Polygonum bistorta Linn. as a green source for synthesis of biocompatible selenium nanoparticles with potent antimicrobial and antioxidant properties.

Here, we report for the first time, green-synthesized selenium nanoparticles (SeNPs) using pharmacologically potent herb of Polygonum bistorta Linn. for multiple biomedical applications. In the study, a facile and an eco-friendly approach is utilized for synthesis of SeNPs using an aqueous roots extract of P. bistorta Linn. followed by extensive characterization via Fourier transform infrared spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Energy Dispersive X-Ray (EDX) analysis. The XRD and FTIR data determine the phase composition and successful capping of plant extract onto the surface of NPs while SEM and TEM micrographic examination reveals the elliptical and spherical morphology of the particles with a mean size of 69 ± 23 nm. After comprehensive characterization, the NPs are investigated for antifungal, antibacterial, antileishmanial, antioxidant, and biocompatibility properties. The study reveals that Polygonum bistorta Linn. synthesized SeNPs exhibit significant antibacterial and antifungal activities with Staphylococcus aureus and Fusarium oxysporum inducing the highest zone of inhibition of 14 ± 1.0 mm and 20 ± 1.2 mm, respectively at the concentration of 40 mg/mL. The NPs are also found to have antiparasitic potential against promastigote and amastigote forms of Leishmania tropica. Furthermore, the NPs are discovered to have excellent potential in neutralizing harmful free radicals thus exhibiting considerable antioxidant potential. Most importantly, Polygonum bistorta Linn. synthesized SeNPs showed substantial compatibility against blood cells in vitro studies, which signifies the nontoxic nature of the NPs. The study thus concludes that medicinally important Polygonum bistorta Linn. roots can be utilized as an eco-friendly, sustainable, and green source for the synthesis of pharmacologically potent selenium nanoparticles.

Probabilistic classification of the severity classes of unhealthy air pollution events.

Air pollution events can be categorized as extreme or non-extreme on the basis of their magnitude of severity. High-risk extreme air pollution events will exert a disastrous effect on the environment. Therefore, public health and policy-making authorities must be able to determine the characteristics of these events. This study proposes a probabilistic machine learning technique for predicting the classification of extreme and non-extreme events on the basis of data features to address the above issue. The use of the naïve Bayes model in the prediction of air pollution classes is proposed to leverage its simplicity as well as high accuracy and efficiency. A case study was conducted on the air pollution index data of Klang, Malaysia, for the period of January 01, 1997, to August 31, 2020. The trained naïve Bayes model achieves high accuracy, sensitivity, and specificity on the training and test datasets. Therefore, the naïve Bayes model can be easily applied in air pollution analysis while providing a promising solution for the accurate and efficient prediction of extreme or non-extreme air pollution events. The findings of this study provide reliable information to public authorities for monitoring and managing sustainable air quality over time.

Global Analysis of Dark- and Heat-Regulated Alternative Splicing in Arabidopsis.

Alternative splicing (AS) is one of the major post-transcriptional regulation mechanisms that contributes to plant responses to various environmental perturbations. Darkness and heat are two common abiotic factors affecting plant growth, yet the involvement and regulation of AS in the plant responses to these signals remain insufficiently examined. In this study, we subjected Arabidopsis seedlings to 6 h of darkness or heat stress and analyzed their transcriptome through short-read RNA sequencing. We revealed that both treatments altered the transcription and AS of a subset of genes yet with different mechanisms. Dark-regulated AS events were found enriched in photosynthesis and light signaling pathways, while heat-regulated AS events were enriched in responses to abiotic stresses but not in heat-responsive genes, which responded primarily through transcriptional regulation. The AS of splicing-related genes (SRGs) was susceptible to both treatments; while dark treatment mostly regulated the AS of these genes, heat had a strong effect on both their transcription and AS. PCR analysis showed that the AS of the Serine/Arginine-rich family gene SR30 was reversely regulated by dark and heat, and heat induced the upregulation of multiple minor SR30 isoforms with intron retention. Our results suggest that AS participates in plant responses to these two abiotic signals and reveal the regulation of splicing regulators during these processes.

