Analysis of the effect of the bovine adenosine triphosphate-binding cassette transporter G2 single nucleotide polymorphism Y581S on transcellular transport of veterinary drugs using new cell culture models

In commercial dairy production, the risk of drug residues and environmental pollutants in milk from ruminants has become an outstanding problem. One of the main determinants of active drug secretion into milk is the ATP-binding cassette transporter G2/breast cancer resistance protein (ABCG2/BCRP). It is located in several organs associated with drug absorption, metabolism, and excretion, and its expression is highly induced during lactation in the mammary gland of ruminants, mice, and humans. As a consequence, potential contamination of milk could expose suckling infants to xenotoxins. In cows, a SNP for this protein affecting quality and quantity of milk production has been described previously (Y581S). In this study, our main purpose was to determine whether this polymorphism has an effect on transcellular transport of veterinary drugs because this could alter substrate pharmacokinetics and milk residues. We stably expressed the wild-type bovine ABCG2 and the Y581S variant in Madin-Darby canine kidney epithelial cells (MDCKII) and MEF3.8 cell lines generating cell models in which the functionality of the bovine transporter could be addressed. Functional studies confirmed the greater functional activity in mitoxantrone accumulation assays for the Y581S variant with a greater relative V(MAX) value (P = 0.040) and showed for the first time that the Y581S variant presents greater transcellular transport of the model ABCG2 substrate nitrofurantoin (P = 0.024) and of 3 veterinary antibiotics, the fluoroquinolone agents enrofloxacin (P = 0.035), danofloxacin (P = 0.001), and difloxacin (P = 0.008), identified as new substrates of the bovine ABCG2. In addition, the inhibitory effect of the macrocyclic lactone ivermectin on the activity of wild-type bovine ABCG2 and the Y581S variant was also confirmed, showing a greater inhibitory potency on the wild-type protein at all the concentrations tested (5 μM, P = 0.017; 10 μM, P = 0.001; 25 μM, P = 0.008; and 50 μM, P = 0.003). Differential transport activity depending on the genotype together with the differential inhibition pattern might have clinical consequences, including changes in substrate pharmacokinetics (and subsequently pharmacodynamics) and more specifically, changes in secretion of ABCG2 substrates into milk, potentially implying important consequences to veterinary therapeutics.     

Histopathologic changes of the ear in canine models of mucopolysaccharidosis types I and VII

Mucopolysaccharidosis (MPS) types I and VII are inborn errors of metabolism caused by mutation of enzymes involved in glycosaminoglycan catabolism, which leads to intralysosomal accumulation of glycosaminoglycans. In children, severe forms of MPS I and VII are characterized by somatic and neurologic manifestations, including a poorly understood hearing loss. The purpose of this study is to describe the age-related histopathologic changes of the ear in spontaneous canine models of MPS I and VII. Pathologic changes in the ear were assessed in MPS I and VII dogs ranging from 1.6 to 9.3 months of age. Paraffin-embedded sections of the whole ear and Epon-embedded semithin sections of the cochlea were examined. The following lesions were blindly scored in the middle and inner ear: inflammation, cells vacuolization, thickening of osseous and membranous structures, perivascular vacuolated macrophages infiltration, and bone resorption. All dogs had lysosomal storage within cells of tympanic membrane, ossicles, tympanic bone and mucosa, cochlear bone, spiral ligament, limbus, and stria vascularis. The MPS I dogs mainly had progressive cochlear lesions. The MPS VII dogs had severe and early middle ear lesions, including chronic otitis media and bone resorption. The MPS I dog only partially recapitulates the pathology seen in humans; specifically, the dog model lacks inflammatory middle ear disease. In contrast, the MPS VII dog has severe inflammatory middle ear disease similar to that reported in the human. In conclusion, the canine MPS VII model appears to be a good model to study MPS VII-related deafness.     

Technical note: Development of an enzyme-linked immunosorbent assay for the determination of florfenicol and thiamphenicol in swine feed

One polyclonal antibody against florfenicol and thiamphenicol was produced and a competitive ELISA was developed for the detection of florfenicol and thiamphenicol in swine feed. The ELISA gave a 50% inhibiting concentration of 1.02 ng/mL for florfenicol. For swine feed fortified with 0.05 to 3.0 mg/kg, the interassay recoveries of florfenicol and thiamphenicol ranged from 86.4 to 118.6%, whereas intraassay recoveries of both drug ranged from 90.1 to 126.5% with less than 15% CV. Results obtained from HPLC-tandem mass spectrometry indicated this ELISA procedure could be used as a convenient method for rapid screening of florfenicol and thiamphenicol in swine feed.     

Nitrous oxide by itself is insufficient to relieve pain due to castration in piglets

