Concentrations of nitric oxide in equine preovulatory follicles before and after administration of human chorionic gonadotropin.

In the present study, follicular fluids of estrous mares treated with saline solution (Control) or nitric oxide synthase (NOS) inhibitors were analyzed for nitric oxide (NO), estradiol-17beta (E2) and progesterone (P4) concentrations before and 36h after administration of human chorionic gonadotropin (hCG). Follicular fluids obtained before (0h) hCG administration from control mares had lower concentrations of NO than those obtained 36h after administration of hCG (58.3+/-17.8 micromol versus 340.4+/-57.7 micromol; P<0.05). A similar pattern was also noted for intrafollicular P4 in control mares, which had lower concentrations of intrafollicular P4 before hCG than 36h post-hCG administration (P<0.05). As expected, E2 concentrations of control follicles sampled before hCG administration were higher than those sampled 36h post-hCG administration (P<0.05). However, the E2 concentrations in follicles of mares treated with the NOS inhibitors N(omega)-nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) did not decrease after hCG administration, unlike those in control mares (P>0.10). In addition, mares treated with NOS inhibitors had lower intrafollicular concentrations of NO and P4 than control mares, both before and after hCG administration (P<0.05). Increased intrafollicular concentrations of NO in control, hCG-stimulated mares provide evidence for the presence of an NO-generating system in the equine preovulatory follicle that is likely upregulated following administration of hCG.

Development of in-vitro-derived bovine embryos in protein-free media: effects of amino acids, glucose, pyruvate, lactate, phosphate and osmotic pressure.

In experiment 1, the effects of a group of either 20 (i.e. glutamine + essential + non-essential) or 11 (i.e. hamster embryo culture medium (HECM)-6) amino acids were evaluated in modified potassium simplex optimised medium (mKSOM) or basic medium (BM)-3. In experiment 2, the effects of glucose, pyruvate, lactate, phosphate or all four substrates were evaluated in low- or high-osmotic pressure BM-3 (255 and 275 mOsmol respectively) containing 20 amino acids (BM-3-20aa). In experiment 1, mKSOM containing 20 amino acids (mKSOM-20aa) supported the highest frequency of total, expanded (Days 7, 8 and 9) and hatched blastocysts. In experiment 2, supplement type affected the frequency of development to at least the morula stage (Day 7), expanded (Day 8), hatched (Day 9) or total blastocysts and cell number per blastocyst. Osmotic pressure affected the frequency of expanded blastocysts (Day 7) and blastocyst cell number. Regardless of the osmotic pressure, BM-3-20aa containing glucose (0.2 mM) supported the highest frequency of blastocyst development. The interaction between supplement type and osmotic pressure was not significant; however, treatment mean differences were more marked in high- than in low-osmotic pressure medium. In conclusion, the beneficial effects of amino acids on in vitro embryo development are influenced by the base medium. Moreover, glucose-containing media supported a higher frequency of embryonic development than pyruvate- and/or phosphate-supplemented media, indicating that glucose plays more important roles in non-energy generating pathways.

Effect of nitric oxide synthase inhabitors on ovulation in hCG-stimulated mares.

Recent studies suggest that nitric oxide (NO) may have a role in regulating ovarian physiology. To investigate the role of NO during ovulation in mares, inhibitors of the nitric oxide synthase (NOS) were administered to estrous mares. Forty cycling mares (20 horses and 20 pony mares) were allotted to one of the three treatment groups. Once a follicle was at least 27 mm in diameter, but smaller than 35 mm, mares were given one of the following treatments: saline solution 0.9% (n = 20, w/v, i.v., every 12 h), Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME; n = 10, 148 micromol/kg, i.v., every 12 h), or aminoguanidine hemisulfate (AG; n = 10, 406 micromol/kg, i.v., every 12 h). When a follicle >30 mm was present on one of the ovaries, ovulation was induced with hCG (2,500 IU, i.v.). The median time of ovulation (+/-6 h) after hCG administration for the treatment groups was 42, 84 and 54 h for mares treated with saline solution, L-NAME and AG, respectively. There was no significant difference between the groups treated with AG or L-NAME (P = 0.06); however, these groups were different from the control group (P < 0.05). The delayed ovulation caused by the administration of NOS inhibitors suggests a role for NO in follicular growth and ovulation in horses.

