Establishment of preliminary reference intervals and cytochemical staining of blood cells in big-bellied seahorses (Hippocampus abdominalis).

BACKGROUND: Despite their popularity, hematology reference intervals (RIs) have not been established in big-bellied seahorses (Hippocampus abdominalis). OBJECTIVE: The objectives of this study were to establish hematologic RIs to compare values between sex in regard to cytochemical staining of blood cells. We also sought to compare white blood cell concentrations using the Natt and Herrick technique vs blood smear estimates. METHODS: Forty-three healthy individuals from the Aquarium du Québec (22 females and 21 males) were included. Normal health status was confirmed by an unremarkable physical examination in five individuals and by necropsy of five other individuals, of which all were excluded from further analyses. Venipuncture was performed from the ventral coccygeal vein in the remaining 33 individuals without anesthesia using heparinized insulin syringes. A blood volume of 0.05 to 0.1 ml was collected to prepare Wright Giemsa-stained blood smears and hematocrits immediately after venipuncture. Whole blood was stored in heparinized Eppendorf tubes to determine red and white blood cell concentrations using the Natt and Herrick technique with a hemocytometer in 10 individuals; these results were compared with blood smear estimates. Additional blood smears were stained with alkaline phosphatase substrate, periodic acid Schiff, and toluidine blue stains. RESULTS: The reference intervals included the packed cell volume (27.4-67.5%), thrombocyte count (19.5-197.7 × 109 /L), and white blood cell (WBC) count (2-54.8 × 109 /L), including neutrophils (1.1-21.3 × 109 /L), lymphocytes (2.7-45.5 × 109 /L), and monocytes (0-2.2 × 109 /L). The WBC hemocytometer counts showed no correlation with blood smear estimates (Spearman's rho = 0.2). There was also no significant difference between the sexes. CONCLUSIONS: These preliminary reference intervals will help assess the health of seahorses.

Evaluation of prolonged immersion in tricaine methanesulfonate for juvenile goldfish (Carassius auratus) euthanasia.

OBJECTIVE: Doses of buffered tricaine methanesulfonate (MS-222) up to 1000 mg/L for 15 minutes are reported inefficient to produce euthanasia in goldfish. The goal of this study was to determine if goldfish can be euthanized by more prolonged immersion in MS-222. ANIMALS: 24 healthy goldfish (weight range: 1 to 10 g) were randomly assigned to 4 groups of 6 fish. PROCEDURES: The first group (G1) was exposed to 500 mg/L buffered MS-222 for 15 minutes then placed in freshwater for 3 hours. The second (G2) and third groups (G3) were exposed to 1000 mg/L of buffered MS-222 for 15 minutes then placed in freshwater for 3 hours and 18 hours respectively. The fourth group (G4) was exposed to 1000 mg/L of buffered MS-222 for 60 minutes then placed in freshwater for 3 hours. Time to cessation and return of operculation were recorded. If the goldfish did not resume operculation, heart rate was evaluated by Doppler ultrasonic flow detector. RESULTS: Median times to apnea were 35 seconds at 1000 mg/L and 65 seconds at 500 mg/L. Re-operculation occurred only in G1 in 5 out of 6 individuals. All fish from G1, 3 fish from G2, 0 fish from G3, 1 fish from G4 had remaining heartbeats at the end of the observation period. CLINICAL RELEVANCE: Overall, a dose of 1000 mg/L of buffered MS-222 for 15 minutes was efficient to euthanize juvenile goldfish at 20 °C. Different fish body mass and water quality parameters might explain different results compared to previous studies.

USE OF SYMMETRIC DIMETHYLARGININE TO DETECT RENAL LESIONS IN FISH: A PRELIMINARY STUDY IN BROOK TROUT (SALVELINUS FONTINALIS).

