Impact of heparin and short term anesthesia on the quantification of cytokines in laboratory mouse plasma

Background

Studies have reported that heparin may be unsuitable as an anticoagulant in human plasma samples when quantifying cytokines using multiplex bead array assays. For mouse samples, multiplex assays have been validated for serum and EDTA-plasma, but it remains to be elucidated whether heparin influences the quantification of cytokines, and if so – to what extent. Furthermore, laboratory mice are often anesthetized for blood sampling, which causes acute stress that may influence circulating cytokine concentrations and thus bias experimental results. The objectives of the present study were to identify whether specific cytokine concentrations varied between heparin-plasma, serum, and EDTA-plasma, and whether short isoflurane anesthesia would influence the concentrations of these cytokines in the circulation. Twenty-three acute phase and pro-inflammatory cytokines were quantified in matched serum, EDTA-plasma, and heparin-plasma samples from anesthetized and unanesthetized male NMRI mice using a multiplex assay. In addition, samples from unanesthetized mice were spiked with three levels of heparin.

Results

The concentrations of five out of 23 cytokines were significantly different between sample types, but only one cytokine (IL-17A) differed between heparin-plasma and serum. When further spiking the heparin-plasma with increasing concentrations of heparin, there was a significant effect on 11 cytokines, where the cytokine recovery could be correlated to the heparin concentration for ten of these cytokines. Anesthesia resulted in lower concentrations of G-CSF, but had no significant impact on the concentrations of the other 22 cytokines.

Conclusion

In mice, heparin seems like a suitable anticoagulant for obtaining plasma for multiplex assays for the cytokines IL-1α, IL-1β, IL-2, IL-6, IL-9, IL-12p40, IL-12p70, IL-13, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNFα, but an effect of heparin in high concentrations should be considered for the cytokines IL-9, IL-12p40, IL-12p70, KC, MCP-1, MIP-1β and RANTES. Short isoflurane anesthesia had significant impact on G-CSF, but none of the other cytokines.     

Effect of Alpha-Tocopherol and Ascorbic Acid on Bovine Oocyte in Vitro Maturation

In vitro culture results in higher oxygen concentrations than in vivo environments, leading to an increased level of reactive oxygen species (ROS) that cause lipid peroxidation of cellular membranes. Alpha-tocopherol (active form of vitamin E) is an antioxidant that protects mammalian cells against lipid peroxidation, which is regenerated by ascorbic acid. The aim of this study was to determine the effect of the addition of alpha-tocopherol and/or ascorbic acid to the maturation medium on bovine oocyte in vitro maturation (IVM) and subsequently on in vitro fertilization (IVF) and embryo development. Cumulus-oocyte complexes (COCs) were matured in Medium 199 (control), and with the addition of alpha-tocopherol and/or ascorbic acid. The concentration of alpha-tocopherol in COCs was determined by high-performance liquid chromatography (HPLC). IVF and in vitro culture (IVC) were carried out in modified synthetic oviductal fluid (mSOF). The quantity of alpha-tocopherol naturally present in COCs diminished by half during IVM (p < 0.05), although in the presence of ascorbic acid it remained constant. A greater amount of alpha-tocopherol was detected in COCs matured in medium supplemented with this antioxidant (p < 0.05), but the addition of alpha-tocopherol plus ascorbic acid maintained higher levels of alpha-tocopherol (p < 0.05). Significant differences were not observed in the percentages of nuclear maturation and fertilization among different treatments. The presence of alpha-tocopherol or ascorbic acid in the maturation medium failed to modify the percentage of blastocysts obtained, unlike the addition of both antioxidants when a significant decrease was observed (p < 0.05). Absorbic acid maintained the antioxidant capacity of the alpha-tocopherol incorporated to COC membranes during IVM. The active form of vitamin E during maturation impaired the acquisition of oocyte developmental competence.     

Osteochondral fragmentation of the plantar/palmar proximal aspect of the proximal phalanx in racing horses

SUMMARY The clinical presentation and outcome of treatment is presented for 26 cases of osteochondral fragmentation of the plantar/palmar proximal aspect of the proximal phalanx. Twenty-three were racing Standardbreds and three were racing Thoroughbreds. The most common reason for presentation was an inability to run straight at high speed. Only eight horses presented for lameness, although on examination 19 were lame. A positive flexion test was recorded in 90% of affected fetlock joints and effusion in 48%. Arthroscopic fragment removal was performed on 23 occasions in 21 horses and arthrotomy in one horse. Of the 16 horses that had returned to racing, 12 had improved their performance, while three showed no improvement, and one was retired for other reasons. In three horses refragmentation occurred after surgery, two of which had improved after initial arthroscopic removal. Degenerative changes within the fetlock joint were detected at surgery in eight horses. Of the four horses treated conservatively, one returned to its previous level of performance temporarily after intra-articular medication, one showed no improvement and two were still resting. Plantar/palmar osteochondral fragmentation of the proximal aspect of the first phalanx is a common cause of low-grade lameness in racing horses, and arthroscopic removal results in improvement in race performance in a high percentage of cases.     

