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.

Dietary n-3 PUFA augments pre-ovulatory follicle turnover and prolificacy in well-fed ewes.

The present study evaluated the effect of dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) on preovulatory follicle (POF) turnover, prolificacy, and endocrine and metabolic milieu in Malpura sheep. Fifty cyclic ewes with 3-3.5 body condition scores on a five-point scale were allocated equally to two groups (n = 25) following estrus synchronization and were supplemented with 0.6 mL/kg body weight of n-3 PUFA-rich fish oil (FO) or palm oil (PO) as control, for 60 d following an acclimatization period of 7 d. All ewes were mated with sexually active rams at the end of the supplementation period. On ultrasonographic ovarian scanning at the last fourth estrus, the mean number of POFs was 77.8% greater (P < 0.01) in FO ewes than in the PO ewes. The proportion of ewes with multiple ovulations two months after the beginning of supplementation was 56% in the FO group as compared to 8% in the PO group. The number of fetuses was 46% higher (P < 0.01) in the FO than in the PO ewes at d 45 of gestation. At lambing, the twinning percent in the FO ewes was three times greater than in the PO ewes (27.3 vs. 9.1%). Plasma cholesterol, estradiol, and insulin concentrations were lower (P < 0.01) in ewes fed with FO than those offered PO group at the end of the feeding period. It was concluded that the dietary supplementation of n-3 PUFA-rich FO in well-fed Malpura ewes improved the number of follicles and ovulation rate which led to an increased prolificacy, accompanied by a reduction of plasma cholesterols, estradiol, and insulin.

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.

Microenvironment of the male and female reproductive tracts regulate sperm fertility: Impact of viscosity, pH, and osmolality.

BACKGROUND: Terminally differentiated mammalian sperm are exposed to gradients of viscosity, pH, and osmolality both in the male and female reproductive tract during their perilous journey to quest the ovum. The complex physicochemical factors play an integral role in preparing sperm for the fertilization process. OBJECTIVES: To elucidate the influence of the reproductive tract microenvironment especially viscosity, pH, and osmolality in regulating sperm functional and fertilization competence. MATERIALS AND METHODS: The data used in this review were collected from the research papers and online databases focusing on the influence of viscosity, pH, and osmolality on sperm function. DISCUSSION: The gradients of viscosity, pH, and osmolality exist across various segments of the male and female reproductive tract. The changes in the viscosity create a physical barrier, pH aid in capacitation and hyperactivation, and the osmotic stress selects a progressive sperm subpopulation for accomplishing fertilization. The sperm function tests are developed based on the concept that the male genotype is the major contributor to the reproductive outcome. However, recent studies demonstrate the significance of sperm genotype-environment interactions that are essentially contributing to reproductive success. Hence, it is imperative to assess the impact of physicochemical stresses and the adaptive ability of the terminally differentiated sperm, which in turn would improve the outcome of the assisted reproductive technologies and male fertility assessment. CONCLUSION: Elucidating the influence of the reproductive tract microenvironment on sperm function provides newer insights into the procedures that need to be adopted for selecting fertile males for breeding, and ejaculates for the assisted reproductive technologies.

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.

Deciphering the complexity of sperm transcriptome reveals genes governing functional membrane and acrosome integrities potentially influence fertility.

