Poor Freezability Research Articles

Effects of hyaluronic acid on sperm parameters, mitochondrial functionand apoptosis of spermatozoa in Simmental bulls with good and poor freezing ability.

Bulls with varying freezability exhibit substantial variation in semen characteristics after cryopreservation. Sperm freezability is positively correlated with membrane cholesterol content, membrane integrity, mitochondrial activityand antioxidant content. The purpose of this study was to determine the optimal concentration of hyaluronic acid (HA) in bull sperm with different cryotolerances. Simmental bulls (n = 10) semen samples were taken and categorized based on their progressive motility (PM) after freeze-thawing: Group I, consisting of bulls (n = 5) with progressive sperm motility ≥45%, was considered good freezability ejaculates (GF), and Group II, including bulls (n = 5) with progressive sperm motility ≤30%, was considered poor freezability ejaculates (PF) bulls. Semen samples were diluted with a Tris-egg-yolk-glycerol (TEYG) extender containing various concentrations of HA: without HA (control), 1 mM HA, 2 mM HAand 4 mM HA. After the freeze-thaw process, sperm kinematics, plasma membrane and acrosome integrity, mitochondrial activityand apoptotic status were evaluated. The addition of 1 mM HA to the diluent of bulls with GF increased PM and linearity (LIN) compared to the control group (p < 0.05). Normal morphology was improved after thawing in the samples treated with 1and 2 mM HA in the GF and PF bullsrespectively. The membrane and acrosome integrity of GF bulls treated with 1 mM HA was significantly (p < 0.05) greater than that of the control groups. Adding 1 mM HA to the extender of bulls with GF and PF improved the proportion of viable cells compared with the highest concentration (4 mM) of HA. The mitochondrial activity of PF bulls treated with 1and 2 mM HA was significantly (p < 0.05) greater than that of the controls and 4 mM HA. Finally, it can be concluded that adding low doses of HA (1 mM) to the TEYG extender of GF and PF bulls ameliorated the post-thaw semen quality.

Variation of existence and location of aquaporin 3 in relation to cryoresistance of ram spermatozoa.

Osmotic changes during the process of freeze-thawing involve changes in the location of aquaporins (AQPs) in membrane domains of spermatozoa. Some AQPs, like aquaporin 3 (AQP3), are linked to sperm cryotolerance in the porcine species. Conspicuous individual variability exists between rams and their ejaculates, which may be classified as displaying good freezability (GFE) or poor freezability (PFE), depending on several endogenous and environmental factors. The present work aimed to examine whether differences in freezability could even involve changes in location and expression of AQP3 in ram spermatozoa. Thirty ejaculates from 10 rams (three of each) were evaluated and subsequently classified as GFE (n = 13) or PFE (n = 17) through a principal component analysis (PCA) and k-means cluster analysis. Spermatozoa were examined for the presence, abundance and distribution of AQP3 by western blot and immunocytochemistry, employing a commercial rabbit polyclonal antibody (AQP3 - ab125219). Although AQP3 was found in the sperm acrosome, midpiece, principal and end piece of the tail in both fresh and after frozen-thawed samples, its highest immunolabeling was found in the mid- and principal piece. In the GFE group, the expression of AQP3 in the mid- and principal piece was greater (P < 0.05) in frozen-thawed samples than in fresh specimens while such differences were not detected in the PFE group. Sperm cryotolerance relates to changes in AQP3 expression and thus AQP3 could be used as a biomarker for cryotolerance. A greater capacity of AQP3 localization in mid- and principal piece of the spermatozoa could be linked to an increase the osmo-adaptative capacity of ejaculates with better capacity to withstand freeze-thawing processes.

