Cross-species oncogenomics offers insight into human muscle-invasive bladder cancer.

BACKGROUND: In humans, muscle-invasive bladder cancer (MIBC) is highly aggressive and associated with a poor prognosis. With a high mutation load and large number of altered genes, strategies to delineate key driver events are necessary. Dogs and cats develop urothelial carcinoma (UC) with histological and clinical similarities to human MIBC. Cattle that graze on bracken fern also develop UC, associated with exposure to the carcinogen ptaquiloside. These species may represent relevant animal models of spontaneous and carcinogen-induced UC that can provide insight into human MIBC. RESULTS: Whole-exome sequencing of domestic canine (n = 87) and feline (n = 23) UC, and comparative analysis with human MIBC reveals a lower mutation rate in animal cases and the absence of APOBEC mutational signatures. A convergence of driver genes (ARID1A, KDM6A, TP53, FAT1, and NRAS) is discovered, along with common focally amplified and deleted genes involved in regulation of the cell cycle and chromatin remodelling. We identify mismatch repair deficiency in a subset of canine and feline UCs with biallelic inactivation of MSH2. Bovine UC (n = 8) is distinctly different; we identify novel mutational signatures which are recapitulated in vitro in human urinary bladder UC cells treated with bracken fern extracts or purified ptaquiloside. CONCLUSION: Canine and feline urinary bladder UC represent relevant models of MIBC in humans, and cross-species analysis can identify evolutionarily conserved driver genes. We characterize mutational signatures in bovine UC associated with bracken fern and ptaquiloside exposure, a human-linked cancer exposure. Our work demonstrates the relevance of cross-species comparative analysis in understanding both human and animal UC.

A novel and simple heat-based method eliminates the highly detrimental effect of xylene deparaffinization on acid-fast stains.

OBJECTIVES: Histopathology is an important method for diagnosing extrapulmonary tuberculosis, yet tissue sections are often negative for mycobacteria after use of acid-fast stain (AFS). This study investigated the mechanism of AFS use and the detrimental effect of histologic processing-in particular, xylene deparaffinization-on AFS and mycobacterial detection. METHODS: The target of the fluorescent Auramine O (AuO) AFS was investigated using triple staining with DNA- and RNA-specific dyes. The effect of xylene deparaffinization on the acid fastness of mycobacteria in cultures or tissue sections was studied using AuO fluorescence as a quantitative marker. The xylene method was compared with a novel, solvent-free projected-hot-air deparaffinization (PHAD). RESULTS: Co-localization of AuO with DNA/RNA stains suggests that intracellular nucleic acids are the true target of AFS, producing highly specific patterns. Xylene reduces mycobacterial fluorescence significantly (P < .0001; moderate effect size, r = 0.33). The PHAD process yielded significantly higher fluorescence than xylene deparaffinization in tissues (P < .0001; large effect size, r = 0.85). CONCLUSIONS: Auramine O can be applied for nucleic acid staining of mycobacteria in tissues producing typical beaded patterns. Acid-fast staining depends heavily on the integrity of the mycobacterial cell wall, which xylene appears to damage. A solvent-free tissue deparaffinization method has the potential to increase mycobacterial detection significantly.

Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Vet-OncoNet: Malignancy Analysis of Neoplasms in Dogs and Cats.

Analysis of canine and feline tumor malignancy data can help clinicians identify high-risk patients and make more accurate decisions. Based on a sample of 16,272 cancer records, including 3266 cats and 13,006 dogs, collected from January 2019 to December 2021 in the Vet-OncoNet Network database, this study aimed to compare the tumor malignancy profile between cats and dogs, considering animal-related factors (sex, age, and breed), topography, and geographic location using a mixed-effects logistic regression model. Cats had a higher proportion of malignant tumors (78.7%) than dogs (46.2%), and the malignancy profile was very different regarding tumors' topographies. The mean age of malignant tumors occurred eight months later than benign ones (9.1, SD = 3.4; 9.8, SD = 3.2), in general. Species (OR = 3.96, 95%CI 3.57: 4.39) and topography (MOR = 4.10) were the two most important determinants of malignancy risk. Female dogs had a higher risk than male dogs (OR = 1.19, 95%CI 1.08: 1.31), which does not appear to be the case in cats (OR = 0.98, 95%CI 0.77: 1.23). Breed contributed significantly to differences in malignancy risk in dogs (MOR = 1.56), particularly in pit bulls and boxers. District of residence was not so relevant in predicting malignancy risk (MOR = 1.14). In both species, the risk of malignancy increased by approximately 20% every three years. It could be hypothesized that species differences in genetic structure may contribute to tumor malignancy.

