Search Results (7)

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality. Full article

Cryopreserved semen is extensively utilized in the artificial insemination (AI) of domestic animals; however, suboptimal conception rates due to oxidative damage following AI continue to pose a challenge. The present study investigated the effects of Pyrroroquinoline Quinone (PQQ), a novel antioxidant, on the semen quality of Holstein bulls during cryopreservation, as well as its potential molecular mechanisms. Semen samples were diluted with varying concentrations of PQQ (0, 50 μmol/L, 100 μmol/L, 150 μmol/L) prior to cryopreservation. Following the freeze–thaw process, a comprehensive evaluation was conducted to assess sperm motility, plasma membrane integrity, acrosome integrity, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and adenosine triphosphate (ATP). Western blot analysis was employed to examine the levels of proteins including PGAM2, CAPZB, CAT, SOD1, and GPX1. Notably, the inclusion of 100 μmol/L PQQ significantly enhanced sperm motility, membrane integrity, and acrosome integrity post freeze–thawing (p < 0.05). Furthermore, the group treated with 100 μmol/L PQQ exhibited reduced levels of MDA and ROS (p < 0.05), while ATP levels were significantly elevated (p < 0.05). Interestingly, treatment with 100 μmol/L PQQ resulted in decreased consumption of PGAM2, CAPZB, CAT, SOD1, and GPX1 proteins in sperm after freeze–thawing, compared to the control group (p < 0.05). These findings indicate that PQQ treatment enhances the quality of bull semen, mitigates oxidative stress damage, and ultimately improves the efficacy of sperm cryopreservation. Full article

Thymoquinone nanoparticles (TQNPs) are broadly utilized in numerous pharmaceutical applications. In the present study, we tested the effects of TQNP supplementation on sperm quality and kinematics, acrosome exocytosis, oxidative biomarkers, apoptosis-like and morphological changes of frozen–thawed buffalo sperm, as well as the fertilizing capacity. Semen was collected from buffalo bulls, diluted (1:10; semen/extender), and divided into five aliquots comprising various concentrations of TQNP 0 (CON), 12.5 (TQNP12.5), 25 (TQNP25), 37.5 (TQNP37.5), and 50 (TQNP50) µg/mL, and then cryopreserved and stored in liquid nitrogen (−196 °C). The results revealed that TQNPs (25 to 50 µg/mL) provided the most optimal results in terms of membrane integrity (p < 0.001) and progressive motility (p < 0.01). In contrast, TQNP50 resulted in a greater post-thawed sperm viability (p = 0.02) compared with other groups. The addition of TQNPs to the extender had no discernible effects on sperm morphology measures. Sperm kinematic motion was significantly improved in the TQNP50 group compared to the control group (p < 0.01). TQNPs effectively reduced the content of H₂O₂ and MDA levels and improved the total antioxidant capacity of post-thawed extended semen (p < 0.01). The addition of TQNP significantly increased the number of intact acrosomes (p < 0.0001) and decreased the number of exocytosed acrosomes (p < 0.0001). A significant reduction in apoptosis-like changes was observed in TQNP groups. The non-return rates of buffalo cows inseminated with TQNP50-treated spermatozoa were higher than those in the control group (p < 0.05; 88% vs. 72%). These findings suggested that the freezing extender supplemented with TQNPs could effectively enhance the cryotolerance and fertility of buffalo sperm. Full article

The purpose of this study was to determine the effect on fresh and post-thaw beef bull semen quality of the supplementation of epidermal growth factor (EGF) to the semen extender at various concentrations (0-control, 50, 100, 200, and 400 ng/mL). For 8 weeks, sperm was collected from four fertile bulls, yielding a total of 32 ejaculates. Semen samples were pooled, diluted with Bullxcell^® extender, and then cooled, equilibrated, and frozen. After thawing, semen was tested for motility and velocity parameters. Furthermore, semen was evaluated for vitality, integrity, mitochondrial and antioxidant (SOD) activities, mucus penetration distance, and in vitro fertilizing capability. The supplementation with EGF prior to cryopreservation improved the total sperm motility at various concentrations over long incubation periods (from 1 to 4 h). Interestingly, EGF addition improved both progressive and rapid motility, particularly at 50, 200, and 400 ng/mL. In addition, EGF, primarily at 200 and 400 ng/mL, significantly increased several velocity parameters after different incubation periods. We can conclude that adding EGF to bull sperm extender before cryopreservation has a positive stimulatory effect on sperm motility without affecting vitality, integrity, or in vitro fertilizing capability. Full article

