Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index.

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility.

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

High-throughput proteomic characterization of seminal plasma from bulls with contrasting semen quality.

Seminal plasma proteins are the major extrinsic factors that can modulate the sperm quality and functions. The present study was carried out to compare the proteomic profiles of seminal plasma from breeding bulls producing good and poor quality semen in an effort to understand the possible proteins associated with semen quality. A total of 910 and 715 proteins were detected in the seminal plasma of poor and good quality semen producing bulls, respectively. A total of 705 proteins were common to both the groups, in which 380 proteins were upregulated and 89 proteins were downregulated in the seminal plasma of poor quality semen, while 236 proteins were co-expressed. The proteins negatively influencing sperm functions such as CCL2, UQCRC2, and SAA1 were among the top ten upregulated proteins in the seminal plasma of poor quality semen. Proteins having a positive role in sperm functions (NGF, EEF1A2, COL1A2, IZUMO4, PRSS1, COL1A1, WFDC2) were among the top ten downregulated proteins in the seminal plasma of poor quality semen. The upregulation of oxidation-reduction process-related proteins, histone proteins (HIST3H2A, H2AFJ, H2AFZ, H2AFX, HIST2H2AB, H2AFV, HIST1H2AC, HIST2H2AC, LOC104975684, LOC524236, LOC614970, LOC529277), and ubiquinol-cytochrome-c reductase proteins (UQCRB, UQCRFS1, UQCRQ, UQCRC1, UQCRC2) indicate deranged oxidation-reduction equilibrium, chromatin condensation and spermatogenesis in poor quality semen producing bulls. The expression of proteins essential for motile cilium (CCDC114, CFAP206, TEKT4), chromatin integrity (PRM2), gamete fusion (IZUMO4, EQTN), hyperactivation, tyrosine phosphorylation, and capacitation [PI3K-Akt signalling pathway-related proteins (COL1A1, COL2A1, COL1A2, SPP1, PDGFA, NGF)] were down regulated in poor quality semen producing bulls. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03474-6.

Seasonal and climatic factors have a significant influence on fertility associated sperm phenomic attributes in crossbred breeding bulls (Bos taurus × Bos indicus).

Although seasonal variations in semen quality and fertility have been studied to a considerable extent in breeding bulls, the effect of climatic variables on sperm functional competency has not been understood in detail. The present study analyzed sperm functional parameters in breeding bulls, over a period of 1 year, and assessed the effect of climatic variables on fertility associated sperm parameters. Seasons were categorized into summer, rainy, autumn, and winter based on the meteorological data. Semen was collected from crossbred bulls (n = 7) across the seasons and evaluated for functional membrane integrity, acrosome reaction status, protamine deficiency, capacitation, and lipid peroxidation status using specific fluorescent probes. The results of the present study revealed that bulls produced higher (p < 0.05) viable and acrosome intact spermatozoa during the autumn. The proportion of uncapacitated spermatozoa was also higher (p < 0.05) during autumn. Further, correlation of sperm functional attributes with environmental variables revealed that sperm viability was significantly (p < 0.05) and negatively correlated with daylength and temperature; acrosomal integrity was significantly (p < 0.05) and negatively correlated with day length; and protamine deficiency had significant (p < 0.05) positive correlation with day length and average temperature, and negative correlation with relative humidity. It was concluded that semen produced during autumn was superior to the semen produced during other seasons in terms of sperm functional competencies required for fertility.

Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa.

Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

The cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Cryopreservation process alters the expression of genes involved in pathways associated with the fertility of bull spermatozoa.

In bovines, cryopreserved semen is used for artificial insemination; however, the fertility of cryopreserved semen is far lower than that of fresh semen. Although cryopreservation alters sperm phenotypic characteristics, its effect on sperm molecular health is not thoroughly understood. The present study applied next-generation sequencing to investigate the effect of cryopreservation on the sperm transcriptomic composition of bull spermatozoa. While freshly ejaculated bull spermatozoa showed 14,280 transcripts, cryopreserved spermatozoa showed only 12,375 transcripts. Comparative analysis revealed that 241 genes were upregulated, 662 genes were downregulated, and 215 genes showed neutral expression in cryopreserved spermatozoa compared to fresh spermatozoa. Gene ontology analysis indicated that the dysregulated transcripts were involved in nucleic acid binding, transcription-specific activity, and protein kinase binding involving protein autophosphorylation, ventricular septum morphogenesis, and organ development. Moreover, the dysregulated genes in cryopreserved spermatozoa were involved in pathways associated with glycogen metabolism, MAPK signalling, embryonic organ morphogenesis, ectodermal placode formation, and regulation of protein auto-phosphorylation. These findings suggest that the cryopreservation process induced alterations in the abundance of sperm transcripts related to potential fertility-associated functions and pathways, which might partly explain the reduced fertility observed with cryopreserved bull spermatozoa.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

Sperm Transcripts Associated With Odorant Binding and Olfactory Transduction Pathways Are Altered in Breeding Bulls Producing Poor-Quality Semen.