Myofibroblastic fibrosarcoma with multifocal osseous metaplasia at the site of equine influenza vaccination

We describe a fibrosarcoma in a 12-year-old Quarterhorse × Arabian gelding as a sequela to equine influenza vaccination. Shortly after the second vaccination, swelling at the site was noticed by the owner and it continued to increase in size over the following 6 months. Biopsy of the mass indicated a fibrosarcoma had developed at the vaccination site. It was approximately 20 cm in diameter and elevated well above the level of the skin. There was no clinical evidence of metastases to the lungs or local lymph nodes. Surgical resection of the mass was performed and the wound healed by first and second intention. Histopathological examination and immunohistochemical staining confirmed a myofibroblastic fibrosarcoma with multifocal osseous metaplasia. To the authors' knowledge, this is the first equine case of a vaccine-associated fibrosarcoma.     

RESEARCH PAPER: Comparison between two methods for cardiac output measurement in propofol‐anesthetized dogs: thermodilution and Doppler

ObjectiveTo compare cardiac output (CO) measured by Doppler echocardiography and thermodilution techniques in spontaneously breathing dogs during continuous infusion of propofol. To do so, CO was obtained using the thermodilution method (CO_TD) and Doppler evaluation of pulmonary flow (CO_DP) and aortic flow (CO_DA).Study designProspective cohort study.AnimalsEight adult dogs weighing 8.3 ± 2.0 kg.MethodsPropofol was used for induction (7.5 ± 1.9 mg kg^?1 IV) followed by a continuous rate infusion at 0.7 mg kg^?1 minute^?1. The animals were positioned in left lateral recumbency on an echocardiography table that allowed for positioning of the transducer at the 3rd and 5th intercostal spaces of the left hemithorax for Doppler evaluation of pulmonary and aortic valves, respectively. CO_DP and CO_DA were calculated from pulmonary and aortic velocity spectra, respectively. A pulmonary artery catheter was inserted via the jugular vein and positioned inside the lumen of the pulmonary artery in order to evaluate CO_TD. The first measurement of CO_TD, CO_DP and CO_DA was performed 30 minutes after beginning continuous infusion (T0) and then at 15‐minute intervals (T15, T30, T45 and T60). Numeric data were submitted to two‐way anova for repeated measurements, Pearson’s correlation coefficient and Bland &; Altman analysis. Data are presented as mean ± SD.ResultsAt T0, CO_TD was lower than CO_DA. CO_DA was higher than CO_TD and CO_DP at T30, T45 and T60. The difference between the CO_TD and CO_DP, when all data were included, was ?0.04 ± 0.22 L minute^?1 and Pearson’s correlation coefficient (r) was 0.86. The difference between the CO_TD and CO_DA was ?0.87 ± 0.54 L minute^?1 and r = 0.69. For CO_TD and CO_DP, the difference was ?0.82 ± 0.59 L minute^?1 and r = 0.61.ConclusionDoppler evaluation of pulmonary flow was a clinically acceptable method for assessing the CO in propofol‐anesthetized dogs.     

Does the Amount of Progesterone in Intravaginal Implants Used to Synchronise Oestrus Affect the Reproductive Performance of Brahman Heifers Artificially Inseminated at a Fixed Time

The study tested the hypothesis that reduced intravaginal implant progesterone (P₄) concentration to synchronise oestrus would increase pregnancy rates to fixed‐time artificial insemination (FTAI) in Bos indicus heifers. Brahman heifers (n = 294; 2 year) were body condition scored (BCS), weighed and scanned for presence of a corpus luteum (CL). Only cyclic heifers were selected and allocated randomly within BCS and 25 kg bodyweight category to one of three P₄ treatment groups. On day 10, heifers received a P₄ implant (CueMate‐1‐pod, 0.78g P₄; CueMate‐2‐pod, 1.56g P₄; or CIDR‐B, 1.9g P₄), 2 mg oestradiol benzoate (ODB) intramuscularly (IM) and 250 ug cloprostenol IM. At day 2, the implant was removed, 250 ug cloprostenol was injected IM and tail paint applied. The heifers received 1 mg ODB 24 h later and were FTAI 48–54 h after implant removal (day 0). Ten randomly selected heifers per group were blood sampled and scanned at days 10, 2, 0 and 6 to define the P₄ profiles pre‐ and post‐FTAI. Heifers were heat‐detected 18–20 days post‐FTAI and oestrous heifers AI’d by the AM/PM rule. Bulls joined the heifers on day 27 post‐FTAI. Transrectal ultrasonography estimated conception date on day 72. Statistical analysis examined the effects of treatment, technician, semen, ovarian status, BCS and liveweight, on pregnancy rate (PR) to FTAI. There was no significant difference (p = 0.362) in PR between treatment groups (CueMate 1‐pod, 36.4%; CueMate 2‐pod, 39.6%: CIDR‐B, 28.3%), but PR was higher in those heifers with increased BCS between FTAI and pregnancy diagnosis (p = 0.005). Thirty‐three per cent of monitor heifers had plasma P₄ concentrations of <1 ng/ml on day 6 after FTAI; only 20% of these conceived vs 60% of heifers with P₄ ≥ 1 ng/ml. In summary, no significant difference in PR was identified between treatments but good BCS and a rising plane of nutrition were critical to PR of these pure grade Brahman heifers in northern Australia.     

