Boar Sperm
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BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Ziyue Qin ◽  
Wencan Wang ◽  
Malik Ahsan Ali ◽  
Yihan Wang ◽  
Yan Zhang ◽  

Abstract Background Cryopreservation induces transcriptomic and epigenetic modifications that strongly impairs sperm quality and function, and thus decrease reproductive performance. N6-methyladenosine (m6A) RNA methylation varies in response to stress and has been implicated in multiple important biological processes, including post-transcriptional fate of mRNA, metabolism, and apoptosis. This study aimed to explore whether cryopreservation induces m6A modification of mRNAs associated with sperm energy metabolism, cryoinjuries, and freezability. Results The mRNA and protein expression of m6A modification enzymes were significantly dysregulated in sperm after cryopreservation. Furthermore, m6A peaks were mainly enriched in coding regions and near stop codons with classical RRACH motifs. The mRNAs containing highly methylated m6A peaks (fts vs. fs) were significantly associated with metabolism and gene expression, while the genes with less methylated m6A peaks were primarily involved in processes regulating RNA metabolism and transcription. Furthermore, the joint analysis of DMMGs and differentially expressed genes indicated that both of these play a vital role in sperm energy metabolism and apoptosis. Conclusions Our study is the first to reveal the dynamic m6A modification of mRNAs in boar sperm during cryopreservation. These epigenetic modifications may affect mRNA expression and are closely related to sperm motility, apoptosis, and metabolism, which will provide novel insights into understanding of the cryoinjuries or freezability of boar sperm during cryopreservation.

Yongjie Xu ◽  
Qiu Han ◽  
Chaofeng Ma ◽  
Yaling Wang ◽  
Pengpeng Zhang ◽  

Sperm cells are of unique elongated structure and function, the development of which is tightly regulated by the existing proteins and the posttranslational modifications (PTM) of these proteins. Based on the phylogenetic relationships of various swine breeds, Yorkshire boar is believed to be distinctly different from Duroc boar. The comprehensive differential proteomics and phosphoproteomics profilings were performed on spermatozoa from both Yorkshire and Duroc boars. By both peptide and PTM peptide quantification followed by statistical analyses, 167 differentially expressed proteins were identified from 1,745 proteins, and 283 differentially expressed phosphopeptides corresponding to 102 unique differentially phosphorylated proteins were measured from 1,140 identified phosphopeptides derived from 363 phosphorylated proteins. The representative results were validated by Western blots. Pathway enrichment analyses revealed that majority of differential expression proteins and differential phosphorylation proteins were primarily concerned with spermatogenesis, male gamete generation, sperm motility, energy metabolism, cilium morphogenesis, axonemal dynein complex assembly, sperm–egg recognition, and capacitation. Remarkably, axonemal dynein complex assembly related proteins, such as SMCP, SUN5, ODF1, AKAP3, and AKAP4 that play a key regulatory role in the sperm physiological functions, were significantly higher in Duroc spermatozoa than that of Yorkshire. Furthermore, phosphorylation of sperm-specific proteins, such as CABYR, ROPN1, CALM1, PRKAR2A, and PRKAR1A, participates in regulation of the boar sperm motility mainly through the cAMP/PKA signal pathway in different breeds, demonstrating that protein phosphorylation may be an important mechanism underlying the sperm diversity. Protein–protein interaction analysis revealed that the 14 overlapped proteins between differential expression proteins and differential phosphorylation proteins potentially played a key role in sperm development and motility of the flagellum, including the proteins ODF1, SMCP, AKAP4, FSIP2, and SUN5. Taken together, these physiologically and functionally differentially expressed proteins (DEPs) and differentially expressed phosphorylated proteins (DPPs) may constitute the proteomic backgrounds between the two different boar breeds. The validation will be performed to delineate the roles of these PTM proteins as modulators of Yorkshire and Duroc boar spermatozoa.

