BackgroundThe Canadian Council on Animal Care and American Veterinary Medical Association classify intraperitoneal (IP) pentobarbital as an acceptable euthanasia method in rats. However, national guidelines do not exist for a recommended dose or volume and IP euthanasia has been described as unreliable, with misinjections leading to variable success in ensuring a timely death. The aims of this study were to assess and improve efficacy and consistency of IP euthanasia.In a randomized, blinded study, 51 adult female Sprague-Dawley rats (170–495 g) received one of four treatments: low-dose low-volume (LL) IP pentobarbital (n = 13, 200 mg/kg pentobarbital), low-dose high-volume (LH) IP pentobarbital (n = 14, 200 mg/kg diluted 1:3 with phosphate buffered saline), high-dose high-volume (HH, n = 14, 800 mg/kg pentobarbital), or saline. Times to loss of righting reflex (LORR) and cessation of heartbeat (CHB) were recorded. To identify misinjections, necropsy examinations were performed on all rats. Video recordings of LL and HH groups were analyzed for pain-associated behaviors. Between-group comparisons were performed with 1-way ANOVA and Games-Howell post hoc tests. Variability in CHB was assessed by calculating the coefficient of variation (CV).ResultsThe fastest euthanasia method (CHB) was HH (283.7 ± 38.0 s), compared with LL (485.8 ± 140.7 s, p = 0.002) and LH (347.7 ± 72.0 s, p = 0.039). Values for CV were: HH, 13.4%; LH, 20.7%; LL, 29.0%. LORR time was longest in LL (139.5 ± 29.6 s), compared with HH (111.6 ± 19.7 s, p = 0.046) and LH (104.2 ± 19.3 s, p = 0.01). Misinjections occurred in 17.0% (7/41) of euthanasia attempts. Pain-associated behavior incidence ranged from 36% (4/11, LL) to 46% (5/11, HH).ConclusionsThese data illustrate refinement of the IP pentobarbital euthanasia technique. Both dose and volume contribute to speed of death, with a dose of 800 mg/kg (HH) being the most effective method. An increase in volume alone does not significantly reduce variability. The proportion of misinjections was similar to that of previous studies.

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Forty cases of equine penile disease were screened with polymerase chain reaction for the presence of papillomaviral DNA. Cases consisted of 20 squamous cell carcinomas (average age of horse, 23.9 years) and 20 non-squamous cell carcinoma diseases (average age of horse, 13.3 years). All horses but one originated from the Northeastern United States. Breeds were not recorded. As based on MY09/MY11 consensus primers, DNA sequences from equine papillomavirus type 2 were amplified from 9 of 20 horses (45%) with penile squamous cell carcinoma and only 1 of 20 horses (5%) with non-squamous cell carcinoma penile disease. Equine papillomavirus type 2 DNA was the only papillomaviral DNA amplified from any of the 40 horses. Tissues from the 10 horses in which papillomaviral DNA was detected by polymerase chain reaction were also screened with in situ hybridization and immunohistochemistry. The presence of papillomavirus was demonstrated in a subset of these by in situ hybridization (6 of 10) and immunohistochemistry (1 of 10). This report describes a possible association between equine penile squamous cell carcinomas and equine papillomavirus type 2. This study is also the first report of equine papillomavirus type 2 infection in North American horses.Keywords genital, horse, equine papillomavirus type 2, polymerase chain reaction, in situ hybridization, immunohistochemistry, squamous cell carcinoma Squamous cell carcinomas (SCCs) constitute approximately 18% of all equine cutaneous neoplasms. 23 Of these, 45% are reported to involve the male external genitalia, 22 and SCCs are the most common neoplasm in this location. 24 Equine penile SCCs (PSCCs) commonly recur after treatment, and the long-term prognosis is poor. 5,12 Whereas ultraviolet light is an accepted cause of SCC in sun-exposed skin in some species, ultraviolet overexposure may be a less likely cause of equine PSCCs because of the ventral location of the male genitalia. Although equine PSCCs have been associated with chronic inflammation and smegma accumulation, the role of these factors in neoplasm development is unclear. 8,25 Approximately 5% of all human cancers and 50% of human PSCCs are caused by papillomavirus (PV) infection.2,16 The aim of this study is to investigate whether equine PSCCs are also associated with PV infection. The rate of detection of PVs in equine PSCCs is compared with the rate of detection in non-SCC equine penile lesions. If PVs are detected more frequently in PSCC than non-SCC lesions, this will provide evidence of a possible association between equine PSCC and PV infection. Materials and MethodsCases of equine penile disease were located by searching the archives of the Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine, Ithaca, New York. Histologic diagnoses were confirmed for this study by one of the authors (J.P.). Ages were recorded, where available. Standard tests for normality were conducted using SAS 9.1. Differentiation between samples from geldings and stallions was not possible,...

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