are consistent with previously published data on strain- and breed-differences pointed out in rats and men. As far as a possible sex-effect is concerned, no clear-cut evidence is likely to be drawn. Finally, P4503A expression was more relevant in small ruminants.     

Anthelmintic resistance in New Zealand: A perspective on recent findings and options for the future

A recent national survey on anthelmintic resistance in cattle and sheep in New Zealand indicated that the magnitude of the problem has increased from very low levels only a few years ago to disturbingly high levels now. There is a particular problem with multiple resistance to all three action families of anthelmintic currently available in Ostertagia (= Teladorsagia) spp in sheep, and to both macrocyclic lactones (ML) and benzimidazoles in Cooperia spp in cattle. The prevalence and extent of resistance indicate that all cattle farmers and most sheep farmers should now be using a combination anthelmintic on most occasions just to achieve effective control of all parasites. Despite this, the presence of resistant parasites has generally not been appreciated by the majority of affected farmers, possibly because most have not formally tested to determine the resistance status of nematodes on their farms.

Anthelmintics will remain the cornerstone of gastrointestinal nematode control in sheep and cattle for the foreseeable future but to ensure their continued effectiveness farmers need to be constantly aware of the need to maintain adequate reservoirs of unselected nematodes, i.e. worms in refugia, to minimise the expansion of the resistant population. High-risk practices in relation to selection of resistance need to be identified and avoided or at least their use limited. These include: treating adult animals where there is no identified need, moving newly- treated animals onto ‘clean’ pasture, and failing to effectively quarantine-drench bought-in animals. None of these are new concepts but many have not been adopted or practised. In particular, sheep farmers should endeavour to avoid treating ewes pre-lambing with long-acting anthelmintics. Farmers needs to negotiate a balance between achieving good parasite control and the sustainability of their control options.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

Estradiol-17β and linseed meal interact to alter visceral organ mass and hormone concentrations from ovariectomized ewes

To evaluate the estrogenic potential of secoisolariciresinol diglycoside (SDG) found in linseed meal (LSM) on visceral organ mass, IGF-I, and thyroid hormone (T₃ and T₄) concentrations, 48 multiparous, ovariectomized ewes (54.6 ± 1.1 kg) were used in a 3 × 4 factorial arrangement. Main effects were length of LSM feeding (0, 1, 7, or 14 d) and length of exposure to estradiol-17β (E₂) implant (0, 6, or 24 h prior to tissue collection). Implanting ewes with E₂ for 24 h increased liver mass relative to empty body weight (EBW; g/kg EBW) compared with ewes implanted for 0 or 6 h (P ≤ 0.03), whereas feeding LSM for 14 d decreased liver mass compared with ewes fed LSM for 1 or 7 d (P ≤ 0.02). There was an LSM × E₂ interaction (P = 0.01) for duodenal mass (g/kg EBW), LSM, and E₂ tended (P = 0.07) to influence the stomach complex mass; however, ileal mass was not affected. Neither LSM nor E₂ affected (P ≥ 0.12) CYP2C or CYP3A mRNA expression or cellularity of the liver. Exogenous E₂ influenced circulating concentrations of IGF-I, T₃, and T₄. The estrogenic or anti-estrogenic potential of LSM is dependent upon the tissue, exposure to E₂, and the duration of LSM feeding. Feeding LSM during gestation, lactation, or during the grow-finish phase warrants further investigation.     

The effect of breed,sex, and drug concentration on the pharmacokinetic profile of ivermectin in cattle

Ivermectin (IVM) is one of the most widely used antiparasitic drugs worldwide and has become the drug of choice for anthelmintic and tick treatment in beef cattle production. It is known that pharmacokinetic parameters are fundamental to the rational use of a drug and food safety and these parameters are influenced by different factors. The aim of this study was to evaluate the pharmacokinetic profile of IVM in Bos indicus, Bos taurus, and crossbreed cattle (B. indicus × B. taurus) kept under same field conditions and the possible impacts of sex and IVM formulation (1% and 3.15%). It was observed that IVM concentration was significantly affected by breed. The plasma concentrations of IVM, AUC, C_max, and t_1/2β were significantly higher in B. indicus compared to B. taurus. Crossbreed animals showed an intermediate profile between European and Indian cattle. No alteration in pharmacokinetics parameters was detected when comparing different gender. Concerning the pharmacokinetic data of IVM formulation, it was verified that T_max, AUC, and t_1/2β were higher in 3.15% IVM animals than those from 1% IVM formulation. The results clearly indicated that the IVM plasma concentrations in B. indicus were higher than that in B. taurus.     