A comprehensive review of protein-based carriers with simple structures for the co-encapsulation of bioactive agents.

The rational design and fabrication of edible codelivery carriers are important to develop functional foods fortified with a plurality of bioactive agents, which may produce synergistic effects in increasing bioactivity and functionality to target specific health benefits. Food proteins possess considerable functional attributes that make them suitable for the delivery of a single bioactive agent in a wide range of platforms. Among the different types of protein-based carriers, protein-ligand nanocomplexes, micro/nanoparticles, and oil-in-water (O/W) emulsions have increasingly attracted attention in the codelivery of multiple bioactive agents, due to the simple and convenient preparation procedure, high stability, matrix compatibility, and dosage flexibility. However, the successful codelivery of bioactive agents with diverse physicochemical properties by using these simple-structure carriers is a daunting task. In this review, some effective strategies such as combined functional properties of proteins, self-assembly, composite, layer-by-layer, and interfacial engineering are introduced to redesign the carrier structure and explore the encapsulation of multiple bioactive agents. It then highlights success stories and challenges in the co-encapsulation of multiple bioactive agents within protein-based carriers with a simple structure. The partition, protection, and release of bioactive agents in these protein-based codelivery carriers are considered and discussed. Finally, safety and application as well as challenges of co-encapsulated bioactive agents in the food industry are also discussed. This work provides a state-of-the-art overview of protein-based particles and O/W emulsions in co-encapsulating bioactive agents, which is essential for the design and development of novel functional foods containing multiple bioactive agents.

Production performance, protein digestibility, gut health and economic efficiency in sexed broilers with super dozing of lysine.

Modern commercial broiler is growing very rapidly and its amino acid requirement is not fulfilling. An experimental trial was conducted to study the effect of super-dosing of lysine in fish meal-based diets (50% fish meal and 50% SBM) on production performance, protein digestibility and economic efficiency in male and female broiler chickens. Four hundred and eighty (480) one-day-old male and female broiler chicks were divided into forty experimental units of 12 birds each. Five levels of dietary lysine i.e. 90, 100, 110, 120 and 130% of Ross-308 recommendation in male and female birds were separately used. Weight gain and feed intake were higher (p < 0.05) in birds received 100 and 110% recommended lysine than other levels. Feed conversion ratio and EPEF were improved (p < 0.05) in birds received 100% recommended lysine than other levels. Higher (p < 0.05) CP digestibility and lower production cost per kg live weight were noted for birds fed diet containing lysine 100, 110 and 120% than 90 and 130%. Male birds had higher (p < 0.05) WG, FI, EPEF, protein digestibility and lower production cost per kg live weight than female birds. In conclusion, lysine levels below 100% and above 110% of Ross recommended levels had poor production performance, protein digestibility and economic efficiency.

Mechanisms of Abscisic Acid-Mediated Drought Stress Responses in Plants.

Drought is one of the major constraints to rain-fed agricultural production, especially under climate change conditions. Plants evolved an array of adaptive strategies that perceive stress stimuli and respond to these stress signals through specific mechanisms. Abscisic acid (ABA) is a premier signal for plants to respond to drought and plays a critical role in plant growth and development. ABA triggers a variety of physiological processes such as stomatal closure, root system modulation, organizing soil microbial communities, activation of transcriptional and post-transcriptional gene expression, and metabolic alterations. Thus, understanding the mechanisms of ABA-mediated drought responses in plants is critical for ensuring crop yield and global food security. In this review, we highlighted how plants adjust ABA perception, transcriptional levels of ABA- and drought-related genes, and regulation of metabolic pathways to alter drought stress responses at both cellular and the whole plant level. Understanding the synergetic role of drought and ABA will strengthen our knowledge to develop stress-resilient crops through integrated advanced biotechnology approaches. This review will elaborate on ABA-mediated drought responses at genetic, biochemical, and molecular levels in plants, which is critical for advancement in stress biology research.