2021 ◽  
Vol 36 (Supplement_1) ◽  
S Galal ◽  
C Jones ◽  
K Coward

Abstract Study question Do solid silica nanoparticles qualify as a new research tool for the in vitro transfer of compounds into gametes prior to Assisted Reproductive Technology (ART). Summary answer Solid silica nanoparticles (SSNPs) could be used as an intra-gamete delivery system to deliver therapeutic biomolecules into gametes prior to ART. What is known already Sperm-mediated gene transfer (SMGT) results in the production of transgenic embryos; however, the success rate of this technique is low. Nanoparticles are an efficient intra-cellular delivery system in vitro. Naturally cell-secreted nanoparticles are involved in the development of gametes. Mesoporous silica nanoparticles have been shown to carry large amounts of compounds and to interact with gametes without toxic effects, thus providing an alternative to naturally secreted nanoparticles. However, this technique is associated with some limitations, such as the size of these nanoparticles. SSNPs can be synthesised on a smaller nanoscale, thus providing higher potential to penetrate gametes and delivering biomolecules. Study design, size, duration This was an experimental in vitro study that investigated the effects of SSNPs on the motility of boar sperm and the degeneration of hamster oocytes, as determined by ooplasm shrinkage. Participants/materials, setting, methods SSNPs (20 nm) were conjugated with fluorescein diacetate–5-maleimide (FDA5M), a fluorescent protein. FDA5M-labelled SSNPS were incubated with boar sperm (N = 3) at 10 and 30µg/ml/107 sperm for four-hours. Motility parameters were assessed by computer-assisted sperm analysis (CASA). Binding potential was evaluated by fluorescent microscopy. Hamster oocytes (7 oocytes/group) were incubated with FDA5M-labelled SSNPs at 100, 150, and 300µg/ml, for two-hours; ooplasm shrinkage was evaluated. Time/matched control sperm was incubated in phosphate-buffered saline and oocytes in KSOM. Main results and the role of chance Exposure to FDA5M-labelled SSNPs did not affect total or progressive sperm motility (P = 0.6735 and 0.9606, respectively), average-path velocity or straight-line velocity after 4-hours of incubation (P = 0.7459 and 0.8696, respectively) compared to controls. SSNPs at 10 µg/ml significantly increased sperm curvilinear velocity after 1-hour (P = 0.0495) and linearity and straightness after 4-hours (P = 0.0389 and 0.0312, respectively). SSNPs at 30 µg/ml significantly increased sperm linearity after 3- and 4-hours (P = 0.0384 and 0.005, respectively). The proportion of sperm showing green fluorescence was significantly higher in the 30µg/ml dose of SSNPs than the 10µg/ml dose after 4-hours (P < 0.00001). In oocytes, the zona pellucida remained morphologically intact and the ooplasm exhibited green fluorescence. The ooplasm of 42% of the oocytes at 300µg/ml showed ooplasm shrinkage (a sign of degeneration); no oocytes showed shrinkage at doses of 100 and 150µg/ml of SSNPs. The green fluorescence in the sperm head and the ooplasm indicated the ability of SSNPs to spontaneously interact non-invasively with these gametes either by surface association or by cell-internalisation. This could provide a safe and non-invasive intra-gamete delivery system for research purposes and clinical therapy. This system could be used to deliver specific agents into gametes prior to ART to improve outcomes. Limitations, reasons for caution The SSNPs are non-biodegradable; it remains unknown as to how gametes or embryos might react with SSNPs over long time periods. The nanotoxicity of SSNPs has not yet been investigated over the long term. SSNPs have still to be tested with embryos to evaluate their effect on embryonic development. Wider implications of the findings: SSNPs could be functionalised to target the nucleus of mammalian gametes and embryos to act as a carrier for oligonucleotides and genes to correct chromosomal abnormalities and to provide genetic therapy in these gametes and embryos to treat hereditary diseases before intra-uterine transfer. Trial registration number Not applicable

Animals ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1939
Yuting Zhang ◽  
Hanlin Liang ◽  
Yan Liu ◽  
Meng Zhao ◽  
Qianqian Xu ◽  

Some potential markers of boar sperm freezability have been found in spermatozoa, but little attention has been paid to seminal plasma. The seminal plasma is composed of secretions from the testis, epididymis, and accessory sex glands. The exposure of spermatozoa to small molecules such as metabolites can affect sperm function. However, details and significance of the seminal plasma metabolome related to boar sperm freezability are unknown. Therefore, the main aim of this study was to explore the differences in the metabolic level of seminal plasma between boars with differential freezability and to explore the candidate biomarkers of semen freezability. A total of 953 metabolites were identified in boar semen plasma by UHPLC-qTOF-MS analysis, and 50 metabolites showed significant change between the GFE group and PFE group. Further, twelve metabolites were subjected to metabolic target analysis, and three metabolites (D-aspartic acid, N-acetyl-L-glutamate (NAG), and inosine) showed differences. In conclusion, there is significant difference in the metabolome of seminal plasma between GFE and PFE individuals. D-aspartic acid, NAG, and inosine in seminal plasma may be potential markers for assessing sperm cryopreservation resistance in boars.

