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.

Promoter polymorphisms in STK35 and IFT27 genes and their associations with boar sperm freezability.

We have shown that STK35 and IFT27 genes are differentially expressed in spermatozoa from boars with good and poor semen freezability (GSF and PSF, respectively). STK35 is a stress-related gene that is implicated in spermatogenesis, whereas IFT27 is a motility-related gene that is mainly involved in intracellular protein transport. In this study we hypothesized that polymorphic variants in the 5'-flanking regulatory regions of STK35 and IFT27 genes could contribute to differences in semen freezability. We also predicted the interactions of the polymorphic variants with transcription factors on the gene promoter activity, using bioinformatics. The 5'-flanking region sequences of the STK35 and IFT27 were PCR amplified and analyzed by Sanger sequencing method. Protein expression in STK35 and IFT27 was determined in pre-freeze (PF) and frozen-thawed (FT) spermatozoa, using western blotting analysis. Sanger sequencing revealed a single nucleotide polymorphism (SNP) rs327863835 (C > T) in STK35 promoter, while two SNPs (rs337563873, A > T; rs331520020, T > C) were detected in IFT27 promoter. STK35 and IFT27 promoter polymorphisms showed significant allele frequency differences between the GSF and PSF groups. Using bioinformatics approaches, we predicted that SNPs resulted in the generation of additional transcription factor binding sites for NFATC2, ELK1 and GR-ß, which appeared to enhance or repress the promoter activity of STK35 or IFT27 in either freezability group. Wide variations in STK35 and IFT27 protein expression were observed among the boars, however, significantly higher protein expression was detected in IFT27 in FT spermatozoa of the GSF group. We suggest that the upstream variants, detected in STK35 and IFT27 promoters, might regulate the transcriptional activity of the genes by affecting their potential binding of transcription factors. The results indicate that the allelic variants in STK35 and IFT27 could be considered as potential genetic markers for predicting boar sperm freezability.

The Usefulness of Sexual Behaviour Assessment at the Beginning of Service to Predict the Suitability of Boars for Artificial Insemination.

Parameters of sexual activity were determined in 49 young boars used for artificial insemination, four times at three-month intervals. The parameters included the time from entering the arena until mounting the phantom; the time from mounting the phantom until achieving erection; the time from achieving full erection until the start of ejaculation; duration of ejaculation; and the number of times the boar mounted the phantom. Characteristics of the ejaculates were also assessed. The libido parameter associated with the greatest efficacy of artificial insemination was the effectiveness of artificial insemination service, the time from entering the arena until the start of ejaculation. The significance of this trait for predicting ejaculation performance was analysed. The libido characteristics were classified into three categories: boars with a short reaction time to the phantom, boars with an intermediate reaction time, and boars with a long reaction time. For these groups, the characteristics of ejaculates collected at the start of the period during which ejaculates were collected and after three, six and nine months were determined. The sexual experience of boars was not associated with the expression of sexual behaviour because young boars during their first three months of ejaculate collections required less time to initiate ejaculation. The ejaculates with the greatest utility were obtained after six months of service. These ejaculates had the largest volume (255.22 mL), and the most insemination doses could be prepared from these ejaculates. On average, more than 23 insemination doses were prepared from ejaculates collected after six months of semen collections, which is about four doses more than from ejaculates collected at the start of artificial insemination service (p < 0.01).The time from entering the arena to beginning ejaculation can be used to predict a boar's future libido. A relationship was shown between the level of libido and ejaculate characteristics. The ejaculates of the boars which needed the longest time to begin ejaculation at the start of semen collections had the greatest sperm concentration and number. In group 3, the boars'ejaculates contained about 6-9 × 109 more sperm than the ejaculates of boars from group 1. After six months of the experimental period, the difference was nearly 15 × 109 sperm (p < 0.05), and after nine months, it exceeded 22 × 109 sperm (p < 0.01).

Fractionated Seminal Plasma of Boar Ejaculates Analyzed by LC-MS/MS: Its Effects on Post-Thaw Semen Quality.