Lack of CpG islands in human unitary pseudogenes and its implication.

CpG islands (CGIs) are aggregation of CpG dinucleotides in the promoters of mammalian genes. These CGIs are present in almost all the housekeeping genes and some tissue-specific genes in the mammalian genome. Extensive research has been done on the prevalence and role of CGIs in protein-coding genes. However, little is known about CGIs in pseudogenes. In the current research project, we focused on CGIs in three main classes of pseudogenes e.g., duplicated pseudogenes (DPGs), processed pseudogenes (PPGs), and unitary pseudogenes (UPGs). We discovered a predominant absence of CGIs in the promoters of all three pseudogenes. We also compared the CGI profile of these pseudogenes with their parent genes and found that unitary pseudogenes (UPGs) differ from the DPGs and PPGs in the sense that in the latter, lack of CGIs is a consequential event while in UPGs, this lack of CGIs in their promoters is not a result of pseudogenization process. We also discussed the implication of the results obtained from this comparison. To our knowledge, this is the first-ever study highlighting this aspect of UPGs throwing new insights into the evolution of genome in general and especially in the context of pseudogenes.

Edible mushroom (Flammulina velutipes) as biosource for silver nanoparticles: from synthesis to diverse biomedical and environmental applications.

The current study reports advanced, ecofriendly and biosynthesized silver NPs for diverse biomedical and environmental applications using Flammulina velutipes as biosource. In the study, a simple aqueous extract of F. velutipes was utilized to reduce the AgNO3 into stable elemental silver (Ag0) at a nanometric scale. The NPs had average size of 21.4 nm, spherical morphology, and were highly stable and pure. The characterized nanoparticles were exploited for a broad range of biomedical applications including bacteriocidal, fungicidal, leishmanicidal, in vitro antialzheimer's, antioxidant, anti-diabetic and biocompatibility studies. Our findings showed that F. velutipes mediated AgNPs exhibited high activity against MDR bacterial strains and spore forming fungal strains. All the tested urinary tract infection bacterial isolates, were resistant to non-coated antibiotics but by applying 1% of the synthesized AgNPs, the bactericidal potential of the tested antibiotics enhanced manifolds. The NPs also exhibited dose-dependent cytotoxic potential against Leishmania tropica with significant LC50 of 248 µg ml-1 for promastigote and 251 µg ml-1 for amastigote forms of the parasite. Furthermore, promising antialzheimer and antidiabetic activities were observed as significant inhibition of α-amylase, α-glucosidase, acetylcholinesterase (AChE) and butrylcholineterase (BChE) were noted. Moreover, remarkable biocompatible nature of the particles was found against human red blood cells. The biosynthesized AgNPs as photocatalyst, also resulted in 98.2% degradation of indigo carmine dye within 140 min. Owing to ecofriendly synthesis, biosafe nature and excellent physicochemical properties F. velutipes AgNPs can be exploited as novel candidates for multifaceted biomedical and environmental applications.

Synthesis, characterization, antimicrobial, antioxidant and computational evaluation of N-acyl-morpholine-4-carbothioamides.