Symmetric dimethylarginine (SDMA) is an early marker for renal lesions in mammals. The objectives of this study were 1) to establish a reference interval of SDMA in healthy brook trout, Salvelinus fontinalis (Mitchill, 1814), and 2) to assess its sensibility as a marker of renal pathology. Plasma SDMA was quantified by liquid chromatography-mass spectrometry in 25 adult brook trout, including 20 fish displaying no renal histologic lesions, and five fish displaying chronic nonactive microscopic granulomas. The fish size (P = 0.30) and weight (P = 0.12) were not statistically different among groups, nor were SDMA values (P = 0.22). However, brook trout without microscopic renal lesions tended to have lower SDMA values (no lesions: mean = 24.9 µg/dL; lesions: mean = 31.4 µg/dL). The reference interval (90% confidence interval [90% CI]) for SDMA concentration in brook trout ranged between 10.0 µg/dL (90% CI: 5.4-14.7) and 39.8 µg/dL (90% CI: 34.8-43.9). These values were higher than those previously reported in other vertebrate species. Further research is needed to evaluate the use of SDMA as a marker of renal function in fishes.

Development and Testing of a Sedation Scale for Use in Rabbits (Oryctolagus cuniculus).

In biomedical research, rabbits are commonly sedated to facilitate a variety of procedures. Developing a sedation assessment scale enables standardization of levels of sedation and comparisons of sedation protocols, and may help in predicting sedation level requirements for different procedures. The goal of this study was to develop a rabbit sedation assessment scale using a psychometric approach. We hypothesized that the sedation scale would have construct validity, good internal consistency, and reliability. In a prospective, randomized, blinded study design, 15 (8 females, 7 males) healthy 1-y-old New Zealand white rabbits received 3 intramuscular treatments: midazolam (0.5 mg/kg; n = 6); midazolam (1.5 mg/kg)-ketamine (5 mg/kg; n = 7); and alfaxalone (4 mg/kg)-dexmedetomidine (0.1 mg/kg)-midazolam (0.2 mg/kg; n = 3). One rabbit received 2 treatments. A sedation scale was developed by using psychometric methods, with assessment performed by 6 independent raters who were blind to treatment. Final sedation scale items included posture, palpebral reflex, orbital tightening, lateral recumbency, loss of righting reflex, supraglottic airway device placement, toe pinch, and general appearance. The scale showed construct validity, good to very good interrater reliability for individual items (6 raters; intraclass correlation coefficient, 0.671 to 0.940), very good intrarater reliability (5 raters; intraclass correlation coefficient, 0.951 to 0.987), and excellent internal consistency (Cronbach α, 0.947). The sedation scale performed well under the conditions tested, suggesting that it can be applied in a wider range of settings (different populations, raters, sedation protocols).

PHARMACOKINETIC, PHARMACODYNAMIC, AND TOXICOLOGY STUDY OF ROBENACOXIB IN RAINBOW TROUT (ONCORHYNCHUS MYKISS).

Postoperative antinociception control in fish is currently suboptimal, as commonly used antiinflammatory drugs last for only a few hours at tested temperatures. Therefore, long-acting anti-inflammatory drugs, such as robenacoxib, could improve the welfare of fish. The pharmacokinetics, duration of antinociceptive action, and potential adverse effects of robenacoxib were evaluated through two prospective randomized blinded trials in rainbow trout (Oncorhynchus mykiss). Six healthy rainbow trout received a single IM administration of robenacoxib (2 mg/kg), and two control fish received the same volume of saline IM. Blood samples were collected at predetermined time points for 5 d. Plasma robenacoxib concentrations were measured using high-performance liquid chromatography-high-resolution hybrid orbitrap mass spectrometry and noncompartmental pharmacokinetic analysis. Ten additional rainbow trout received an intralabial injection of 0.05 ml of 2% acetic acid following a previously validated nociceptive model. The treated group (n = 6) received 2 mg/kg of robenacoxib IM and the control group (n = 4) received an equivalent volume of saline IM. The behavior, appetite, and opercular rate of the fish were evaluated every hour for 5 h, then once daily for 3 d. All 12 treated trout and 6 controls underwent histopathologic evaluation. Average maximum plasma concentration (Cmax) was 329.9 ± 137.3 ng/ml observed at 2.1 ± 0.7 h (Tmax) and terminal half-life was 12.6 ± 2.27 h. Plasma concentrations described as antinociceptive in domestic carnivores were measured for 3-4 d. This dose was associated with a significant decrease in rocking behavior (P = 0.017). No adverse effects were detected clinically nor on histopathology. Robenacoxib administered IM at 2 mg/kg appears to be safe and may provide an antinociceptive effect in rainbow trout. This study presents a new therapeutic option to provide long-lasting antinociception in rainbow trout.