This study aimed to characterize the protein composition of fractionated seminal plasma (SP) by liquid chromatography mass spectrometry (LC-MS/MS) analysis and investigate its effects on survival of frozen-thaw (FT) boar spermatozoa following storage. Seminal plasma (SP) was fractionated by gel filtration chromatography to give two fractions, SP1 with more than 40 kDa (>40 kDa) and SP2 with less than 40 kDa (<40 kDa). SP1 and SP2 were subjected to LC-MS/MS and bioinformatics analysis. Following cryopreservation, FT boar semen (n = 7) was thawed in Beltsville Thawing Solution (BTS), BTS + SP1 or BTS + SP2, stored at different periods and subjected to post-thaw (PT) quality assessment. A total of 52 and 22 abundant proteins were detected in SP1 and SP2, respectively. FN1, ANGPTL1, and KIF15 proteins were more abundance in SP1, whereas a high abundance of spermadhesins (PSP-I and PSP-II) was detected in SP2. Proteins of the fractionated SP were involved in various biological processes, such as cell motility and signal transduction. The dominant pathway of SP1 proteins was the apelin signaling pathway (GNA13, MEF2D, SPHK2, and MEF2C), whereas a pathway related to lysosome (CTSH, CTSB, and NPC2) was mainly represented by SP2 proteins. In most of the boars, significantly higher motility characteristics, membrane integrity, and viability were observed in FT spermatozoa exposed to SP1 or SP2 compared with BTS. The results of our study confirm that a combination of several proteins from the fractionated SP exerted beneficial effects on the sperm membrane, resulting in improved quality characteristics following PT storage.

Effects of breed and ejaculate volume on sperm morphology and semen parameters of boars.

The aim of the study was to determine the relation between the semen quality, frequency of sperm defects, sperm dimensions and shape, and the ejaculate volume of Large White and Landrace boars. A total of 648 ejaculates collected from 31 Large White and 30 Landrace boars were divided into three groups according to the criterion of the ejaculate volume. In this study Landrace boars produced ejaculates with higher volume, sperm concentration, and total numbers of spermatozoa than Large White boars. Landrace boars also showed a lower frequency of sperm with morphological abnormalities (P < 0.05). Landrace boars sperm had larger heads, which were by 0.15 µm longer, and by a larger perimeter and area (P < 0.05). Landrace boar spermatozoa also had a longer flagellum and were generally larger and by 2.07 µm longer than Large White boar sperm (P < 0.05). Significant differences were also found in the shape of sperm of the two breeds (P < 0.05). Landrace boars sperm had more elongated heads, and the ratio of head size to flagellum length was lower than in Large White boars sperm (P < 0.05). Sperm from ejaculates with low volume had a shorter flagellum and a greater head length/flagellum length ratio than sperm from medium- and high-volume ejaculates (P < 0.05).

Molecular Characterisation of Uterine Endometrial Proteins during Early Stages of Pregnancy in Pigs by MALDI TOF/TOF.

The molecular mechanism underlying embryonic implantation is vital to understand the correct communications between endometrium and developing conceptus during early stages of pregnancy. This study's objective was to determine molecular changes in the uterine endometrial proteome during the preimplantation and peri-implantation between 9 days (9D), 12 days (12D), and 16 days (16D) of pregnant Polish Large White (PLW) gilts. 2DE-MALDI-TOF/TOF and ClueGOTM approaches were employed to analyse the biological networks and molecular changes in porcine endometrial proteome during maternal recognition of pregnancy. A total of sixteen differentially expressed proteins (DEPs) were identified using 2-DE gels and MALDI-TOF/TOF mass spectrometry. Comparison between 9D and 12D of pregnancy identified APOA1, CAPZB, LDHB, CCT5, ANXA4, CFB, TTR upregulated DEPs, and ANXA5, SMS downregulated DEPs. Comparison between 9D and 16D of pregnancy identified HP, APOA1, ACTB, CCT5, ANXA4, CFB upregulated DEPs and ANXA5, SMS, LDHB, ACTR3, HP, ENO3, OAT downregulated DEPs. However, a comparison between 12D and 16D of pregnancy identified HP, ACTB upregulated DEPs, and CRYM, ANXA4, ANXA5, CAPZB, LDHB, ACTR3, CCT5, ENO3, OAT, TTR down-regulated DEPs. Outcomes of this study revealed key proteins and their interactions with metabolic pathways involved in the recognition and establishment of early pregnancy in PLW gilts.

Gene promoter polymorphisms in boar spermatozoa differing in freezability.