Cross Species Analysis and Comparison of Tumors in Dogs and Cats, by Age, Sex, Topography and Main Morphologies. Data from Vet-OncoNet.

The animal cancer burden is essential for the translational value of companion animals in comparative oncology. The present work aims to describe, analyze, and compare frequencies and associations of tumors in dogs and cats based on the Animal Cancer Registry created by Vet-OncoNet. With 9079 registries, regarding 2019 and 2020, 81% (n = 7355) belonged to dogs. In comparison, cats have a general one-year right advance in the mean age of cancer diagnosis compared to dogs. The multivariate topography group analysis shows a distinct pattern between the two species: dogs have higher odds of cancer in the genito-urinary system, spleen, soft tissue tumors and skin, while cats show higher odds for tumors in the eyes, digestive organs, nasal cavity, lymph nodes, bones and mammary glands. Regarding morphologies, dogs are overrepresented in mast cell tumors (MCT), melanomas, and hemangiosarcomas. While cats are overrepresented in fibrosarcomas, lymphomas (T and B-cell), in malignant mammary tumors, and squamous cell carcinoma (SCC). Females have greater odds only in the mammary gland, with males having greater odds in six of twelve topographies. This study is the first outcome of continuous animal cancer registration studies in Portugal.

Evaluation of Commercial Myxomatosis Vaccines against Recombinant Myxoma Virus (ha-MYXV) in Iberian Hare and Wild Rabbit.

The recent emergence of a new myxoma virus capable of causing disease in the Iberian hare (Lepus granatensis) has resulted in numerous outbreaks with high mortality leading to the reduction, or even the disappearance, of many local populations of this wild species in the Iberian Peninsula. Currently, the available vaccines that prevent myxomatosis in domestic rabbits caused by classic strains of myxoma virus have not been assessed for use in Iberian hares. The main objective of this study was to evaluate the efficacy of commercial rabbit vaccines in Iberian hares and wild rabbits against the natural recombinant myxoma virus (ha-MYXV), bearing in mind its application in specific scenarios where capture is possible, such as genetic reserves. The study used a limited number of animals (pilot study), 15 Iberian hares and 10 wild rabbits. Hares were vaccinated with Mixohipra-FSA vaccine (Hipra) and Mixohipra-H vaccine (Hipra) using two different doses, and rabbits were vaccinated with the Mixohipra-H vaccine or the Nobivac Myxo-RHD PLUS (MSD Animal Health) using the recommended doses for domestic rabbits. After the vaccination trials, the animals were challenged with a wild type strain of ha-MYXV. The results showed that no protection to ha-MYXV challenge was afforded when a commercial dose of Mixohipra-FSA or Mixohipra-H vaccine was used in hares. However, the application of a higher dose of Mixohipra-FSA vaccine may induce protection and could possibly be used to counteract the accelerated decrease of wild hare populations due to ha-MYXV emergence. The two commercial vaccines (Mixohipra-H and Nobivac Myxo-RHD PLUS) tested in wild rabbits were fully protective against ha-MYXV infection. This knowledge gives more insights into ha-MYXV management in hares and rabbits and emphasises the importance of developing a vaccine capable of protecting wild populations of Iberian hare and wild rabbit towards MYXV and ha-MYXV strains.

A Quadruplex qPCR for Detection and Differentiation of Classic and Natural Recombinant Myxoma Virus Strains of Leporids.

A natural recombinant myxoma virus (referred to as ha-MYXV or MYXV-Tol08/18) emerged in the Iberian hare (Lepus granatensis) and the European rabbit (Oryctolagus cuniculus) in late 2018 and mid-2020, respectively. This new virus is genetically distinct from classic myxoma virus (MYXV) strains that caused myxomatosis in rabbits until then, by acquiring an additional 2.8 Kbp insert within the m009L gene that disrupted it into ORFs m009L-a and m009L-b. To distinguish ha-MYXV from classic MYXV strains, we developed a robust qPCR multiplex technique that combines the amplification of the m000.5L/R duplicated gene, conserved in all myxoma virus strains including ha-MYXV, with the amplification of two other genes targeted by the real-time PCR systems designed during this study, specific either for classic MYXV or ha-MYXV strains. The first system targets the boundaries between ORFs m009L-a and m009L-b, only contiguous in classic strains, while the second amplifies a fragment within gene m060L, only present in recombinant MYXV strains. All amplification reactions were validated and normalized by a fourth PCR system directed to a housekeeping gene (18S rRNA) conserved in eukaryotic organisms, including hares and rabbits. The multiplex PCR (mPCR) technique described here was optimized for Taqman® and Evagreen® systems allowing the detection of as few as nine copies of viral DNA in the sample with an efficiency > 93%. This real-time multiplex is the first fast method available for the differential diagnosis between classic and recombinant MYXV strains, also allowing the detection of co-infections. The system proves to be an essential and effective tool for monitoring the geographical spread of ha-MYXV in the hare and wild rabbit populations, supporting the management of both species in the field.