The present research paper reports the convenient synthesis, successful characterization, in vitro antibacterial, antifungal, antioxidant potency and biocompatibility of N-acyl-morpholine-4-carbothioamides (5a-5j). The biocompatible derivatives were found to be highly active against the tested bacterial and fungal strains. Moreover, some of the screened N-acyl-morpholine-4-carbothioamides exhibited excellent antioxidant potential. Docking simulation provided additional information about possibilities of their inhibitory potential against RNA. It has been predicted by in silico investigation of the binding pattern that compounds 5a and 5j can serve as the potential surrogate for design of novel and potent antibacterial agents. The results for the in vitro bioassays were promising with the identification of compounds 5a and 5j as the lead and selective candidate for RNA inhibition. Results of the docking computations further ascertained the inhibitory potential of compound 5a. Based on the in silico studies, it can be suggested that compounds 5a and 5j can serve as a structural model for the design of antibacterial agents with better inhibitory potential. Binding mode of compound 5j inside the active site of RNA in 3D space. 5j displayed highest antibacterial potential than the reference drug ampicillin with ZOI 10.50 mm against Staphylococcus aureus. 5j also displayed highest antifungal potential than the reference drug amphotericin B with ZOI 18.20 mm against Fusarium solani.

Detection of lead in soil implying sample heating and laser-induced breakdown spectroscopy.

The emission line intensities enhancement and sensitivity of laser-induced breakdown spectroscopy (LIBS) has been a subject of great interest for the last several years to improve the detection of the trace elements in soil and other environmental samples. Among several other methodologies, LIBS of the heated targets is emerging as one of the effective techniques to achieve the objective. We have investigated the effect of target heating (room temperature, 100°C, and 200°C) on the emission enhancement and plasma parameters of the laser-produced plasma on the soil sample containing 80 ppm lead. In addition, the limit of detection (LOD) of lead in soil has been determined at a fixed target temperature (200°C) and with varying lead concentration (20 ppm, 80 ppm, and 100 ppm) in the soil samples. With increasing the target temperature, not only do the emission line intensities, the excitation temperature, and electron number density increase, but also the spectral lines of Pb emerge, which were absent in the soil spectra recorded at room temperature. The limit of detection of lead in LIBS of the heated soil target has been determined as 3.8 ppm. This study reveals the potential application of the LIBS of a pre-heated target for the detection of lead with an improved LOD in the environmental sample.

Central Role for Adipocyte Na,K-ATPase Oxidant Amplification Loop in the Pathogenesis of Experimental Uremic Cardiomyopathy.

BACKGROUND: Oxidative stress in adipocyte plays a central role in the pathogenesis of obesity as well as in the associated cardiovascular complications. The putative uremic toxin indoxyl sulfate induces oxidative stress and dramatically alters adipocyte phenotype in vitro. Mice that have undergone partial nephrectomy serve as an experimental model of uremic cardiomyopathy. This study examined the effects on adipocytes of administering a peptide that reduces oxidative stress to the mouse model. METHODS: A lentivirus vector introduced the peptide NaKtide with an adiponectin promoter into the mouse model of experimental uremic cardiomyopathy, intraperitoneally. Then adipocyte-specific expression of the peptide was assessed for mice fed a standard diet compared with mice fed a western diet enriched in fat and fructose. RESULTS: Partial nephrectomy induced cardiomyopathy and anemia in the mice, introducing oxidant stress and an altered molecular phenotype of adipocytes that increased production of systemic inflammatory cytokines instead of accumulating lipids, within 4 weeks. Consumption of a western diet significantly worsened the adipocyte oxidant stress, but expression of NaKtide in adipocytes completely prevented the worsening. The peptide-carrying lentivirus achieved comparable expression in skeletal muscle, but did not ameliorate the disease phenotype. CONCLUSIONS: Adipocyte-specific expression of NaKtide, introduced with a lentiviral vector, significantly ameliorated adipocyte dysfunction and uremic cardiomyopathy in partially nephrectomized mice. These data suggest that the redox state of adipocytes controls the development of uremic cardiomyopathy in mice subjected to partial nephrectomy. If confirmed in humans, the oxidative state of adipocytes may be a therapeutic target in chronic renal failure.

Application of stacking hybrid machine learning algorithms in delineating multi-type flooding in Bangladesh.