Single nucleotide polymorphisms (SNPs) in the 5'-flanking regulatory regions of genes could affect their expression levels. This is a follow-up study aimed to identify polymorphic variants in the 5'-flanking regulatory regions of genes expressed in boar spermatozoa, and to predict the interactions of such variants with transcription factors (TFs) on the gene promoter activity, using bioinformatics. Five and six boars were classified as having good and poor semen freezability (GSF and PSF, respectively) according to post-thaw (PT) assessment of sperm motility and membrane integrity characteristics. The 5'-flanking region sequences of the 14 genes (FOS, NFATC3, EAF2, FGF-14, BAMBI, RAB33B, CKS2, LARS2, SLC25A16, ACADM, CPT2, CCT3, DTD2 and CCDC85A) were PCR amplified and analyzed by Sanger sequencing method. A total of 32 polymorphic variants were identified in the 5'-flanking regions of the genes, including 4 insertion/deletion (indel) polymorphisms, and 8 unknown (novel) SNPs. Multiple sequence alignment analysis revealed a 26-bp indel variant in the 5'-flanking region of the LARS2 gene, which showed greater protein expression in spermatozoa from boars of the PSF group. It was found that 17 polymorphic variants, observed in the differentially expressed (DE) genes, showed significant allele frequency differences between the GSF and PSF groups. Polymorphic variants in the 5'-flanking regulatory regions of the genes contributed to the decrease or increase in the binding affinity for different testis-specific TFs, such as SMAD1, NF-1, FOXMI, RXRA, STAT4 and C/EBPß. This study provides more insights into the mechanisms responsible for variations in transcriptional activity in promoters of genes expressed in boar spermatozoa. The allelic variants are promising genetic markers for predicting the freezability of boar spermatozoa.

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.

Identification of Differentially Expressed Gene Transcripts in Porcine Endometrium during Early Stages of Pregnancy.

During the early stages of pregnancy, the uterine endometrium undergoes dramatic morphologic and functional changes accompanied with dynamic variation in gene expression. Pregnancy-stage specific differentially expressed gene (DEG)-transcript-probes were investigated and identified by comparing endometrium transcriptome at 9th day (9D), 12th day (12D) and 16th day (16D) of early pregnancy in Polish large-white (PLW) gilts. Endometrium comparisons between 9D-vs-12D, 9D-vs-16D and 12D-vs-16D of early pregnancy identified 6049, 374 and 6034 highly significant DEG-transcript-probes (p < 0.001; >2 FC). GO term enrichment analysis identified commonly shared upregulated endometrial DEG-transcript-probes (p < 0.001; >2 FC), that were regulating the gene functions of anatomic structure development and transport (TG), DNA-binding and methyltransferase activity (ZBTB2), ion-binding and kinase activity (CKM), cell proliferation and apoptosis activity (IL1B). Downregulated DEG-transcript-probes (p < 0.001; >2 FC) were involved in regulating the gene functions of phosphatase activity (PTPN11), TC616413 gene-transcript and Sus-scrofa LOC100525539. Moreover, blastn comparison of microarray-probes sequences against sus-scrofa11 assembly identified commonly shared upregulated endometrial DEG-transcript-probes (E < 0.06; >2 FC), that were regulating the gene functions of reproduction and growth (SELENOP), cytoskeleton organization and kinase activity (CDC42BPA), phosphatase activity (MINPP1), enzyme-binding and cell-population proliferation (VAV3), cancer-susceptibility candidate gene (CASC4), cytoskeletal protein-binding (COBLL1), ion-binding, enzyme regulator activity (ACAP2) Downregulated endometrial DEG-transcript-probes (E < 0.06; >2FC) were involved in regulating the gene functions of signal-transduction (TMEM33), catabolic and metabolic processes (KLHL15). Microarray validation experiment on selected candidate genes showed complementarity to significant endometrial DEG-transcript-probes responsible for the regulation of immune response (IL1B, S100A11), lipid metabolism (FABP3, PPARG), cell-adhesion (ITGAV), angiogenesis (IL1B), intercellular transmission (NMB), cell-adhesion (OPN) and response to stimuli (RBP4) was confirmed by RT-PCR. This study provides a clue that identified pregnancy-stage specific microarray transcript probes could be considered as candidate genes for recognition and establishment of early pregnancy in the pig.

Gene Polymorphisms in Boar Spermatozoa and Their Associations with Post-Thaw Semen Quality.

