Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H₂DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa.

The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl^® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

Bacterial contamination of semen is an often overlooked, yet important, factor contributing to decreased sperm vitality. Understanding the impact of bacterial presence on sperm structural integrity and functional activity may assist the development of effective strategies to prevent, or manage, bacteriospermia in the breeding practice. The aim of this study was to describe the bacterial profiles of ram semen (n = 35), and we also focused on the associations between bacteriospermia, sperm structure, and function, as well as oxidative and inflammatory characteristics of semen. For a better insight, the samples were divided into three groups, according to the breeds used in the study: native Wallachian (NW), improved Wallachian (IW), and Slovak dairy (SD) breeds. The results showed a significantly lower motility and membrane integrity in the NW group in comparison to the IW and SD groups, which was accompanied by a significantly higher concentration of leukocytes, increased reactive oxygen species (ROS) generation, and subsequent oxidative insults to the sperm lipids and proteins. Accordingly, the NW group presented with the highest bacterial load, in which Staphylococcus and Escherichia were the predominant representatives. The Pearson correlation analysis uncovered positive relationships amongst the bacterial load and leukocytospermia (r = 0.613), the extent of lipid peroxidation (r = 0.598), protein oxidation (r = 0.514), and DNA fragmentation (r = 0.638). Furthermore, positive correlations were found between the bacterial load and pro-inflammatory molecules, such as the C-reactive protein (r = 0.592), interleukin 1 (r = 0.709), and interleukin 6 (r = 0.474), indicating a possible involvement of the immune response in the process of bacteriospermia. Overall, our data indicate that ram semen quality may be equally affected by the bacterial load and diversity. Furthermore, we can assume that the presence of bacteria in ejaculates triggers inflammatory processes, causes ROS overproduction, and, thereby, contributes to alterations in the sperm structure, while at the same time compromising the fertilization ability of male gametes.

Commercial turkey breeding relies almost entirely on artificial insemination, yet avian sperm are unusually vulnerable to cooling and freezing injury. As a result, extender chemistry and processing steps, especially low-temperature equilibration, are pivotal for post-thaw performance. We evaluated how extender choice, paired with equilibration time, shapes the post-thaw quality of turkey semen. Ejaculates were diluted in Beltsville, Sperm Motility Medium (SMM), Botucrio, or Kobidil⁺, then equilibrated for 20 or 40 min before freezing; samples were cryostored for one month and assessed immediately after thawing. The outcomes included motility/kinematics, membrane integrity, mitochondrial activity and membrane potential, apoptosis/necrosis, reactive oxygen species (ROS), DNA fragmentation, and bacteriological load. Overall, 20 min equilibration improved post-thaw motility and membrane integrity, and reduced DNA fragmentation and ROS. Among extenders, Beltsville delivered the best overall sperm quality. Considering the extender × time interaction, Beltsville, Botucrio, and Kobidil⁺ performed best at 20 min, whereas SMM performed best at 40 min. Thus, Beltsville and SMM provide strong, time-specific options for turkey semen cryopreservation—Beltsville at 20 min and SMM at 40 min.