Athina Basioura ◽  
Georgios Tsousis ◽  
Constantin M. Boscos ◽  
Ioannis A. Tsakmakidis

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1813
Ilias Michos ◽  
Maria Tsantarliotou ◽  
Constantin M. Boscos ◽  
Georgios Tsousis ◽  
Athina Basioura ◽  

This study aimed to evaluate boar sperm characteristics and proteins, in relation to their importance regarding in vivo fertility. Sixty-five ejaculates were used and 468 sows (parity ≥ 2) were inseminated. Sperm CASA kinetics, morphology, viability, DNA fragmentation, mitochondrial membrane potential, sperm membrane biochemical activity (HOST) and sperm proteins (Heat Shock Protein 90-HSP90, glutathione peroxidase-5-GPX5, Osteopontin 70-OPN70) were assessed and related to field fertility (number of live-born piglets—NLBP, litter size ≥ 12 piglets—LS, farrowing rate—FR). Statistical analysis was conducted with simple and multiple regression models. Simple regression analysis showed that immotile sperm (IM) significantly affected the NLBP and LS, explaining 6.7% and 6.5% of their variation, respectively. The HOST positive spermatozoa significantly affected the NLBP and LS, explaining 24.5% and 7.8% of their variation, respectively. Similarly, sperm with activated mitochondria significantly affected the NLBP, explaining 13.5% of its variation. Moreover, the OPN70 affected LS and FR, explaining 7.5% and 10.8% of their variation, respectively. Sperm GPX5 protein affected FR, explaining 6.7% of its variation. Multiple regression analysis showed that the combination of IM and/OPN70 explains 13.0% of the variation regarding LS, and the combination of GPX5 and OPN70 explains 13.6% of the variation regarding FR. In conclusion, the estimation of parameters IM, membrane biochemical activity, mitochondrial membrane potential, OPN and GPX5 can provide useful information regarding semen doses for field fertility.

2021 ◽  
Vol 52 (6) ◽  
Janyaporn Rungruangsak ◽  
Junpen Suwimonteerabutr ◽  
Kakanang Buranaamnuay ◽  
Arun Chankrachang ◽  
Padet Tummaruk

Coomassie blue staining has been reported as an effective and inexpensive method for evaluating the acrosome integrity of spermatozoa, though to date its use to evaluate cryopreserved boar sperm has not been reported. Moreover, there is no information concerning the agreement between Coomassie blue staining and fluorescein isothiocyanate conjugated peanut agglutinin and ethidium homodimer (FITC-PNA/EthD-1) methods for assessing sperm acrosome integrity for any species. The current study was performed to determine the efficacy and agreement between Coomassie blue and FITC-PNA/EthD-1 staining methods for evaluating the acrosome integrity of frozen-thawed boar sperm. A total of 25 semen samples were cryopreserved using lactose-egg yolk-based extender and loaded into 0.5 PVC-French straws. Sperm motility and motion characteristics were determined using a computer-assisted sperm analysis system. Sperm viability and plasma membrane integrity were evaluated using the SYBR-14/EthD-1 and hypo-osmotic swelling test, respectively. Acrosome integrity of frozen-thawed boar sperm was evaluated using both FITC-PNA/EthD-1 and Coomassie blue staining to assess the association between sperm acrosome integrity and agreement between these two methods. The average percent acrosome integrity of frozen-thawed boar sperm as determined by FITC-PNA/ EthD-1 and Coomassie blue staining was 48.8 ± 12.6% and 52.6 ± 13.6%, respectively (P>0.05). Interestingly, Coomassie blue staining found a correlation between sperm viability and acrosome integrity (r=0.609, P=0.002), while FITC-PNA/EthD-1 staining did not (P>0.05). However, the acrosome integrity of frozen-thawed boar sperm evaluated by FITC-PNA/ EthD-1 and Coomassie blue staining was significantly correlated (r=0.448, P=0.025, n=25). The Bland-Altman plot determined that this agreement was acceptable. In conclusion, the acrosome integrity of the frozen-thawed boar sperm assessed via Coomassie blue staining was significantly correlated with that obtained via the FITC-PNA/EthD-1 staining method, and the two methods showed good agreement. Moreover, the significant association between the acrosome integrity of frozen-thawed boar sperm determined by Coomassie blue staining with other sperm quality parameters indicates that this is an effective method for assessing the acrosome integrity of frozen-thawed sperm in pigs.