Cimetidine inhibits nitric oxide associated nitrate production in a soft-tissue inflammation model in the horse.

Cimetidine (CIM) is an H2-receptor antagonist that has been used in racehorses in an attempt to reduce the occurrence of stress-related gastric ulceration. It has also been shown to produce several useful effects other than its gastric acid suppression properties. Further, it is a well documented antagonist of cytochrome P-450 (CYP) mediated oxygenation reactions. Nitric oxide (NO), a recently discovered mediator or modifier of numerous physiological functions, is generated by several forms of nitric oxide synthase (NOS), one of which is inducible (iNOS). Inducible NOS, expressed in neutrophils and macrophages as part of the inflammatory response to noxious stimuli, contains both a CYP and a CYP reductase domain. Because of the similarity of structure of iNOS and CYP, it was decided to determine whether CIM could reduce NO production, using a carrageenan inflammation model in the horse. Two experiments were conducted. In Trial 1, six female Thoroughbred horses each had three tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into three treatments: 0.9% NaCl (NaCI), CIM (3 mg/kg), and aminoguanidine (AG; 25 mg/kg), an inhibitor of iNOS. Each mare received three i.v. injections 12 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and tissue chamber fluid (TCF) were collected serially. Concentrations of NO3- (the major metabolite of NO), albumin, total protein, CIM and AG were measured and complete cell counts and differentials were conducted. Trial 2 also used six female Thoroughbred horses implanted with at least two tissue chambers inserted subcutaneously on the sides of the neck. The study was divided into two treatments: NaCl (0.9%) and CIM (6 mg/kg). Each mare received seven i.v. injections of either NaCl or CIM 8 h apart prior to instillation of 1 mL of carrageenan into the test chamber. Blood and TCF were collected serially as before, and analysed for NO3- and CIM content. Areas under the curve (AUC) of the different parameters were calculated for the periods of -1-1, -1-3 and -1-7 days (Trial 1) and -2-1 for Trial 2. Absolute values were also compared at 4, 8 and 12 h postcarrageenan. Saline treatment did not reduce the elevated concentrations of NO3- in either plasma or TCF. Plasma, test chamber and control chamber NO3-concentrations rose from 0 to 12 h, and were very similar in all three sampled fluids. Cimetidine significantly (P< or =0.05) decreased NO3- production in plasma over the periods of -1-1, -1-3, and -1-7 days post inflammation when compared to NaCl treatment in Trial 1. Aminoguanidine and CIM decreased NO3-production in TCF for the periods -1-1, 1-3, and -1-7 days post inflammation in Trial 1 and -2-1 for Trial 2. Both CIM and AG also significantly reduced NO3-concentrations in plasma and TCF at 12 h postinitiation (Trials 1 and 2). Thus CIM, at the doses studied, was capable of reducing NO3- concentrations in this model as effectively as AG, a relatively specific inhibitor of iNOS activity.

The regulation of drugs and medicines in horse racing in the United States. The Association of Racing Commissioners International Uniform Classification of Foreign Substances Guidelines.

