Tracking main environmental factors masking a minor steroidal doping effect using metabolomic analysis of horse urine by liquid chromatography-high-resolution mass spectrometry.

There is an urgent need to implement holistic and untargeted doping control protocols with improved discriminatory power, compared to conventional methods that only target doping agents. Metabolomics, which aims to characterize all metabolites present in biological matrices, could fulfill this need. In this context, the aim of this study was to evaluate the impact of environmental factors on the ability to obtain a metabolic signature of stanozolol administration in horse doping situation. Urine samples from 16 horses breeded in two different places were collected over a one-year period, before, during and seven months after the administration of stanozolol, a horse doping agent. Metabolomic analysis was performed using ultra-high pressure reverse phase liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (MS). Results showed a major impact of the nutritional regimen, drug administration (for de-worming purpose) and breeding place on the metabolite profiles of horse urines, which hampered the detection of metabolic perturbations induced by stanozolol administration. After having used MS/MS experiments to characterize some MS features related to these environmental factors, we showed that highlighting and then removing the features impacted by these confounding factors before performing supervised multivariate statistical analyses could address this issue. In conclusion, adequate consideration should be given to environmental and physiological factors; otherwise, they can emerge as confounding factors and conceal doping administration.

PHARMACOKINETICS OF A SUSTAINED-RELEASE FORMULATION OF MELOXICAM AFTER SUBCUTANEOUS ADMINISTRATION TO AMERICAN FLAMINGOS ( PHOENICOPTERUS RUBER).

Meloxicam is commonly used in avian medicine to relieve pain and inflammation, but the recommended dosing frequency can be multiple times per day, which can contribute to stress during convalescence. In this study, the pharmacokinetics of a sustained-release formulation of meloxicam were determined after subcutaneous administration of a single 3-mg/kg dose to eight healthy adult American flamingos ( Phoenicopterus ruber). Blood samples were collected before (time 0) and at 0.5, 1, 4, 8, 12, 24, 48, 96, and 120 hr after drug administration. Analysis of meloxicam in plasma samples was conducted with the use of reversed-phase high-performance liquid chromatography, and pharmacokinetic parameters were calculated by noncompartmental analysis. Plasma concentrations reached a mean maximum (±standard deviation) of 7.65 (±2.39) µg/ml at 0.56 (±0.18) hr with a terminal half-life of 1.76 (±1.41) hr. Based on these findings, this sustained-release formulation of meloxicam does not extend the interval between treatments as compared to the regular formulation, so it is not recommended in American flamingos at this time.

Associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits in dairy cows.

The aim of this study was to investigate associations between pathogen-specific cases of subclinical mastitis and milk yield, quality, protein composition, and cheese-making traits. Forty-one multibreed herds were selected for the study, and composite milk samples were collected from 1,508 cows belonging to 3 specialized dairy breeds (Holstein Friesian, Brown Swiss, and Jersey) and 3 dual-purpose breeds of Alpine origin (Simmental, Rendena, and Grey Alpine). Milk composition [i.e., fat, protein, casein, lactose, pH, urea, and somatic cell count (SCC)] was analyzed, and separation of protein fractions was performed by reversed-phase high performance liquid chromatography. Eleven coagulation traits were measured: 5 traditional milk coagulation properties [time from rennet addition to milk gelation (RCT, min), curd-firming rate as the time to a curd firmness (CF) of 20 mm (k20, min), and CF at 30, 45, and 60 min from rennet addition (a30, a45, and a60, mm)], and 6 new curd firming and syneresis traits [potential asymptotical CF at an infinite time (CFP, mm), curd-firming instant rate constant (kCF, % × min-1), curd syneresis instant rate constant (kSR, % × min-1), modeled RCT (RCTeq, min), maximum CF value (CFmax, mm), and time at CFmax (tmax, min)]. We also measured 3 cheese yield traits, expressing the weights of total fresh curd (%CYCURD), dry matter (%CYSOLIDS), and water (%CYWATER) in the curd as percentages of the weight of the processed milk, and 4 nutrient recovery traits (RECPROTEIN, RECFAT, RECSOLIDS, and RECENERGY), representing the percentage ratio between each nutrient in the curd and milk. Milk samples with SCC > 100,000 cells/mL were subjected to bacteriological examination. All samples were divided into 7 clusters of udder health (UH) status: healthy (cows with milk SCC < 100,000 cells/mL and uncultured); culture-negative samples with low, medium, or high SCC; and culture-positive samples divided into contagious, environmental, and opportunistic intramammary infection (IMI). Data were analyzed using a linear mixed model. Significant variations in the casein to protein ratio and lactose content were observed in all culture-positive samples and in culture-negative samples with medium to high SCC compared to normal milk. No differences were observed among contagious, environmental, and opportunistic pathogens, suggesting an effect of inflammation rather than infection. The greatest impairment in milk quantity and composition, clotting ability, and cheese production was observed in the 2 UH status groups with the highest milk SCC (i.e., contagious IMI and culture-negative samples with high SCC), revealing a discrepancy between the bacteriological results and inflammatory status, and thus confirming the importance of SCC as an indicator of udder health and milk quality.

