Search Results (9)

ProAKAP4, a precursor of AKAP4 (A-kinase anchor protein) found in the flagellum of mammalian and non-mammalian spermatozoa, serves as a structural protein with established correlations to motility parameters across diverse species. This study aimed to determine the proAKAP4 level evolution in thawed stallion semen over a 3 h period, examining its correlation with motility descriptors and mitochondrial membrane potential. Utilizing sixteen ejaculates from four French warmblood stallions, this study involved maintaining thawed samples at 37 °C for 3 h, conducting proAKAP4 enzyme-linked immunosorbent assays (ELISA), computer-assisted sperm analysis (CASA), and mitochondrial membrane potential by JC-1 probe and flow cytometry at 0, 1, and 3 h post-thawing. The findings indicate significant positive correlations (p ≤ 0.05) between proAKAP4 levels and sperm total or progressive motility at all time points analyzed. Spermatozoa velocity descriptors (VAP, VCL, VSL) and spermatozoa lateral head displacement (ALH) display positive correlations (p ≤ 0.05) with ProAKAP4 at the 0 h post-thawing. ProAKAP4 concentration exhibits no discernible difference between batches with or without a cryoprotectant. Notably, proAKAP4 consumption remains insignificant within the initial hour after thawing but becomes significant (p ≤ 0.05) between 1 and 3 h post-thawing. In summary, proAKAP4 demonstrates positive correlations with total and progressive motility in stallion semen for up to 3 h after thawing, albeit showing a noticeable decrease starting from the first hour post-thawing, indicating a progressive consumption as a result of spermatozoa motile activity. Full article

Artificial insemination (AI) is used frequently in the breeding of sport horses, apart from Thoroughbreds. Most AIs are carried out with cooled semen rather than frozen semen because of the difficulties in identifying a protocol that is suitable for freezing most ejaculates and the necessity to inseminate close to ovulation because of the short life of the thawed spermatozoa. More widespread use of frozen semen would improve biosecurity, allow greater choice of stallions, and offer more flexibility when managing deliveries of semen to the stud. It would even decrease the amount of antibiotics used in semen extenders, since the volume of frozen semen is smaller than when cooled semen is inseminated. However, there is considerable variability in the cryosurvival of spermatozoa from different stallions, leading to the classification of stallions as good or bad freezers. Improvements could be made at the level of stallion nutrition, the semen collection regimen, the extender, the removal of seminal plasma, and the cooling protocol, among others. Stallion sperm membranes are highly susceptible to lipid peroxidation, but research on antioxidants has failed to identify an additive that would benefit all stallions. In the future, biomarkers for sperm freezability could be used as an aid in identifying suitable ejaculates for cryopreservation. Full article

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation. Full article

Artificial insemination is an important assisted reproductive technology that has been applied in several mammalian species. However, successful cryopreservation of semen of South American camelids has been limited, hindering the commercial application of artificial insemination in alpaca species. In this scenario, the addition of antioxidants to semen extenders provides a strategy to improve the freezability of mammalian sperm. Bioactive metabolites from natural extracts of black maca have shown valuable antioxidant properties. Thus, the objective of this study was to evaluate the effect of the addition of atomized black maca in the freezing medium of epididymal spermatozoa of alpacas. Fifteen pairs of epididymis were collected from a local slaughterhouse. Each sample was divided into six groups: (1) fresh, (2) yolk medium (YM), (3) 10 mg/mL maca, (4) 20 mg/mL maca, (5) 30 mg/mL maca, and (6) resveratrol (as an antioxidant control). Sperm cryopreservation was performed through the slow freezing method. Markers associated with functionality, such as motility, viability, and plasma membrane integrity, as well as markers associated with oxidative damage, such as DNA integrity, total ROS production, and mitochondrial function, were analyzed. The results show that the supplementation with black maca (20 mg/mL) improved the sperm motility, viability, plasma membrane integrity, and mitochondrial function evaluated according to an index of formazan deposits. Similarly, the ROS production decreased with maca at 20 mg/mL, although the DNA integrity did not show any differences among the groups. These results suggest that maca at 20 mg/mL has cytoprotective effects during freezing/thawing of epididymal sperm of alpaca species. Further research will be focused on assessing the effects of maca supplementation on semen extenders by using biomolecular markers (proAKAP4) associated with fertility. Full article

ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyzed. Spermatic motility descriptors were obtained by the CASA system, and four motile subpopulations (SP) with specific motility patterns were statistically identified. ProAKAP4 concentrations were evaluated by ELISA. The relationship between motility descriptors of sperm subpopulations and proAKAP4 concentrations was evaluated. Following a hierarchical cluster statistical analysis, ejaculates were divided into two groups according to their proAKAP4 concentrations, either having low proAKAP4 concentrations (5.06–35.61 ng/10M spz; n = 23) or high (39.92–82.23 ng/10M spz; n = 25) proAKAP4 concentrations (p < 0.001). ProAKAP4 concentrations were positively correlated (p < 0.05) with total and progressive motility, as well as with parameters of velocity. ProAKAP4 amount also showed a negative correlation (p < 0.05) with sperm motile subpopulation number 3, which was the subpopulation with the lowest velocity parameters. In conclusion, proAKAP4 concentration in stallion semen positively reflects sperm progressive motility with the functional velocity kinematic descriptors. Concentrations of proAKAP4 higher than 37.77 ng/10M spz were correlated with a very good quality frozen/thawed stallion semen. Full article

