Clenbuterol in the horse: urinary concentrations determined by ELISA and GC/MS after clinical doses

Clenbuterol is a beta2 agonist/antagonist bronchodilator marketed as Ventipulmin and is the only member of this group of drugs approved by the US Food and Drug Administration (FDA) for use in horses. Clenbuterol is a class 3 drug in the Association of Racing Commissioners International (ARCI) classification system; therefore, its identification in postrace samples may lead to sanctions. Recently, the sensitivity of postrace testing for clenbuterol has been substantially increased. The objective of this study was to determine the 'detection times' for clenbuterol after administration of an oral clinical dose (0.8 g/kg, b.i.d.) of Ventipulmin syrup. Five horses received oral clenbuterol (0.8 g/kg, b.i.d.) for 10 days, and urine concentrations of clenbuterol were determined by an enhanced enzyme-linked immunoabsorbent assay (ELISA) test and gas chromatography/mass spectrometric (GC/MS) analysis by two different methods for 30 days after administration. Twenty-four hours after the last administration, urine concentrations of apparent clenbuterol, as measured by ELISA, averaged about 500 ng/mL, dropping to about 1 ng/mL by day 5 posttreatment. However, there was a later transient increase in the mean concentrations of apparent clenbuterol in urine, peaking at 7 ng/mL on day 10 postadministration. The urine samples were also analysed using mass spectral quantification of both the trimethylsilyl (TMS) and methane boronic acid (MBA) derivatives of clenbuterol. Analysis using the TMS method showed that, at 24 h after the last administration, the mean concentration of recovered clenbuterol was about 22 ng/mL. Thereafter, clenbuterol concentrations fell below the limit of detection of the TMS-method by day 5 after administration but became transiently detectable again at day 10, with a mean concentration of about 1 ng/mL. Derivatization with MBA offers significant advantages over TMS for the mass spectral detection of clenbuterol, primarily because MBA derivatization yields a high molecular weight base peak of 243 m/z, which is ideal for quantitative purposes. Therefore, mass spectral analyses of selected urine samples, including the transient peak on day 10, were repeated using MBA derivatization, and comparable results were obtained. The results show that clenbuterol was undetectable in horse urine by day 5 after administration. However, an unexpected secondary peak of clenbuterol was observed at day 10 after administration that averaged approximately 1 ng/mL. Because of this secondary peak, the detection time for clenbuterol (0.8 g/kg, b.i.d. x 10 days) is at least 11 days if the threshold for detection is set at 1 ng/mL.     

The importation of the black rhinoceros (Diceros bicornis) from Zimbabwe into Australia

SUMMARY The paper describes a program to import and breed black rhinoceros ex situ at Western Plains Zoo in Australia. Nine rhinoceros (7 females and 2 males) captured in 1992 in Chete National Park, Zimbabwe, were transported to Australia via Cocos Island. The veterinary treatment of the animals before and during quarantine in Zimbabwe and on Cocos (Keeling) Islands is described. Three animals died; an adult male on Cocos Islands and a juvenile male and an adult female at Western Plains Zoo, Dubbo, New South Wales. The juvenile male died as a result of trauma sustained shortly after arrival and the two adults after developing a severe hepatopathy. The group of 6 females and an additional 4 males imported from the USA in 1994 have adapted well to captivity and to the climate and environment of central west New South Wales.     

Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine.

Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-administration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which isoxsuprine is recovered has never been established, although it has long been assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Using ESI/MS/MS positive mode as an analytical tool, urine samples collected 4-8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions. Titration of this material with increasing concentrations of sodium acetate yielded m/z peaks consistent with the presence of monosodium and disodium isoxsuprine-glucuronide complexes, suggesting that the starting material was a dipotassium-isoxsuprine-glucuronide complex. Electrospray ionization mass spectrometry negative mode disclosed the presence of a m/z 476 peak that declined following enzymatic hydrolysis and resulted in the concomitant appearance of peaks at m/z 300 and 175. The resulting peaks were consistent with the presence of isoxsuprine (m/z 300) and a glucuronic acid residue (m/z 175). Examination of the daughter ion spectrum of this putative isoxsuprine-glucuronide m/z 476 peak showed overlap of many peaks with those of similar spectra of authentic morphine-3- and morphine-6-glucuronides, suggesting they were derived from glucuronic acid conjugation. These data suggest that isoxsuprine occurs in post-administration urine samples as an isoxsuprine-glucuronide conjugate and also, under some circumstances, as an isoxsuprine-glucuronide-dipotassium complex.     

Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine

Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight horses yielded a peak urine concentration of apparent bupivacaine of 23.3 ng/mL 2 h after injection as determined with enzyme-linked immunosorbent assay (ELISA) screening. The major metabolite recovered from beta-glucuronidase-treated equine urine after dosing with bupivacaine is a hydroxybupivacaine, either 3-hydroxybupivacaine, 4-hydroxybupivacaine, or a mixture of the two. To determine which positional isomer occurs in the horse, 4-hydroxybupivacaine was obtained from Maxxam Analytics, Inc., and 3-hydroxybupivacaine was synthesized, purified, and characterized. Furthermore, a quantitative mass spectrometric method was developed for the metabolite as recovered from horse urine. Following subcutaneous injection of the HNED of bupivacaine, the concentration of the hydroxybupivacaine recovered from horse urine reached a peak of 27.4 ng/mL at 4 h after administration as measured by gas chromatography/mass spectrometry (GC/MS). It was also unequivocally demonstrated with ion chromatography that the hydroxybupivacaine metabolite found in horse urine is exclusively 3-hydroxybupivacaine and not 4-hydroxybupivacaine. The mean pH of the 4-h urine samples was 7.21; the mean urine creatinine was 209.5 mg/dL; and the mean urine specific gravity was 1.028. There was no apparent effect of pH, urine creatinine concentration, or specific gravity on the concentration of 3-hydroxybupivacaine recovered. The concentration of bupivacaine or its metabolites after administration of a HNED dose are detectable by mass spectrometric techniques. This study also suggests that recovery of concentrations less than approximately 30 ng/mL of 3-hydroxybupivacaine from postrace urine samples is unlikely to be associated with a recent local anaesthetic effect of bupivacaine.     

Mepivacaine: its pharmacological effects and their relationship to analytical findings in the horse

Mepivacaine is a local anaesthetic drug that is widely used in equine medicine and is classified by the Association of Racing Commissioners International (ARCI) as a Class 2 foreign substance that may cause regulators to impose significant penalties if residues are identified in post-race urine samples. Therefore, an analytical/pharmacological database was developed for this agent and its metabolites. Using an abaxial sesamoid local anaesthetic model, it was determined that the highest no-effect dose (HNED) for its local anaesthetic effect was 2 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, it was determined that subcutaneous (s.c.) administration of the HNED of mepivacaine to eight horses yielded a peak urinary concentration of apparent mepivacaine of 63 ng/mL 2 h after injection. The major identified metabolite recovered from equine urine after dosing with mepivacaine is 3-hydroxymepivacaine. Therefore, 3-hydroxymepivacaine was synthesized, purified and characterized, and a quantitative mass spectrometric method was developed for this metabolite as isolated from horse urine. Following subcutaneous injection of the HNED of mepivacaine, the concentration of 3-hydroxymepivacaine recovered from horse urine reached a peak of about 64.6 ng/mL at 4 h after administration as measured by GC/MS. The concentration of mepivacaine or its metabolites after administration of a HNED dose are detectable by mass spectral techniques. Within the limits of this research, the study suggests that recovery of concentrations less than about 65 ng/mL of 3-hydroxymepivacaine from post-race urine samples may not be associated with a recent LA effect of mepivacaine.     

Reproductive Parameters of Mangalarga Marchador Mares in a Commercial Embryo Transfer Programme

The objective of this study is to evaluate the reproductive efficiency in donors and recipient Mangalarga Marchador mares in commercial programmes of embryo transfer (ET) and the effects of some reproductive characteristics and ET methodology on conception rates in the recipient mares. A total of 1140 flushing procedures were performed and 830 embryos (72.8%) were recovered. There were no differences between the rates of embryonic recovery in the different breeding seasons (p > 0.05) and 92.8% of the recovered embryos were 8–9 days old. There was no difference in the embryonic recovery regarding the collection order from the first to the ninth embryo collection along the breeding season, as well as among mares inseminated during the foal heat or subsequent cycles (p > 0.05). Pregnancy rates observed in the total period of all reproductive seasons at 15, 30, 45 and 60 days of pregnancy were 73.4, 69.9, 66.7 and 64.5%, respectively. Differences in pregnancy rate and early embryonic loss rates were not observed between embryos transferred immediately after collection (66.8% and 13.5%) and embryos transported at room temperature for periods of <1 h (62.9% and 14.4%; p > 0.05). Pregnancy rates were higher when the interval between ovulations of donor and recipient mares remained between ?3 and ?2 days (p < 0.05), and the lowest rates were observed for intervals of ?6 days (p < 0.05) with intermediary values for intervals of ?1, 0 and +1 (p > 0.05). Embryonic loss rates, however, did not differ between intervals of ovulation’s synchronism between donor and recipient mares (p > 0.05). This flexibilization in the ovulatory synchronism between donor and recipient mares optimizes the use of recipient mares, thus reducing costs and facilitating management of horse breeding farms.     

Comparison of bone healing,as assessed by computed tomography,following tibial tuberosity advancement in dogs with and without autogenous cancellous bone grafts

Abstract

AIMS: To objectively compare measures of bone healing, using computed tomography (CT) in dogs following bilateral tibial tuberosity advancement (TTA), between tibiae treated with and without autogenous cancellous bone grafts.