The primary reason for developing the ARCI Uniform Classification of Foreign Substances was to give stewards and other racing regulators guidelines to assist them in understanding the relative performance effects and general offensiveness to the Rules of Racing of various drugs and medications. As such, these guidelines have been very useful in the world of racing regulation--officially or unofficially--because this classification system, for the first time, places a relative number on the inappropriateness of any one of more than 750 agents appearing in forensic samples taken from racing horses. The guidelines set up by this system established the first framework for dialogue among veterinary pharmacologists reviewing these drugs. Prior to development of the guidelines, pharmacologists had their own opinions about these agents and their effects on performance. The guidelines, however, established a framework for discussion, and there has been surprising unanimity about the classification of each of these agents. Not only does this classification system provide a useful basis for dialogue among experts, it is also useful for regulators, horsemen and other laymen, most of whom have little training or experience with drugs and their effects on horses. The system is easily understandable and communicates the relative possibility of any classified substance to affect the performance of a horse. Consequently, the system has made it possible for laymen to understand the degree of impropriety of all drugs and medicines with which they may have contact. Grouping a large number of drugs into specific classes has greatly facilitated discussion about regulations and penalties, and the classification system is related to proposed penalty guidelines which were developed in parallel. With regard to penalties for Class 1 agents, it is easy to assign and defend substantial penalties after examining the guideline statement describing the possible performance effects of this group of agents as well as the fact that they have no well recognized therapeutic role. Similarly, the relatively modest effects of class 4 and 5 agents, combined with the fact that these groups encompass a large number of well recognized therapeutic agents, helps in understanding the possible presence of trace levels of these agents in post-race samples. In summary, the ARCI Uniform Classification of Foreign Substances Guidelines condenses data on drugs and medications and places them into a simple five class system. This system has made it possible to confidently discuss the regulatory implications of the identification of any one of the approximately 750 classified substances potentially found in forensic samples from a performance horse. As such it facilitates both the development and implementation of more understandable and equitable regulatory processes.

Characterization of a sterile soft-tissue inflammation model in thoroughbred horses.

This paper describes the use of subcutaneously-placed tissue chambers as a sterile soft-tissue inflammation model in Thoroughbred horses. Acute, non-immune inflammation was initiated by injecting a sterile lambda carrageenan solution into a tissue chamber. This model was used to study the temporal changes in oxygen and carbon dioxide tensions, pH, bicarbonate, protein, albumin, prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) concentrations, cell counts and differential counts in tissue fluid from inflamed tissue chambers and control chambers. Skin temperatures over control and inflamed chambers were also compared. Carrageenan-induced inflammation resulted in significant increases in tissue-fluid carbon dioxide tension, leucocyte count, albumin, and PGE2 and LTB4 concentrations. It also resulted in a significant decrease in tissue fluid pH and HCO3-concentration. Inflammation did not result in significant changes in tissue-fluid protein concentration, differential cell counts or skin temperature over the chambers. The use of this type of tissue chamber is well-suited for studying the pathophysiology of a self-contained, non-immune inflammatory process. The model described in this paper could prove to be very useful in studies of the distribution of anti-inflammatory drugs and the effects of such drugs on various aspects of the inflammatory process.

Single and multiple dose pharmacokinetics of gentamicin administered intravenously and intramuscularly in adult conditioned thoroughbred mares.

The pharmacokinetics of gentamicin following single and multiple intravenous and intramuscular doses were compared in a two phase, randomised cross-over study in horses. Gentamicin was administered to 6 healthy, conditioned Thoroughbred mares at a dosage of 3.3 mg/kg body weight every 12 hours for 5 intravenous or intramuscular consecutive treatments. Equal numbers of horses were treated by either route during each phase. There was a wash-out period of 5 days between phases. During each phase serial blood samples were collected from each mare immediately before treatment and at 16 intervals following the first and fifth administrations. Blood samples were also collected immediately before treatment and at 30 and 60 minutes following doses 2 through to 4. Gentamicin plasma concentrations were determined by fluorescence polarisation immunoassay. Plasma gentamicin concentration versus time data for both single and multiple doses by either route was best described by a 2 compartmental open model with first order rate constants. A distribution half-life (T1/2 alpha) of 0.1 +/- 0.1 hours, terminal half-life (T1/2 beta) of 1.2 +/- 0.2 hours, mean residence time (MRT) of 1.4 +/- 0.1 hours and total body clearance (ClB) of 1.4 +/- 0.2 ml/kg/min were observed following multidose gentamicin intravenous administration. The volume of distribution at steady state (Vdss) was 117.6 +/- 10.8 ml/kg. No significant differences (P > 0.05) were observed for any of the parameters between single or multiple doses for either route of administration. Except for AUC, significant (P < or = 0.05) differences were observed between multiple intravenous and intramuscular treatments for all pharmacokinetic parameters determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Non-steroidal anti-inflammatory drugs in domestic animals: II Disposition and clinical indications.