Functional sperm quality markers to predict bull fertility have been actively investigated. Among them, proAKAP4, which is the precursor of AKAP4, the main structural protein in the fibrous sheath of spermatozoa; appears to be promising, especially since spermatozoa lacking AKAP4 expression were shown to be immotile, abnormal, and infertile. In this study, the objective was to evaluate proAKAP4 concentration values with the classic sperm motility descriptors and fertility outcomes (NRR at 90 days) in post-thawed conditions of 10 bulls’ semen. ProAKAP4 expression was confirmed by Western blotting and proAKAP4 concentrations were determined by ELISA. Variations in proAKAP4 concentrations were observed independently of the motility sperm descriptors measured using computer-assisted semen analysis (CASA). A ProAKAP4 concentration of 38.67 ± 8.55 ng/10 million spermatozoa was obtained as a statistical mean of all samples. Threshold values of proAKAP4 were then determined between 19.96 to 96.95 ng/10 million spermatozoa. ProAKAP4 concentrations were positively correlated with progressive motility and the linearity coefficient. The sperm showing the lowest progressive motility were the samples exhibiting proAKAP4 concentrations below 20 ng/10 million spermatozoa. Furthermore, proAKAP4 concentrations were significantly higher in bulls with a higher NRR in the field. Our results demonstrate a correlation between the semen concentration of proAKAP4 and NRR-90d (p = 0.05) in post-thawed bull semen, highlighting the potential of proAKAP4 as a predictive marker of bull fertility. Full article

The aim of this study was to determine the concentration of proAKAP4 and other indicators of cryopotential of spermatozoa cryopreserved in extender with Holothuroidea extract addition. Nine Holstein Friesian bulls, 3.5 years old, of known health status, were used for the study. The animals were kept and fed equally. Semen was collected once a week using an artificial vagina. The commercially available Holothuroidea extract was used as a supplement to the commercial extender (0, 2, 4 and 6 µL/mL) before the freezing/thawing process. The viability, motility, motion parameters, and acrosome integrity of the sperm were analyzed with (test) or without (control) extract samples. Furthermore, the concentration of the proAKAP4 biomarker in frozen sperm was assessed. It was shown that the addition of 4 and 6 µL of the extract may have a positive effect on the quality parameters of the sperm after thawing. The results indicate that extender supplementation with the above extract modulates (increases) the concentration of proAKAP4 in sperm at all tested levels. Additionally, this indicator has become helpful in identifying sperm of poor biological quality. Moreover, it has been proven that the proAKAP4 biomarker can be successfully used to evaluate the effectiveness of the use of various extenders for semen cryopreservation. Full article

Myocardial infarction (MI) is a leading cause of maladaptive cardiac remodeling and heart failure. In the damaged heart, loss of function is mainly due to cardiomyocyte death and remodeling of the cardiac tissue. The current study shows that A-kinase anchoring protein 2 (AKAP2) orchestrates cellular processes favoring cardioprotection in infarcted hearts. Induction of AKAP2 knockout (KO) in cardiomyocytes of adult mice increases infarct size and exacerbates cardiac dysfunction after MI, as visualized by increased left ventricular dilation and reduced fractional shortening and ejection fraction. In cardiomyocytes, AKAP2 forms a signaling complex with PKA and the steroid receptor co-activator 3 (Src3). Upon activation of cAMP signaling, the AKAP2/PKA/Src3 complex favors PKA-mediated phosphorylation and activation of estrogen receptor α (ERα). This results in the upregulation of ER-dependent genes involved in protection against apoptosis and angiogenesis, including Bcl2 and the vascular endothelial growth factor a (VEGFa). In line with these findings, cardiomyocyte-specific AKAP2 KO reduces Bcl2 and VEGFa expression, increases myocardial apoptosis and impairs the formation of new blood vessels in infarcted hearts. Collectively, our findings suggest that AKAP2 organizes a transcriptional complex that mediates pro-angiogenic and anti-apoptotic responses that protect infarcted hearts. Full article

To improve artificial insemination protocols in ovine species it is crucial to optimize sperm quality evaluation after preservation technologies. Emerging technologies based on novel biomolecules and related to redox balance and proteins involved in sperm motility such as ProAKAP4 could be successfully applied in ram sperm evaluation. In this work, a multiparametric analysis of fresh, cooled, and cryopreserved ram sperm was performed at different complexity levels. Samples were evaluated in terms of motility (total motility, progressive motility, and curvilinear velocity), viability, apoptosis, content of reactive oxygen species, oxidation‒reduction potential, and ProAKAP4 expression and concentration. As expected, cryopreserved samples showed a significant decrease of sperm quality (p < 0.05), evidencing different freezability classes among samples that were detected by ProAKAP4 analyses. However, in cooled sperm no differences were found concerning motility, viability, apoptosis, ROS content, and redox balance compared to fresh sperm that could explain the reported decrease in fertility rates. However, although the proportion of sperm ProAKAP4 positive-cells remained unaltered in cooled sperm compared to fresh control, the concentration of this protein significantly decreased (p < 0.05) in cooled samples. This altered protein level could contribute to the decrease in fertility rates of cooled samples detected by some authors. More importantly, ProAKAP4 can be established as a promising diagnostic parameter of sperm quality allowing us to optimize sperm conservation protocols and finally improve artificial insemination in ovine species. Full article