Genetic markers have been used to assess the freezability of semen. With the advancement in molecular genetic techniques, it is possible to assess the relationships between sperm functions and gene polymorphisms. In this study, variant calling analysis of RNA-Seq datasets was used to identify single nucleotide polymorphisms (SNPs) in boar spermatozoa and to explore the associations between SNPs and post-thaw semen quality. Assessment of post-thaw sperm quality characteristics showed that 21 boars were considered as having good semen freezability (GSF), while 19 boars were classified as having poor semen freezability (PSF). Variant calling demonstrated that most of the polymorphisms (67%) detected in boar spermatozoa were at the 3'-untranslated regions (3'-UTRs). Analysis of SNP abundance in various functional gene categories showed that gene ontology (GO) terms were related to response to stress, motility, metabolism, reproduction, and embryo development. Genomic DNA was isolated from sperm samples of 40 boars. Forty SNPs were selected and genotyped, and several SNPs were significantly associated with motility and membrane integrity of frozen-thawed (FT) spermatozoa. Polymorphism in SCLT1 gene was associated with significantly higher motility and plasma membrane integrity of FT spermatozoa from boars of the GSF group compared with those of the PSF group. Likewise, polymorphisms in MAP3K20, MS4A2, and ROBO1 genes were significantly associated with reduced cryo-induced lipid peroxidation and DNA damage of FT spermatozoa from boars of the GSF group. Candidate genes with significant SNP associations, including APPL1, PLBD1, FBXO16, EML5, RAB3C, OXSR1, PRICKLE1, and MAP3K20 genes, represent potential markers for post-thaw semen quality, and they might be relevant for future improvement in the selection procedure of boars for cryopreservation. The findings of this study provide evidence indicating that polymorphisms in genes expressed in spermatozoa could be considered as factors associated with post-thaw semen quality.

Proteome of cat semen obtained after urethral catheterization.

The binding of seminal plasma (SP) proteins by spermatozoa plays an important role in the regulation of sperm epididymal maturation, motility gaining in female reproductive tracts and sperm-egg interaction. The aim of the study was to analyze the SP and sperm extracts proteome of cat (Felis catus) semen. The seminal plasma and spermatozoa were obtained by urethra catheterization from 10 male cats. Proteins were extracted using RIPA buffer and separated by electrophoresis (SDS-PAGE). The gels were analyzed using MultiAnalyst software. The proteins were subsequently analyzed using NanoUPLC-Q-TOF/MS. UniProt database-supported identification resulted in 106 proteins identified in the cat SP and 98 proteins in the extracts of spermatozoa. Based on a gene ontology analysis, dominant molecular functions of feline SP proteins were binding, catalytic, and antioxidant activity (56%, 33%, and 11% of cases, respectively). The molecular functions of sperm extracts proteins were mainly involved in catalytic activity (41%) and binding (23%). The proteins present in both, the SP and spermatozoa's extracts, were: serum albumin (ALB), semenogelin 2 (SEMG 2), clusterin (CLU), lactoferrin (LTF), prostatic acid phosphatase (ACPP), prolactin inducible protein (PIP), negative elongation factor E (NELF-E) and ectonucleotide pyrophosphatase (ENPP3). Protein-protein interactions analysis showed significant connection for 12 proteins in the cat semen. The seminal plasma proteins which, with high probability score, participate in important metabolic pathways are: glutathione peroxidases (GPx5 and 6), prostatic acid phosphatase (ACPP), ß-hexosaminidase (HEXB), polymeric immunoglobulin receptor (pIgR) and serpin family F member 1 (SERPINF1). For sperm protein extracts it were: pyruvate dehydrogenase (PDHB), succinate-CoA-ligase (SUCLA2), malate dehydrogenase (MDH2), ATP synthase F1 subunit alpha (ATP5F1A) and tubulin beta (TUBB).

Single nucleotide polymorphism discovery in bovine liver using RNA-seq technology.

