Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most commonly used classes of drugs in both human and veterinary medicine. However, many clinical side effects have been observed, especially when treatment has been prolonged. While the anti-inflammatory efficacy and safety of repeated administration of firocoxib (Previcox®), which is a selective NSAID COX-2 inhibitor, has been evaluated for short-term use (one to fourteen days), its clinical relevance for longer-term use is not known. As a preliminary study, healthy, adult male and female horses (n = 7) were treated with firocoxib for 40 days concomitant with the collection of blood samples encompassing treatment to assess hematological and biochemical endpoints. Daily oral administration of firocoxib was performed with one 57 mg tablet/animal (0.11-0.14 mg/kg), which was crushed and mixed with feed. Blood samples were collected one day before treatment (D0 or basal sample), during (D10, D20, D30, and D40), and after treatment (D55 and D70). Results indicated some hematological and biochemical effects were significantly reduced (p < 0.05) towards the end of treatment on D40 relative to pre-treatment or baseline values on D0. Post-treatment, all values returned to pre-treatment values within 30 days without any apparent clinical adversities. In conclusion, while these preliminary results are favorable for prolonged use of firocoxib in horses, future studies are required to evaluate the efficacy of prolonged use accompanied with other clinically relevant endpoints in healthy as well as injured or diseased animals.

The evaluation of hematological and plasma biochemical parameters and the subsequent establishment of reference intervals facilitate the diagnosis of the health status of animals. This work aimed to determine the blood parameters of wild specimens of the stingrays Potamotrygon motoro and Potamotrygon orbignyi from the lower Solimões River region, Amazonas, Brazil. One hundred forty-one stingrays were captured, 92 specimens of P. motoro and 49 of P. orbignyi, of both sexes and at different stages of development. No effect of sex was observed on the blood parameters of juvenile animals for both species. P. motoro neonates presented a distinct hematological and biochemical profile, with significantly lower hematocrit values, hemoglobina, number of erythrocytes, mean corpuscular hemoglobin concentration, monocytes, plasma glucose, total proteins, albumin, and globulin. On the other hand, total cholesterol and urea levels were significantly higher in this same group compared to juveniles of the same species. Comparison between species revealed lower values of triglycerides and total cholesterol in P. orbignyi of both sexes. The results obtained are pioneering for these Amazonian species in white water environments and will serve as a basis for evaluating the health status of wild stingrays. Thus, from the analysis of the blood of the P. motoro and P. orbignyi stingrays, it was possible to observe good health conditions.

BACKGROUND: Although widely used, the ADVIA 120 hematology analyzer has not been previously validated for determining the differential leukocyte count in goats.
OBJECTIVE: The aim of this study was to compare the differential leukocyte counts provided by the ADVIA 120 (A-diff) and the manual method (M-Diff) in goats.
METHODS: EDTA blood samples that were analyzed within 4 h of collection were used in the study. The following exclusion criteria were applied: inappropriately filled tubes or tubes containing clots, erroneous ADVIA peroxidase cytograms, and blood smears of poor quality. The A-Diff was compared with the M-Diff performed by two independent observers on 200 leukocytes.
RESULTS: Forty samples were included after previously excluding eight samples. The correlation between the A-Diff and M-Diff was very strong for eosinophils (r = .870, p < .001) and strong for lymphocytes (r = .796, p < .001) and neutrophils (r = .730, p < .001), while no significant correlation was observed for monocytes (r = .026, p = .872). The Passing-Bablok regression analyses revealed statistically significant constant errors for neutrophils (5.83%; 95% confidence interval [CI]: 0.41%, 12.18%) and eosinophils (1.89%; 95% CI: 1.17%, 2.71%). Bland-Altman analyses showed a statistically significant negative bias for lymphocytes (-5.0%) and a statistically significant positive bias for eosinophils (2.2%). The very low basophil percentages precluded a meaningful method comparison.
CONCLUSIONS: The ADVIA 120 overall demonstrated good performance for the differential WBC count in goats under the conditions of this study. Therefore, it can be considered suitable for routine hematologic screening in goats. Nonetheless, it should be emphasized that any abnormal result should be confirmed with a blood smear evaluation.

We present the results of an in situ study of a set of blood parameters in adult marsh frogs (Pelophylax ridibundus (Pallas 1771) from populations inhabiting the largest system of rice fields in Bulgaria, the Tsalapitsa rice fields (TRF), under chronic stress conditions. This study was conducted in spring 2022 to assess the health status of TRF frogs compared to that of frogs occupying a reference site (RS). Furthermore, this study also compared the results obtained for the TRF population with those obtained in a study conducted at the exact same location with P. ridibundus individuals in 2013 (Zhelev et al. 2018). This comparison highlights the potential effects of persistent use of agrochemicals (pesticides and fertilizers) on the marsh frogs of later generations. Our results suggest that the general health of marsh frogs in the polluted site (PS) in southern Bulgaria has severely deteriorated. Frogs of both sexes were anemic with weakened immune systems compared to those living in the RS. The long-term use of agrochemicals in the PS affected males to a greater extent than it did females. Statistically significant hypochromia was observed in males, combined with general leukopenia, neutrophilia, lymphopenia, monocytosis, eosinophilia, and higher neutrophil/lymphocyte (N/L) ratios.

