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Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

ABSTRACT: The objective of this study was to determine the relationship of enzymatic (superoxide dismutase, SOD; glutathione peroxidase, GPX; catalase, CAT; and paraoxonase type 1, PON1) and non-enzymatic antioxidants (measured in terms of: Trolox equivalent antioxidant capacity, TEAC; cupric-reducing antioxidant capacity, CUPRAC; and ferric-reducing ability of plasma, FRAP), as well as the oxidative stress index (OSI) in seminal plasma (SP) with the resilience of horse sperm to freeze-thawing. Twenty-one ejaculates (one per individual) were collected and split into two aliquots: the first was used to harvest the SP and assess the activity levels of antioxidants and the OSI, and the second one was cryopreserved. The following post-thaw sperm quality parameters were evaluated: sperm motility, plasma membrane and acrosome integrity, mitochondrial membrane potential, intracellular levels of reactive oxygen species (ROS), and plasma membrane lipid disorder. Based on post-thaw total motility (TM) and plasma membrane integrity (SYBR14⁺/PI^-), ejaculates were hierarchically (p < 0.001) clustered into two groups of good (GFE) and poor (PFE) freezability. The SP activity levels of PON1, SOD, and TEAC were higher (p < 0.05) in GFE than in PFE, showing a positive relationship (p < 0.05) with some sperm motility parameters and with plasma membrane (PON1 and TEAC) and acrosome (SOD and TEAC) integrity. In contrast, OSI was higher (p < 0.05) in the SP of PFE than in that of GFE, and was negatively correlated (p < 0.05) to some sperm motility parameters and to plasma membrane and acrosome integrity, and positively (p < 0.05) to the percentage of viable sperm with high plasma membrane lipid disorder. In conclusion, enzymatic (PON1 and SOD) and non-enzymatic (TEAC) antioxidants of SP are related to horse sperm cryotolerance. In addition, our results suggest that PON1 could be one of the main antioxidant enzymes involved in the control of ROS in this species. Further investigation is needed to confirm the potential use of these SP-antioxidants and OSI to predict sperm cryotolerance in horses.

SUBMITTER: Catalan J

PROVIDER: S-EPMC9311553 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study investigated whether the activities of the antioxidant components of donkey seminal plasma (SP)-both enzymatic (superoxide dismutase (SOD), catalase-like (CAT), glutathione peroxidase-like (GPX), and paraoxonase type 1 (PON1)) and non-enzymatic (measured in terms of total thiol, copper-reducing antioxidant capacity (CUPRAC), ferric-reducing ability of plasma (FRAP), and Trolox equivalent antioxidant capacity (TEAC))-and oxidative stress index (OSI) are related to sperm cryotolerance. For this purpose, 15 ejaculates from jackasses (one per individual) were collected and split into two aliquots. The first one was used for measuring the activities levels of enzymatic and non-enzymatic antioxidants and OSI in SP, whereas the other aliquot was cryopreserved. Before cryopreservation, sperm quality parameters (concentration, motility, and viability) were evaluated. After thawing, sperm motility, plasma membrane integrity, lipid disorder, mitochondrial membrane potential, reactive oxygen species (ROS), and calcium intracellular levels were also determined. Based on the percentages of total motility (TM) and of sperm with an intact plasma membrane (SYBR14+/PI-) after thawing, samples were classified as good-freezability (GFE) or poor-freezability (PFE) ejaculates through cluster analyses. The SP activity levels of enzymatic (SOD and PON1) and non-enzymatic antioxidants (CUPRAC, FRAP, and TEAC) were higher (p < 0.05) in GFE than in PFE, whereas SP-OSI was higher (p < 0.05) in PFE than in GFE. In addition, the activity levels of SOD, PON1, GPX, CUPRAC, FRAP, and TEAC were positively (p < 0.05) related to post-thaw sperm motility and plasma membrane integrity and negatively to intracellular ROS levels. The SP-OSI was negatively correlated (p < 0.05) to post-thaw sperm quality parameters and positively to intracellular ROS levels. It can thus be concluded that donkey SP antioxidants are related to sperm cryotolerance and that measurements of antioxidants PON1, SOD, CUPRAC, FRAP, and TEAC, as well as SP-OSI, could be used as markers of sperm cryotolerance. Further research addressing the relationship of these antioxidants and SP-OSI with sperm cryotolerance and their potential use as freezing markers is warranted.

