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Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation.

ABSTRACT: Metabolomic approaches allow the study of downstream gene expression events since metabolites are considered as the products of cell signaling pathways. For this reason, many studies in humans have already been conducted to determine the influence of the metabolites present in seminal plasma (SP) on sperm physiology, and to identify putative biomarkers. However, in livestock species, these relationships are yet to be uncovered. Thus, the present study aimed to explore: (i) if concentrations of metabolites in pig SP are related to sperm quality and functionality, and (ii) if they could predict the sperm resilience to liquid storage at 17°C. To this end, 28 ejaculates were individually collected and split into three aliquots: one was used for SP analysis through nuclear magnetic resonance (NMR) spectroscopy; another served for the evaluation of sperm concentration and morphology; and the last one was utilized to determine sperm functionality parameters using computer-assisted sperm analysis (CASA) and flow cytometry after 0 h and 72 h of liquid-storage at 17°C. NMR analysis allowed the identification and quantification of 23 metabolites present in pig SP which, except for fumarate, were not observed to follow a breed-dependent behavior. Moreover, specific relationships between metabolites and sperm variables were identified: (i) glutamate, methanol, trimethylamine N-oxide, carnitine, and isoleucine were seen to be related to some sperm quality and functionality parameters evaluated immediately after semen collection; (ii) leucine, hypotaurine, carnitine and isoleucine were found to be associated to the sperm ability to withstand liquid storage; and (iii) Bayesian multiple regression models allowed the identification of metabolite patterns for specific sperm parameters at both 0 h and 72 h. The identification of these relationships opens up the possibility of further investigating these metabolites as potential sperm functional biomarkers.

SUBMITTER: Mateo-Otero Y

PROVIDER: S-EPMC8194698 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMetabolomic approaches, which include the study of low molecular weight molecules, are an emerging -omics technology useful for identification of biomarkers. In this field, nuclear magnetic resonance (NMR) spectroscopy has already been used to uncover (in) fertility biomarkers in the seminal plasma (SP) of several mammalian species. However, NMR studies profiling the porcine SP metabolome to uncover in vivo fertility biomarkers are yet to be carried out. Thus, this study aimed to evaluate the putative relationship between SP-metabolites and in vivo fertility outcomes. To this end, 24 entire ejaculates (three ejaculates per boar) were collected from artificial insemination (AI)-boars throughout a year (one ejaculate every 4 months). Immediately after collection, ejaculates were centrifuged to obtain SP-samples, which were stored for subsequent metabolomic analysis by NMR spectroscopy. Fertility outcomes from 1525 inseminations were recorded over a year, including farrowing rate, litter size, stillbirths per litter and the duration of pregnancy.ResultsA total of 24 metabolites were identified and quantified in all SP-samples. Receiver operating characteristic (ROC) curve analysis showed that lactate levels in SP had discriminative capacity for farrowing rate (area under the curve [AUC] = 0.764) while carnitine (AUC = 0.847), hypotaurine (AUC = 0.819), sn-glycero-3-phosphocholine (AUC = 0.833), glutamate (AUC = 0.799) and glucose (AUC = 0.750) showed it for litter size. Similarly, citrate (AUC = 0.743), creatine (AUC = 0.812), phenylalanine (AUC = 0.750), tyrosine (AUC = 0.753) and malonate (AUC = 0.868) levels had discriminative capacity for stillbirths per litter; and malonate (AUC = 0.767) and fumarate (AUC = 0.868) levels for gestation length.ConclusionsThe assessment of selected SP-metabolites in ejaculates through NMR spectroscopy could be considered as a promising non-invasive tool to predict in vivo fertility outcomes in pigs. Moreover, supplementing AI-doses with specific metabolites should also be envisaged as a way to improve their fertility potential.

Project description:Aldose reductase B1 (AKR1B1), a NADPH-dependent enzyme that belongs to the aldo-keto reductase protein superfamily, has been reported to be involved in both male and female reproductive physiology. The objectives of this study were: (1) to evaluate the concentration of SP-AKR1B1 in pig ejaculate fractions; (2) to describe the immunohistochemical localization of AKR1B1 alongside the boar genital tract; (3) to evaluate the relationship between SP-AKR1B1 and sperm quality/functionality parameters. Ejaculates from seven boars (one ejaculate per boar) were collected in separate portions [the first 10 mL of the sperm rich fraction (SRF-P1), the rest of the SRF (SRF-P2), and the post-SRF (PSRF)], and the concentration of SP-AKR1B1 was assessed using an enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry and immunoblotting targeting was conducted in the reproductive tissues of these boars. Additionally, the entire ejaculates of 14 boars (one ejaculate per boar) were collected and split into three separate aliquots for: (i) SP-AKR1B1 quantification; (ii) assessment of sperm concentration and morphology; and (iii) evaluation of sperm quality and functionality parameters upon ejaculate collection (0 h) and after 72 h of liquid storage at 17°C. Concentration of AKR1B1 in the SP of SRF-P1 (458.2 ± 116.33 ng/mL) was lower (P < 0.05) than that of SRF-P2 (1105.0 ± 229.80 ng/mL) and PSRF (1342.4 ± 260.18 ng/mL). Monomeric and dimeric AKR1B1 forms were expressed alongside the reproductive tissues, except in the bulbourethral glands. No relationship between SP-AKR1B1 and sperm quality/functionality parameters was observed either at 0 h or after 72 h of storage at 17°C. In conclusion, AKR1B1 is expressed in the reproductive organs of boars (except bulbourethral glands) and a higher concentration is found in the PSRF suggesting that seminal vesicles would be the main secretory source. However, this enzyme does not appear to be related to sperm quality/functionality or to the sperm ability to withstand liquid storage at 17°C.

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: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 (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.

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Metabolite Profiling of Pig Seminal Plasma Identifies Potential Biomarkers for Sperm Resilience to Liquid Preservation.

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