Comparative evaluation of staining techniques in thawed cryopreserved dog semen

Abstract

Background: Accurate assessment of sperm morphology is essential for evaluating the quality of cryopreserved canine semen used in artificial insemination and for improving cryopreservation protocols. This study compared six staining techniques, Eosin, Eosin–Nigrosin, Diff-Quick®, Hemacolor®, Spermac®, and Formol-citrate Bengal Rose, for light-microscopic evaluation of frozen–thawed canine spermatozoa. Methods: Semen from ten dogs was thawed, divided into four aliquots, and either left untreated or exposed to thermal stress at 6 °C, 18 °C, or 37 °C for two hours to induce morphological variation. A total of 360 slides and 960 evaluations were performed immediately after staining and again after 24 h, 7 days, and 3 months to assess staining quality and stability over time. Results: Eosin produced stable staining for up to three months and was the most economical method, though its initial detail recognition was lower than that of Spermac® and Formol-citrate Bengal Rose. Eosin–Nigrosin showed reduced contrast and detail. Diff-Quick® provided better contrast than Eosin–Nigrosin, while Hemacolor® maintained consistent quality regardless of stress treatment or storage duration. Spermac® yielded the highest initial morphological detail but deteriorated during storage. Formol-citrate Bengal Rose combined high detail recognition with stable staining throughout the study. Cryopreservation increased looped tails, and incubation at 37 °C markedly elevated pathological sperm and rudimentary tails. Conclusions: All six staining methods were suitable for evaluating and archiving frozen–thawed canine semen. Formol-citrate Bengal Rose and Spermac® offered the best detail, while Eosin provided a cost-effective option with excellent long-term stability.

Extracellular vesicles (EVs) have become a key area of research, as scientists study their role in various biological processes. These vesicles appear to play a key role in the use of mesenchymal signaling cells (MSCs, formerly known as mesenchymal stem cells) to treat various diseases, such as osteoarthritis (OA), and other degenerative conditions. In our experiments, we examined EVs formed by canine mesenchymal signaling cells (MSCs) to identify them according to current guidelines and define their content, particularly the microRNA (miRNA) they contain, for future research projects. After obtaining the EVs, we demonstrated via Western blotting and transmission electron microscopy that the nanoparticles visible in the nanotracking analysis were positive for CD9 and ALIX and positive for CD9 and CD81, respectively. Markers for nanoparticles that do not represent extracellular particles—tested here as cytochrome C for mitochondrial particles and histones for nuclear particles—were negative. Finally, we detected a total of 85 different miRNAs in the negative controls. To determine the potential influence of various cell stimulations intended to induce osteoarthritis (e.g., interleukin-1β stimulation) or a possible treatment (e.g., shockwave therapy), or the influence of ITS prior to extracellular vesicle extraction, we detected a total of 208 different miRNAs. These results demonstrate how canine EVs from MSCs can be detected in vitro and how the EVs’ miRNA profile changes after stimulation of the producing cells. This information may provide valuable insight into the understanding and treatment of osteoarthritis. Additionally, we demonstrated that using ITS instead of FCS to produce EVs should be reconsidered due to the significant change in miRNA expression levels.