Deciphering sperm transcriptome is the key to understanding the molecular mechanisms governing peri-fertilization, embryonic development, and pregnancy establishment. This study aimed to profile sperm transcriptome to identify signature transcripts regulating male fertility. Semen samples were collected from 47 bulls with varied fertility rates. The sperm total RNA was isolated (n = 8) and subjected to transcriptome sequencing. Based on the expression pattern obtained from RNA profiling, the bulls were grouped (p = 0.03) into high-fertile and sub-fertile, and signature transcripts controlling sperm functions and fertility were identified. The results were validated using the OMIM database, qPCR, and sperm function tests. The sperm contains 1100 to 1700 intact transcripts, of which BCL2L11 and CAPZA3 were abundant and associated (p < 0.05) with spermatogenesis and post-embryonic organ morphogenesis. The upregulated genes in the acrosome integrity and functional membrane integrity groups had a close association with the fertility rate. The biological functions of these upregulated genes (p < 0.05) in the high-fertile bulls were associated with spermatogenesis (AFF4 and BRIP1), sperm motility (AK6 and ATP6V1G3), capacitation and zona binding (AGFG1), embryo development (TCF7 and AKIRIN2), and placental development (KRT19). The transcripts involved in pathways regulating embryonic development such as translation (EEF1B2 and MTIF3, p = 8.87E-05) and nonsense-mediated decay (RPL23 and RPL7A, p = 5.01E-27) were upregulated in high-fertile bulls. The identified transcripts may significantly impact oocyte function, embryogenesis, trophectoderm development, and pregnancy establishment. In addition, the study also reveals that the genes governing sperm functional membrane integrity and acrosome integrity have a prospective effect on male 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.

IGF-1 treatment during in vitro maturation improves developmental potential of ovine oocytes through the regulation of PI3K/Akt and apoptosis signaling.

This study aimed to assess the effect of the insulin-like grow factor 1 (IGF-1) treatment during in vitro maturation on the gene expression and developmental ability of ovine oocytes. Ovine cumulus-oocyte complexes (COC) were matured in vitro without (control) or with the supplementation of IGF-1 (100 ng/ml) and then subjected to in vitro fertilization and culture. The rate of oocyte maturation and embryo development was recorded and expression of the selected genes (involved in the PI3K/Akt and apoptosis signaling) was assessed in the matured oocytes. The IGF-1 treatment significantly (p < .05) improved the oocyte maturation rate (%) as compared to the control (81.5 ± 2.40 vs. 73.6 ± 0.94). Similarly, as compared to the control, the IGF-1 treatment significantly (p < .05) improved the rate (%) of cleavage (54.7 ± 1.58 vs. 67.2 ± 3.65) and the formation of 4-8 cell embryos (30.7 ± 2.89 vs. 44.1 ± 4.01) and morula (20.7 ± 2.08 vs. 32.8 ± 2.78). The IGF-1 treatment significantly (p < .05) upregulated the expression of IGF1R, PI3KR1, AKT1 and BCL2 and downregulated the expression of GSK3ß, FOXO3 and CASP9 in the matured oocytes. In conclusion, the IGF-1 treatment significantly improved the developmental competence of ovine oocytes through the regulation of the PI3K/Akt and apoptosis signaling.

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.

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.

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.

Sub-fertility in crossbred bulls: deciphering testicular level transcriptomic alterations between zebu (Bos indicus) and crossbred (Bos taurus x Bos indicus) bulls.