Most non-steroidal anti-inflammatory drugs (NSAID's) have similar absorption and disposition characteristics. Absorption after oral administration is good, extensive plasma protein binding results in a small volume of distribution, and excretion of metabolites occurs mainly in the urine after hepatic biotransformation of the active drug. The main clinical indications for use of NSAID's are anti-inflammatory, analgesic and antipyretic, and an increasing list of new indications are continually being found. The disposition, pharmacokinetics and clinical indications of non-steroidal anti-inflammatory drugs in domestic animals are reviewed.

Consideration of sheep as a minor species: comparison of drug metabolism and disposition with other domestic ruminants.

The processes which determine bioequivalence of a given drug among species are many, and include absorption from a site of administration, renal, biliary and intestinal elimination, sequestration (in particular, binding to proteins or other macromolecules), distribution and redistribution, biotransformation, and receptor population density and uniqueness. The present review is limited to physiologic and pharmacologic parameters which affect drug distribution, elimination, and metabolism, primarily because these are areas where sufficient data is available to make comparisons between sheep and other ruminants. The literature suggest a high degree of similarity among domestic ruminants in the distribution and elimination of drugs that are not metabolized but eliminated by passive processes such as renal glomerular filtration. Although the data are not as uniform as when conducted under rigorous control in the same laboratory setting, it is possible to predict very similar pharmacokinetic profiles for many of the antimicrobials between cattle, sheep and goats. Metabolic scaling of kinetic parameters could be predicted in comparative studies conducted under well-controlled conditions, in which case the greatest similarities would occur among the small ruminants with values being somewhat dissimilar between sheep or goats and cattle. Plasma protein binding of drugs appears to be very similar among the ruminants, and its influence on drug distribution and elimination does not appear to vary appreciably between cattle and sheep. There is, however, very little definitive data which describes binding parameters, and there is very little data of any kind on binding to caprine albumin other plasma proteins of the goat. Sheep apparently differ from cattle, however, in transcortin concentrations, and this could affect the distribution and elimination of prednisolone or any other synthetic steroid which exhibits high affinity binding to this transport protein. Among the drug substances for which comparative information is available, there is very little to suggest qualitative differences in routes of metabolism among ruminants. There appears to be a remarkable degree of similarity in both major and minor pathways of drug metabolism among these species, and in fact, no documentable differences of a qualitative nature have been found. It should be noted, however, that documentable differences would only be established in a direct comparison of species conducted in the same laboratory under carefully controlled conditions. Such conditions would necessarily include assurance of no recent exposure to enzyme inducing agents or inhibitors or to agents which could deplete endogenous substances necessary to the enzymatic process.(ABSTRACT TRUNCATED AT 400 WORDS)

Matrix solid phase dispersion (MSPD) isolation and liquid chromatographic determination of furazolidone in pork muscle tissue.

