Capacitation is a crucial sperm maturation process occurring in vivo in the female reproductive tract, enabling spermatozoa to fertilize the oocyte. In vitro, capacitation can be induced using defined capacitation media (CM), with further assessment of protein tyrosine phosphorylation (PTyr) patterns widely used as a marker to evaluate sperm capacitation. This review critically examines the factors influencing PTyr detection in boar spermatozoa variability introduced by different methodological approaches. Discrepancies in PTyr patterns may be a result of different sperm handling, including preservation methods, selection techniques, and capacitation protocols. Semen extenders, which may contain unknown components, can affect the variability in capacitation status. Selection techniques commonly employed to isolate viable spermatozoa may initiate different capacitation regulatory pathways, resulting in variability in analyzed sperm subpopulations and inconsistencies in PTyr detection. Similarly, the lack of standardization in CM composition significantly impacts capacitation outcomes. Fixation protocols further increase variability in PTyr pattern detection, as aldehydic fixatives potentially alter protein structures, while alcohol-based fixatives cause protein aggregation and plasma membrane disruption. While PTyr immunofluorescence remains a valuable tool for capacitation assessment, its reliability is limited by methodological variability. Mimicking in vivo conditions is crucial, and even minor modifications in the sperm capacitation process may provide inconsistent results in PTyr patterns across studies. This review offers valuable insights into often-disregarded methodological details and highlights the need for improved for better standardization of capacitation protocols. The uniform methodological approach improves reproducibility and reliability in capacitation studies and stimulates further investigation leading to the discovery of alternative additional markers to determine the capacitation status in mammalian spermatozoa.

• Keywords
• Capacitation media, Immunofluorescence, Protein phosphorylation, Reproduction, Signaling pathway, Sperm fixation,
• MeSH
• Semen Analysis veterinary   MeSH
• Phosphorylation MeSH
• Phosphotyrosine * metabolism   MeSH
• Sperm Capacitation * physiology   MeSH
• Swine physiology   MeSH
• Spermatozoa * physiology   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Phosphotyrosine * MeSH

Uncaria tomentosa (UT) or cat's claw, is a vine belonging to the Rubiaceae family and native to South and Central America. Various parts of the plant, including bark, showed many therapeutic activities (e.g., antioxidant and antibacterial), but the in vitro effects on gametes have still not been investigated. During boar semen storage for artificial insemination purposes, oxidative stress and bacterial contamination negatively affect sperm quality. In this study, we evaluated the tolerance of boar sperm to UT ethanolic extract at four concentrations (1.6 to 0.025 μg/mL). The analyses were carried out on sperm samples under oxidative stress, induced by H2O2 and Fe2+/Ascorbate, and during 96 h of semen storage at 17°C. The antibacterial activity of the extract (1,024 to 8 μg/mL) was tested against commercial strains and bacteria isolated from the semen. The treatments ranging from 0.4 to 0.025 μg/mL protected sperm membrane (p < 0.05) and preserved some kinetic parameters in samples under oxidative stress (Fe2+/Ascorbate). During semen storage, the extract did not show any cytotoxicity, and mean values of some sperm parameters were higher than the control group, although not significant (p > 0.05). All tested Gram-positive bacteria exhibited growth inhibition. The most frequently isolated Gram-negative bacteria from semen (i.e., Citrobacter koseri, Pseudomonas aeruginosa, Stenotrophomonas maltophilia) also showed complete growth inhibition, while the remaining strains showed a partial decrease in growth. Taken together, our findings show that Uncaria tomentosa is a promising plant-based additive for boar semen storage.

• Keywords
• antibiotics, antioxidant, cat's claw, oxidative stress, pig, plant extract, secondary metabolites, semen storage,
• Publication type
• Journal Article MeSH

• Keywords
• antioxidants, artificial insemination, biomarkers, domestic animals, oxidative stress, semen analyses, semen preservation, ultrasonography,
• Publication type
• Editorial MeSH

This study was aimed to determine the effect of CaCl2 on the motility and viability of stallion spermatozoa during different incubation temperatures. Experimental samples were prepared by diluting the ejaculates (n = 10) from three uniformly housed and fed breeding stallions with six different concentrations of CaCl2 (A: 0.1125, B: 0.225, C: 0.45, D: 0.938, E: 1.25, and F: 1.875 mg/mL). The control samples (CON) were prepared by diluting ejaculate only with physiological solution. Samples were divided into two aliquots for analyses at different storage temperatures (5 °C and 37 °C). The motility parameters were analysed by Computer Assisted Semen Analysis system at several time intervals (0, 1, 2 and 3 h) and the viability was assessed using a mitochondrial toxicity test (MTT) realized at the end of incubation at both temperatures. Addition of CaCl2 to stallion semen showed significant effect on motility parameters, especially in the highest concentrations at 5 °C. Significant objectionable effect of CaCl2 on both total and progressive motility was observed at temperature 37 °C compared to control sample. However, results of velocity curved line in samples C, D and F at time 1 h and also at time 2 h in sample F showed significant positive effect of CaCl2. Sperm viability in experimental samples did not show a significant difference compared to the control at either 5 °C or 37 °C. The results of this study did not confirm essential effect of calcium on reproductive parameters of stallion. To conclude, our study demonstrated that the effect of CaCl2 on stallion sperm motility differs in a dose-dependent manner; however, the overall impact on motility parameters does not seem to be beneficial.

• Keywords
• CaCl2, Motility, Spermatozoa, Stallion, Viability,
• Publication type
• Journal Article MeSH

AIM/OBJECTIVES: This study examines the in vitro impact of an ethanolic extract derived from Bryonia laciniosa seeds on the Gir bull (Bos indicus) spermatozoa. The objective is to thoroughly assess the effects of the seed extract on the physiological parameters of bull spermatozoa, followed by evaluating its effects on X and Y-bearing spermatozoa and its impact on gene expression through transcriptome profiling. MATERIAL METHOD: For this study, one Gir bull was selected, and 12 ejaculates were collected at one-week time intervals. Sperm cells were isolated from each ejaculate and incubated with varying concentrations of the ethanolic extract. The physiological parameters of the spermatozoa were assessed using Computer Assisted Semen Analysis (CASA) and compared with control groups to evaluate the extract's effects on sperm quality and motility. RESULTS AND DISCUSSION: At a concentration of 18 mg/mL B. laciniosa extract, we noticed a statistically significant 16.4% increase in sperm motility (p = 0.0065). In order to understand the specific effect on X and Y-bearing spermatozoa, motile and non-motile sperm separated by glass wool column method and further evaluated for quantification of X and Y-bearing sperm in all samples by ddPCR. To understand the effect of B. laciniosa extract on spermatozoa at the molecular level, whole transcriptome profiling was carried out using Illumina MiSeq. Transcriptome profiling revealed 81 genes that were expressed differently between the group treated with the extract and the control group. The current investigation revealed an increase in the expression of TLX1, CRYGB, KLF13, and ZAR1 transcripts, which play a role in embryonic development. In addition, several genes have been identified that are involved in sperm motility, such GSK3B, LAPRS, MAPK1, CAMK2B, and AQP7. The findings exhibited the therapeutic effectiveness of B. laciniosa seeds in augmenting fertility through a synergistic blend of activities, including enhanced sperm motility and positive influence on embryogenesis.

• Keywords
• Bryonia laciniosa, CASA, Shivlingi, bull spermatozoa, transcriptomics,
• Publication type
• Journal Article MeSH