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0251973
Yoku Kato ◽  
Satheesh Kumar ◽  
Christian Lessard ◽  
Janice L. Bailey

In boar sperm, we have previously shown that capacitation is associated with the appearance of the p32 tyrosine phosphoprotein complex. The principal tyrosine phosphoprotein involved in this complex is the acrosin-binding protein (ACRBP), which regulates the autoconversion of proacrosin to intermediate forms of acrosin in both boar and mouse sperm. However, the complete biological role of ACRBP has not yet been elucidated. In this study, we tested the hypothesis that tyrosine phophorylation and the presence of the ACRBP in the sperm head are largely necessary to induce capacitation, the acrosome reaction (AR) and sperm-zona pellucida (ZP) binding, all of which are necessary steps for fertilization. In vitro fertilization (IVF) was performed using matured porcine oocytes and pre-capacitated boar sperm cultured with anti-phosphotyrosine antibodies or antibodies against ACRBP. Anti-ACRBP antibodies reduced capacitation and spontaneous AR (P<0.05). Sperm-ZP binding declined in the presence of anti-phosphotyrosine or anti-ACRBP antibodies. The localisation of anti-ACRBP antibodies on the sperm head, reduced the ability of the sperm to undergo the AR in response to solubilized ZP or by inhibiting the sarco/endoplasmic reticulum Ca2+-ATPase. These results support our hypothesis that tyrosine phosphorylated proteins and ACRBP are present upon the sperm surface in order to participate in sperm-ZP binding, and that ACRBP upon the surface of the sperm head facilitates capacitation and the AR in the porcine.

2021 ◽  
Vol 99 (Supplement_1) ◽  
pp. 211-211
Kayode B Balogun ◽  
Griffin Nicholls ◽  
Olujide Sokunbi ◽  
Kara R Stewart

Abstract Improvements in the post-thaw quality of frozen semen could allow increased utilization of this technology in the swine industry. The objective of this study was to investigate the effects of natural honey inclusion in semen extender and freezing media on motility, mobility, and morphology of cryopreserved boar sperm. Ejaculates from 6 terminal cross-bred boars were collected using the gloved-hand technique for 3 weeks and used in a 2 x 3 factorial study design. Following collection, semen samples were incubated overnight in dilution extender with and without natural honey (D0: Androhep Plus; D1: Androhep Plus + 0.25% honey). The following day, the semen samples were cooled to 4 C in LEY cooling extender then frozen in freezing media containing 93% cooling extender + 6% glycerol + 1% Equex-STM Paste (F1), or freezing media with natural honey replacing 50% of the glycerol (F2) or 50% of the Equex-STM paste (F3). Semen samples were frozen using a controlled-rate freezer and stored in liquid nitrogen. Two straws per treatment for each boar were thawed and semen quality assessed. The inclusion of natural honey in dilution extender had no effect on post-thaw motility (P=0.733), progressive motility (P=0.562), or other mobility parameters (0.995≤P≥0.081). However, D1 had a higher percentage of normal acrosomes (P=0.001) and morphologically normal cells (P&lt; 0.001) resulting from lower tail abnormalities compared to D0 (P=0.006). Post-thaw motility (P&lt; 0.001) and progressive motility (P&lt; 0.001) were increased in F3 compared to both F2 and F1. F1 had reduced normal acrosomes (P=0.009) and morphologically normal cells (P&lt; 0.001) resulting from higher tail abnormalities (P&lt; 0.001). In conclusion, the inclusion of natural honey, at 0.25%, in dilution extender helps maintain normal sperm and acrosome morphology, and replacing 50% Equex-STM Paste with honey in freezing extender improves post-thaw sperm motility and progressive motility of frozen-thawed boar semen.

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