Modulation of ruminal and intestinal fermentation by medicinal plants and zinc from different sources

Two experiments were conducted on sheep to determine the effect of dietary supplementation with zinc and a medicinal plant mixture on haematological parameters and microbial activity in the rumen and large intestine. In Experiment 1, 24 male lambs were randomly divided into four groups: One group was fed an unsupplemented basal diet (control), and three groups were fed a diet supplemented with 70 mg Zn/kg diet in the form of Zn sulphate (ZnSO₄), a Zn‐chelate of glycine hydrate (Zn‐Gly) or a Zn‐proteinate (Zn‐Pro), for five months. The ruminal content was collected separately from each lamb, and batch cultures of ruminal fluid were incubated in vitro with mixture of medicinal plants (Mix) with different roughage:concentrate ratios (800:200 and 400:600, w/w). Bioactive compounds in Mix were quantified by UPLC/MS/MS. In Experiment 2, four sheep were fed a diet consisting of meadow hay and barley grain (400:600, w/w), with Zn‐Gly (70 mg Zn/kg diet), Mix (10% replacement of meadow hay) or Zn‐Gly and Mix (Zn‐Gly‐Mix) as supplements in a Latin square design. Mix decreased total gas (p < 0.001) and methane (p < 0.01) production in vitro. In Experiment 1, caecal isobutyrate and isovalerate concentrations varied among the dietary treatments (p < 0.01). The isovalerate concentration of the zinc‐supplemented groups in the distal colon was higher (p < 0.001) compared with the control. In Experiment 2, the molar proportion of isobutyrate was the highest in the faeces of the sheep fed the diet with Zn‐Gly‐Mix (p < 0.01). The plasma zinc concentration was higher in the groups fed a diet supplemented with zinc (p < 0.001). The haematological profile and antioxidant status did not differ between the dietary groups (p > 0.05). The diets containing medicinal plants and organic zinc thus helped to modulate the characteristics of fermentation in ruminants.     

A comparison of vitamin B12 levels in the liver and serum of sheep receiving treatments used to correct cobalt deficiency

The efficacies of four methods, used for the prophylaxis of cobalt deficiency in sheep as measured by the elevation of liver and serum vitamin B₁₂ levels, were compared in marginally deficient sheep over 14 weeks. The methods used were weekly drenches of either cobalt sulphate or cobalt chelate (EDTA); three-weekly injections of hydroxocobalamin, and ruminal cobalt pellets.

On the basis of elevated liver and serum vitamin B₁₂ levels, chelated cobalt was shown to be available to rumen microflora for the synthesis of vitamin B12. However, at no stage were liver and serum vitamin B₁₂ levels of sheep receiving the chelate significantly different from those receiving the same amount of cobalt as the sulphate.

After five, three-weekly injections of hydroxocabalamin liver vitamin B₁₂ levels were significantly higher (p < 0.01) than for the other treatments, with the exception of cobalt sulphate.

Cobalt pellets led to an initial rapid and significant rise in serum vitamin B₁₂ when compared with the other treatments. However, at four weeks there was no significant difference between treatment groups for serum vitamin B₁₂. Fourteen weeks after the administration of cobalt pellets, serum and liver. vitamin B₁₂ levels in this group were not significantly different from those of untreated sheep. At this time, three out of 12 sheep had lost their pellets.     

