Mammary gland tumors and pyometra are important health hazards in canines and are frequently associated with ovarian hormonal abnormalities¹. Ovariohysterectomy performed at an early age, minimizes the prolonged exposure of uterus and mammary tissues to reproductive hormones and thereby reduces the risk of developing both mammary gland tumors and pyometra¹. Surgical excision is the first line of treatment for both mammary gland tumors and pyometra, in order to curtain the hormonal influence, which arrest the tumor recurrence. In present study infected uterus and mammary gland tumors were removed simultaneously and recorded the clinical pathology, serum clinical parameters, hormonal profile, cytology, histopathology and immunohistochemistry.

Three female bitches with abnormal mammary masses and concurrently with pyometra were brought for the treatment to Veterinary Clinical Complex, Veterinary College and Research Institute, TANUVAS, Namakkal, Tamil Nadu. Age 9, 8 and 9 years respectively were included in the study. Blood samples for haemato-biochemistry and fine needle aspirations were obtained for clinical pathology. Among three bitches, both mammary tumor mass and uterus were removed from Spid, only mammary tumor mass from Labrador Retriever and Spid cross due to severe anaemia (Packed cell volume – 24.9%) was unfit for surgical intervention.

Peripheral blood samples were collected by venipuncture in K₃EDTA coated vacutainers. The haematology parameters such as haemoglobin, packed cell volume, total erythrocyte count, erythrocytic indices, total and absolute leukocyte count, differential leukocyte counts and platelet counts were analysed by using VetScan HM2, USA. Serum separated were subjected for evaluation of biochemical parameters such as albumin, total protein, glucose, cholesterol, triglycerides, blood urea nitrogen, creatinine and electrolytes (calcium, phosphorus, magnesium, sodium, potassium and chloride) by using A50 Biosystems, India. Serum hormone profile of estradiol, progesterone and testosterone were analysed by radioimmunoassay² using¹²⁵ I labelled antigen and antibody coated tubes with standards (Immunotech, Radiova, Czech Republic) and radioactivity was measured by using Gamma Counter, Stratec, Germany.

Plain thoracic and abdominal radiographs were taken in left lateral view for each animal to assess the metastases in the lungs and pathology of uterus, respectively using Wipro GE 525 DX X ray unit with exposure of 65-80 kVp, 24-30 mAs and source image distance of 100 cm were employed. The exposed film was processed with Carestream Vitaflex computerised radiography X ray film processor. Fine needle aspirates of the tumor masses were used for preparation of cytological smears and stained with supravital stains.

The mammary tumor masses and infected uterus from affected dogs were removed by aseptic surgical procedures under general anaesthesia. Gross observations such as location, weight, size, length and width were recorded. Representative pieces of tumor mass was collected and fixed in 10 percent formalin. They were processed, embedded in paraffin, sectioned at 3-5µm thickness and stained with haematoxylin and eosin (H&E)³.

The paraffin embedded tissue sections were taken on 0.01% Poly-L-lysine coated slides for immunohistochemical demonstration of pan-cytokeratin and vimentin. The slides was placed in coplin jar with 1M Tris EDTA (pH 9) for antigen retrieval in pressure cooker for 15 min at 120˚C. Quenching of peroxidase was carried out by incubating with peroxide block (3 per cent hydrogen peroxide in water) for 10 min. Tissue sections were incubated with primary antibodies (ready to use procured from Pathnsitu biotechnologies, Hyderabad) for pan-cytokeratin and vimentin for 30 min at room temperature and followed by HRPO conjugated secondary antibody for 30 min at room temperature and then counterstained with Mayer’s haematoxylin⁴.

The mean values of haematological parameters are presented in Table. The blood picture showed moderate anaemia and leukocytosis with left shift. The other haematological parameters falls within normal range. The mean values of serum biochemical parameters and sex steroid hormones are presented in Table. Serum biochemistry showed decreased protein and electrolyte levels and increased cholesterol, blood urea nitrogen and creatinine values. Serum hormone levels of estradiol and progesterone were increased with no alteration in testosterone. Radiography revealed moderately distended uterus with partial displacement of intestine (Fig. 1).