Variation in food resources can result in dramatic fluctuations in the body condition of animals dependent on those resources. Decreases in body mass can disrupt patterns of energy allocation and impose stress, thereby altering immune function. In this study, we investigated links between changes in body mass of captive cane toads (Rhinella marina), their circulating white blood cell populations, and their performance in immune assays. Captive toads that lost weight over a three-month period had increased levels of monocytes and heterophils and reduced levels of eosinophils. Basophil and lymphocyte levels were unrelated to changes in mass. Because individuals that lost mass had higher heterophil levels but stable lymphocyte levels, the ratio of these cell types was also higher, partially consistent with a stress response. Phagocytic ability of whole blood was higher in toads that lost mass, owing to increased circulating levels of phagocytic cells. Other measures of immune performance were unrelated to mass change. These results highlight the challenges faced by invasive species as they expand their range into novel environments which may impose substantial seasonal changes in food availability that were not present in the native range. Individuals facing energy restrictions may shift their immune function towards more economical and general avenues of combating pathogens. This article is part of the theme issue \'Amphibian immunity: stress, disease and ecoimmunology\'.

Chronic otitis media, inflammation of the middle ear, is a sequel to acute otitis media in ∼8% of children. Chronic otitis media with effusion is the most common cause of childhood deafness and is characterised by effusion of white blood cells into the auditory bulla cavity. Skull flat bones have trans-cortical vessels which are responsible for the majority of blood flow in and out of the bone. In experimental models of stroke and aseptic meningitis there is preferential recruitment of myeloid cells (neutrophils and monocytes) from the marrow in skull flat bones. We report trans-cortical vessels in the mouse temporal bone connect to the bulla mucosal vasculature and potentially represent a means to recruit myeloid cells directly into the inflamed bulla. The mutant mouse strains Junbo (Mecom Jbo/+ ) and Jeff (Fbxo11 Jf/+ ) develop chronic otitis spontaneously; Mecom Jbo/+ mice have highly cellular neutrophil (90%) rich bulla exudates whereas Fbxo11 Jf/+ mice have low cellularity serous effusions (5% neutrophils) indicating differing demand for neutrophil recruitment. However we found peripheral leukograms of Mecom Jbo/+ and Fbxo11 Jf/+ mice are similar to their respective wild-type littermate controls with healthy bullae and infer preferential mobilization of myeloid cells from temporal bulla bone marrow may mitigate the need for a systemic inflammatory reaction. The cytokines, chemokines and haematopoietic factors found in the inflamed bulla represent candidate signalling molecules for myeloid cell mobilization from temporal bone marrow. The density of white blood cells in the bulla cavity is positively correlated with extent of mucosal thickening in Mecom Jbo/+ , Fbxo11 Jf/+ , and Eda Ta mice and is accompanied by changes in epithelial populations and bone remodelling. In Mecom Jbo/+ mice there was a positive correlation between bulla cavity WBC numbers and total bacterial load. The degree of inflammation varies between contralateral bullae and between mutant mice of different ages suggesting inflammation may wax and wane and may be re-initiated by a new wave of bacterial infection. Clearance of white blood cells and inflammatory stimuli from the bulla cavity is impaired and this may create a pro-inflammatory feedback loop which further exacerbates otitis media and delays its resolution.

Hematological parameters can provide key information to an animal health status. However, this information is usually hard to obtain. Here, we described the hematological parameters of Leptodactylus podicipinus in the Brazilian Pantanal. We measured red blood cell morphometrics, erythrogram, and leukogram. We also tested for phylogenetic signal in the erythrogram and leukogram of 48 frog species from 15 families, testing if body size explains their variation. Lymphocytes were the most abundant leukocytes (>60%) in L. podicipinus, followed by neutrophils (∼10%). Given that L. podicipinus is an abundant and widely distributed species in central Brazil, knowing its hematological pattern can help establish a baseline and improve its use as a bioindicator of environmental degradation. Mean corpuscular hemoglobin and value contributed more to the phylomorphospace of erythrogram, in which Leptodactylus spp. and Hypsiboas raniceps had lower values of these variables, whereas Bufotes viridis and Hyla arborea had high values. The phylogenetic signal was spread throughout the dimensions of the leukogram phylomorphospace. The variables that most contributed to it were total leukocytes counts, lymphocytes, and neutrophils. We also found a moderate phylogenetic signal for both the erythrogram and leukogram. Accordingly, body size accounted for a low proportion of variation in both the leukogram (4.7%) and erythrogram (0.57%). By applying phylogenetic comparative methods to hematological parameters, our results add a new perspective on the evolution of blood cell physiology in frogs.