Floods are among the most devastating natural hazards in Bangladesh. The country experiences multi-type floods (i.e., fluvial, flash, pluvial, and surge floods) every year. However, areas prone to multi-type floods have not yet been assessed on a national scale. Here, we used locally weighted linear regression (LWLR), random subspace (RSS), reduced error pruning tree (REPTree), random forest (RF), and M5P model tree algorithms in a hybrid ensemble to assess multi-type flood probabilities at a national scale in Bangladesh. We used historical flood data (1988-2020), remote sensing images (e.g., MODIS, Landsat 5-8, and Sentinel-1), and topography, hydrogeology, and environmental datasets to train and validate the proposed algorithms. According to the results, the stacking ensemble machine learning LWLR-RF algorithm performed better than the other algorithms in predicting flood probabilities, with R2 = 0.967-0.999, MAE = 0.022-0.117, RMSE = 0.029-0.148, RAE = 4.48-23.38%, and RRSE = 5.8829.69% for the training and testing datasets. Furthermore, true skill statistics (TSS: 0.929-0.967), corrected classified instances (CCI: 96.45-98.35), area under the curve (AUC: 0.983-0.997), and Gini coefficients (0.966-0.994) were computed to validate the constructed (LWLR-RF) multi-type flood probability maps. The maps constructed via the LWLR-RF algorithm revealed that the proportions of different categories of flooding areas in Bangladesh are fluvial flooding 1.50%, 5.71%, 12.66%, and 13.77% of the total land area; flash floods of 4.16%, 8.90%, 11.11%, and 5.07%; pluvial flooding: 5.72%, 3.25%, 5.07%, and 0.90%; and surge flooding, 1.69%, 1.04%, 0.52%, and 8.64% of the total land area, respectively. These percentages represent low, medium, high, and very high probabilities of flooding. The findings can guide future flood risk management and sustainable land-use planning in the study area.

Determination of pesticide residues in dates using UHPLC-QqQ-MS/MS: method development and validation.

A modified, efficient, and sensitive acetate-buffered QuEChERS extraction method was developed for the quantitative study of 16 commonly applied multiclass pesticides on date palm fruit. The date palm fruit samples were rehydrated by adding water during comminution. Samples were extracted with acidified acetonitrile, buffered with acetate salt. To minimize the matrix interferences, clean-up of the rehydrated samples was optimized by comparison with different sorbents (alumina, silica gel, florisil, primary secondary amine (PSA), and chitosan). The method validation parameters were evaluated as per European Union (EU) guidelines (SANTE/12682/2019). For 16 pesticides, % recovery of 69 to 121.8% with an associated precision (RSD ≤ 20%) was achieved at the fortification levels that were 0.5 to 2 times of European Union maximum residue limits (EU-MRLs). The validated method was successfully employed for the analysis of date palm fruit samples (n = 20) collected from various markets. Forty percent (40%) of samples (n = 8) were found to be contaminated with various pesticides. The most frequently detected residues were carbofuran, carbaryl, metalaxyl, tebuconazole, triazophos, and pyriproxyfen. The concentration of all the detected pesticides in real samples was below the EU-MRLs.

National survey of intraoperative mitomycin C use during trabeculectomy surgery in the UK.

PURPOSE: Mitomycin C is a routinely used antimetabolite which effectively limits the scarring process. Conventionally, the intra-operative technique of MMC delivery during trabeculectomy is the direct application of the soaked sponges. The aim of this study is to evaluate the current practice of delivering MMC during trabeculectomy and to see the practices related to a retained MMC swab during trabeculectomy in the UK. METHODS: An electronic questionnaire of 8 questions regarding the intraoperative use of MMC during trabeculectomy surgery was emailed to 69 ophthalmologist members of the UK and Eire glaucoma society (UKEGS) through email in July 2019. RESULTS: 97.2% of the surveyed glaucoma surgeons said they use MMC during trabeculectomy routinely, while 2.9% said they never use MMC for trabeculectomy. In reply to a question 'Have you ever had a retained MMC-soaked sponge/fragment of sponge', 11.76% replied 'yes', 88.24% said 'No'. In response to what technique was employed at the time of retained MMC sponges, all the respondents said they had employed the same technique of 'multiple individual sponges'. 47.1% surgeons said they use multiple individual sponges on being asked what technique they currently employ for delivering MMC during trabeculectomy surgery, while 14.7% surgeons use multiple sponges linked with a 'necklace suture' and 5.9% inject MMC. CONCLUSION: Our survey suggests the intra-operative MMC use in the UK is high and that unintentional MMC-soaked swab retention does occur, albeit rarely. A preference of using the MMC-soaked swabs as a delivery method of MMC is seen in our survey.