BACKGROUND: RNA-seq is a useful next-generation sequencing (NGS) technology that has been widely used to understand mammalian transcriptome architecture and function. In this study, a breed-specific RNA-seq experiment was utilized to detect putative single nucleotide polymorphisms (SNPs) in liver tissue of young bulls of the Polish Red, Polish Holstein-Friesian (HF) and Hereford breeds, and to understand the genomic variation in the three cattle breeds that may reflect differences in production traits. RESULTS: The RNA-seq experiment on bovine liver produced 107,114,4072 raw paired-end reads, with an average of approximately 60 million paired-end reads per library. Breed-wise, a total of 345.06, 290.04 and 436.03 million paired-end reads were obtained from the Polish Red, Polish HF, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed that 81.35%, 82.81% and 84.21% of the mapped sequencing reads were properly paired to the Polish Red, Polish HF, and Hereford breeds, respectively. This study identified 5,641,401 SNPs and insertion and deletion (indel) positions expressed in the bovine liver with an average of 313,411 SNPs and indel per young bull. Following the removal of the indel mutations, a total of 195,3804, 152,7120 and 205,3184 raw SNPs expressed in bovine liver were identified for the Polish Red, Polish HF, and Hereford breeds, respectively. Breed-wise, three highly reliable breed-specific SNP-databases (SNP-dbs) with 31,562, 24,945 and 28,194 SNP records were constructed for the Polish Red, Polish HF, and Hereford breeds, respectively. Using a combination of stringent parameters of a minimum depth of ≥10 mapping reads that support the polymorphic nucleotide base and 100% SNP ratio, 4,368, 3,780 and 3,800 SNP records were detected in the Polish Red, Polish HF, and Hereford breeds, respectively. The SNP detections using RNA-seq data were successfully validated by kompetitive allele-specific PCR (KASPTM) SNP genotyping assay. The comprehensive QTL/CG analysis of 110 QTL/CG with RNA-seq data identified 20 monomorphic SNP hit loci (CARTPT, GAD1, GDF5, GHRH, GHRL, GRB10, IGFBPL1, IGFL1, LEP, LHX4, MC4R, MSTN, NKAIN1, PLAG1, POU1F1, SDR16C5, SH2B2, TOX, UCP3 and WNT10B) in all three cattle breeds. However, six SNP loci (CCSER1, GHR, KCNIP4, MTSS1, EGFR and NSMCE2) were identified as highly polymorphic among the cattle breeds. CONCLUSIONS: This study identified breed-specific SNPs with greater SNP ratio and excellent mapping coverage, as well as monomorphic and highly polymorphic putative SNP loci within QTL/CGs of bovine liver tissue. A breed-specific SNP-db constructed for bovine liver yielded nearly six million SNPs. In addition, a KASPTM SNP genotyping assay, as a reliable cost-effective method, successfully validated the breed-specific putative SNPs originating from the RNA-seq experiments.

Metabolic Activity of Boar Semen Stored in Different Extenders Supplemented with Ostrich Egg Yolk Lipoproteins.

INTRODUCTION: The aim of this study was to evaluate the effect of lipoprotein fraction isolated from ostrich egg yolk (LPFo) on the metabolic activity of boar spermatozoa following liquid semen storage in different extenders and temperatures. MATERIAL AND METHODS: Boar ejaculates were extended in Androhep, Beltsville thawing solution (BTS), and Martín-Rillo and Alias (MR-A) without (control) or with the addition of LPFo and stored for three days at either 5°C or 16°C. The analysed sperm parameters included total motility (TMOT), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), oxygen consumption, and adenosine triphosphate (ATP) production. RESULTS: The sperm metabolic activity seemed to be higher in the LPFo-based extenders following storage for three days, irrespective of the storage temperature. Compared with the LPFo-free extenders, significantly higher (P < 0.05) sperm PMI and MMP were observed in BTS and MR-A extenders supplemented with LPFo during storage for three days at 5°C. Spermatozoa stored in the BTS-LPFo extender exhibited higher (P < 0.05) TMOT and oxygen consumption, whereas higher (P < 0.05) PMI was observed in spermatozoa stored in Androhep-LPFo and MR-A-LPFo for three days at 16°C. No significant differences (P > 0.05) in ATP content were observed between the LPFo-free and LPFo-based extenders during storage. CONCLUSIONS: Supplementation of LPFo to semen extenders had varying effects on the metabolic activity of boar spermatozoa stored at different temperatures. It can be suggested that the interactions of various components of the extenders and seminal plasma with LPFo exert beneficial effects on the sperm metabolic activity during liquid storage of boar semen.

Single Nucleotide Polymorphism Discovery in Bovine Pituitary Gland Using RNA-Seq Technology.