Spitz and Labrador Retriever dogs showed the tumor mass in left inguinal mammary gland (Fig. 2, 3), whereas, it was observed in right inguinal mammary gland in Spid cross dog. The infected uterus removed surgically was edematous, turgid and the lumen was filled with about 200 ml of yellowish creamy pus. The weight and size of the tumor masses excised from Spid were 40g and 5cm×4cm and Labrador 20g and 4 cm × 2 cm. The consistency of the tumor masses were hard or firm and rarely indurated. The indurated tumors were often cartilaginous produce gritty sound on cutting. Fine needle aspiration cytology of tumor masses revealed sheet of neoplastic cells with anisocytosis, anisokaryosis, hyperchromasia, increased nuclear to cytoplasmic ratio, nuclear molding and basophilic cytoplasm (Fig. 4).

Histopathological examination of tumor mass from Spid and Labrador Retriever revealed mixed mammary carcinoma. It was characterised by neoplastic population of glandular epithelial cells, fibroblasts and chondroid cells (Fig. 5). The glandular epithelial cells were highly proliferative and comprised of numerous secretory acini surrounded by fibroblasts. Acini showed presence of eosinophilic secretions and they were lined by 2-3 layers of neoplastic cells. Neoplastic cells revealed spherical to oval shaped darkly stained nuclei with multiple mitotic figures. Fibroblasts were spindle shaped with fusiform shaped nuclei exhibiting wavy appearance. Chondroid cells exhibited foci of cartilagenous area with no atypia and degeneration (Fig. 6). It also revealed small clusters of neoplastic cells in a chondroid matrix surrounded by epithelial compartment in the form of cystic tubules (Fig. 7). These tubules were lined by a single layer of neoplastic cells and luminal surface showing scanty secretions mixed with numerous neoplastic and inflammatory cells. Angiogenesis with formation of new blood vessels was noticed in few areas. It was also characterised by metaplastic transformation of chondrocytes to osteocytes with islands of tumor osteoid or bony trabeculae which is attempting to form medullary cavity in the peripheral parts of the cartilagenous plates (Fig. 8).

Immunohistochemical examination of tumor masses revealed strong positive expression to pan-cytokeratin and vimentin. Luminal epithelial cells showed strong (5+) positive cytoplasmic expression to pan-cytokeratin (Fig. 9) whereas mesenchymal compartments such as chondroid cells showed moderate (3+) positive cytoplasmic expression to vimentin (Fig. 10).

The present study discussed the pathological features of canine mixed mammary tumor with concurrent occurrence of pyometra. Clinical signs of abdominal distension, vomition, polyuria, polydipsia, vaginal discharge recorded in this study were in accordance with earlier reports^5,6. Less marked haematological changes such as moderate anaemia and leukocytosis with left shift might be due to open type pyometra⁵. Hypoproteinemia was not in accordance with earlier findings⁵ who reported hyperproteinemia which might be due to toxaemia induced hepatocellular damage. Electrolyte disturbances might be due to abnormal calcitonin secretion in mammary mesenchymal tumors. The present study showed two-fold elevation of blood urea nitrogen and four-fold elevation of creatinine were similar to previous report⁶. The increased cholesterol could be related to fatty diet which predisposes the animal to develop mammary tumors by altering the hormonal levels.

Further, the present study confirmed that elevated estradiol and progesterone are responsible for the development of mammary tumor and pyometra, respectively^7,8. Cytological findings of morphological variation of tumor cells were well compared⁹. Mixed type carcinoma was consistent with reports of earlier findings¹⁰. Immunohistochemistry revealed positive cytoplasmic expression to pan-cytokeratin and vimentin were in accordance with previous study^11,12. Histopathology and immunohistochemistry of tumor masses confirmed that the bitches were affected with malignant mammary tumors. Clinical pathological parameters of anaemia, leukocytosis with left shift, elevated blood urea nitrogen, creatinine and progesterone which might be due to pyometra, whereas decreased protein and electrolyte levels and increased cholesterol and estradiol which could be due to mammary tumor. Concurrent removal of both uterus and tumor in the present study eliminated the hormonal source which was highly helpful in preventing the recurrence rate.

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