Physical-chemical and biological characterization of different preparations of equine chorionic gonadotropin.

Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical-chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (tR= 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone.

Isolation and immunomodulatory activity of bursal peptide, a novel bursal peptide from the chicken bursa of Fabricius.

The bursa of Fabricius (BF), which is unique to birds, serves as the central humoral immune organ and plays a significant role in B lymphocyte differentiation. In this study, a new bursal peptide (BP-IV) was isolated from BF, which promoted colony-forming unit pre-B formation and regulated B cell differentiation. BP-IV also exerted immunomodulatory effects on antigen-specific immune responses via both humoral and cellular immunity in chicken and mice that had been immunized with inactivated avian influenza virus (AIV; H9N2 subtype), including enhancing AIV-specific antibody and cytokine production. The results of this study provided novel insights into the use of a potential candidate reagent for B cell development and future immuno-pharmacological use.

Pharmacokinetics of meloxicam in koalas (Phascolarctos cinereus) after intravenous, subcutaneous and oral administration.

The pharmacokinetic profile of meloxicam in clinically healthy koalas (n = 15) was investigated. Single doses of meloxicam were administered intravenously (i.v.) (0.4 mg/kg; n = 5), subcutaneously (s.c.) (0.2 mg/kg; n = 1) or orally (0.2 mg/kg; n = 3), and multiple doses were administered to two groups of koalas via the oral or s.c. routes (n = 3 for both routes) with a loading dose of 0.2 mg/kg for day 1 followed by 0.1 mg/kg s.i.d for a further 3 days. Plasma meloxicam concentrations were quantified by high-performance liquid chromatography. Following i.v. administration, meloxicam exhibited a rapid clearance (CL) of 0.44 ± 0.20 (SD) L/h/kg, a volume of distribution at terminal phase (Vz ) of 0.72 ± 0.22 L/kg and a volume of distribution at steady state (Vss ) of 0.22 ± 0.12 L/kg. Median plasma terminal half-life (t(1/2)) was 1.19 h (range 0.71-1.62 h). Following oral administration either from single or repeated doses, only maximum peak plasma concentration (C(max) 0.013 ± 0.001 and 0.014 ± 0.001 µg/mL, respectively) was measurable [limit of quantitation (LOQ) >0.01 µg/mL] between 4-8 h. Oral bioavailability was negligible in koalas. Plasma protein binding of meloxicam was ~98%. Three meloxicam metabolites were detected in plasma with one identified as the 5-hydroxy methyl derivative. This study demonstrated that koalas exhibited rapid CL and extremely poor oral bioavailability compared with other eutherian species. Accordingly, the currently recommended dose regimen of meloxicam for this species appears inadequate.

Short communication: CSN1S1-CSN3 (α(S1)-κ-casein) composite genotypes affect detailed milk protein composition of Mediterranean water buffalo.

The aim of the study was to investigate the effect of composite CSN1S1-CSN3 [α(S1)-κ-casein (CN)] genotype on milk protein composition in Mediterranean water buffalo. Content of α(S1)-CN, α(S2)-CN, ß-CN, γ-CN, κ-CN, glycosylated and unglycosylated κ-CN, α-lactalbumin, and ß-lactoglobulin was measured by reversed-phase HPLC using 621 individual milk samples. Genotypes at CSN1S1 and CSN3 were also obtained by reversed-phase HPLC. Two alleles were detected at CSN1S1 (corresponding to the A and B variants, O62823: p.Leu193Ser,) and at CSN3 (corresponding to the X1 and X2 variants, CAP12622.1: p.Ile156Thr). Increased proportions of α(S1)-CN in total casein (TCN) were associated with genotypes carrying CSN1S1 A. Genotypes associated with a marked decrease of the proportion of α(S1)-CN in TCN (composite genotypes AB-X1X1 and BB-X1X2) were associated with marked increases in the proportion of α(S2)-CN. In addition, composite genotypes carrying the X1 allele at CSN3 were associated with a greater proportion of α(S2)-CN in TCN relative to those carrying CSN3 X2. Composite genotypes greatly affected also the variability of ratios of κ-CN to TCN, with genotypes carrying the X1 allele at CSN3 being associated with decreased ratios. The decreased content of glycosylated κ-CN associated with CSN3 X1 was responsible for the overall lower content of total κ-CN in milk of X1-carrying animals. Increasing the frequency of specific genotypes might be an effective way to alter milk protein composition, namely the proportion of α(S1)-CN, α(S2)-CN, and κ-CN in TCN, and the degree of glycosylation of κ-CN.