Nutritive value of sorghum silage for sheep

This study compared ensiled sorghum (ES) from two varieties (first and second cuts) with corn silage (CS) for chemical analysis, total-tract nutrient digestibility, ruminal variables and blood biochemistry metabolites in sheep. Five experimental silages representing first cut ES var. Pegah (PS1), second cut ES var. Pegah (PS2), first cut ES var. Speedfeed (SS1), second cut ES var. Speedfeed (SS2) and CS were assessed for chemical analysis, silage fermentation and anti-quality compounds. Diets consisted of lucerne hay, silage and a premix (at the ratio of 53.5:46:0.5 on a dry matter [DM] basis). Feed consumption, total-tract nutrient digestibility, microbial nitrogen supply (MNS), ruminal variables and blood biochemistry metabolites were determined in a Latin square design of five periods with five male sheep. Compared to sorghum, CS had lower (p < 0.01) concentrations of ash, water-soluble carbohydrates and crude protein, but higher starch, lignin (sa), non-structural carbohydrates and apparent nutrient digestibility. The measurements of pH, lactic acid, NH₃-N and volatile fatty acids (VFA) confirmed that all silages were well-preserved. The anti-quality compound levels in the silages were below the safe limits for ruminants. No differences were observed in feed consumption (p > 0.05) and total-tract digestibility of DM and nutrients (p > 0.05). Rumen pH, NH₃-N, VFA concentrations and acetate to propionate ratio showed no difference among treatments (p > 0.05). Sheep fed on sorghum silage showed a decrease (p < 0.01) in total protozoa, and particularly in the Entodiniae population. There were no differences in cellulolytic bacteria counts (p > 0.05) among treatments. Urinary N, MNS and blood biochemistry metabolites showed no difference among treatments (p > 0.05). Silages from both cuts of sorghum had comparable total-tract nutrient digestibility to CS, therefore, these varieties could be used as a more drought resilient silage source in the feeding of sheep.     

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.     

Oxygen uptake and net flux of metabolites by splanchnic tissues of sheep in response to short‐term mesenteric infusion of nitrogenous compounds

This study was conducted in an attempt to quantify the impact of N load on splanchnic tissues metabolism of sheep. The trial was conducted with four male sheep (45 ± 2.5 kg body weight (BW)) surgically implanted with chronic indwelling catheters into the portal, hepatic and mesenteric veins. Blood flow and metabolic flux through portal‐drained viscera (PDV), liver and total splanchnic tissues (ST) were measured daily following a 4 × 4 Latin Square experimental design, where sheep were continually infused into the mesenteric vein with a physiological saline (0.15 m NaCl) solution during 90 min followed by the infusion, during more 120 min, of either solution: physiological saline (control), 0.250 m NH₄HCO₃, 0.250 m L‐alanine or 0.125 m L‐arginine, all of them infused at a rate of 1.5 ml/min to provide 375 μmol N/min. During the treatment infusion period, the net removal of ammonia N and the net production of urea N by liver were higher (p < .05) in NH₄HCO₃ infused sheep_. Based on oxygen consumption, and on average of all treatments, the heat produced by liver and ST was on average 6 and 14 kcal/kg BW representing 16% and 38% of the metabolizable energy intake respectively. Linear relationships between variables indicated that gluconeogenesis and ureagenesis occurred concomitantly and both processes accounted for approximately 50% of total liver energy expenditure, two‐thirds of it associated with gluconeogenesis. The results of the current study did not present clear evidence of the expected energy costs associated with ammonia N, alanine or arginine metabolism by liver. However, they indicated that gluconeogenesis is on average a more energy expensive process than ureagenesis.     

Pharmacokinetic behaviour and anthelmintic efficacy of 1-n-butyl carbamoyl oxfendazole given by intramuscular injection

Oxfendazole (OFZ) was chemically modified to 1-n-butyl carbamoyl OFZ (C₄-OFZ) in an attempt to improve the solubility of OFZ and enable it to be administered by injection. After intramuscular injection to sheep and cattle, C₄-OFZ was metabolised to OFZ which resulted in higher plasma OFZ concentrations that persisted for a considerably longer period than those observed following administration of OFZ orally. The anthelmintic efficacy of injected C₄-OFZ was tested, in sheep, against strains ofTrichostrongylus colubriformis,Haemonchus contortus andOstertagia circumcincta, which were highly resistant to benzimidazoles. In all cases, the C₄-OFZ treatment showed a significant improvement in efficacy over the conventional oral OFZ drench.