Effect of slow-cooling, cooled storage, and centrifugation prior to cryopreservation on post-thaw spermatological parameters in stallions classified as good or poor freezers

Effect of slow-cooling, cooled storage, and centrifugation prior to cryopreservation on post-thaw spermatological parameters in stallions classified as good or poor freezers

Effect of post-thaw supplementation of seminal plasma to the cryopreserved spermatozoa of Malabari bucks on in vivo fertility

The study was conducted to assess the effect of post-thaw supplementation of seminal plasma to the cryopreserved spermatozoa of Malabari bucks on in vivo fertility. The ejaculates collected from Malabari bucks of good (n=3) and poor (n=3) semen freezability were pooled separately and cryopreserved. After thawing, seminal plasma from good semen freezability bucks was supplemented in such a way that group I (GFSP) included thawed semen of good freezability with supplementation, group II (GF) included thawed semen of good freezability without supplementation, group III (PFSP) included thawed semen of poor freezability with supplementation and group IV (PF) included thawed semen of poor freezability without supplementation. After a period of incubation for 10 min. at 37°C, the semen from each group was used for transcervical insemination of Malabari does of proven fertility. The results showed that there was a significant improvement in the conception rate with post-thaw supplementation. Percent of animals kidded also differed significantly in the four groups. But, prolificacy did not differ in GF and GFSP.

TMT-based comparative proteomic analysis of Dezhou donkey spermatozoa related to freezability

The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation. Our recent study shows that the freezability of donkey semen is related to the seminal plasma proteome. In this study, we aimed to identify the different abundance sperm proteins in good freezability ejaculates (GFEs) and poor freezability ejaculates (PFEs) using a Tandem Mass Tag (TMT) peptide labeling coupled with LC-MS/MS approach. A total of 2682 proteins were identified, among which 58 were significantly up-regulated in GFEs and 16 were down-regulated compared with PFEs. Bioinformatic analysis results revealed that the majority of different abundance proteins (DAPs) participated in copper and calcium binding, regulation of RNA biosynthetic process, positive regulation of innate immune response, and negative regulation of programmed cell death. KEGG pathway enrichment analysis showed the up-regulated proteins in GF group were mainly involved in N-Glycan biosynthesis and protein processing in endoplasmic reticulum. Our study was the first to analyze the proteome of sperm from donkey ejaculates with different freezabilities. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability. SignificanceCryopreserved semen has been widely used in assisted reproductive technology. However, semen cryopreservation is a damaging process, which can cause oxidative stress, reduce sperm motility and motility. There are differences in sperm freezability reported to exist between or within breeds, and even between fractions coming from the same ejaculate. The freezability difference between donkey ejaculates is a limiting factor of sperm cryopreservation.The mechanisms that affect the freezing difference in sperm quality remain to be investigated, and freezability differences was found to be related to protein composition of spermatozoa. Some protein markers that can indicate good freezability or poor freezability semen have been identified in mammals. Until now, there is no information about the relationship between donkey spermatozoa proteome and freezability. Additional novel biomarkers of semen freezability in donkey spermatozoa are also needed. The identified candidate proteins might be used to explore the molecular mechanism related to donkey sperm cryotolerance and might improve the screening of jacks with good sperm freezability.

Evaluation of metabolomic profiling and oxidative stress markers of bull seminal plasma and their relationship with sperm motility before and after thawing

Mechanisms underlying differentiation of good or poor freezability ejaculates in bull are still poorly understood. This study aims at investigating oxidative stress parameters and metabolomic profiling of seminal plasma (SP) in relation to bull semen motility before and after cryopreservation. Semen samples were collected by artificial vagina from 20 bulls aged from 10 to 12 mo. An aliquot of semen was centrifuged to obtain SP (stored at -80°C), another was diluted 1:1 with a pre-warmed extender and cryopreserved. Motility assessment (CASA) was performed on fresh and thawed semen (0 and 3 h after thawing). Semen samples were divided into good freezers (GF) and bad freezers (BF) groups according to thawed semen motility, with cutoff values as follows: TM ≥ 60% and PM ≥ 30%. SP was used for oxidative stress markers analysis by spectroscopy techniques and metabolomic profile by nuclear magnetic resonance (NMR). The relationship between NMR spectra and semen quality was evaluated by the PCA/PLS-DA analysis. General linear models and mixed linear models were used to evaluate the relationship between seminal plasma oxidative stress parameters and semen quality. A clear separation between GF and BF ejaculates was observed on the first two principal components of the variance (PC1 vs PC2) of NMR spectra of SP. The increase of advanced oxidative protein products (AOPP) and thiols concentrations on SP were significantly related with higher straightness (STR), beat cross frequency (BCF) and linearity (LIN) and the decrease of amplitude of lateral head displacement (ALH) of fresh semen. Thiols and AOPP concentrations on SP were positively related to semen freezability. In addition, higher AOPP concentration was significantly related to higher TM, PM and LIN of thawed spermatozoa, whereas higher levels of thiols in SP concentration were positively related to the increment of sperm PM after thawing. These findings suggested that SP composition can modulate bull semen freezabilty. Further analyses could elucidate which metabolites and oxidative stress parameters could be used as biomarkers of good semen quality after cryopreservation.