Health screening for a translocation of captive-reared tuatara (Sphenodon punctatus) to an island refuge

AIMS: To screen tuatara undergoing translocation from a captive crèche to an island refuge for evidence of health and known diseases, and apply basic epidemiological techniques to assess the significance of disease test results.

METHODS: Tuatara (n=353) were physically examined and samples were taken from a random selection (n=30) for estimated white cell counts, screening for haemoparasites, and culture for Salmonella, Yersinia, Aeromonas and Campylobacter spp. Direct faecal smears were carried out on-site, and faecal floats were later performed to assess levels of endoparasitism with helminths and protozoa (n=69). Modified Ziehl-Neelsen staining was used to screen faecal smears, and positive specimens were further screened using an immunofluorescence antibody (IFA) test for Cryptosporidium oocysts.

RESULTS: There was no evidence of external parasites on any of the animals examined and only one animal had a gross abnormality. All estimated white cell counts were in the range 2.8– 17.5 × 10⁹/L. No haemoparasites were observed. There were no enteric pathogens cultured, indicating the intestinal carriage of these bacteria in the tuatara was 9.4%. Of the 69 individual faecal samples examined, 12 (17%) had unidentified coccidial oocysts, 21 (30%) had nematode ova of various kinds, and 12 (17%) had intestinal carriage of motile protozoa consistent with Trichomonas spp and another unidentified organism. Nineteen (28%) tuatara had acid-fast oocysts present; however, IFA staining failed to detect any Cryptosporidium oocysts.

CONCLUSIONS: Our understanding of the diversity of gastrointestinal endoparasites affecting tuatara is inadequate as many of the parasite ova seen could not be identified. This is the first record of tuatara as a host for Trichomonas spp of protozoa in the gastrointestinal tract.     

Dermatomycosis caused by Paranannizziopsis australasiensis in five tuatara (Sphenodon punctatus) and a coastal bearded dragon (Pogona barbata) in a zoological collection in New Zealand

CASE HISTORY: Health monitoring of tuatara (Sphenodon punctatus) at Auckland Zoo between 2001 and 2009 showed that 58/93 tuatara had been affected by dermatitis of unknown origin. From 2011 onwards, cases of suspected fungal dermatitis underwent extensive diagnostic investigations.

CLINCAL FINDINGS: Six cases of dermatomycosis were attributed to Paranannizziopsis australasiensis, five in tuatara and one in a coastal bearded dragon (Pogona barbata). Cases presented typically as raised, yellow to brown encrustations on the skin. Severe cases progressed to necrotising ulcerative dermatitis, and in the bearded dragon to fatal systemic mycosis. Following topical and systemic treatments, lesions resolved in all five tuatara.

LABORATORY FINDINGS: Histopathological examination of skin biopsy samples revealed dermatitis with intralesional septate branching hyphae. Fungal culture yielded isolates morphologically resembling Chrysosporium species, and isolates were submitted for molecular confirmation and sequencing of DNA.

DIAGNOSIS: All six cases were confirmed as dermatitis due to infection with P. australasiensis, on the basis of fungal culture and DNA sequencing of isolates.

CLINICAL RELEVANCE: These are the first reported cases of dermatomycosis associated with P. australasiensis infection in tuatara, and the first cases in which systemic therapeutic agents have been used in the treatment of such disease. Tuatara at the Auckland Zoo are now routinely examined every 3 months and tissue samples from any lesions sent for histopathology and fungal culture. Further work to elucidate the epidemiology and significance of P. australasiensis infections in reptiles in New Zealand is important for both welfare and conservation purposes.     

Risk factors for nuisance barking in dogs

Objective   To determine the key variables related to the environment, animal and owner that influence nuisance barking by dogs in a city with a subtropical climate.
Design and Population   A case–control survey of dog owners in Brisbane, Queensland, was conducted using a questionnaire investigating key variables connected to nuisance barking. Owners of dogs exhibiting nuisance barking were obtained from a list of dogs being treated in a Brisbane behaviour clinic, and those of control dogs were selected from a telephone directory.
Results   Univariate analysis showed that animal, owner and environmental factors all potentially influence the occurrence of nuisance barking. Multivariate analysis identified the following factors, with the relevant odds ratios (OR) as significant: age of the dog (young dog vs old dog, OR 11.2); multiple dogs in the household vs single (OR 5.6); origin of the dog (home bred vs obtained from breeder or friend, OR 4.0); type of dog, (herding vs other types, OR 3.2) and dog with access to the home vs dog without access (OR 2.5).
Conclusion   The greatest risk for nuisance barking occurs with a young dog of the herding type that is home bred and with access to the house in a multiple dog household.