Association between milk protein gene variants and protein composition traits in dairy cattle.

The objective of this study was to identify DNA markers in the 4 casein genes (CSN1S1, CSN1S2, CSN2, and CSN3) and the 2 major whey protein genes (LALBA and LGB) that show associations with milk protein profile measured by reverse-phase HPLC. Fifty-three single nucleotide polymorphisms (SNP) were genotyped for cows in a unique resource population consisting of purebred Holstein and (Holstein × Jersey) × Holstein crossbred animals. Seven traits were analyzed, including concentrations of α(S)-casein (CN), ß-CN, κ-CN, α-lactalbumin, ß-lactoglobulin, and 2 additional secondary traits, the total concentration of the above 5 milk proteins and the α(S)-CN to ß-CN ratio. A substantial fraction of phenotypic variation could be explained by the additive genetic component for the 7 milk protein composition traits studied. Moreover, several SNP were significantly associated with all examined traits at an experiment-wise error rate of 0.05, except for α-lactalbumin. Importantly, the significant SNP explained a large proportion of the phenotypic variation of milk protein composition. Our findings could be used for selecting animals that produce milk with desired composition or desired processing and manufacturing properties.

Effect of graded levels of niacin supplementation of a semipurified diet on energy and nitrogen balance, growth performance, diarrhea occurrence, and niacin metabolite excretion by growing swine.

Thirty-six crossbred barrows with an average initial age of 42 d and BW of 13.8 kg were placed in individual metabolism crates in a 35-d experiment to evaluate the supplementation of a semipurified diet with graded levels of crystalline niacin. Response criteria were energy and N balance, growth performance, occurrence of niacin deficiency diarrhea, and urinary excretion of the niacin metabolite N(1)-methyl-2-pyridone-5-carboxylamide (PYR). The basal diet met the true ileal Trp requirement of growing swine, and supplementation with 6, 10, 14, 18, 22, or 44 mg of niacin/kg made 6 treatments. Pigs were observed for scours twice daily, and pig BW and feed consumption were determined weekly. Total urine collections and fecal grab samples were made twice daily from each pig from d 28 to 35. Pigs fed the diet containing 14 mg of niacin/kg absorbed and retained more (P < 0.05) grams of N/d, had a greater N digestibility (%, P < 0.05), a greater ADFI and ADG (P < 0.10), and no diarrhea (P < 0.05) compared with pigs fed the diet containing 6 mg of niacin/kg, and pigs fed the diet containing 10 mg of niacin/kg were intermediate in ADG. There were no additional improvements in the response criteria with niacin supplementation greater than 14 mg/kg. Urinary PYR criteria (mg/L and mg/d) were greater (P < 0.001) for pigs fed the diet containing 44 mg of niacin/kg than for pigs fed the diets containing 6 to 22 mg of niacin/kg. However, urinary PYR criteria for pigs fed the diets containing 6 to 22 mg of niacin/kg did not differ from each other, indicating that PYR was not a sensitive indicator of niacin status for growing swine. Niacin treatment did not affect the percentages of N retained/N absorbed, N retained/N intake, DE, or ME. In conclusion, 14 mg of crystalline niacin/kg of semipurified diet adequate in Trp was the minimum concentration of niacin that maximized N utilization and growth performance, and prevented niacin deficiency diarrhea of growing swine in the current experiment. Because practical feed ingredients may be sources of available endogenous niacin, supplementation of practical diets with 100% of the current NRC requirement for niacin should provide adequate niacin for growing swine.

Enrofloxacin-based therapeutic strategy for the prevention of endometritis in susceptible mares.