The freezability of Mediterranean buffalo sperm is associated with lysine succinylation and lipid metabolism.

Semen cryopreservation is used for the propagation of variety among species and domestic breeding. Mitochondria are implicated in sperm freezability, and their proteins are prone to succinylation, but the relationship between sperm freezability and mitochondrial protein succinylation is unclear. In this study, six bulls were classified as having good or poor freezability ejaculates (GFE or PFE, each 3 bulls). The fresh sperm mitochondrial membrane potential (MMP) and pan succinylation level of the two groups were first detected. Then the lysine succinylome and fatty acid content of the two groups were analyzed using label-free LC-MS/MS and GC-MS/MS in multiple reaction monitoring (MRM) modes, respectively. The results indicated that the GFE sperm had significantly higher MMPs than the PFE group (p <0.05). A total of 1393 succinylation sites corresponding to 426 proteins were assessed and 5 succinylated peptides of the GFE group were markedly upregulated, while 3 were significantly downregulated (FC > 2.0- < 0.5 and p-value < 0.05) when compared to the PFE group. Forty-six succinylated proteins were identified to have consistent presence/absence expression. The upregulated succinylated proteins in the GFE sperm were enriched in lipid metabolic processes. A total of 31 fatty acids were further subjected to quantitative analysis of which 23 including arachidic (C20:0), linolenic (C18:3n3), and docosahexaenoic acids (C22:6n3) were decreased in GFE sperm when compared with PFE (p <0.05). These results suggest that lysine succinylation can potentially influence the sperm freezability of Mediterranean buffaloes through mitochondrial lipid metabolism. This novel study provides our understanding of sperm succinylation and the molecular basis for the mechanism of sperm freezability.

Albumin (ALB) and protein disulfide isomerase family A member 4 (PDIA4) are novel markers to predict sperm freezability of Erhualian boar

The frozen semen of Erhualian pig can promote the continuous improvement of commercial pigs, but currently, frozen semen fails to satisfy the practical application requirement. Oxidative damage is one of the crucial factors affecting the quality of frozen semen; besides, there are individual differences in boar sperm freezability. Based on the previous analysis of the proteomic differences of Erhualian boar sperm with different freezability, two differentially abundant proteins (DAPs) in boar sperm, albumin (ALB) and protein disulfide isomerase family A member 4 (PDIA4), were selected as the research objects in the current study. It is assumed that redox-related proteins ALB and PDIA4 can be used as markers to predict Erhualian boar sperm freezability. We cryopreserved the semen of 14 Erhualian boars. According to the difference of frozen semen quality, boars with good and poor freezability ejaculates (GFE and PFE, n = 3) were selected respectively. The relative contents of ALB and PDIA4 in GFE and PFE were analyzed by Western blot, and the localization patterns of ALB and PDIA4 in pre-frozen and frozen-thawed sperm were detected by immunofluorescence. The results showed that the abundances of ALB and PDIA4 in GFE were significantly higher than PFE, and there was a significant correlation between the relative contents of ALB and PDIA4 and frozen-thawed sperm quality parameters. Additionally, the freezing process had no effect on the localization patterns of ALB and PDIA4 in spermatozoa. In conclusion, these results suggest that ALB and PDIA4 are related to boar sperm cryotolerance and may be used as novel freezability markers.

Expression of TXNRD1, HSPA4L and ATP1B1 Genes Associated with the Freezability of Boar Sperm.

