Poor Semen Freezability Research Articles

Post-thaw quality of Malabari buck semen with different freezing resilience

The study was conducted to assess the difference in post-thaw quality parameters of Malabari buck spermatozoa of good and poor semen freezability. The ejaculates collected from Malabari bucks of good (n=3) and poor (n=3) semen freezability were pooled separately as group A and group B, respectively after the initial evaluation. The extended semen in each group was processed and cryopreserved by the conventional method of static vapour freezing. After thawing, the quality of semen in both groups was assessed in terms of progressive motility, viability, morphological abnormalities, acrosome integrity, functional membrane integrity and lipid peroxidation status (MDA) of spermatozoa. Results showed that there was no significant difference between the fresh semen characteristics of group A and B bucks except for concentration, progressive motility and functional membrane integrity of spermatozoa. But, post-thaw quality of groups A and B differed significantly in terms of progressive motility (48.89 ± 0.64 % vs 18.06 ± 1.08 %), viability (55.17 ± 0.57 % vs 26.33 ± 0.95 %), acrosome integrity (62.44 ± 0.62 % vs 33.42 ± 0.34 %), functional membrane integrity (47.00 ± 0.36 % vs 23.36 ± 0.63 %) and MDA concentration (7.83 ± 0.29 nmol/mL vs 13.95 ± 0.08 nmol/mL) whereas morphological abnormalities (2.11 ± 0.08 % vs 2.39 ± 0.09 %) did not differ significantly. The findings of the present study showed that even with similar fresh semen characteristics, spermatozoa from different Malabari bucks have varying degrees of cryosurvivability.

Expression and Role of PIWI Proteins and piRNAs in Reproduction of Water Buffalo (Bubalus bubalis, Linn.)

High-fertile and productive dairy animals are important to satisfy the growing population's demand. Sire fertility is one of the essential factors that regulate the overall pregnancy rate of dairy herds. However, sire fertility varies from 10 to 90%, suggesting that male fertility largely accounts for varying fertility levels across the herd. Sub-fertile bulls and females should be identified and discarded promptly to improve the dairy herd's productivity. The most dominant factors implicated in culling are poor semen quality, poor semen freezability (<35% post-thaw motility), and poor libido for the bulls and hard breeders for females that cause huge economic loss to the raisers. Understanding the basic mechanism of male and female fertility has undergone tremendous change in recent times owing to the advancement of molecular tools judging the essential molecules responsible for fertility. Presently, a new molecular niche has surfaced in testes, strongly influencing the fertilization potential of spermatozoa. Over the last decade, there has arrived a conclusion that out of several factors, piRNA and PIWI proteins are largely implicated in regulating the vital aspects of fertility and embryogenesis. While this development is advancing in other animals, very limited information is available on PIWI protein and piRNAs in large animals. Except for a few sporadic information on PIWI protein in cattle, very limited information is available on piRNAs and PIWI protein in regulation with buffalo bull fertility and growth of embryos of buffaloes, posting a huge demand for research.

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-β, 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.

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.

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/EBPβ. 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.

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.

Transcriptome analysis of boar spermatozoa with different freezability using RNA-Seq