Project description:Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P<0.05) on the percentage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was a positive correlation between seminal plasma concentrations of valine, isoleucine lysine, leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat ('good freezer') and Black-Red Andaluza ('bad freezer') showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed (14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay revealed that freezing/thawing procedures in presence of seminal plasma provoked higher DNA fragmentation in most of the breeds, with a positive correlation between seminal alanine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed effect on sperm viability was highly reduced. It is concluded that specific seminal plasma amino acids were associated with post-thaw percentage of viable sperm and DNA integrity. The removal of seminal plasma decreases the variability of the results and DNA fragmentation damages.

Project description:Seminal plasma (SP) is the natural environment for spermatozoa and contains a number of components, especially proteins important for successful sperm maturation and fertilization. Nevertheless, in standard frozen stallion insemination doses production, SP is completely removed and is replaced by a semen extender. In the present study, we analyzed the effects of the selected seminal plasma protein groups that might play an important role in reducing the detrimental effects on spermatozoa during the cryopreservation process. SP proteins were separated according to their ability to bind to heparin into heparin-binding (Hep+) and heparin-non-binding (Hep-) fractions. The addition of three concentrations-125, 250, and 500 µg/mL-of each protein fraction was tested. After thawing, the following parameters were assessed: sperm motility (by CASA), plasma membrane integrity (PI staining), and acrosomal membrane integrity (PNA staining) using flow cytometry, and capacitation status (anti-phosphotyrosine antibody) using imaging-based flow cytometry. Our results showed that SP protein fractions had a significant effect on the kinematic parameters of spermatozoa and on a proportion of their subpopulations. The 125 µg/mL of Hep+ protein fraction resulted in increased linearity (LIN) and straightness (STR), moreover, with the highest values of sperm velocities (VAP, VSL), also this group contained the highest proportion of the fast sperm subpopulation. In contrast, the highest percentage of slow subpopulation was in the groups with 500 µg/mL of Hep+ fraction and 250 µg/mL of Hep- fraction. Interestingly, acrosomal membrane integrity was also highest in the groups with Hep+ fraction in concentrations of 125 µg/mL. Our results showed that the addition of protein fractions did not significantly affect the plasma membrane integrity and capacitation status of stallion spermatozoa. Moreover, our results confirmed that the effect of SP proteins on the sperm functionality is concentration-dependent, as has been reported for other species. Our study significantly contributes to the lack of studies dealing with possible use of specific stallion SP fractions in the complex puzzle of the improvement of cryopreservation protocols. It is clear that improvement in this field still needs more outputs from future studies, which should be focused on the effect of individual SP proteins on other sperm functional parameters with further implication on the success of artificial insemination in in vivo conditions.

Project description:Molecular mechanisms underlying sperm motility have not been fully explained, particularly in chickens. The objective was to identify seminal plasma proteins associated with chicken sperm motility by comparing the seminal plasma proteomic profile of roosters with low sperm motility (LSM, n = 4) and high sperm motility (HSM, n = 4). Using a label-free MS-based method, a total of 522 seminal plasma proteins were identified, including 386 (∼74%) previously reported and 136 novel ones. A total of 70 differentially abundant proteins were defined, including 48 more-abundant, 15 less-abundant, and seven proteins unique to the LSM group (specific proteins). Key secretory proteins like less-abundant adhesion G-protein coupled receptor G2 (ADGRG2) and more-abundant serine peptidase inhibitor Kazal-type 2 (SPINK2) in the LSM suggested that the corresponding secretory tissues played a crucial role in maintaining sperm motility. Majority (80%) of the more-abundant and five specific proteins were annotated to the cytoplasmic domain which might be a result of higher plasma membrane damage and acrosome dysfunction in LSM. Additionally, more-abundant mitochondrial proteins were detected in LSM seminal plasma associated with lower spermatozoa mitochondrial membrane potential (ΔΨm) and ATP concentrations. Further studies showed that the spermatozoa might be suffering from oxidative stress, as the amount of spermatozoa reactive oxygen species (ROS) were largely enhanced, seminal malondialdehyde (MDA) concentrations were increased, and the seminal plasma total antioxidant capacity (T-AOC) were decreased. Our study provides an additional catalogue of chicken seminal plasma proteome and supports the idea that seminal plasma could be as an indicator of spermatozoa physiology. More-abundant of acrosome, mitochondria and sperm cytoskeleton proteins in the seminal plasma could be a marker of sperm dysfunction and loss of motility. The degeneration of spermatozoa caused by the reduced seminal T-AOC and enhanced oxidative stress might be potential determinants of low sperm motility. These results could extend our understanding of sperm motility and sperm physiology regulation.

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Seminal Plasma Antioxidants Are Related to Sperm Cryotolerance in the Horse.

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