Spermatozoa carries a reservoir of mRNAs regulating sperm functions and fertilizing potential. Although it is well recognized that a considerable proportion of high genetic merit breeding bulls produce poor-quality semen, the transcriptomic alterations in spermatozoa from such bulls are not understood. In the present study, comparative high-throughput transcriptomic profiling of spermatozoa from good and poor-quality semen-producing bulls was carried out to identify the transcripts associated with semen quality. Using next-generation sequencing (NGS), we identified 11,632 transcripts in Holstein Friesian bull spermatozoa; after total hit normalization, a total of 544 transcripts were detected, of which 185 transcripts were common to both good and poor-quality semen, while 181 sperm transcripts were unique to good quality semen, and 178 transcripts were unique to poor-quality semen. Among the co-expressed transcripts, 31 were upregulated, while 108 were downregulated, and 46 were neutrally expressed in poor-quality semen. Bioinformatics analysis revealed that the dysregulated transcripts were predominantly involved in molecular function, such as olfactory receptor activity and odor binding, and in biological process, such as detection of chemical stimulus involved in sensory perception, sensory perception of smell, signal transduction, and signal synaptic transmission. Since a majority of the dysregulated transcripts were involved in the olfactory pathway (85% of enriched dysregulated genes were involved in this pathway), the expression of selected five transcripts associated with this pathway (OR2T11, OR10S1, ORIL3, OR5M11, and PRRX1) were validated using real-time qPCR, and it was found that their transcriptional abundance followed the same trend as observed in NGS; the sperm transcriptional abundance of OR2T11 and OR10S1 differed significantly (p < 0.05) between good and poor-quality semen. It is concluded that poor-quality semen showed altered expression of transcripts associated with olfactory receptors and pathways indicating the relationship between olfactory pathway and semen quality in bulls.

Epididymosomes: A potential male fertility influencer.

During transit and storage in epididymis, spermatozoa undergo final maturation, acquire motility, functional competence and the ability to fertilise an oocyte. Epididymal secretions contain a complex biochemical milieu of diverse inorganic ions, proteins, metabolites and other molecules. Since it is believed that spermatozoa are translationally silent, proteins appearing in them are thought to be synthesised elsewhere, including epididymis, and then incorporated to the cells. One of the important mechanisms suggested to be involved in transfer of epididymal secretions to spermatozoa is through exosomes called epididymosomes. Epididymosomes released from the epididymal epithelium contain proteins, noncoding RNAs and distinct set of lipids that are transferred to spermatozoa while they pass through the different epididymal regions. Owing to the importance of these molecules for sperm maturation and fertilising ability, research on epididymosomes has gained increasing attention during the last decade. This review is focused on epididymosomes, with emphasis on recent advances in the understanding of mechanisms of epididymosomal cargo transfer to spermatozoa and potential roles of epididymosomes in sperm function and beyond. Possibilities of utilising the molecular signatures of epididymosomes as a tool for male fertility assessment are also discussed.

Sperm phenotypic characteristics and oviduct binding ability are altered in breeding bulls with high sperm DNA fragmentation index.

In the present study, we standardized an in vitro oviduct explants model for cattle and assessed the oviduct explants binding ability and phenotypic characteristics of spermatozoa obtained from breeding bulls with high- and low-sperm DNA fragmentation index (DFI%). Cryopreserved spermatozoa from Holstein Friesian crossbred breeding bulls (n = 45) with known field fertility were assessed for DFI% and were classified into either high DFI% or low DFI% category. Flow cytometry was used to assess sperm membrane integrity, acrosome reaction status, mitochondrial membrane potential and intracellular calcium concentrations. It was found that spermatozoa from bulls with low DFI% had significantly higher (P < 0.05) membrane integrity, acrosome intactness, and mitochondrial membrane potential. To assess the sperm oviduct binding ability, oviduct explants were prepared by incubating the oviduct cells overnight in TCM-199 medium at 38.5 °C under 5% CO2. Different sperm concentrations and times of incubation were evaluated and found that 2 million spermatozoa and 1-h incubation yielded high binding index (BI). The BI was also significantly (P < 0.01) higher (>2 times) in the bulls with low-DFI% as compared to high DFI% bulls. The correlation between binding index and DFI% was negative and significant (r = -0.528; P < 0.05). Further, the binding index was positively correlated with conception rate (r = 0.703), intact sperm membrane (r = 0.631) and mitochondrial membrane potential (r = 0.609). It is inferred that sperm phenotypic characteristics and oviduct binding ability are impaired in breeding bulls with high sperm DFI%, which might be associated with low conception rates in these bulls.

Comparative Transcriptomic Analysis of Spermatozoa From High- and Low-Fertile Crossbred Bulls: Implications for Fertility Prediction.

Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.

Supplementation of a combination of herbs improves immunity, uterine cleansing and facilitate early resumption of ovarian cyclicity: A study on post-partum dairy buffaloes.