Surgical castration is performed on most male piglets in the United States. However, castration is painful and analgesics have been considered to relieve pain. Inhalant gases with analgesic properties allow for a fast induction, have short-term and reversible effects, and are a needle-free option. Nitrous oxide (N(2)O; "laughing gas") has been widely used in human surgery and dental offices as an analgesic, sedative, and anxiolytic drug, yet N(2)O has not been thoroughly investigated for use in farm animals. We hypothesized that the analgesic effect of N(2)O could reduce the pain experienced by piglets during or immediately after castration. Twenty-four male piglets, from 12 litters, were castrated at 3 d of age. One piglet received N(2)O and a littermate received air as a control. After 150 s of exposure to the gas, castration was performed while the piglet remained exposed to the gas. Agitation scores and total vocalization length were recorded during castration. Behavioral observations were continued for 3 d postcastration by using a 5-min scan-sampling method for 4 h the first morning and for 2-h periods in the morning and afternoon of each day thereafter. Body weight gain was measured on the day before castration, at 3 d postcastration, and at weaning. Data were analyzed using a mixed model in SAS (Cary, NC). Nitrous oxide successfully induced anesthesia in all N(2)O piglets, as validated by a skin pinch test and the loss of the palpebral reflex. Total vocalization length was shorter in piglets receiving N(2)O during the induction phase (P = 0.003) but was not different during castration itself because piglets receiving N(2)O awoke and vocalized as much as control piglets (P = 0.87). Agitation scores during the whole procedure were reduced in piglets receiving N(2)O in both frequency (P = 0.005) and intensity (P = 0.026). For 2 h after castration, piglets receiving N(2)O displayed less huddling behavior than did control piglets (P = 0.01). Over the 3 d, piglets receiving N(2)O performed more tail wagging (P = 0.02) and tended to show fewer sleep spasms (P = 0.06) than did control piglets. Piglets given N(2)O tended to have a reduced growth rate compared with control piglets at 3 d postcastration and at weaning (P = 0.05 and P = 0.06, respectively). Nitrous oxide was effective in inducing anesthesia in neonatal piglets during handling. Nonetheless, its analgesic effects appeared insufficient in preventing castration-induced pain.     

Anatomy of the collateral ligaments of the feline elbow joint: functional implications

Cats show a higher capability to supinate their forearms than dogs. This suggests a special arrangement of the collateral ligaments of the feline elbow joint. Therefore, the course of the ligaments was examined in 13 adult cats. The size of the ligaments was measured, and effects of passive joint movements were studied. Ligaments of five additional cats were examined histologically. The lateral collateral ligament (LCL) had a superficial and deep part, both originating from the humerus. The free humeral portion of the LCL was short and contained fibrous cartilage. Fibre bundles of the deep part inserted into the annular ligament, while the remaining deep fibres and the superficial part inserted with a long antebrachial portion on the radius. The medial collateral ligament (MCL) originated from the humeral epicondyle and divided into cranial and caudal parts. The caudal part inserted medioproximally on the ulna, while the cranial part attached primarily with a long thin part to the caudal aspect of the radius. During supination, the MCL loosened thus allowing medial widening of the joint space, up to 2 mm. A specific feature of the feline elbow is the long thin part of the MCL. Its course through a special furrow distal to the medial coronoid causes the tightening of the feline MCL during pronation. Apart from that, the feline collateral ligaments combine the features of both human and canine cubital anatomy. This explains the range of supination in cats, which is intermediate between humans and dogs.     

Modulation of nicotinamide adenine dinucleotide phosphate oxidase activity through sequential posttranslational modifications of p22 phagocytic oxidase during capacitation and acrosome reaction in goat spermatozoa

Superoxide anion radical, produced in low quantities, plays a positive role in sperm function. Spermatozoa produce superoxide anion radical during posttesticular development, which shows an abrupt increase during capacitation. The NAD phosphate oxidase (NOX) family members NOX2 and NOX5 are the 2 enzymes implicated in superoxide production in spermatozoa. We examined the organization of NOX2 in goat spermatozoa during epididymal maturation, capacitation, and acrosome reaction. Spermatozoa from testis, caput epididymidis, corpus epididymidis, and cauda epididymidis possessed components of the phagocytic oxidase (PHOX; i.e., gp91phox, p22phox, p67phox, p47phox, p40phox), and ras-related C3 botulinum toxin substrate 1/2 (Rac1/2) on spermatozoa, and their concentrations did not show significant alterations during epididymal maturation. During capacitation in vitro, p22phox underwent Thr-phosphorylation, which resulted in a mobility shift of the corresponding band toward greater molecular mass. The Rac1/2 also showed a mobility shift from 32 to 23 kDa during capacitation. During progesterone-induced acrosome reaction, the spermatozoa experienced a total loss of p22phox and p47phox. The p47phox, but not p22phox, was detected in the exocytic vesicles of the acrosome. The Thr-phosphorylated form of p22phox was ubiquitinated and degraded through proteasome-mediated pathways in goat sperm cell lysates. Thus, Thr phosphorylation of p22phox acts as a regulatory switch in goat spermatozoa that transiently activates the NOX2 system during capacitation and subsequently directs it for degradation through the ubiiquitin-proteasomal pathway during progesterone-induced acrosome reaction.     

Molecular forms of selected antioxidant enzymes in dog semen--electrophoretical identification

The aim of the study was the electrophoretical identification of molecular forms of selected antioxidant enzymes in dog semen. Ejaculates to be studied were chosen from five dogs, aged from two to eight years. Polyacrylamide gel electrophoresis was carried out under non-denaturing conditions and then gels were stained for the activity of the following enzymes: superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Sperm homogenates and all fractions (pre-spermatic, spermatic and post-spermatic) of dog ejaculate demonstrated one protein band with SOD activity characterized by low electrophoretic mobility. Based on the confirmed sensitivity to H2O2, it can be assumed that the detected SOD is an enzyme containing ions of Zn2+ and Cu2+ (Cu,Zn SOD). In sperm homogenates one protein band with GPx activity was characterized by high electrophoretic mobility, whereas in the spermatic and post-spermatic fractions of dog ejaculate three protein bands with different (low, medium and high) electrophoretic mobility were identified. CAT molecular forms were not found in either sperm homogenates or in the analyzed fractions of ejaculate.