Identification of hub genes and their expression profiling for predicting buffalo (Bubalus bubalis) semen quality and fertility.

Sperm transcriptomics provide insights into subtle differences in sperm fertilization competence. For predicting the success of complex traits like male fertility, identification of hub genes involved in various sperm functions are essential. The bulls from the transcriptome profiled samples (n = 21), were grouped into good and poor progressive motility (PM), acrosome integrity (AI), functional membrane integrity (FMI) and fertility rate (FR) groups. The up-regulated genes identified in each group were 87, 470, 1715 and 36, respectively. Gene networks were constructed using up- and down-regulated genes from each group. The top clusters from the upregulated gene networks of the PM, AI, FMI and FR groups were involved in tyrosine kinase (FDR = 1.61E-11), apoptosis (FDR = 1.65E-8), translation (FDR = 2.2E-16) and ribosomal pathway (FDR = 1.98E-21), respectively. From the clusters, the hub genes were identified and validated in a fresh set of semen samples (n = 12) using RT-qPCR. Importantly, the genes (fold change) RPL36AL (14.99) in AI, EIF5A (54.32) in FMI, and RPLP0 (8.55) and RPS28 (13.42) in FR were significantly (p < 0.05) up-regulated. The study suggests that the expression levels of MAPK3 (PM), RPL36AL + RPS27A or RPL36AL + EXT2 (AI), RPL36AL or RPS27A (FMI) and RPS18 + RPS28 (FR) are potential markers for diagnosing the semen quality and fertility status of bulls which can be used for the breeding program.

Relationship among seminal antigenicity, antioxidant status and metabolically active sperm from Holstein-Friesian (Bos taurus) bulls.

Sperm antigenicity has been implicated as a regulatory factor for acquiring fertilizing competence in the female reproductive tract. Overt immune response against the sperm proteins leads to idiopathic infertility. Hence, the aim of the study was to evaluate the influence of the auto-antigenic potential of sperm on the antioxidant status, metabolic activities and reactive oxygen species (ROS) in bovine. Semen from Holstein-Friesian bulls (n = 15) was collected and classified into higher (HA, n = 8) and lower (LA, n = 7) antigenic groups based on micro-titer agglutination assay. The neat semen was subjected to the evaluation of bacterial load, leukocyte count, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lipid peroxidation (LPO) levels. Antioxidant activities in seminal plasma and intracellular ROS levels in the post-thawed sperm were estimated. The number of leukocytes was lower (p < .05) in the HA than the LA semen. The percentage of metabolically active sperm was higher (p < .05) in HA than the LA group. The activities of total non-enzymatic antioxidant, superoxide dismutase (SOD) and catalase (CAT) were higher (p < .05) while glutathione peroxidase activity was lower (p < .05) in the seminal plasma of LA group. The LPO levels of neat sperm and the percentage sperm positive for intracellular ROS in the cryopreserved sample were lower (p < .05) in the HA group. Auto-antigenic levels were positively correlated with the percentage of metabolically active sperm (r = 0.73, p < .01). However, the seminal auto-antigenicity was negatively (p < .05) correlated with the levels of SOD (r=-0.66), CAT (r=-0.72), LPO (r=-0.602) and intracellular ROS (r=-0.835). The findings were represented in graphical abstract. It is inferred that the higher auto-antigenic levels protect the quality of bovine semen by promoting sperm metabolism and lowering ROS and LPO levels.

Cryostress induces fragmentation and alters the abundance of sperm transcripts associated with fertilizing competence and reproductive processes in buffalo.

The study aimed to assess the influence of cryostress on RNA integrity and functional significance in sperm fertilizing ability. The fresh and post-thawed buffalo sperm (n = 6 each) samples were evaluated for their functional attributes, and sperm total RNA was subjected to transcriptome sequencing followed by validation using real-time PCR and dot blot. Overall, 6911 genes had an expression of FPKM > 1, and among these 431 genes were abundantly expressed (FPKM > 20) in buffalo sperm. These abundantly expressed genes regulate reproductive functions such as sperm motility (TEKT2, SPEM1, and PRM3, FDR = 1.10E-08), fertilization (EQTN, PLCZ1, and SPESP1, FDR = 7.25E-06) and the developmental process involved in reproduction (SPACA1, TNP1, and YBX2, FDR = 7.21E-06). Cryopreservation significantly (p < 0.05) affected the structural and functional membrane integrities of sperm. The expression levels of transcripts that regulate the metabolic activities and fertility-related functions were compromised during cryopreservation. Interestingly, cryostress induces the expression of genes involved (p < 0.05) in chemokine signaling (CX3CL1, CCL20, and CXCR4), G-protein coupled receptor binding (ADRB1, EDN1, and BRS3), translation (RPS28, MRPL28, and RPL18A), oxidative phosphorylation (ND1, ND2, and COX2), response to reactive oxygen species (GLRX2, HYAL2, and EDN1), and immune responses (CX3CL1, CCL26, and TBXA2R). These precociously expressed genes during cryopreservation alter the signaling mechanisms that govern sperm functional competence and can impact fertilization and early embryonic development.