It is important to evaluate the role of captivity as a potential stressor. An understanding of stress responses to capture and transition to captivity may inform the limitations of laboratory studies on wild animals, aid in understanding the consequences of introducing animals into captive environments, and help predict which species may be successful invasives. We investigated physiological effects of captivity by comparing at-capture blood variables in wild Burmese pythons (Python bivittatus) in Florida to pythons recently brought into captivity (1-109 days). We conducted an acute restraint test by collecting samples at baseline (immediately at handling) and one hour post-restraint across wild field-sampled (n = 19) and recently-captive (n = 33) pythons to evaluate fluctuations in plasma corticosterone, bacterial killing ability, antibody response, leukogram, and serpentovirus infection. We observed higher baseline plasma corticosterone and monocytes in recently captive compared to wild snakes, which both subsided in snakes held for a longer time in captivity, and a mild decrease in lymphocytes in the middle of the captivity period. Functional immunity and viral infection were not affected by captivity, and pythons maintained restraint-induced responses in corticosterone, heterophil to lymphocyte ratio, and monocyte counts throughout captivity. Prevalence for serpentovirus was 50%, though infection status was related to sampling date rather than captivity, indicating that viral infection may be seasonal. The history of Burmese python as a common captive animal for research and pet trade, as well as its general resilience to effects of capture and short-term captivity, may contribute to its invasion success in Florida.

The northwest Atlantic leatherback sea turtle (Dermochelys coriacea) population is exhibiting decreasing trends along numerous nesting beaches. Since population health and viability are inherently linked, it is important to establish species- and life-stage class-specific blood analyte reference intervals (RIs) so that effects of future disturbances on organismal health can be better understood. For hatchling leatherbacks, the objectives of this study were to (1) establish RIs for morphometrics and blood analytes; (2) evaluate correlations between hatchling morphometrics, blood analytes, and hatching success; and (3) determine temporal trends in hatchling morphometrics and blood analytes across nesting season. Blood samples were collected from 176 naturally emerging leatherback hatchlings from 18 clutches. Reference intervals were established for morphometrics and blood analytes. Negative relationships were noted between hatchling mass and packed cell volume, total white blood cells, heterophils, lymphocytes, and total protein and between body condition index (BCI) and immature red blood cells (RBC), RBC polychromasia and anisocytosis, and total protein. Clutch deposition date showed positive relationships with lymphocytes and total protein, and negative relationships with hatchling mass and BCI. Hatching success was positively correlated with mass, and negatively with total protein and glucose, suggesting that nutritional provisions in eggs, incubation time, and/or metabolic rates could change later in the season and affect survivorship. These various observed correlations provide evidence for increased physiological stress (e.g., inflammation, subclinical dehydration) in hatchlings emerging later in nesting season, presumably due to increased nest temperatures or other environmental factors (e.g., moisture/rainfall). Data reported herein provide morphometric and blood analyte data for leatherback hatchlings and will allow for future investigations into spatiotemporal trends and responses to various stressors.

BACKGROUND: The hematology of turtles of the Amazon, such as the Arrau turtle (Podocnemis expansa), has not been well described in the literature. Referencing how to collect and analyze blood samples is fundamental for the medical practice of these exotic animals.
OBJECTIVE: The aim of this study was to evaluate the hematologic parameters of Arrau turtles of the Amazon to determine the best anticoagulant (EDTA, citrate, or sodium heparin) and cytologic stain (Giemsa, Quick Panoptic, or Rosenfeld) for use with this species.
METHODS: Blood samples from eight turtles were collected. Three blood smears were made using blood without anticoagulant, and the rest of the sample was distributed in three tubes, containing EDTA, citrate, and sodium heparin, for erythrogram and leukogram analyses.
RESULTS: All blood samples anticoagulated with EDTA showed marked hemolysis. Blood collected with citrate showed minimal hemolysis, and blood collected with sodium heparin did not have any hemolysis. Some analyses were not performed on blood containing EDTA because of the intense hemolysis. The erythrocyte variables were obtained and showed better results for blood samples in heparin. Blood smears without anticoagulant were appropriate for differential leukocyte counts with the three stain types, while blood with any of the anticoagulants made it difficult to differentiate leukocytes.
CONCLUSIONS: Sodium heparin showed the best results and should be the anticoagulant of choice for hematologic studies in P expansa. All cytologic stains were efficient, allowing adequate identification of distinct cell groups. Leukocyte differentiation was assured and had better contrast using blood smears made from blood without an anticoagulant.