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Abstract

Objective—To evaluate whether urine supernatant contains amplifiable DNA and to determine factors that influence genotyping of samples from racehorses after storage and transportation.

Sample Population—580 urine, 279 whole blood, and 40 plasma samples obtained from 261 Thoroughbreds and Standardbreds.

Procedures—Genomic DNA was isolated from stored blood and urine samples collected from racehorses after competition. Quantified DNA was evaluated to determine whether 5 equine microsatellite loci (VHL20, HTG4, AHT4, HMS6, and HMS7) could be amplified by use of PCR techniques. Fragment size of each amplified locus was determined by use of capillary electrophoresis.

Results—High–molecular-weight and amplifiable DNA were recovered from refrigerated blood samples, but recovery from urine varied. Deoxyribonucleic acid was recovered from both urine supernatant and sediment. Freeze-thaw cycles of urine caused accumulation of amplifiable DNA in the supernatant and clearance of naked DNA. Repeated freeze-thaw cycles significantly decreased DNA yield and induced DNA degradation, which resulted in failure to detect microsatellite loci. Select drugs detected in test samples did not affect PCR amplification. Contaminants in DNA isolates inhibited PCR amplification and resulted in partial microsatellite profiles.

Conclusions and Clinical Relevance—Properly stored urine and blood samples were successfully genotyped, but subjecting urine to freeze-thaw cycles was most detrimental to the integrity of DNA. Increasing the volume of urine used improved recovery of DNA.

Full access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To determine the effects of prolonged administration of the oral NSAIDs phenylbutazone and firocoxib on concentrations of cytokines and growth factors in platelet-rich plasma (PRP) and autologous protein solution (APS).

ANIMALS

6 adult University owned horses.

METHODS

Horses were randomized to receive phenylbutazone (1 g, orally, q 12 h) or firocoxib (57 mg, orally, q 24 h) for 6 days. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before the administration of NSAIDs and at 7 days (1 day following cessation of NSAIDs). Horses underwent a two-week washout period, during which blood was obtained at 14 days and 21 days. The protocol was repeated with a crossover design. PRP and APS were analyzed for concentrations of platelets, leukocytes, and several cytokines (IL-1β, IL-10, IL-6, IL-8, and tumor necrosis factor-α) and growth factors (PDGF, FGF-2, and TGF-β1) using immunoassays. Plasma was evaluated for drug concentrations.

RESULTS

No significant differences existed in concentrations of growth factors and cytokines before or after prolonged administration of NSAIDs. There were significant differences in concentrations of leukocytes and platelets in PRP compared to APS, with higher concentrations of leukocytes at the day 7 time point (T) in APS (phenylbutazone) and in concentrations of platelets in APS at T0 (firocoxib) and in APS at T7 (phenylbutazone).

CLINICAL RELEVANCE

Veterinarians can recommend the administration of these oral NSAIDs prior to obtaining blood for PRP and APS provided a single-day washout period is instituted.

Open access
in American Journal of Veterinary Research

Abstract

OBJECTIVE

To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP).

ANIMALS

6 adult university-owned horses.

METHODS

For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-β1, IL-1β, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays.

RESULTS

There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP.

CLINICAL RELEVANCE

These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Open access