Semen freezability is associated with genetic markers, and there is a diverse set of sperm transcripts that have been attributed to various cellular functions. RNA-Seq was performed to compare the transcript profiles of spermatozoa from boars with different semen freezability. We examined ejaculates from the Polish large white (PLW) boars that were classified as having good and poor semen freezability (GSF and PSF, respectively; n = 3 boars per group) by assessing post-thaw motility characteristics, mitochondrial membrane potential, plasma membrane and acrosome integrity. Total RNA was isolated from fresh spermatozoa from boars of the GSF and PSF groups and subjected to RNA-Seq (Illumina NextSeq 500 platform). Transcript abundance was assessed with the DESeq2, DESeq, and EdgeR Bioconductor R packages, and varying numbers of differentially expressed gene (DEG) transcripts were detected in the spermatozoa of each boar. Using RNA-Seq, we identified several genes associated with inflammation and apoptosis (FOS, NFATC3, ITGAL, EAF2 and ZDHHC14), spermatogenesis (FGF-14 and BAMBI), autophagy (RAB33B), protein phosphorylation (PTPRU and PTPN2) and energy metabolism (ND6 and ACADM) that were predominantly up-regulated in poor freezability ejaculates. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) validated the transcript expression levels detected by RNA-Seq and thus confirmed the reliability of this technique. Subsequent validation with western blotting showed that the expression of three proteins was in accordance with the transcript abundance. Overall, we demonstrated that the up-regulation of the DEG transcripts in spermatozoa was associated with poor semen freezability. We suggest that spermatozoa transcriptome profiling provides a foundation to further elucidate the relevance of sperm-related transcripts on cryo-survival. The sperm-related transcripts, namely FOS, NFATC3, EAF2, BAMBI, PTPRU, PTPN2, ND6 and ACADM, are potential markers for predicting the freezability of boar semen.

Effect of post-thaw supplementation of fractionated seminal plasma on survival of boar spermatozoa.

In our recent study we demonstrated that the holding of fresh semen in fractionated seminal plasma (SP1, >40 kDa; SP2, ⟨40 kDa), obtained by gel filtration chromatography, significantly improved the sperm quality characteristics following cryopreservation (Wasilewska-Sakowska et al. 2019). In this study we investigated the effect of post-thaw (PT) supplementation of fractionated SP (SP1 and SP2) on the survival of spermatozoa from boars with good and poor semen freezability, GSF and PSF, respectively. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis showed distinct differences in the protein profiles of SP1 and SP2 from boars with GSF or PSF regarding the number of protein spots. Sperm motility characteristics and the motion patterns, assessed using the computer-assisted sperm analysis (CASA) system, were markedly higher in PT semen supplemented with SP1 and SP2 from boars with GSF. Post-thaw supplementation of either SP1 or SP2 from boars with GSF significantly improved mitochondrial function, plasma membrane and acrosome integrity, and viability during storage. The findings of this study have confirmed that the presence of protective protein components in varying abundance in either fractionated SP from boars with good freezability ejaculates significantly improved the sperm survival following PT storage.

Total RNA quality in boar spermatozoa with different freezability.

In this study the quality of total RNA, isolated from fresh spermatozoa, was compared between boars with good and poor semen freezability (GSF and PSF, respectively). Semen from 3 boars with GSF exhibited significantly higher total motility, mitochondrial function, plasma membrane integrity and reduced lipid peroxidation compared with 3 boars with PSF after cryo- preservation. There were variations in the quality of RNA isolated from spermatozoa of boars with GSF and PSF. Boars with GSF exhibited mainly full-length, intact RNA, whereas substantial amounts of degraded RNA were detected in spermatozoa from boars with PSF. Further under- standing of the biological relevance of RNAs in sperm function is critical to improve the freezabil- ity of boar semen.

Culling pattern of Karan Fries (crossbred) males in an organised herd

Information on culling pattern of animals helps in developing effective breeding plans for genetic improvement of the herd. Data on 292 reserved KF males out of 1740 male calves born, spread over a period of sixteen years from 1997 to 2012, were collected based on pedigree performance. The data were analysed using descriptive statistics considering the period of birth, season of birth, genetic group and age group. Study revealed increase in culling percentage with advancement in age. Culling in KF males for the different periods (1997-2000, P-1; 2001-2004, P-2; 2005-2008, P-3 and 2009-2012, P-4) were 75.93, 92.16, 64.44 and 28.26 percent, respectively. Maximum culling was observed in males born in summer season (76.12%) which could be due to inclement weather conditions exposure in young age and insufficient quality green leguminous fodder in subsequent months. Generally, upto the age of 3 years, culling in KF males were maximum in interbred and minimum in F1 but with advancing age, culling rate was found higher in F1 genetic group KF males where reproductive problems were the main causes. High culling rate in &lt;1.5 yrs age groups was observed due to poor growth (3.42%) while in adult bulls of above 1.5yrs of age, main reasons of culling was surplus (33.22%) followed by poor semen quality (10.27%), poor libido (7.53%), and poor semen freezability (4.45%). It can be concluded that culling pattern study can help in formulation of better management strategy to reduce the problems associated with culling and better economic return for the farm.