Enrofloxacin (EFX) is often used empirically to prevent uterine infections in mares in order to improve efficiency on Commercial Embryo Transfer Farms. This study investigated the uterine distribution of EFX and its metabolite ciprofloxacin (CFX) in mares and assessed the minimal inhibitory concentrations (MIC) of EFX against various common pathogens as a basis for establishing a rational dosing schedule. Plasma and uterine pharmacokinetic (PK) studies were performed in two groups (n = 5) of healthy mares following intravenous (i.v.) administration of EFX at either 2.5 and at 5 mg/kg bodyweight. Plasma and endometrial tissue samples, taken before for up to 48 h after treatment were analysed by Reverse Phase HPLC. MIC values for wild strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (beta-haemolytic streptococci) ranged from 0.25-2 and 1.5-3.0 microg/mL respectively. In terms of tissue distribution, the sum of the endometrial concentrations of the parent drug (EFX) and its active metabolite (CFX) (in terms of AUC), exceeded those in plasma by 249% and 941% following administration of EFX at 2.5 and 5 mg/kg respectively. After i.v. treatment with EFX at 5 mg/kg, endometrial concentrations of EFX and CFX above the MIC value were detected for 36-48 and 22-43 h posttreatment for Gram-negative and -positive isolates respectively. Concentrations above MIC were maintained for much shorter periods at the lower (2.5 mg/kg) treatment dose. Based on these results, a conventional dose (5 mg/kg) of EFX given prebreeding followed by two further doses at 36-48 h postbreeding are proposed as a rational strategy for using of EFX as a preventative therapy against a variety of common bacterial strains associated with equine endometritis.

Characterization of cerebrospinal fluid monoamine metabolites in peripubertal chimpanzees (Pan troglodytes).

BACKGROUND: Assessment of cerebrospinal (CSF) monoamine metabolites 5-hydroxyindoeacetic acid (5-HIAA) and homovanillic acid (HVA), and the serotonin precursor tryptophan (TRP), in chimpanzees may help in understanding the neurobiology underlying aggressive, impulsive behavior in humans and non-human primates. METHODS: Two CSF samples were obtained from 11 peripubertal chimpanzees 8 months apart and were assayed for monoamine metabolite and TRP concentrations. RESULTS: Substantial inter-individual stability was observed for 5-HIAA (n = 11; r = 0.83, P < 0.001) and HVA (r = 0.91, P < 0.001). Females had significantly higher concentrations of 5-HIAA compared to males (F(1,8) = 7.31; P < 0.05). Levels of 5-HIAA (r = -0.62, P < 0.05), HVA (r = -0.86, P < 0.001) and TRP levels (r = -0.67; P < 0.05) decreased with age. CONCLUSION: Close parallels were observed between chimpanzees and humans with respect to absolute levels, sex effects, ontogeny, and 5-HIAA-HVA correlations, supporting the potential utility of the measures in understanding relationships between monoamine functioning and behavior in chimpanzees and humans.

Polymeric nanoparticles as an oral delivery system for biocontrol agents for the brushtail possum (Trichosurus vulpecula).

AIM: To investigate polymeric nanoparticles as an oral delivery system for protein biocontrol agents for control of the brushtail possum, Trichosurus vulpecula. METHODS: Insulin-loaded poly(ethyl 2-cyanoacrylate) (PECA) nanoparticles were prepared using interfacial polymerisation, and characterised for size, zeta potential, and efficiency of encapsulation of insulin. In-vitro release of insulin-loaded PECA nanoparticles was quantified using reverse-phase high-pressure liquid chromatography (HPLC). The in-vivo pharmacokinetics of insulin in PECA nanoparticles was investigated following I/V administration, and when injected directly into the caecum alone or in conjunction with the permeation enhancer EDTA. Blood samples were collected at intervals from -5 to 180 minutes, and the concentration of insulin in plasma was quantified using a radioimmunoassay (RIA) validated for possum plasma. RESULTS: Poly(ethyl 2-cyanoacrylate) nanoparticles were produced with a uniform particle size of 200-300 nm, and the mean entrapment of insulin was 78%. In-vitro release of insulin from the PECA nanoparticles was controlled, although incomplete, and approximately 30% of the insulin remained entrapped. The bioavailability of insulin when administered in a PECA nanoparticulate formulation injected directly into the caecum was <1%, and was not increased by addition of the permeation enhancer. CONCLUSIONS: The nanoparticulate formulations investigated as part of this study resulted in low bioavailability of the peptide insulin in the brushtail possum.