The aim of this study was to evaluate the influence of Hoechst 33342 (H-42) concentration and of the male donor on the efficiency of sex-sorting procedure in canine spermatozoa. Semen samples from six dogs (three ejaculates/dog) were diluted to 100 × 10(6) sperm/ml, split into four aliquots, stained with increasing H-42 concentrations (5, 7.5, 10 and 12.5 μl, respectively) and sorted by flow cytometry. The rates of non-viable (FDA+), oriented (OS) and selected spermatozoa (SS), as well as the average sorting rates (SR, sorted spermatozoa/s), were used to determine the sorting efficiency. The effects of the sorting procedure on the quality of sorted spermatozoa were evaluated in terms of total motility (TM), percentage of viable spermatozoa (spermatozoa with membrane and acrosomal integrity) and percentage of spermatozoa with reacted/damaged acrosomes. X- and Y-chromosome-bearing sperm populations were identified in all of the samples stained with 7.5, 10 and 12.5 μl of H-42, while these two populations were only identified in 77.5% of samples stained with 5 μl. The values of OS, SS and SR were influenced by the male donor (p < 0.01) but not by the H-42 concentration used. The quality of sorted sperm samples immediately after sorting was similar to that of fresh samples, while centrifugation resulted in significant reduction (p < 0.05) in TM and in the percentage of viable spermatozoa and a significant increase (p < 0.01) in the percentage of spermatozoa with damage/reacted acrosomes. In conclusion, the sex-sorting of canine spermatozoa by flow cytometry can be performed successfully using H-42 concentrations between 7.5 and 12.5 μl. The efficiency of the sorting procedure varies based on the dog from which the sperm sample derives.

The aim of this study was to evaluate the effects of rapid cooling prior to freezing on frozen-thawed canine sperm quality. In experiment 1, centrifuged ejaculates from 6 dogs were pooled, split into 4 aliquots and cryopreserved by the Uppsala procedure using different cooling rates (control, cooling speed 18 C/90 min and average cooling rate 0.2 C/min; rapid, cooling speed 18 C/8 min and average cooling rate 2.25 C/min) in combination with 2 glycerol addition protocols (fractionated or unfractionated). In experiment 2, centrifuged ejaculates from 4 dogs were processed individually using the same cooling rates described in experiment 1 in combination with an unfractionated glycerol addition protocol. Each of the experiments was replicated 5 times. Sperm quality was evaluated after 30 and 150 min of post-thawing incubation at 38 C. Total motility (TM), progressive motility (PM) and quality of movement parameters were assessed using a computerized system, and sperm viability (spermatozoa with intact plasma and acrosome membranes) was assessed using flow cytometry (H-42/PI/FITC-PNA). Values for TM, PM, viable spermatozoa and the quality of movement parameters after thawing were not significantly affected by the cooling rate. The interaction between the cooling rate and the added glycerol protocol was not significant. There were significant differences among the males (P<0.01) in the sperm quality parameters evaluated after thawing. The interaction between the males and the cooling rate was not significant. In conclusion, canine spermatozoa can be cryopreserved using the Uppsala method at an average cooling rate of 2.25 C/min prior to freezing together with addition of fractionated or unfractionated glycerol.

No abstract

A thirteen‐year‐old female intact mixed dog was presented to the emergency service with a history of vomiting and apathy. The bitch was under treatment of prednisone for chronic cough. The patient was depressed and showed abdominal pain. Abdominal radiographs revealed an increased liver size, a rounded structure of gas opacity located within the liver and a soft tissue opacity mass in the caudal abdomen. The ultrasonographic findings of the abdomen were consistent with emphysematous hepatic abscess (HA), vacuolar hepatopathy, focal peritonitis and pyometra. Surgical drainage, lavage and omentalisation of the liver abscess were performed, followed by an ovariohysterectomy. Twelve hours after surgery, the patient died due to the suspected multiple organ failure. Culture of the abscess and uterine fluid revealed presence of Burkholderia cepacia. To the authors’ knowledge, this report represented the first description of HA presumably caused by the haematogeneous spread of Burkholderia cepacia from a uterine infection.

Granulosa cell tumour is a common ovarian pathology in bitches. Depending on its hormonal production, this tumour often induces persistent oestrus signs and uterine disorders. Chronic treatment with progesterone contraceptives has been associated with endometrial mineralisation in bitches. The authors describe the case of a three-year-old female entire mastiff dog with a one-year history of chronic hyperoestrogenism signs and anorexia. Serum biochemistry and haematology revealed moderate leucocytosis. Abdominal ultrasound showed dilated uterine horns with anechoic fluid in the lumen, small cystic structures, and multiple stippled hyperechoic lesions with mild acoustic shadowing and comet tail artefact along the endometrial wall. Furthermore, cystic structures were observed in the right ovary. After surgery, histopathological study revealed the presence of endometrial hyperplasia-pyometra complex, endometrial mineralisation and a granulosa cell tumour. To the best of the authors’ knowledge, this is the first report of endometrial mineralisation associated with ovarian granulosa cell tumour in a bitch.