METHODS: Ten dogs with bilateral cranial cruciate ligament disease requiring surgical stabilisation were prospectively recruited to undergo single-session bilateral TTA, with only one, randomly assigned, tibia receiving bone graft in the osteotomy deficit. Bone healing at the osteotomy site was assessed using CT performed 38–70 days post-operatively. CT images were evaluated using both objective measurements of osseous bridging and subjective evaluation by six radiologists. Repeated measures ANOVA was used to compare the objective outcomes between the grafted and non-grafted tibiae.

RESULTS: The mean percentage of the osteotomy deficit bridged at the lateral cortex was greater in grafted (77.6, SD 35.2%) compared to non-grafted (63.0, SD 36.5%) tibiae (p=0.001), but did not differ at the medial cortex (p=0.1). The mean minimum callus width was greater in grafted (7.2, SD 3.3 mm) compared to non-grafted (3.6, SD 2.9 mm) tibiae (p<0.001). There was no difference in mean attenuation (measured in Hounsfield units) of the callus between grafted and non-grafted tibiae (p=0.5). The grafted tibia was deemed to have superior bone healing in 50/60 subjective assessments made by radiologists.

CONCLUSIONS: Superior osseous bridging was detected by CT analysis following TTA using autogenous cancellous bone grafts compared with no graft. This was shown by greater bridging percentage at the lateral cortex and formation of a broader callus. Qualitative assessments made by six radiologists also supported the conclusion that bone healing was improved by use of autogenous cancellous bone graft. CT was a useful method for assessing evidence of bone healing following TTA.

CLINICAL RELEVANCE: These findings justify the application of autogenous cancellous bone graft to augment healing following TTA in dogs.     

Epidemiology of Bovine Venereal Campylobacteriosis: Geographic Distribution and Recent Advances in Molecular Diagnostic Techniques

Bovine venereal campylobacteriosis (BVC) is a major cause of economic loss to the cattle industries in different parts of the world. Camplylobacter fetus subsp. venerealis (Cfv), the main causative agent of BVC, is highly adapted to the genital tract of cattle and is transmitted by carrier bulls. However, infertility and abortions can also be caused by the intestinal pathogens C. fetus subsp. fetus (Cff), and C. jenuni, which are not venereally transmitted. Bovine venereal campylobacteriosis, caused by Cfv associated with lowered fertility, embryo mortality and abortion, repeated returns to service, reduced pregnancy rates and extended calving intervals, has the highest prevalence in developing countries where natural breeding in cattle is widely practised. The epidemiology, pathogenesis and diagnosis of the disease have been the subject of previous reviews. The main focus of this review is to highlight the epidemiology of this disease with particular reference to geographical distribution and recent advances in molecular diagnostic techniques. It is hoped that further research interest of scientists will be stimulated with a view to finding lasting solutions to the reproductive problems associated with the disease for better livestock productivity, particularly in developing endemic countries.     

Growth, Survival, and Body Composition of Cage-Cultured Nile Tilapia Oreochromis niloticus Fed Pelleted and Unpelleted Distillers Grains with Solubles in Polyculture with Freshwater Prawn Macrobrachium rosenbergii

Abstract.— A 12‐wk feeding trial was conducted in cages with juvenile Nile tilapia Oreochromis niloticus to evaluate distillers grains with solubles (DDGS) as a direct feed, the effects of pelleting on its utilization, and the compatibility of caged tilapia and prawns in polyculture. Nine 1.0‐m³ cages were stocked with 200 juvenile (26 ± 0.9 g) tilapia. Cages were suspended in a 0.2‐ha pond stocked with juvenile freshwater prawns Macrobrachium rosenbergii at 40,000/ha. Three replicate cages were randomly assigned to each dietary treatment. In one dietary treatment DDGS was fed as an unpelleted loose grain ration (26% protein). In a second dietary treatment fish were fed DDGS that had been steam‐pelleted (23% protein). Fish in a third dietary treatment were fed a commercial catfish diet (31% protein) for comparison. After 12 wk, individual weight, individual length, and specific growth rate were significantly higher (P < 0.05) and feed conversion ratio was significantly lower (P < 0.05) for fish fed the commercial catfish diet than for fish fed either unpelleted or pelleted DDGS. Specific growth rate was significantly higher (P < 0.05) for fish fed pelleted DDGS than for fish fed unpelleted DDGS. Survival did not differ significantly (P > 0.05) among treatments (>95%). Although growth was increased in fish fed the commercial diet, their cost of production (<0.66/kg gain) was significantly higher (P < 0.05) than in fish fed unpelleted and pelleted DDGS (<0.26/ kg gain and <0.37/kg gain, respectively). The costs of gain in fish fed unpelleted DDGS was significantly lower (P < 0.05) than in fish fed the pelleted DDGS. Prawn production was 1,449 kg/ha and addition of tilapia in polyculture increased total pond productivity approximately 81 %. These data suggest that DDGS provides economical growth in tilapia when fed as a direct feed and that polyculture of tilapia may improve overall pond efficiency in freshwater prawn production ponds, even at temperate latitudes.