X chromosome-linked genes in the mature sperm influence semen quality and fertility of breeding bulls.

The role of sperm expressed X-linked genes on bull fertility has not been studied in detail. The objective of the present study was to assess the influence of X-linked genes on the sperm functional parameters and field fertility rate in the Holstein Friesian cattle (n = 12) and Murrah buffalo (n = 7) bulls. The enrichment analysis (cattle = 8; buffalo = 8) of the X-linked genes was carried out using retrospective RNA-seq data and mRNA expression levels of functionally relevant genes were validated using the RT-qPCR. The mRNA expression levels of these genes were functionally associated with sperm attributes and field fertility rate. The sperm transcriptome studies revealed that the total number of expressed genes and the transcript content of the X-linked genes in the mature sperm were very low in both species, and only 23.31% of these genes were commonly expressed between them. The transcript pool corresponding to the X-linked genes represents embryonic organ development (p = 0.03) and reproduction (p = 0.02) processes in cattle and buffalo sperm, respectively. The mRNA expression levels of X-linked genes, RPL10 and ZCCHC13 in cattle; AKAP4, TSPAN6, RPL10 and RPS4X in buffalo were significantly (p < 0.05) correlated with sperm kinematics. Importantly, the mRNA expression levels of the genes RPL10 (r = -0.68) and RPS4X (r = 0.81) had a significant correlation with the field fertility rate in cattle and buffalo, respectively. Multivariate regression models and receiver operating curve analysis suggest that the mRNA expression levels of X-linked genes may be useful in predicting bull fertility. The study indicates that sperm-expressed X-linked genes influence semen quality and field fertility rate in both cattle and buffalo.

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus.

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.

Current scenario and challenges ahead in application of spermatogonial stem cell technology in livestock.

PURPOSE: Spermatogonial stem cells (SSCs) are the source for the mature male gamete. SSC technology in humans is mainly focusing on preserving fertility in cancer patients. Whereas in livestock, it is used for mining the factors associated with male fertility. The review discusses the present status of SSC biology, methodologies developed for in vitro culture, and challenges ahead in establishing SSC technology for the propagation of superior germplasm with special reference to livestock. METHOD: Published literatures from PubMed and Google Scholar on topics of SSCs isolation, purification, characterization, short and long-term culture of SSCs, stemness maintenance, epigenetic modifications of SSCs, growth factors, and SSC cryopreservation and transplantation were used for the study. RESULT: The fine-tuning of SSC isolation and culture conditions with special reference to feeder cells, growth factors, and additives need to be refined for livestock. An insight into the molecular mechanisms involved in maintaining stemness and proliferation of SSCs could facilitate the dissemination of superior germplasm through transplantation and transgenesis. The epigenetic influence on the composition and expression of the biomolecules during in vitro differentiation of cultured cells is essential for sustaining fertility. The development of surrogate males through gene-editing will be historic achievement for the foothold of the SSCs technology. CONCLUSION: Detailed studies on the species-specific factors regulating the stemness and differentiation of the SSCs are required for the development of a long-term culture system and in vitro spermatogenesis in livestock. Epigenetic changes in the SSCs during in vitro culture have to be elucidated for the successful application of SSCs for improving the productivity of the animals.

Supraphysiological concentration of urea affects the functional competence of Holstein-Friesian (Bos taurus) sperm.

To understand the effects of urea on sperm functional attributes, fresh bull semen (n = 12) was subjected to four different concentrations (mg/mL) of urea to mimic the physiological (0.04 and 0.13), supraphysiological (0.43) concentrations and control (0 mg/mL). Sperm membrane integrity, kinematics, chromatin integrity, and mitochondrial membrane potential were assessed at different time points (before incubation, 0, 1, 2, and 4 h) of incubation. The concentration of urea in serum and seminal plasma was estimated and correlated with the ejaculate rejection rate and sperm functional attributes. The relative expression of urea transporter gene transcripts (UT-A and UT-B) was assessed in sperm and testis (control) using real-time PCR. The supraphysiological concentration of urea affected sperm kinematics, viability, functional membrane integrity, and acrosome integrity within 1 h of incubation (p < 0.05). Sperm head area decreased (p < 0.05) at 0 h and subsequently increased at 1 h of incubation in all media except supraphysiological (0.43 mg/dL) concentration of urea. Seminal plasma urea concentration showed a significant negative correlation with sperm motility, membrane integrity, and mitochondrial membrane potential (p < 0.05), but had a positive correlation with the ejaculate rejection rate (r = 0.69). Relative expression of the urea transporter genes revealed that UT-A was expressed only in the testis. In contrast, UT-B was expressed in both the testis and sperm, suggesting UT-B's role in regulating urea transport in sperm. At a supraphysiological level, urea adversely affected sperm functional attributes, osmoadaptation and may affect fertility.