A method for the isolation and liquid chromatographic (LC) determination of furazolidone in pork muscle tissue is presented. Blank or furazolidone-fortified pork muscle tissue samples (0.5 g) were blended with octadecylsilyl (C18, 18% load, endcapped, 2 g) derivatized silica. A column made from C18/pork matrix was first washed with hexane (8 mL), followed by elution of furazolidone with ethyl acetate. The ethyl acetate extract was then passed through an activated alumina column. The eluate contained furazolidone that was free from interfering compounds when analyzed by LC with UV detection (photodiode array, 365 nm). Detector response with increasing concentrations of furazolidone isolated from fortified samples was linear (r = 0.998 +/- 0.002) with an average percentage recovery of 89.5 +/- 8.1% for the concentration range (7.8-250 ng/g) examined and resulted in a minimum detectable limit of 390 pg on column, and a detector response of more than 5 times baseline noise. The inter-assay variability was 9.9 +/- 5.4% with an intra-assay variability of 1.5%.

Pharmacokinetics and elimination of salicylic acid in rabbits.

Sodium salicylate was administered to rabbits in order to compare its disposition with that in other major and minor agricultural species. A dose of 44 mg/kg was given orally (p.o.) or intravenously (i.v.), and plasma and urine samples were collected for 36 h and 96 h, respectively. The majority of the drug was excreted as salicylic acid (SA) within 12 h. The major metabolites following an oral dose were salicyluric acid (SUA) and the glucuronide conjugates of SA and SUA. Following i.v. dosing, sulfate conjugates of both SA and SUA were also evident. Both SA and SUA were detected in plasma. Following i.v. administration, SA was distributed with a Vss of 0.249 +/- 0.082 l/kg and cleared at a rate of 0.0432 +/- 0.006 l/h/kg. The biological half-life, calculated from the terminal disposition-rate constant, was 4.3 h (i.v.) or 9.7 h (p.o.). The urinary elimination pattern of SA and metabolites in the rabbit was similar to that previously reported by our laboratories for cattle and goats, although total recovery of the administered dose was not as high as for the latter two species. However, the volume of distribution was larger than for cattle and goats, and rabbits cleared the drug more slowly than those species. As a consequence, the biological half-life was eight to ten times longer than in the ruminants studied previously.

Pharmacokinetics of gentamicin in newborn to 30-day-old foals.

Gentamicin sulfate, equivalent to 4 mg of gentamicin base/kg of body weight, was administered IV to 6 Thoroughbred foals on day 1 (12 to 24 hours of age) and at 5, 10, 15, and 30 days after birth. On day 40 after parturition, gentamicin was given to the mares at a dosage similar to that used in foals. Decay of serum gentamicin concentrations was best described by a 2-compartment model. Among foals, the overall elimination rate constant at 30 days of age was significantly (P less than 0.05) greater than at days 1, 10, and 15. There was, however, no difference in the overall elimination rate constant between foals and mares. The volume of distribution (Vd), determined on the basis of total area under the disposition curve, did not change between day 1 and day 30. Mean values of Vd of foals were between 1.5 and 2.5 times higher than the mean Vd of the mares; however, only values from the foals at days 5 and 10 were significantly greater. Both age and interindividual differences were reflected in the total body clearance (ClB) of gentamicin. Total body clearance of gentamicin of foals on day 1 was less than that of foals on days 5, 10, and 30. Additionally, C1B of gentamicin on day 15 was less than that on day 30. There was no significant difference between ClB of foals and mares except for the day-30 group, which had a higher clearance rate than did the adults. Protein binding of gentamicin was less than 30% in all groups, and there were no apparent age-related differences.

Matrix solid phase dispersion isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue.

A method for isolation and liquid chromatographic determination of oxytetracycline in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and oxytetracycline-fortified fish muscle tissue samples (0.5 g) were blended with octadecylsllyl (C18, 40 microns, 18% load, endcapped) derivatized silica packing material (2 g) containing 0.05 g each of oxalic acid and disodium ethylenediaminetetraacetate. A column made from the C18/fish tissue matrix was first washed with hexane (8 mL), following which the oxytetracycline was eluted with acetonitrile-methanol (1 + 1, v/v) containing 0.06% w/v each of butylated hydroxyanisole and butylated hydroxytoluene. The eluate contained oxytetracycline analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array set at 365 nm). Standard curves for oxytetracycline isolated from fortified samples were linear (0.998 +/- 0.002) with an average absolute percentage recovery of 80.9 +/- 6.6% for the concentration range (50, 100, 200, 400, 800, 1600, and 3200 ng/g) examined. The interassay variability was 11.3 +/- 5.2% with an intra-assay variability of 1.1%.