Examination of bovine pituitary gland transcriptome by strand-specific RNA-seq allows detection of putative single nucleotide polymorphisms (SNPs) within potential candidate genes (CGs) or QTLs regions as well as to understand the genomics variations that contribute to economic trait. Here we report a breed-specific model to successfully perform the detection of SNPs in the pituitary gland of young growing bulls representing Polish Holstein-Friesian (HF), Polish Red, and Hereford breeds at three developmental ages viz., six months, nine months, and twelve months. A total of 18 bovine pituitary gland polyA transcriptome libraries were prepared and sequenced using the Illumina NextSeq 500 platform. Sequenced FastQ databases of all 18 young bulls were submitted to NCBI-SRA database with NCBI-SRA accession numbers SRS1296732. For the investigated young bulls, a total of 113,882,3098 raw paired-end reads with a length of 156 bases were obtained, resulting in an approximately 63 million paired-end reads per library. Breed-wise, a total of 515.38, 215.39, and 408.04 million paired-end reads were obtained for Polish HF, Polish Red, and Hereford breeds, respectively. Burrows-Wheeler Aligner (BWA) read alignments showed 93.04%, 94.39%, and 83.46% of the mapped sequencing reads were properly paired to the Polish HF, Polish Red, and Hereford breeds, respectively. Constructed breed-specific SNP-db of three cattle breeds yielded at 13,775,885 SNPs. On an average 765,326 breed-specific SNPs per young bull were identified. Using two stringent filtering parameters, i.e., a minimum 10 SNP reads per base with an accuracy ≥ 90% and a minimum 10 SNP reads per base with an accuracy = 100%, SNP-db records were trimmed to construct a highly reliable SNP-db. This resulted in a reduction of 95,7% and 96,4% cut-off mark of constructed raw SNP-db. Finally, SNP discoveries using RNA-Seq data were validated by KASP™ SNP genotyping assay. The comprehensive QTLs/CGs analysis of 76 QTLs/CGs with RNA-seq data identified KCNIP4, CCSER1, DPP6, MAP3K5 and GHR CGs with highest SNPs hit loci in all three breeds and developmental ages. However, CAST CG with more than 100 SNPs hits were observed only in Polish HF and Hereford breeds.These findings are important for identification and construction of novel tissue specific SNP-db and breed specific SNP-db dataset by screening of putative SNPs according to QTL db and candidate genes for bovine growth and reproduction traits, one can develop genomic selection strategies for growth and reproductive traits.

The antigenic character of zinc-binding proteins and their localization in boar reproductive tract--a preliminary study.

In this study immunoelectrophoretic and double immunodiffusion analyses were used to investigate the antigenic character of zinc-binding proteins (ZnBPs), whereas the indirect immunofluorescence technique was used to identify their origin in boar reproductive tract. The mmunoelectrophoretic analysis of ZnBPs of the seminal plasma resulted in the appearance of three antigenic protein complexes, while specific immunoreactivity patterns of the anti-ZnBP serum were detected by double immunodiffusion analysis. Indirect immunofluorescence technique confirmed that ZnBPs were secreted by different reproductive tract tissues, suggesting their contributions to the seminal plasma.

Seasonal changes in antioxidant defence systems in seminal plasma and fluids of the boar reproductive tract.

This study aimed to analyze seasonal variations in the antioxidant defence systems of the seminal plasma and fluids of the cauda epididymis and vesicular glands of the boar. The analyzed antioxidants included superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and total L-glutathione (GSH+GSSG). Seasonal changes in total protein content and total antioxidant status (TAS) of the seminal plasma and reproductive fluids were also analyzed. Compared with the spring-summer period, total protein content in the seminal plasma was significantly higher during the autumn-winter period. Among the antioxidants analyzed, only SOD activity showed marked seasonal variations, being significantly higher during the spring-summer period. Likewise, the fluid of the cauda epididymis exhibited greater SOD and CAT activity during the spring-summer period, whereas TAS levels were markedly higher during the autumn-winter period. Neither GPx activity nor total GSH+GSSG content in the cauda epididymal fluid was significantly affected by the seasonal periods. The vesicular gland fluid exhibited an approximately 4-fold greater level of SOD activity during the autumn-winter period, as compared with the spring-summer period. By contrast, greater CAT and GPx activity, and a higher level of total GSH+GSSG were observed in the vesicular gland fluid during the spring-summer period. In conclusion, the findings of this study indicate that seasonal variations could have varying effects on the antioxidant defence systems in the seminal plasma and fluids of the boar reproductive tract.