Establishment of bioinformatics pipeline for deciphering the biological complexities of fragmented sperm transcriptome.

Despite the development of several tools for the analysis of the transcriptome data, non-availability of a standard pipeline for analyzing the low quality and fragmented mRNA samples pose a major challenge to the computational molecular biologist for effective interpretation of the data. Hence the present study aimed to establish a bioinformatics pipeline for analyzing the biologically fragmented sperm RNA. Sperm transcriptome data (2 x 75 PE sequencing) generated from bulls (n = 8) of high-fertile (n = 4) and low-fertile (n = 4) classified based on the fertility rate (41.52 ± 1.07 vs 36.04 ± 1.04%) were analyzed with different bioinformatics tools for alignment, quantitation, and differential gene expression studies. TopHat2 was effectual compared to HISAT2 and STAR for sperm mRNA due to the higher exonic (6% vs 2%) mapping percentage and quantitating the low expressed genes. TopHat2 also had significantly strong correlation with STAR (0.871, p = 0.05) and HISAT2 (0.933, p = 0.01). TopHat2 and Cufflinks combo quantitated the number of genes higher than the other combinations. Among the tools (Cuffdiff, DESeq, DESeq2, edgeR, and limma) used for the differential gene expression analysis, edgeR and limma identified the largest number of significantly differentially expressed genes (p < 0.05) with biological relevance. The concordance analysis concurred that edgeR had an edge over the other tools. It also identified a higher number (9.5%) of fertility-related genes to be differentially expressed between the two groups. The present study established that TopHat2, Cufflinks, and edgeR as a suitable pipeline for the analysis of fragmented mRNA from bovine spermatozoa.

Seminal antigenicity affects mitochondrial membrane potential and acrosome reaction ability of the spermatozoa during cryopreservation.

The objective of this study was to assess the influence of spermatozoa surface antigenic proteins on the functional competence of bovine neat and frozen-thawed semen. The breeding bulls (n = 38) were screened for seminal antigenic levels in neat semen based on the agglutination titrations with anti-sperm antibody (ASA). Bulls having high (n = 8) and low (n = 7) antigenic levels were selected and spermatozoa functional parameters were analyzed in neat and frozen-thawed semen samples. In neat semen, kinematics such as straightness (73.6 ± 1.0 and 66.9 ± 1.5%), linearity (48.6 ± 1.2 and 40.1 ± 3.9%), curvilinear velocity (103.3 ± 2.6 and 93.4 ± 3.8 µm/s), straight-line velocity (65.7 ± 2.6 and 53.7 ± 2.2 µm/s) and average path velocity (53.8 ± 2.5 and 39.8 ± 2.3 µm/s) were significantly high (p < 0.05) in samples with lower antigenicity. The percentage of spermatozoa that can penetrate mucus (49.9 ± 2.3 and 37.1 ± 3.2) was significantly higher in semen samples with low ASAs. The total motile (84.0 ± 2.5 and 86.0 ± 1.5) and progressive motile (68.4 ± 3.7 and 69.2 ± 1.6) spermatozoa were higher in neat semen samples with higher antigenicity. A significantly (p < 0.05) higher mitochondrial membrane potential was observed in neat (82.5 ± 2.8 and 69.0 ± 2.0%) and post-thaw (28 ± 5. 6 and 16 ± 3.7%) samples of the lower antigenic group. The percentage of acrosome-reacted spermatozoa was significantly (p < 0.05) higher in neat (58.7 ± 2.9 and 52.6 ± 1.8), but reduced significantly (p < 0.05) in post-thaw (32.0 ± 2.0 and 48.0 ± 2.6) semen of higher antigenic groups. The study reveals that higher seminal antigenicity reduces mitochondrial membrane potential and acrosome reaction ability in post-thaw spermatozoa.

Elucidating the processes and pathways enriched in buffalo sperm proteome in regulating semen quality.