BACKGROUND: The incidence of poor semen quality and sub-fertility/infertility is higher in crossbred as compared to Zebu males. Several attempts have been made to understand the possible reasons for higher incidence of fertility problems in crossbred males, at sperm phenotype, proteome and genome level but with variable results. Since the quality of the ejaculated spermatozoa is determined by the testicular environment, assessing the testicular transcriptome between these breeds would help in identifying the possible mechanisms associated with infertility in crossbred bulls. However, such information is not available. We performed global transcriptomic profiling of testicular tissue from crossbred and Zebu bulls using Agilent Bos taurus GXP 8X60k AMADID: 29411 array. To the best of our knowledge, this is the first study comparing the testicular mRNAs between crossbred and Zebu bulls. RESULTS: Out of the 14,419 transcripts detected in bovine testis, 1466 were differentially expressed between crossbred and Zebu bulls, in which 1038 were upregulated and 428 were downregulated in crossbred bulls. PI4KB and DPY19L2 genes, reported to be involved in sperm capacitation and acrosome formation respectively, were among the top 10 downregulated transcripts in crossbred testis. Genes involved in ubiquitination and proteolysis were upregulated, while genes involved in cell proliferation, stem cell differentiation, stem cell population maintenance, steroidogenesis, WNT signalling, protein localization to plasma membrane, endocannabinoid signalling, heparin binding, cAMP metabolism and GABA receptor activity were downregulated in crossbred testis. Among the 10 genes validated using qPCR, expression of CCNYL, SOX2, MSMB, SPATA7, TNP1, TNP2 and CRISP2 followed the same trend as observed in microarray analysis with SPATA7 being significantly downregulated and transition proteins (TNP1, TNP2) being significantly upregulated in crossbred bulls. CONCLUSIONS: Abundant proteolysis by ubiquitination and downregulation of WNT signaling, cell proliferation, differentiation and steroidogenesis might be associated with higher incidence of poor semen quality and/or sub-fertility/infertility in crossbred bulls as compared to Zebu bulls. Downregulation of SPATA7 (Spermatogenesis Associated 7) and upregulation of transition proteins (TNP1 and TNP2) in crossbred bull testis might be associated with impaired spermatogenesis processes including improper chromatin compaction in crossbred bulls.

Immuno-histological mapping and functional association of seminal proteins in testis and excurrent ducts with sperm function in buffalo.

The region-specific expression of seminal proteins in testis and excurrent duct system determines the quality and function of the spermatozoa. In the present study, localization and expression of some of the seminal proteins such as insulin-like growth factor receptor 1ß (IGF-1Rß), phosphatidylethanolamine-binding protein 4 (PEBP4), α-tubulin and tissue factor pathway inhibitor 2 (TFPI2) were carried out in testis, excurrent duct system and spermatozoa of buffalo. IGF-1Rß was localized in the cells of the seminiferous tubules of the testis, except in primary spermatocytes. The PEBP4 was localized only in the elongated spermatid, whereas α-tubulin and TFPI2 proteins were localized in all cells of the seminiferous tubule including spermatocyte. In the buffalo spermatozoa, IGF-1Rß, PEBP4, α-tubulin and TFPI2 were localized in the acrosome region, the post-acrosomal region till the tail end, post-acrosome to the entire tail region and the equatorial region, respectively. The study indicates that IGF-1R, α-tubulin and PEBP4 proteins regulate spermatogenesis, whereas TFPI2 may be involved during the zona binding process of the buffalo spermatozoa.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls.

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P < 0.05), while protamine deficiency and lipid peroxidation were higher (P < 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P < 0.05 and fold change >1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.

Impact of environmental contaminants on reproductive health of male domestic ruminants: a review.

Environmental contaminants are gaining more attention in the livestock sector lately due to their harmful effects on productivity and fertility of livestock. Recent research indicates that many domestic ruminants are becoming subfertile/infertile due to confounding reasons associated with management. Contaminants like metals, metalloids, herbicides, pesticides, insecticides, chemicals, or natural contaminants are present everywhere in day to day life and are becoming a threat to the livestock. Studies on a broad-spectrum of animals suggest that high doses of acute or low doses of chronic exposure to the contaminants lead to disruption of multi-organs/systems including reproductive function. The lowered reproductive efficiency in animals is attributed to the endocrine disruptor activities of the environmental contaminants on the gonads, affecting gametogenesis and steroidogenesis. In vitro studies on testicular cells and the semen suggest that spermatozoa are more susceptible to damage by environmental contaminants. The quality of the semen happens to be a critical factor in the livestock industry. Contaminants affecting gametogenesis and steroidogenesis may lead to devastating consequences to the livestock reproduction, and thus the production. However, there is a lack of collective data on the effect of such environmental contaminants on the fertility of male domestic ruminants. This review discusses the studies related to the impact of environmental contaminants on male fertility in large (bull and buffalo) and small (sheep and goat) ruminants by focusing on the underlying molecular interactions between the contaminants and gonads.