Interactions of egg yolk lipoprotein fraction with boar spermatozoa assessed with a fluorescent membrane probe.

The interactions of a fluorescent membrane probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), with boar spermatozoa were followed through the use of lipoprotein fraction of ostrich egg yolk (LPFo). Semen samples, extended in Kortowo 3 (K3) extender, were supplemented with 2% or 5% LPFo and stored for 3h at 16 degrees C. Additionally, cold shock-treated spermatozoa (1h at 4 degrees C) were stored in K3 extender supplemented with LPFo for 3h at 16 degrees C. In each boar, the fluorescent enhancement of ANS was observed in K3-extended semen supplemented with LPFo, prior to storage. Following storage, there was a significant increase in LPFo-ANS fluorescence, particularly in the sperm membrane overlying the head and midpiece regions. There were significant differences among the boars with respect to the sperm populations defined by the LPFo-ANS fluorescence. Sperm viability was not significantly affected during the storage period. Furthermore, the proportions of spermatozoa defined by the different patterns of LPFo-ANS fluorescence were low and remained unchanged after storage of cold shock-treated spermatozoa with 2% or 5% LPFo, suggesting irreversible damage to the sperm membrane architecture. These findings indicate that the ANS fluorescent probe could be used to shed more light on the nature of the interactions between LPFo and sperm membrane following semen preservation. Such valuable information could contribute to the development of an optimal protocol for cryopreservation of boar semen.

Characteristics of spermatozoa of whole ejaculate and sperm-rich fraction of dog semen following exposure to media varying in osmolality.

This study aimed to analyze the effects of different osmolalities on the characteristics of spermatozoa originating from whole ejaculates (WE; including the prostatic fluid) and the sperm-rich fractions (SRF). Ejaculates, collected from four mixed-breed dogs, were exposed for 10 min at room temperature to Tris-fructose-citrate (TFC) solution with osmolality ranging from 150 to 1100 mOsm. After treatment spermatozoa were evaluated by microscopic analysis of motility and fluorescent assessments of plasma membrane integrity (carboxyfluorescein diacetate and propidium iodide, CFDA/PI) and mitochondrial function (rhodamine 123, R123). Irrespective of the sperm source, there was a complete loss of motility when spermatozoa were exposed to TFC solution with 1100 mOsm. There were no marked differences in the sperm characteristics between media with 300 and 350 mOsm, regardless of the ejaculate collection procedure. However, a marked reduction in motility of spermatozoa retrieved either from the WE or SRF was observed after exposure to different anisosmotic conditions (150, 550 and 800 mOsm). In all dogs, spermatozoa from the WE exhibited greater osmotolerance in terms of plasma membrane integrity and mitochondrial function when exposed to anisosmotic conditions (150, 550, 800 and 1100 mOsm). There were inter-dog variations in response to various osmotic conditions. The findings of this study indicated that spermatozoa from the WE tolerated exposure to a wider range of osmolality than those from the SRF. It seemed that the presence of prostatic fluid of dog semen rendered the sperm membrane structures less susceptible to osmotic stress.

Application of biochemical markers for identification of biological properties of animal semen.

The use of biochemical markers for identification of biological properties of semen will help to develop new criteria that are accurate and objective in predicting and improving male fertility. Understanding and controlling the mechanisms involved in fertility is a key challenge, which is of fundamental importance in successful animal reproductive performance. Moreover, unraveling the unique molecular mechanism associated with sperm function might have considerable diagnostic value in the evaluation of male infertility. This review offered insights into some recent achievements and provided perspectives for possible applications of the biochemical markers of semen.

Proteomics of boar seminal plasma - current studies and possibility of their application in biotechnology of animal reproduction.

Proteomics is critical to identify the properties and functions of proteins involved in the mechanism regulating the male reproductive tract function. This approach is important in male fertility assessment and clinical diagnosis of the physiological state of individual reproductive organs. Proteomics also provides a tool to understand the interactions of seminal plasma proteins with spermatozoa, which could provide a useful model for studying ligand-cell interaction occurring at the sperm cell surface. This review covers a selection of advances in the realm of functional proteomics of boar seminal plasma proteins and is focused on some fundamental proteomic technologies. Also, this review explores key themes in proteomics and their application in animal reproductive techniques.