Sperm carries a reservoir of proteins regulating the molecular functions to attain functional competence. Semen samples collected from buffalo bulls were assessed for sperm functional attributes (n = 11) and proteome profiling (n = 6). Sperm proteins were extracted and profiled by employing LC-MS/MS. Overall, the buffalo sperm contained 1365 proteins, of which 458 were common between the groups. The unique proteins were 477 and 430 in good and poor quality semen, respectively. In the whole proteome of buffalo sperm, sexual reproduction with phosphatidylethanolamine-binding protein1 (PEBP1), fetuin-B (FETUB) and acrosin (ACR) was the most enriched (p = 8.44E-19) biological process, also with thermogenesis (p = 0.003), oocyte meiosis (p = 0.007) and vascular smooth muscle contraction (p = 0.009) apart from metabolic pathways. In good quality semen, mesenchyme migration (p = 1.24E-07) and morphogenesis (p = 0.001) were abundant biological processes. In good quality semen, the fluid shear stress (p = 0.01) and, in poor quality semen, valine, leucine and isoleucine degradation (p = 3.8E-05) pathways were enriched. In good quality semen, 7 proteins were significantly (p < 0.05) upregulated and 33 proteins were significantly (p < 0.05) downregulated. On validating the abundantly expressed sperm proteins, serine protease inhibitor Kazal-type 2-like (SPINK2; 2.17-fold) and neddylin (NEDD8; 1.13-fold) were upregulated and YBX2 was downregulated (0.41-fold) in good quality semen as compared with poor quality semen (1-fold). The present findings revealed the importance of sperm proteins in oocyte maturation, fertilization process and early embryonic development. The variations in the proteomic composition can be used as potential markers for the selection of breeding bulls.

Orchestrating the expression levels of sperm mRNAs reveals CCDC174 as an important determinant of semen quality and bull fertility.

Bulls with acceptable semen quality vary in actual field fertility and this can be elucidated by studying the expression levels of mRNAs in the sperm. The present study aimed at assessing the variations in the sperm gene expression levels of PRM1, CCDC174, RPL36A, TMCO2, SWI5 and OIT3 in bulls differing in fertility status. Frozen semen samples from Holstein-Friesian bulls were classified into high-fertile (n = 8, average field conception rate = 46.1 ± 0.51, p < 0.001) and sub-fertile (n = 7, average field conception rate = 39.4 ± 0.69) groups. In the post-thaw semen samples, sperm kinematics, structural and functional membrane integrities, mitochondrial membrane potential and chromatin distribution were analyzed. The sperm total RNA was subjected to gene expression studies by Real-Time PCR. Multivariate regression analysis was performed using gene expression levels and conception rates. The sperm functional attributes did not differ significantly between the groups. The relative mRNA levels (fold change) of CCDC174 (6.20), RPL36A (4.66), SWI5 (1.86) and OIT3 (1.53) were higher in high-fertile bulls. Further, the expression level of the CCDC174 gene was significantly (p = 0.02) up-regulated in high-fertile bulls. The fertility prediction multivariate model with genes, CCDC174, RPL36A, TMCO2 and OIT3 had the maximum coefficient of determination (R2 = 0.68) with the field conception rate. This model had 93.3% bull fertility prediction accuracy with 100% sensitivity and 87.5% specificity. The study suggests that the expression level of CCDC174 can be used as a potential marker for assessing bull fertility.

Dietary boron supplementation enhances sperm quality and immunity through influencing the associated biochemical parameters and modulating the genes expression at testicular tissue.

