Proteomic Analysis of Intracellular and Membrane-Associated Fractions of Canine (Canis lupus familiaris) Epididymal Spermatozoa and Sperm Structure Separation.

This study was provided for proteomic analysis of intracellular and membrane-associated fractions of canine (Canis lupus familiaris) epididymal spermatozoa and additionally to find optimal sonication parameters for the epididymal sperm morphological structure separation and sperm protein isolation. Sperm samples were collected from 15 dogs. Sperm protein fractions: intracellular (SIPs) and membrane-associated (SMAPs) were isolated. After sonication, sperm morphology was evaluated using Spermac Stain™. The sperm protein fractions were analyzed using gel electrophoresis (SDS-PAGE) and nanoliquid chromatography coupled to quadrupole time-of-flight mass spectrometry (NanoLC-Q-TOF/MS). UniProt database-supported identification resulted in 42 proteins identified in the SIPs and 153 proteins in the SMAPs. Differentially abundant proteins (DAPs) were found in SIPs and SMAPs. Based on a gene ontology analysis, the dominant molecular functions of SIPs were catalytic activity (50%) and binding (28%). Hydrolase activity (33%) and transferase activity (21%) functions were dominant for SMAPs. Bioinformatic analysis of SIPs and SMAPs showed their participation in important metabolic pathways in epididymal sperm, which may suggest their potential as sperm quality biomarkers. The use of sonication 150 W, 10 min, may be recommended for the separation of dog epididymal sperm heads, tails, acrosomes and the protein isolation.

Effect of Extender Supplementation with Low-molecular-weight Antioxidants on Selected Quality Parameters of Cryopreserved Canine Spermatozoa.

Introduction The addition of low-molecular-weight antioxidants during the freezing process improves post-thaw sperm quality. The high antioxidant potential of cryopreserved semen could have a positive effect on the motility, viability, and energy status of sperm cells and their ability to bind to the zona pellucida of oocytes. The aim of the study was to determine the effects of different concentrations and combinations of vitamins E and C in a semen extender on selected quality parameters of frozen-thawed canine spermatozoa. Material and Methods The experimental material was the semen of four mixed-breed dogs. Sperm viability (motility, plasma membrane integrity, and mitochondrial function) was examined at 0, 60, and 120 min in semen samples supplemented with the extender and in the controls. Results Combined supplementation with vitamins C + E at a concentration of 200 + 200 µM /1 × 109 spermatozoa had the most profound effect on total sperm motility, linear motility, and the percentage of spermatozoa with intact plasma membrane and active mitochondria. Conclusion The synergistic activity of vitamins E and C had a more beneficial influence on the quality of frozen-thawed sperm than these non-enzymatic antioxidants applied separately.

Characteristics of spermatozoa of whole ejaculate and sperm-rich fraction of dog semen following exposure to media varying in osmolality.

This study aimed to analyze the effects of different osmolalities on the characteristics of spermatozoa originating from whole ejaculates (WE; including the prostatic fluid) and the sperm-rich fractions (SRF). Ejaculates, collected from four mixed-breed dogs, were exposed for 10 min at room temperature to Tris-fructose-citrate (TFC) solution with osmolality ranging from 150 to 1100 mOsm. After treatment spermatozoa were evaluated by microscopic analysis of motility and fluorescent assessments of plasma membrane integrity (carboxyfluorescein diacetate and propidium iodide, CFDA/PI) and mitochondrial function (rhodamine 123, R123). Irrespective of the sperm source, there was a complete loss of motility when spermatozoa were exposed to TFC solution with 1100 mOsm. There were no marked differences in the sperm characteristics between media with 300 and 350 mOsm, regardless of the ejaculate collection procedure. However, a marked reduction in motility of spermatozoa retrieved either from the WE or SRF was observed after exposure to different anisosmotic conditions (150, 550 and 800 mOsm). In all dogs, spermatozoa from the WE exhibited greater osmotolerance in terms of plasma membrane integrity and mitochondrial function when exposed to anisosmotic conditions (150, 550, 800 and 1100 mOsm). There were inter-dog variations in response to various osmotic conditions. The findings of this study indicated that spermatozoa from the WE tolerated exposure to a wider range of osmolality than those from the SRF. It seemed that the presence of prostatic fluid of dog semen rendered the sperm membrane structures less susceptible to osmotic stress.