Advancing canine mammary tumor diagnostics: Unraveling the diagnostic potential of Cytokeratin 19 through droplet digital PCR analysis.

Cytokeratin 19 (CK19) is a complex intracytoplasmic cytoskeletal protein primarily localized in the ducts of the mammary gland and skin epithelial cells. In humans, the expression of CK19 gene within circulating tumor cells (CTCs) extracted from blood samples of breast cancer patients reflects tumor cell activity, offering valuable insights for predicting early metastatic relapse or monitoring treatment effectiveness. However, knowledge of serum tumor markers is limited in veterinary oncology. Recently, droplet digital PCR (ddPCR), has been employed to explore rare target genes due to its heightened sensitivity and accuracy as a novel molecular diagnostic tool. The objectives of this study were to investigate the expression of the CK19 mRNA in CTCs, non-neoplastic mammary tissues, and both benign and malignant canine mammary tumors (CMTs) through ddPCR analysis. In Study I, we optimized the discard volume for blood samples to reduce CK19 contamination from skin epithelial cells post-venipuncture. The results revealed that discarding the initial 3 mL of blood was adequate and effective in eliminating CK19 mRNA contamination. In Study II, after the removal of the initial 3 mL of blood, we investigated CK19 mRNA-positive CTCs in the peripheral blood of normal healthy dogs, including those with benign and malignant CMTs. Intriguingly, CK19 mRNA was undetectable in all blood samples. The expression of CK19 mRNA in mammary tissues was investigated in Study III. The copy number (CN) ratios of the CK19 gene in non-neoplastic mammary tissues (14.77 ± 14.65) were significantly higher (P < 0.05) than those in benign (4.23 ± 3.35) and malignant groups (6.56 ± 5.64). Notably, no difference was observed between the benign and malignant groups. In conclusion, CK19 mRNA appeared unlikely to be a suitable candidate as a biomarker in the peripheral blood of CMTs, while the CN ratio in mammary tissues could serve as a potential discriminator between non-neoplastic and CMT groups, complementing the gold standard of histopathological examination.

Validation of droplet digital PCR for cytokeratin 19 mRNA detection in canine peripheral blood and mammary gland.

In humans, peripheral blood cytokeratin 19 (CK19) mRNA-positive circulating tumor cells (CTCs) was utilized to identify early-stage breast cancer patients with micrometastatic disease who are at risk for disease progression and monitor treatment response in patients with advanced disease. To our knowledge, there has been little research regarding CK19 in canine mammary tumors (CMTs) using molecular methods. A droplet digital PCR (ddPCR) is proposed as a precise and sensitive quantification of nucleic acid targets. Hence, this study aimed to validate a newly designed assay for CK19 detection in canine blood and mammary tissue, along with the reference gene HPRT, by ddPCR. All primers and probes showed a precise match with the exon region of target genes. The assay exhibited PCR efficacy of 90.4% and 91.0% for CK19 and HPRT amplifications with linearity, respectively. The annealing temperature (Ta) for duplex ddPCR was 55 °C, providing the highest concentrations of both genes tested by the synthetic plasmid DNA. The limit of detection (LOD) of CK19 and HPRT were 2.16 ± 1.27 and 2.44 ± 1.31 copies/µL, respectively. Finally, the ddPCR assay was validated with canine peripheral blood, non-neoplastic mammary tissues and spiked samples. Our findings provide a new platform for CK19 studies in CMT diagnosis through blood and mammary tissues.

Impact of gonadal status on ultrasonographic renal parenchymal dimensions in healthy cats.

OBJECTIVES: The aim of this study was to evaluate the effect of gonadal status on ultrasonographic renal parenchymal dimensions in healthy cats. METHODS: Forty healthy cats (10 intact males, 10 intact females, 10 castrated males and 10 spayed females) presented to the Division of Obstetrics, Gynecology and Reproduction, and the Diagnostic Imaging Unit at The Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University. They were ultrasonographically examined to assess renal length, aortic luminal diameter, cortical thickness and medullary thickness. RESULTS: Regardless of gonadal status, the renal length, aortic luminal diameter, cortical thickness and medulla thickness of males were greater than those of females (P <0.05). In general, neutered cats had thicker medullae (0.36 ± 0.08 cm) and higher mean renal length:aortic luminal diameter ratio (12.15 ± 1.48) than intact cats (0.32 ± 0.08 cm and 11.22 ± 1.37 cm, respectively) (P <0.05), but no differences were observed in renal length, cortical thickness or aortic luminal diameter. Interestingly, when comparing between sexes with relatively equal body weight, only sex had an impact on renal length. CONCLUSIONS AND RELEVANCE: Gonadal status has an effect on medullary thickness and mean renal length:aortic luminal diameter ratio.

Optimal concentration of Rho-associated coiled-coil kinase (ROCK) inhibitor improved sperm membrane functionality and fertilizing ability of cryopreserved-thawed feline sperm.

Sperm cryopreservation induces irreversible loss of viability and fertilizing ability. This study aimed at examining the effects of Rho-associated, coiled-coil kinase (ROCK) inhibitor on quality of frozen-thawed feline sperm. Ejaculated semen from individual cats (n = 6) was examined for the expression of LIMK1 and LIMK2 mediated ROCK cascade. The effects of ROCK inhibitor during cooling and cryopreservation on sperm quality and fertilizing ability were also examined. Feline sperm were treated with different concentrations of ROCK inhibitor (10, 20 and 40 µM) during cooling at 4 °C and cryopreservation. Sperm cooled and conventionally cryopreserved without ROCK inhibitor (0 µM) served as a control group. The ROCK cascade was confirmed in feline sperm as they expressed mRNA of LIMK1 and LIMK2 genes. Cryopreservation significantly reduced sperm quality in terms of viability (91.63 ± 3.96 vs. 60.11 ± 8.93), progressive motility (91.67 ± 3.54 vs. 46.67 ± 8.66) and acrosome integrity (93.49 ± 3.64 vs. 63.81 ± 5.31) for fresh and frozen-thawed sperm, respectively (p < 0.05). The positive effects of ROCK inhibitor on sperm quality were pronounced at 1 and 3 h post-thaw. ROCK inhibitor at 10 µM significantly improved sperm motility and membrane functionality compared to those observed in a control group (0 µM) (p < 0.05). In vitro fertilization revealed that supplement ROCK inhibitor at 10 µM during cryopreservation significantly improved in vitro fertilizing ability of the frozen-thawed sperm (p < 0.05). However, it did not subsequently increase morula and blastocyst rates (p > 0.05). Increased concentrations of ROCK inhibitor to 20 and 40 µM did not further improve the quality of frozen-thawed sperm. In conclusion, an optimal concentration (10 µM) of the ROCK inhibitor added into cooling medium could improve post-thaw sperm quality.