For years, compounds of natural origin have been the subject of extensive biomedical research due to very interesting, new ingredients potentially useful for various pharmaceutical, medical and industrial applications. The therapeutic properties and healing benefits of sea cucumbers may result from the presence of numerous, biologically active ingredients. Sperm subjected to processing and subsequent storage at low temperatures experience a number of damage, including the loss of the integrity of the cytoplasmic membrane, DNA and acrosome defragmentation. Therefore, the aim of this experiment was to investigate the cytoprotective potential of sea cucumber extract against cryopreserved sperm and semen fertility rate. Commercially available sea cucumber extract was taken from the cellulose shell, then 790 mg of powder was weighed out and placed in 3 glass tubes containing, respectively: 10 mL of water-glycerin solution (WG), water-ethanol (EC), glycerin-ethanol (GE), glycerin-DMSO (DG). Tubes were mixed with vortex for 3 min, then placed in a water bath and incubated for 16 h at 40 °C. Six simmental bulls, 3 years old, of known health status were used for the experiment. Semen was collected from each male once a week (for 18 weeks) using an artificial vagina. After an initial assessment of semen quality, the ejaculates were pooled to eliminate individual differences between males, then diluted to a final concentration of 80 × 10⁶ sperm/mL with a commercial extender (Optixcell, IMV, L’Aigle, France) and divided into 16 equal samples. Control (C) without additive, the test samples contained 2, 4, 6, 8 and 10 µL WG, 2, 4, 6, 8 and 10 µL WE, 2, 4, 6, 8 and 10 µL GE, 2, 4, 6, 8 and 10 µL DG. Semen was frozen/thawed and assessed for motility, viability, DNA defragmentation, mitochondrial membrane potential and acrosome integrity. It was shown a positive effect of water-glycerin (WG) and glycerine-ethanol (GE) extracts on the efficiency of sperm preservation at low temperatures. Established that, depending on the type of prepared extract, the sea cucumber can have both cytoprotective (WG, GE, WE) and cytotoxic (DG) effects. Moreover, too high concentrations of the extract can adversely affect the sperm in terms of parameters such as viability, motility, mitochondrial potential, and the integrity of the acrosome or DNA of cells. The present study, thanks to the use of model animals to study the cytoprotective potential of the sea cucumber extract, proves that it can be a potential candidate for use in semen cryopreservation technology to improve the efficiency of storage at low temperatures. Further research is needed to optimize the composition of individual types of extracts and their effect on sperm. The highest effectiveness of female fertilization was observed when doses from GE groups (2 and 4) were used for insemination. The results of this analysis prove that the addition of the tested extract may improve the fertilization efficiency. Full article

The ejaculate is heterogenous and sperm sub-populations with different kinematic patterns can be identified in various species. Nevertheless, although these sub-populations are statistically well defined, the statistical differences are not always relevant. The aim of the present study was to characterize kinematic sub-populations in sperm from two bovine species, and diluted with different commercial extenders, and to determine the statistical relevance of sub-populations through Bayesian analysis. Semen from 10 bulls was evaluated after thawing. An ISAS^®v1 computer-assisted sperm analysis (CASA)-Mot system was employed with an image acquisition rate of 50 Hz and ISAS^®D4C20 counting chambers. Sub-populations of motile spermatozoa were characterized using multivariate procedures such as principal components (PCs) analysis and clustering methods (k-means model). Four different sperm sub-populations were identified from three PCs that involved progressiveness, velocity, and cell undulatory movement. The proportions of the different sperm sub-populations varied with the extender used and in the two species. Despite a statistical difference (p < 0.05) between extenders, the Bayesian analysis confirmed that only one of them (Triladyl^®) presented relevant differences in kinematic patterns when compared with Tris-EY and OptiXcell^®. Extenders differed in the proportion of sperm cells in each of the kinematic sub-populations. Similar patterns were identified in Bos taurus and Bos indicus. Bayesian results indicate that sub-populations SP₁, SP₂, and SP₃ were different for PC criteria and these differences were relevant. For velocity, linearity, and progressiveness, the SP₄ did not show a relevant difference regarding the other sperm sub-populations. The classical approach of clustering or sperm subpopulation thus may not have a direct biological meaning. Therefore, the biological relevance of sperm sub-populations needs to be reevaluated. Full article

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 μg/mL of enrofloxacin or 0.0625 μg/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 × 10⁶ colony forming units (CFU)/mL or 3.24 × 10⁸ CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p < 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI. Full article