Cryopreservation is associated with increased oxidative stress, which is responsible for sperm damage. We analyzed the effect of cryopreservation on mRNA and protein expression of thioredoxin reductase 1 (TXNRD1), heat shock protein family A (HSP 70) member 4 like (HSPA4L) and sodium/potassium-transporting ATPase subunit beta-1 (ATP1B1) genes in boar sperm with different freezability. Boars were classified as having good and poor semen freezability (GSF and PSF, respectively), according to the assessment of post-thaw sperm motility. Total RNA was isolated from fresh pre-freeze (PF) and frozen-thawed (FT) sperm from five boars of the GSF and PSF groups, respectively. Quantification of TXNRD1, HSPA4L and ATP1B1 gene expression was performed by RT-qPCR analysis. Proteins extracted from sperm were subjected to Western blotting and SDS-PAGE analyses. Poor freezability ejaculates were characterized by significantly higher relative mRNA expression levels of TXNRD1 and HSPA4L in FT sperm compared with the fresh PF sperm. Furthermore, the relative mRNA expression level of ATP1B1 was significantly higher in the fresh PF sperm of the GSF group. Western blotting analysis revealed significantly higher relative expression of TXNRD1 protein in the fresh PF sperm of the GSF group, while HSPA4L protein expression was markedly increased in FT sperm of the PSF group. Electrophoretic and densitometric analyses revealed a higher number of proteins in the fresh PF and FT sperm of the PSF and GSF groups, respectively. The results of this study indicate that ATP1B1 mRNA expression in the fresh PF sperm is a promising cryotolerance marker, while the variations of TXNRD1 and HSPA4L protein expression in the fresh PF or FT sperm provide useful information that may help to elucidate their biological significance in cryo-damage.

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

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.

Impact of Seminal Plasma Antioxidants on Donkey Sperm Cryotolerance.

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.

Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n=40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I=very low ubiquitin intensity and IV=very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p≤.05 was considered significant. We observed that PF stallions showed higher percentages (p<.05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p<.05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p<.05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

Physical characteristics of Malabari buck semen and sperm characteristics of semen with good and poor freezability

Physical characteristics of Malabari buck semen and sperm characteristics of semen with good and poor freezability

Razlika između proteina sjemene plazme i proteina sperme za dobru i lošu sposobnost smrzavanja ejakulata nerasta

The present study was performed to compare the expression of sperm proteins, i.e. triosephosphate isomerase (TPI) and acrosin binding protein (ACRBP) and seminal plasma proteins, i.e. glutathione peroxidase 5 (GPX5) and fibronectin 1 (FN1), in boar semen with good, moderate and poor freezability. The study was conducted by determining the protein contents in 32 sperm samples and 38 seminal plasma samples of semen. The ejaculated semen was divided into two portions: the first portion was centrifuged to separate the pellet of sperm from the seminal plasma and the second portion was cryopreserved. After thawing, the ejaculates were classified into three groups according to their post-thawed sperm motility: good (60.2 ± 1.7%), moderate (29.3 ± 2.0%) and poor (16.6 ± 2.2%) freezabilities. The expressions of GPX5 and FN1 in seminal plasma and TPI and ACRBP in sperm were determined using Western blot analysis. It was found that, for sperm proteins, the level of TPI was negatively correlated with the post-thawed total sperm motility (r = -0.38, P = 0.029). For seminal plasma proteins, the level of FN1 in the seminal plasma was positively correlated with the post-thawed total sperm motility (r = 0.37, P = 0.021) and progressive motility (r = 0.39, P = 0.016). The expression of GPX5 was not correlated with any of the frozen–thawed sperm qualities (P &amp;gt; 0.05). In conclusions, boar semen containing a high level of FN1 in seminal plasma has better freezability. Frozen–thawed sperm motility was positively correlated with the level of FN1 in boar seminal plasma and negatively correlated with TPI in boar spermatozoa.

Cryotolerance of equine spermatozoa correlates with specific fatty acid pattern: A pilot study