A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies.

In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.

A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit.

Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.

Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus).

In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains' ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.

Establishment of a bioluminescent canine B-cell lymphoma xenograft model for monitoring tumor progression and treatment response in preclinical studies.

Canine diffuse large B-cell lymphoma (DLBCL) is one of the most common cancers in dogs which shares remarkable similarities with its human counterpart, making the dog an excellent model for the investigation of novel therapeutic agents. However, the integration of canine lymphoma in comparative studies has been limited due in part to the lack of suitable xenograft mouse models for preclinical studies. To overcome these limitations, we established and characterized a localized subcutaneous bioluminescent canine DLBCL xenograft mouse model. The canine CLBL-1 cell line stably expressing the luciferase and green fluorescent protein reporters was generated and used to establish the xenograft tumor model. A pilot study was first conducted with three different cell densities (0.1×10(6), 0.5×10(6) and 1×10(6) cells) in SCID mice. All mice presented homogeneous tumor induction within eight days after subcutaneous injection, with a 100% engraftment efficiency and no significant differences were observed among groups. The tumors were highly aggressive and localized at the site of inoculation and reproduced histological features and immunophenotype consistent with canine DLBCL. Importantly, xenograft tumors were detected and quantified by bioluminescent imaging. To assess response to therapy, a therapeutic study with a histone deacetylase inhibitor, panobinostat, was performed. The results demonstrated that panobinostat (20 mg/kg) efficiently inhibited tumor growth and that bioluminescent imaging allowed the monitorization and quantification of tumor response to therapy. In summary, this study provides a bioluminescence canine DLBCL model that offers high engraftment efficiency, preservation of tumor features, and noninvasive monitoring of tumor progression, validating the model as a promising preclinical tool for both veterinary and human medicine.

Comparison of Histological and Proliferation Features of Canine Oral Squamous Cell Carcinoma Based on Intraoral Location: 36 Cases.

Grade and labeling indices for immunohistochemical tumor proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) were evaluated in 36 cases of canine oral squamous cell carcinoma (OSCC) based upon intraoral location. Grade was significantly associated with location ( P = .035). Grade II tumors were most frequently diagnosed. Grade I tumors were identified in the gingiva and the buccal mucosa, and grade III tumors were seen in the gingiva and the tonsillar region. Animals with tumors arising from the tonsils and of the tongue tended to be older ( P = .007), and those in the former group were more likely to have metastatic disease at the time of diagnosis ( P = .001). Mean expression of PCNA and Ki-67 proliferation index (PI) for all tumors were 62.54% and 50.70%, respectively, and there was a statistical significant association between the 2 variables ( R = .70; P < .001). Proliferation index was not associated with any of the intraoral locations evaluated, but higher PCNA PI was significantly associated with grade ( P = .031). Ki-67 PI was significantly associated with lymph node metastasis at the time of diagnosis, especially for OSCC of gingival location ( P = .028). The results obtained in this study are preliminary but clinically relevant, since they provide information that can explain differences in biologic behavior among intraoral locations and contribute to more accurate tumor staging to support the choice for different treatment strategies available for OSCC.

Late prostatic metastasis of an uveal melanoma in a miniature Schnauzer dog.

This manuscript describes a previously unreported clinical case of canine uveal melanoma in a miniature Schnauzer dog with an unusual location of metastasis (prostate) and delayed occurrence (3 years after primary tumor diagnosis and enucleation). Immunohistochemical labeling of both tumors with Melan A, Ki-67, and c-kit added some valuable information.

Mitochondrial genetic diversity, selection and recombination in a canine transmissible cancer.