Antiviral activity of hexapeptides derived from conserved regions of bacterial proteases against HCV NS3 protease.

The main cause of hepatitis C is hepatitis C virus or HCV and for the cure of hepatitis C, NS3/4A protease has been found an important and emerging target. A number of HCV NS3/4A protease inhibitors have been discovered which have shown subsequent reduction in reducing the viral load leading to this infection however they are still undergoing clinical trials for improvement. Bacterial proteases are of great pharmaceutical importance and have a key role in various biological processes and in life cycle of several pathogens. The current study was planned to explore hexapeptides derived from conserved regions of bacterial proteases for their potential in blocking the NS3 protease activity of HCV which would finally inhibit HCV multiplication. For this, a novel protease gene nprB was isolated from a thermophilic bacterium Streptomyces thermovulgaris and bioinformatics analyses were performed. PCR amplification and sequencing of nprB gene indicated an open reading frame of 178 aa (20191.18 Dalton).The peptide GGVHIN was the top ranked with minimum S-score of -17.21) followed by hexapeptides VDAHAN, GVGREA, GALNES and VHINSS with their S-scores of -14.73, -13.78, -10.72 and -10.70, respectively. A phylogram was also reconstructed to reveal evolutionary relationships of nprB with its various homologs. The provided data will serve as a background to further reveal pharmaceutical and biotechnological importance of this novel protease gene from S. thermovulgaris in future.

Novel N-Acyl-1H-imidazole-1-carbothioamides: Design, Synthesis, Biological and Computational Studies.

The present study reports the convenient synthesis, spectroscopic characterization, bio-assays and computational evaluation of a novel series of N-acyl-1H-imidazole-1-carbothioamides. The screened derivatives displayed excellent antioxidant activity, moderate antibacterial and antifungal potential. The screened derivatives were found to be highly biocompatible against hRBCs. Molecular docking ascertained the mechanism and mode of action towards the molecular target delineating that ligands and complexes were stabilized at the active site by electrostatic and hydrophobic forces in accordance to the corresponding experimental results. Docking simulation provided additional information about the possibilities of inhibitory potential of the compounds against RNA. Computational evaluation predicted that N-acyl-1H-imidazole-1-carbothioamides 5c and 5g can serve as potential surrogates for hit to lead generation and design of novel antioxidant and antibacterial agents.

Intensity-duration-frequency approach for risk assessment of air pollution events.

Intensity-duration-frequency (IDF) curves can serve as useful tools in risk assessment of extreme environmental events. Thus, this study proposes an IDF approach for evaluating the risk of expected occurrences of extreme air pollution as measured by an air pollution index (API). Hourly data of Klang city in Malaysia from 1997 to 2016 are analyzed. For each year, a block maxima size is determined based on four different monsoon seasons. Generalized extreme value (GEV) distribution is used as a model to represent the probabilistic behavior of maximum intensity of the API, which is derived from each block. Based on the GEV model, the IDF curves are developed to estimate the extreme pollution intensities that correspond to various duration hours and return periods. Considering the IDF curves, we found that for any duration hour, the magnitude of pollution intensity tends to be high in parallel with increasing return periods. In fact, a high-intensity pollution event that poses a high risk of affecting the environment is less frequent than low-intensity pollution. In conclusion, the IDF curves provide a good basis for decision makers to assess the expected risk of extreme pollution events in the future.