Sperm cryopreservation represents a powerful tool for horse breeding. To improve the efficiency of artificial insemination in the horse using cryopreserved spermatozoa, an adequate understanding of the underlying biophysical properties that affect sperm cryosurvival needs to be reached yet. In this pilot study, we described isolation and analysis of the main fatty acids from sperms of stallions classified as good and poor freezers (7 GF and 5 PF, according to sperm motility and viability, before and after cryopreservation). Fatty acid profiles were only assessed in pre-thaw sperms. Eight main fatty acids were identified, using gas chromatography, and their contents were expressed as percentage of the total lipid content. We found that lauric, myristic and oleic acid (C12:0, C14:0 and C18:1n9c) turned out to be about 2-fold more abundant in the sperm cells of the GFs compared with PFs. Moreover, we described for the first time the presence of a very high amount of a trans geometrical isomer of linoleic acid, linolelaidic acid (C18:2n6t), in pre-thaw PF spermatozoa. Notably, we found in fresh sperms of PF stallions a ratio of unsaturated fatty acids to saturated fatty acids which was twice that of those of GF group, suggesting a positive effect of a high saturated-to-unsaturated fatty acid ratio for the “freezability” of equine spermatozoa. Finally, principal component analysis (PCA) confirmed the relationships between specific fatty acids and cryotolerance of equine spermatozoa, also providing a graphical classification and additional information about the dominant variables governing the classification process.

Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals.

Sperm cryopreservation is a powerful tool for the livestock breeding program. Several technical attempts have been made to enhance the efficiency of spermatozoa cryopreservation in different farm animal species. However, it is well-recognized that mammalian spermatozoa are susceptible to cryo-injury caused by cryopreservation processes. Moreover, the factors leading to cryo-injuries are complicated, and the cryo-damage mechanism has not been methodically explained until now, which directly influences the quality of frozen–thawed spermatozoa. Currently, the various OMICS technologies in sperm cryo-biology have been conducted, particularly proteomics and transcriptomics studies. It has contributed while exploring the molecular alterations caused by cryopreservation, identification of various freezability markers and specific proteins that could be added to semen diluents before cryopreservation to improve sperm cryo-survival. Therefore, understanding the cryo-injury mechanism of spermatozoa is essential for the optimization of current cryopreservation processes. Recently, the application of newly-emerged proteomics and transcriptomics technologies to study the effects of cryopreservation on sperm is becoming a hotspot. This review detailed an updated overview of OMICS elements involved in sperm cryo-tolerance and freeze-thawed quality. While also detailed a mechanism of sperm cryo-injury and utilizing OMICS technology that assesses the sperm freezability potential biomarkers as well as the accurate classification between the excellent and poor freezer breeding candidate.

The structure of subpopulations of stallion spermatozoa after thawing differs between good and poor freezers

The aim of this study was to evaluate differences in the presence of sperm subpopulations in frozen-thawed semen in stallions with different freezability. The motility of individual spermatozoa of 24 stallions from 15 breeds was evaluated using computer-assisted sperm analysis (CASA) immediately after thawing (T0) and after 30 min of incubation (T30). In accordance with our previous studies, samples were initially divided based on their total motility into categories of good (GF) and poor (PF) freezers. K-means cluster analysis of kinematic parameters of spermatozoa was used to divide motile sperm (n = 57 630) into three subpopulations. Analysis of variance was used to evaluate differences in the subpopulations between GF and PF stallions at the times of incubation T0 and T30. Statistically significant differences were found in most kinematic parameters between PF and GF stallions as well as between the times of incubation T0 and T30 (P &amp;lt; 0.05). Spermatozoa of good freezers are represented more frequently in the fast and medium fast subpopulations and are faster and more linear than those of poor freezers (P &amp;lt; 0.05). Sperm from PF stallions were more strongly affected by longer incubation. The percentage of sperm in the fast and medium fast subpopulations was lower in samples from PF stallions, but assessment of the motility parameters in particular sperm subpopulations revealed that these sperm had good velocity. Poor freezer samples had lower sperm quality due to a reduced total proportion of motile sperm, and these samples were more sensitive to prolonged time after thawing. Thus, an efficient sperm selection method or a special insemination technique should be used for obtaining doses from stallions with poor freezability. Our study showed that the CASA system and cluster analysis are promising tools for better understanding the significant differences in the individual stallion freezability, and further research should be focused on their application in the field.

Regulatory Potential of Long Non-Coding RNAs (lncRNAs) in Boar Spermatozoa with Good and Poor Freezability.