Effect of Multiple Freezing of Stallion Semen on Sperm Quality and Fertility

To increase the efficiency of equine semen, it could be useful to split the artificial insemination dose and refreeze the redundant spermatozoa. In experiment I, semen of 10 sires of the Hanoverian breed, with poor and good semen freezability, was collected by artificial vagina, centrifuged, extended in INRA82 at 400 × 106 sperm/mL, and automatically frozen. After this first routinely applied freezing program, semen from each stallion was thawed, resuspended in INRA82 at 40 × 106 sperm/mL, filled in 0.5-mL straws, and refrozen. These steps were repeated, and sperm concentration was adjusted to 20 × 106 sperm/mL after a third freezing cycle. Regardless of stallion freezability group, sperm motility and sperm membrane integrity (FITC/PNA-Syto-PI-stain) decreased stepwise after first, second, and third freezing (62.3% ± 9.35, 24.0% ± 15.4, 3.3% ± 4.34, P ≤ .05; 29.6% ± 8.64, 14.9% ± 6.38, 8.3% ± 3.24, P ≤ .05), whereas the percentage of acrosome-reacted cells increased (19.5% ± 7.59, 23.9% ± 8.51, 29.2% ± 6.58, P ≤ .05). Sperm chromatin integrity was unaffected after multiple freeze/thaw cycles (DFI value: 18.6% ± 6.6, 17.2% ± 6.84, 17.1% ± 7.21, P > .05). In experiment II estrous, Hanoverian warmblood mares were inseminated with a total of 200 × 106 spermatozoa of two stallions with either good or poor semen freezability originating from the first, second, and third freeze/thaw cycle. First-cycle pregnancy rates were 4/10, 40%; 1/10, 10%; and 0/10, 0%. In conclusion, as expected, sperm viability of stallion spermatozoa significantly decreases as a consequence of multiple freezing. However, sperm chromatin integrity was not affected. Pregnancy rates after insemination of mares with refrozen semen are reduced.

Subfertility problems leading to disposal of breeding bulls.

Subfertility problems are encountered frequently in the cattle and buffalo bulls commercially maintained for semen production in dairy farms and under field conditions for natural insemination. Reports are scarce on the incidence of subfertility in breeding bulls, especially in India. The objective of the present study was to assess the incidence of the male reproductive anomalies leading to disposal of bovine bulls at GADVASU dairy farm, Ludhiana, Punjab (India). Data on frequency of various subfertility and disposal pattern of bulls maintained at the dairy farm, GADVASU, were collected for 12 yrs (1999 to 2010) and compiled from different record registers. Percentage of bulls that produced freezable semen (out of reserved ones) was less in cattle (25.641%) as compared to that of buffalo (30.4%). Various subfertility traits like poor libido and unacceptable seminal profile were found to be the significant reasons (p<0.01) for culling of the breeding bulls. Inadequate sex drive and poor semen quality were the main contributing factors for bull disposal in cattle whereas poor semen freezability was most frequently observed in buffalo bulls. All the male reproductive traits were significantly different (p<0.05) for the periods of birth, except for semen volume, initial motility (IM), age at last semen collection (ALSC) and age at disposal. The ages at first and last semen collection as well as freezing (i.e. AFSC, ALSC and AFSF, ALSF, respectively) and age at disposal (AD) were higher in buffalo. The spermatological parameters and semen production period (SPP) were higher in cattle. The age at first semen donation and breeding period could be reduced by introducing the bulls to training at an early age. The results revealed an increasing trend in individual motility (IM) while semen volume, AFSC, AFSF, AD, FSPP, SPP, ALSC and ALSF showed a decreasing, however, not a definite trend, over the periods. The semen donation traits like, AFSF, of the cattle and buffalo bulls could be predicted from the AFSC, using prediction equation derived in the present study.