INTRODUCTION: Dietary boron improves immune and antioxidant status and calcium metabolism in mammals. However, till date the effects of dietary boron supplementation on male reproduction, especially on sperm production and sperm quality in farm animals are not documented. OBJECTIVE: The present study was aimed to investigate the influence of dietary boron on semen production, semen quality, immunity and molecular changes in the testis, blood and seminal plasma and to assess the interrelationship with other minerals in male goats. METHODOLOGY: The study was conducted in 21 adult male goats divided into 3 groups (control, boron and selenium supplemented groups, n = 7 each). In boron group, boron was supplemented at 40 ppm and in selenium group, selenium was supplemented at 1 ppm over and above the basal level. In control group, only the basal diet was fed without supplementary boron or selenium. The feeding trial was carried out for 60 days. Selenium was taken as a positive control for the dietary boron supplementation experiment. Following feeding trials, the sperm concentration, kinematics and functional attributes, immunity and molecular level changes in the testis, biomolecular changes in the blood and seminal plasma and also interrelationship with other minerals were studied. RESULTS: The average sperm concentration (million/ml) and the total sperm production (million/ejaculate) were significantly (p < 0.05) increased in boron supplemented group when compared to selenium and control groups. The boron levels in blood plasma (r = 0.65) and seminal plasma (r = 0.54) showed a positive correlation with sperm progressive motility. Blood and seminal plasma metabolic biomarker namely, aspartate aminotransferase (AST) (p < 0.01) was significantly lower in the boron and selenium supplemented group than control, while alanine aminotransferase (ALT) (p < 0.05) was significantly lower in the boron supplemented group than selenium and control group. There was a significant increase in the mRNA expression of serine proteinase inhibitor (SERPIN) and interferon γ (IFNγ) in the testis of boron supplemented than the control group. Boron supplementation up-regulated the immune-regulatory gene, interleukin 2 (IL2) and antioxidant gene, catalase (CAT) in the peripheral blood mononuclear cells (PBMC). On contrary, toll-like receptor 2 (TLR2) mRNA expression was significantly (p < 0.05) down-regulated in boron and selenium supplemented groups. CONCLUSION: The study revealed that dietary boron supplementation increased the sperm output, sperm motility and enhanced the immune and antioxidant defense capacity in male goats. The improved semen quality can be attributed to enhanced expression of testicular SERPIN, a crucial protein for the regulation of spermatogenesis process.

Isolation and enrichment of putative spermatogonial stem cells from ram (Ovis aries) testis.

The present study aimed to isolate and enrich putative SSCs from ram testes, which are positive for promyelocytic leukaemia zinc-finger protein (PLZF). The putative SSCs were isolated using a combination of enzymes with different concentrations, collagenase (1 and 2 mg/ml), hyaluronidase (1 mg/ml) and trypsin (0.25 and 0.5 mg/ml). The isolated SSCs were purified using an extracellular matrix such as laminin (20 µg/ml), DSA-lectin (5 µg/ml) and gelatin (0.2%) in combination with BSA (0.5 mg/ml). The number of putative SSCs/ tubule was significantly (p < 0.05) higher in prepubertal (3.1 ±â€¯0.51) and adult (3.45 ±â€¯0.58) than the number of gonocytes/tubule in neonatal (0.59 ±â€¯0.03) testis. Optimum enzyme combinations required for isolation of putative SSCs from prepubertal testis (collagenase; 2 mg/ml and trypsin; 0.5 mg/ml) were different from adult testis (collagenase; 1 mg/ml, trypsin; 0.25 mg/ml and hyaluronidase; 1 mg/ml). Though the number of putative SSCs/tubule was comparable in prepubertal and adult animals, a significantly (p < 0.05) higher percentage of putative SSCs (7.33 Vs 0.47%) were isolated from prepubertal testis than the adult. Differential plating using laminin along with BSA resulted in a significantly (p < 0.05) higher number of putative SSCs. The enzyme combinations suitable for isolation of putative SSCs from prepubertal testis are different from adult ram testis and the laminin has been found to be effective for purification of putative SSCs from testicular cells isolates.

IGF1 stabilizes sperm membrane proteins to reduce cryoinjury and maintain post-thaw sperm motility in buffalo (Bubalus bubalis) spermatozoa.

Insulin like growth factor 1 (IGF1) in the seminal plasma is reported to improve sperm motility by reducing oxidative stress. The present study was conducted to assess the effect of addition of IGF1 on sperm function and protein composition during cryopreservation process. Semen samples were collected from six Murrah buffaloes (2 ejaculates from each animal) and diluted (80 million/ml) in tris egg yolk extender and divided into control, T1, T2 and T3, groups supplemented with 0, 50, 100 and 150 ng of IGF1/mL, respectively. The semen was filled in straws (250 µL) and straws from each group were divided into two batches. One batch was processed for freezing and another batch was incubated at 4 °C for 4 h. The sperm kinematic and functional parameters were studied in both the batches. A significant (P < 0.05) positive effect of IGF1 was observed on functional membrane integrity (%) during incubation at 4 °C for 4 h in T3 as compared to control group. The spermatozoa (%) positive for structural membrane integrity, mitochondrial membrane potential and the metabolic activity in post-thaw semen were significantly (P < 0.05) high in T3 than the control group. The acrosomal integrity was significantly (P < 0.05) higher in T2 group as compared to control. The proteins (kDa) of 17.3 with pI 4.2 (calmodulin), 11.3 with pI 6.5 (dermcidin) and 18.1 with pI 5.5 (sperm acrosome membrane associated protein3) were protected in IGF1 group. The study suggests that IGF1 can be added to the extender for improving cryosurvial of buffalo spermatozoa.