Canine transmissible venereal tumour (CTVT) is a clonally transmissible cancer that originated approximately 11,000 years ago and affects dogs worldwide. Despite the clonal origin of the CTVT nuclear genome, CTVT mitochondrial genomes (mtDNAs) have been acquired by periodic capture from transient hosts. We sequenced 449 complete mtDNAs from a global population of CTVTs, and show that mtDNA horizontal transfer has occurred at least five times, delineating five tumour clades whose distributions track two millennia of dog global migration. Negative selection has operated to prevent accumulation of deleterious mutations in captured mtDNA, and recombination has caused occasional mtDNA re-assortment. These findings implicate functional mtDNA as a driver of CTVT global metastatic spread, further highlighting the important role of mtDNA in cancer evolution.

Notch signaling dynamics in the adult healthy prostate and in prostatic tumor development.

BACKGROUND: The Notch signaling pathway has been implicated in prostate development, maintenance and tumorigenesis by its key role in cell-fate determination, differentiation and proliferation. Therefore, we proposed to analyze Notch family members transcription and expression, including ligands (Dll1, 3, 4 and Jagged1 and 2), receptors (Notch1-4) and effectors (Hes1, 2, 5 and Hey1, 2, L), in both normal and tumor bearing mouse prostates to better understand the dynamics of Notch signaling in prostate tumorigenesis. METHODS: Wild type mice and transgenic adenocarcinoma of the mouse prostate model (TRAMP) mice were sacrificed at 18, 24 or 30 weeks of age and the prostates collected and processed for either whole prostate or prostate cell specific populations mRNA analysis and for protein expression analysis by immunohistochemistry and immunofluorescence. RESULTS: We observed that Dll1 and Dll4 are expressed in the luminal compartment of the mouse healthy prostate, whereas Jagged2 expression is restricted to the basal and stromal compartment. Additionally, Notch2 and Notch4 are normally expressed in the prostate luminal compartment while Notch2 and Notch3 are also expressed in the stromal layer of the healthy prostate. As prostate tumor development takes place, there is up-regulation of Notch components. Particularly, the prostate tumor lesions have increased expression of Jagged1 and 2, of Notch3 and of Hey1. We have also detected the presence of activated Notch3 in prostatic tumors that co-express Jagged1 and ultimately the Hey1 effector. CONCLUSIONS: Taken together our results point out the Notch axis Jagged1-2/Notch3/Hey1 to be important for prostate tumor development and worthy of additional functional studies and validation in human clinical disease.

Endothelial Jagged1 promotes solid tumor growth through both pro-angiogenic and angiocrine functions.

Angiogenesis is an essential process required for tumor growth and progression. The Notch signaling pathway has been identified as a key regulator of the neo-angiogenic process. Jagged-1 (Jag1) is a Notch ligand required for embryonic and retinal vascular development, which direct contribution to the regulation of tumor angiogenesis remains to be fully characterized. The current study addresses the role of endothelial Jagged1-mediated Notch signaling in the context of tumoral angiogenesis in two different mouse tumor models: subcutaneous Lewis Lung Carcinoma (LLC) tumor transplants and the autochthonous Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP). The role of endothelial Jagged1 in tumor growth and neo-angiogenesis was investigated with endothelial-specific Jag1 gain- and loss-of-function mouse mutants (eJag1OE and eJag1cKO). By modulating levels of endothelial Jag1, we observed that this ligand regulates tumor vessel density, branching, and perivascular maturation, thus affecting tumor vascular perfusion. The pro-angiogenic function is exerted by its ability to positively regulate levels of Vegfr-2 while negatively regulating Vegfr-1. Additionally, endothelial Jagged1 appears to exert an angiocrine function possibly by activating Notch3/Hey1 in tumor cells, promoting proliferation, survival and epithelial-to-mesenchymal transition (EMT), potentiating tumor development. These findings provide valuable mechanistic insights into the role of endothelial Jagged1 in promoting solid tumor development and support the notion that it may constitute a promising target for cancer therapy.

Detection and quantification of bovine papillomavirus type 2 in urinary bladders and lymph nodes in cases of Bovine Enzootic Hematuria from the endemic region of Azores.