Matrix solid phase dispersion isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue.

A method for the isolation and liquid chromatographic determination of sulfadimethoxine in catfish (Ictalurus punctatus) muscle tissue is presented. Blank control and sulfadimethoxine-fortified fish muscle tissue samples (0.5 g) were blended with octadecyisilyl (C18, 40 micrograms, 18% load, endcapped) derivatized silica packing material. A column made from the C18/fish tissue blend was first washed with hexane (8 mL), following which the sulfadimethoxine was eluted with dichloromethane (8 mL). The eluant contained sulfadimethoxine analyte that was free from interfering compounds when analyzed by liquid chromatography with UV detection (photodiode array, 270 nm). Standard curves for sulfadimethoxine isolated from fortified samples were linear (0.999 +/- 0.001) with an average relative percentage recovery of 101.1 +/- 4.2% for the concentration range (50, 100, 200, 400, 800, and 1600 ng/g) examined using sulfamethoxazole as the internal standard. The interassay variability was 10.7 +/- 8.2% with an intra-assay variability of 2.2%.

Matrix solid phase dispersion isolation and liquid chromatographic determination of five benzimidazole anthelmintics in fortified beef liver.

A multiresidue method for isolation and liquid chromatographic determination of 5 benzimidazole anthelmintics (thiabendazole, oxfendazole, mebendazole, albendazole, and fenbendazole) in beef liver tissue is presented. Blank or benzimidazole-fortified liver samples (0.5 g) were blended with octadecylsilyl derivatized silica packing material (C18, 18% load, endcapped, 2 g). A column made from the C18/liver matrix was first washed with hexane (8 mL), following which the benzimidazoles were eluted with acetonitrile. The acetonitrile extract was then passed through an activated alumina column. The eluate contained benzimidazole analytes that were free from interfering compounds as determined by UV detection (photodiode array, 290 nm). Correlation coefficients of standard curves for individual benzimidazoles isolated from fortified samples, using internal standardization, were linear (0.996 +/- 0.002 to 0.999 +/- 0.001) with average relative percentage recoveries from 62.0 +/- 6.7 to 86.8 +/- 8.6% for the concentration range (100-3200 ng/g) examined. The interassay variability was 7.0 +/- 4.1 to 12.9 +/- 10.2% with an intra-assay variability from 2.2 to 4.0%.

Elimination of salicylic acid in goats and cattle.

Sodium salicylate was administered to cattle and goats IV and PO according to a crossover design. Total urinary excretion of SA and its metabolites was measured for 3 days after dosing. Salicyluric acid (SUA) was the only metabolite detected in urine of either species. Recovery of sodium salicylate and SUA in goats amounted to 67.9 and 34.6% of the dose, respectively, after IV administration. After oral dosing, total recoveries were 30.2% (sodium salicylate) and 71.7% (SUA) of dose. By comparison, cattle excreted significantly (P less than 0.05) less sodium salicylate (54.0%) and more SUA (49.9%) after IV dosing. The same pattern was observed after oral administration, wherein cattle excreted less than 12% as sodium salicylate and more than 99% as SUA. In both species, almost 90% of the drug excreted as sodium salicylate was found in urine within the first 12 hours after an IV dose and within 24 hours after oral dosing. The excretion of SUA was somewhat slower in both species, especially after oral administration. The data suggested that there were only quantitative differences in the metabolism and elimination of sodium salicylate between the 2 species, with cattle excreting a higher proportion of the drug as the glycine conjugate SUA.