Long non-coding RNAs (lncRNAs) are suggested to play an important role in the sperm biological processes. We performed de novo transcriptome assembly to characterize lncRNAs in spermatozoa, and to investigate the role of the potential target genes of the differentially expressed lncRNAs (DElncRNAs) in sperm freezability. We detected approximately 4007 DElncRNAs, which were differentially expressed in spermatozoa from boars classified as having good and poor semen freezability (GSF and PSF, respectively). Most of the DElncRNAs were upregulated in boars of the PSF group and appeared to significantly affect the sperm’s response to the cryopreservation conditions. Furthermore, we predicted that the potential target genes were regulated by DElncRNAs in cis or trans. It was found that DElncRNAs of both freezability groups had potential cis- and trans-regulatory effects on different protein-coding genes, such as COX7A2L, TXNDC8 and SOX-7. Gene Ontology (GO) enrichment revealed that the DElncRNA target genes are associated with numerous biological processes, including signal transduction, response to stress, cell death (apoptosis), motility and embryo development. Significant differences in the de novo assembled transcriptome expression profiles of the DElncRNAs between the freezability groups were confirmed by quantitative real-time PCR analysis. This study reveals the potential effects of protein-coding genes of DElncRNAs on sperm functions, which could contribute to further research on their relevance in semen freezability.

Cellular and Functional Physiopathology of Bull Sperm With Altered Sperm Freezability.

The objective of this study was to ascertain the cellular and functional parameters as well as ROS related changes in sperm from bulls with varied sperm freezability phenotypes. Using principal component analysis (PCA), the variables were reduced to two principal components, of which PC1 explained 48% of the variance, and PC2 explained 24% of the variance, and clustered animals into two distinct groups of good freezability (GF) and poor freezability (PF). In ROS associated pathophysiology, there were more dead superoxide anion positive (Dead SO+) sperm in GF bulls than those in PF (15.72 and 12.00%; P = 0.024), and that Dead SO+ and live hydrogen positive cells (live H2O2+) were positively correlated with freezability, respectively (R2 = 0.55, P < 0.0130) and (rs = 0.63, P = 0.0498). Related to sperm functional integrity, sperm from PF bulls had greater dead intact acrosome (DIAC) than those from GF bulls (26.29 and 16.10%; P = 0.028) whereas sperm from GF bulls tended to have greater live intact acrosome (LIAC) than those from PF bulls (64.47 and 50.05%; P = 0.084). Sperm with dead reacted acrosome (DRAC) in PF bulls were greater compared to those in GF (19.27 and 11.48%; P = 0.007). While DIAC (R2 = 0.56, P = 0.0124) and DRAC (R2 = 0.57, P < 0.0111) were negatively correlated with freezability phenotype, LIAC (R2 = 0.36, P = 0.0628) was positively correlated. Protamine deficiency (PRM) was similar between sperm from GF and PF bulls (7.20 and 0.64%; P = 0.206) and (rs = 0.70, P = 0.0251) was correlated with freezability. Sperm characteristics associated with cryotolerance are important for advancing both fundamental andrology and assisted reproductive technologies across mammals.

Lyophilized seminal plasma can improve stallion semen freezability

The aim of this study was to evaluate the effect of lyophilized seminal plasma (LSP) on stallion semen freezability. Seminal plasma from 30 stallions was lyophilized to obtain a pool of LSP. Fifteen ejaculates from five stallions were supplemented before freezing with 0 mg/mL (Control), 1.44 mg/mL (LSP1), 5.04 mg/mL (LSP2) or 8.68 mg/ mL (LSP3) of LSP. Total antioxidant capacity (TAC) of LSP was assessed using Oxygen Radical Absorbance Capacity (ORAC) assay. Post-thaw motility and kinetics, sperm viability, normal morphology and membrane integrity were evaluated. Completely randomized mixed models were fitted for data analyses. The results was analyzed based on freezability of semen samples. TAC for LSP pool was 13679.4±911.6 μmol Trolox 100/g (ORAC units). Semen supplementation with LSP1 and LSP2 showed a positive effect on post-thaw total motility and membrane integrity. Supplementation with LSP3 showed a decrease in post-thaw total and progressive motility, straight line velocity and sperm viability. For poor freezability semen samples, supplementation with LSP1 and LSP2, showed higher post-thaw total motility and membrane integrity than good freezability semen samples. In conclusion, supplementation with LSP can improve the post-thaw seminal quality of stallion semen with poor freezability.