Modeling the transition behaviors of PM10 pollution index.

Modeling and evaluating the behavior of particulate matter (PM10) is an important step in obtaining valuable information that can serve as a basis for environmental risk management, planning, and controlling the adverse effects of air pollution. This study proposes the use of a Markov chain model as an alternative approach for deriving relevant insights and understanding of PM10 data. Using first- and higher-order Markov chains, we analyzed daily PM10 index data for the city of Klang, Malaysia and found the Markov chain model to fit the PM10 data well. Based on the fitted model, we comprehensively describe the stochastic behaviors in the PM10 index based on the properties of the Markov chain, including its states classification, ergodic properties, long-term behaviors, and mean return times. Overall, this study concludes that the Markov chain model provides a good alternative technique for obtaining valuable information from different perspectives for the analysis of PM10 data.

Isolation and characterization of nprB, a novel protease from Streptomyces thermovulgaris.

Bacterial proteases are of great pharmaceutical importance and have a key role in various biological processes and in life cycle of several pathogens. New technology used for rational protein engineering as well improved delivery options will expand the potential pharmaceutical applications of proteases. The catalytic proteases belong to metalloproteases (EC.3.4.24) that comprise thermo lysine. The metalloproteases and their homologs have many important biotechnological and therapeutic applications. In the present study, a novel protease gene nprB was isolated from a thermophilic bacterium Streptomyces thermovulgaris and bioinformatics analyses were performed. PCR amplification and sequencing of nprB gene indicated an open reading frame of 178 aa (20191.18 Dalton). Based on protein sequence homology as well as conserved motifs and PTF domain the protein is characterized as a thermo lysine-like protease and is a member of M4 family of metalloproteases. Different bioinformatics tools such as ProtParam, SOPMA, signalP4.1 and ProDom from the ExPAsy server were used for structural and functional analyses. A phylogram was also reconstructed to reveal evolutionary relationships of nprB with its various homologs. The provided data will serve as a background to further reveal pharmaceutical and biotechnological importance of this novel protease gene from S. thermovulgaris in future.

Comparative proteomic analysis of high- and low-fertile buffalo bull spermatozoa for identification of fertility-associated proteins.

The present study identified few potential proteins in the spermatozoa of buffalo bulls that can be used as an aid in fertility determination through comparative proteomics. The sperm proteome of high-fertile buffalo bulls was compared with that of low-fertile buffalo bulls using two-dimensional difference gel electrophoresis (2D-DIGE), and the differentially expressed proteins were identified through mass spectrometric method. The protein interaction network and the functional bioinformatics analysis of differentially expressed proteins were also carried out. In the spermatozoa of high-fertile bulls, 10 proteins were found overexpressed and 15 proteins were underexpressed at the level of twofold or more (p ≤ 0.05). The proteins overexpressed in high-fertile spermatozoa were PDZD8, GTF2F2, ZNF397, KIZ, LOH12CR1, ACRBP, PRSS37, CYP11B2, F13A1 and SPO11, whereas those overexpressed in low-fertile spermatozoa were MT1A, ATP5F1, CS, TCRB, PRODH2, HARS, IDH3A, SRPK3, Uncharacterized protein C9orf9 homolog isoform X4, TUBB2B, GPR4, PMP2, CTSL1, TPPP2 and EGFL6. The differential expression ranged from 2.0- to 6.1-fold between the two groups, where CYP11B2 was high abundant in high-fertile spermatozoa and MT1A was highly abundant in low-fertile spermatozoa. Most of the proteins overexpressed in low-fertile spermatozoa were related to energy metabolism and capacitation factors, pointing out the possible role of pre-mature capacitation and cryo-damages in reducing the fertility of cryopreserved buffalo spermatozoa.