Bovine Enzootic Hematuria (BEH) is a disease with a severe impact on production indexes and characterized by the development of bovine urinary bladder tumors, particularly in the Azores archipelago. The purpose of this study was to investigate and quantify BPV2 tissue distribution in bovine urinary bladder tumors, normal bladders, and iliac lymph nodes of cattle from the Azores. A real-time PCR system targeting the L1 gene was developed and allowed for the specific detection of the virus. BPV2 DNA was detected in a large proportion of the samples tested, both from neoplastic and healthy tissues, indicating that this virus is very prevalent in the bovine population of the Azores. Moreover, all types of tissues tested were positive, confirming a wide viral distribution within the infected animal. Bovine cutaneous papillomas sampled from Portuguese mainland dairy cattle were used as controls. Viral load ranged between 2.2×10(4) copies/cell in the skin papillomas, and 0.0002 copies/cell in the urinary bladders tumors from the Azores. This is the first report presenting quantitative data on BPV2 infection in bovine urinary bladder lesions from the Azores. This approach will provide a useful tool to evaluate the role of BPV2 not only in the pathogenesis BEH but also in cell transformation mechanisms.

Influence of temperature and humidity manipulation on chicken embryonic development.

BACKGROUND: Temperature and relative humidity (RH) are very important factors affecting embryo development, hatchability, and posthatch performance. This study aimed at characterizing embryonic metabolic and behavioural response to a harsh incubation environment generated by manipulations (elevations and drops) in these two key factors. This study was aimed at establishing patterns of metabolic and behavioural response, as well as mortality and the development of malformations, all of which can potentially be used in monitoring incubating operations and diagnosing problems with faulty equipment. RESULTS: Of all the parameters monitored throughout embryonic development the ones shown to be most affected were: albumen-weight to egg-weight ratio (AR); yolk-weight to egg-weight ratio (YR); embryo-weight to egg-weight ratio (ER); heart rate (HR); voluntary movements per minute (VMM); mortality rates; malformation prevalence and type. The most significant changes in the evolution of AR and YR throughout incubation involved delay and reduction in the amplitude of the expected drop in albumen and yolk levels, reflecting lower nutrient consumption by the embryo. ER tended to grow more slowly and remain lower than the established normal, especially in embryos challenged with temperature treatments. HR and VMM were considered to be strong indicators of embryonic stress, as all treatments applied resulted in elevated heart rate and decreased embryo movement. Mortality rates for both temperature-related treatments were higher during the first four days of incubation. Changes in relative humidity have produced less radical effects on mortality. Malformation rates were higher for embryos subjected to high incubation temperatures and were most prominently related to the abdominal wall, head, skull and limbs. Overall, manipulations in environmental (incubator) temperature during incubation produced more drastic changes in embryo development than humidity-related manipulations, especially where mortality and malformation rates were concerned. CONCLUSIONS: This paper describes changes in embryonic metabolism and behaviour, as well as in mortality and malformation rates, in response to manipulations in environmental temperature and relative humidity. Together with further studies, these patterns may prove helpful in the diagnosis of equipment malfunctions relating to temperature or relative humidity.

Amelanotic uveal cyst in a Yorkshire terrier dog.

The diagnostic challenge presented by an amelanotic uveal cyst with an atypical appearance in a 9-year-old Yorkshire terrier dog is reported. The dog was presented with a peculiar cystic neoformation adherent to the edge of the pupil of the right eye. The cyst wall was attached to the pupillary margin and it was bean-shaped, measuring approximately 4.5 × 2.5 mm. It was white in colour with several red striations and a small brown spot in the middle, which conferred on it a peculiar appearance. The cyst could not be transilluminated and partially impaired vision. Apart from that, the ophthalmic exam revealed no other abnormalities and the eye showed no signs of inflammation. Ocular ultrasound revealed the cystic nature of the neoformation. During paracentesis of the anterior chamber, the cyst was deflated and both the cyst wall and fluid were aspirated. The tissue obtained was sent for a histological examination and was considered as corresponding to a uveal cyst. The dog improved from the post-operative uveitis without any complication and after 24 months of follow-up showed no recurrences.

Uterine adenocarcinoma with pulmonary, liver and mesentery metastasis in a holstein cow.

The clinical and pathology features of a cow with uterine adenocarcinoma and multiple metastasis are described. Weight loss, inappetence, mild respiratory signs, and reduced milk yield were evident on clinical examination. Grossly deformed uterus, enlarged iliac lymph nodes, and rosary arranged nodules in the mesentery were felt by rectal palpation. Right side laparotomy revealed numerous small masses covering the omentum, and mesentery. Euthanasia was performed. Necropsy and histopathology exam revealed a uterine adenocarcinoma with multiple pulmonary, liver and mesentery metastasis. Uterine adenocarcinoma with metastasis should be included in the differential diagnosis of cows showing weight loss and mild respiratory distress and palpation of numerous firm nodules in the mesentery should be suggestive of neoplasias' metastasis.