ETHNOPHARMACOLOGICAL RELEVANCE: A combination of Trachyspermum ammi L., Curcuma longa L., Cuminum cyminum L., Trigonella foenum-graecum L., Foeniculum vulgare Mill., Anethum graveolens L and Zingiber officinale Roscoe is used as immunity booster and reproductive efficiency enhancing agents in folklore medicine. AIM OF THE STUDY: The present study aimed to assess the immunomodulatory, uterine cleansing and reproduction enhancing effects of polyherbal mixture in post-partum buffaloes. MATERIALS AND METHODS: Enzyme linked immunosorbent assay (ELISA) was used to investigate the effects of polyherbal mixture feeding on for quantification of neutrophil functions and blood progesterone hormone estimation. Ultrasonography was used to assess the status of uterine involution, fluid in uterus and ovarian follicular status. Quantitative real time PCR (qRT-PCR) was used to measure the expression of chemokine genes CXCR1, CXCR2 AND IL-8. Artificial insemination with cryopreserved semen was used to breed the animals. Reproductive efficiency parameters were assessed using standard calculation methods. RESULTS: Neutrophil functions and transcriptional abundance of chemokine genes were significantly (P < 0.05) higher in buffaloes supplemented with polyherbal mixture compared to buffaloes in control group. The rate of cervical and uterine involution was significantly (P < 0.05) higher in treatment group compared to control group. The service period was shorter, days to first insemination was earlier and the number of services per conception was lower in buffaloes supplemented with polyherbal mixture compared to the buffaloes in control group. The proportion of buffaloes with large ovarian follicles within 28 days of post-partum was also significantly (P < 0.05) higher in treatment group compared to the control group. CONCLUSIONS: The polyherbal mixture used in the study improved the immunity of the buffaloes, facilitated early involution of cervix and uterus, efficient cleansing of lochia and improved subsequent fertility. It has the potential to be used in dairy animals for improving post-partum reproductive efficiency.

Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic bulls.

Spermatozoa produced during winter are superior in terms of phenotypic characteristics and oviduct explants binding ability in the water buffalo (Bubalus bubalis).

Although reduced reproductive efficiency during summer has been well documented in buffaloes, the reason for the same is yet to be understood. The present study was conducted to identify the subtle differences in sperm phenotypic characteristics (motility, membrane integrity, acrosome reaction and lipid peroxidation status), oviduct binding ability and expression of fertility-associated genes (AK 1, ATP5D, CatSper 1, Cytochrome P450 aromatase, SPP1 and PEBP1) between winter and summer seasons in buffaloes. Cryopreserved spermatozoa from 6 Murrah buffalo bulls (3 ejaculates/bull/season) were utilized for the study. Real-time quantitative PCR was performed for assessing the expression patterns of select fertility-associated genes. The proportion of motile and membrane intact spermatozoa was significantly higher (p < .05) in winter as compared to summer ejaculates. The proportion of moribund and lipid peroxidized spermatozoa was significantly lower (p < .05) in winter ejaculates as compared to summer. The sperm-oviduct binding index was significantly lower (p < .01) when spermatozoa from summer ejaculates were used as compared to winter ejaculates. The expression of fertility-associated genes did not differ significantly between the two seasons except for PEPB1; the transcriptional abundance of PEPB1 was significantly (p < .05) lower in summer as compared to winter season. It was inferred that buffalo spermatozoa produced during winter season were superior in terms of cryotolerance, membrane and acrosome integrity, lipid peroxidation status and the ability to bind with oviduct explants.

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.

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility.

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = -0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.

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.

Growth performance and rumen development in Malabari kids reared under different production systems.

In order to investigate the effect of rearing systems on growth and rumen development in Malabari male kids, 14 pre-weaned Malabari male kids of uniform morphological characters were randomly divided into two equal groups as T1 and T2. Both the groups were reared intensively for 12 weeks whereby kids under T1 group were allowed to suckle their dams and provided green grass ad libitum. The kids under T2 were weaned at the age of 7 days and provided formulated semi-solid broiler goat concentrate diet through the feeding bottle and were not offered any grass/roughage. Parameters like live weight gain, daily weight gain and body measurements were studied to evaluate the growth performance. However, gastrointestinal tract morphometric studies and qualitative morphological analysis of rumen papillae were undertaken to measure the rumen development. The daily weight gain of kids under T2 was significantly (P ≤ 0.01) higher than the daily weight gain of kids under T1. Also, the body measurements like height at withers, heart girth and body length were significantly (P ≤ 0.01) higher in T2 than in T1. The rumen and abomasum were comparatively more developed in T2 than in T1. The morphology of rumen papillae in both groups was different in size, shape and colour. The length, width, density and surface area of rumen papillae among kids in T2 were significantly (P ≤ 0.01) higher than those kids in T1. The results of the present study indicated that the feeding of semi-solid broiler goat concentrate diet improved growth performance and early rumen development in kids.