Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 µM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 µM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 µM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Proteomic and molecular analyses to understand the promotive effect of safranal on soybean growth under salt stress.

Safranal is a free radical scavenger and useful as an antioxidant molecule; however, its promotive role in soybean is not explored. Salt stress decreased soybean growth and safranal improved it even if under salt stress. To study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive­oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking. SIGNIFICANCE: To study the positive mechanism of safranal on soybean growth, a proteomic approach was used. According to functional categorization, oppositely changed proteins were further confirmed using biochemical techniques. Actin and calcium-dependent protein kinase decreased in soybean root and hypocotyl, respectively, under salt stress and increased with safranal application. Xyloglucan endotransglucosylase/ hydrolase increased in soybean root under salt stress but decreased with safranal application. Peroxidase increased under salt stress and further enhanced by safranal application in soybean root. Actin, RuvB-like helicase, and protein kinase domain-containing protein were upregulated under salt stress and further enhanced by safranal application under salt stress. Dynamin GTPase was downregulated under salt stress but recovered with safranal application under salt stress. Glutathione peroxidase and PfkB domain-containing protein were upregulated by safranal application under salt stress in soybean root. These results suggest that safranal improves soybean growth through the regulation of cell wall and nuclear proteins along with reactive­oxygen species scavenging system. Furthermore, it might promote salt-stress tolerance through the regulation of membrane proteins involved in endocytosis and post-Golgi trafficking.

Screening and validation of salt-stress responsive cg-SSR markers in wheat (Triticum aestivum L.) germplasm of Pakistan.

BACKGROUND: Soil salinity has been affecting wheat production worldwide over past few decades. Evaluation of wheat genotypes for salinity tolerance at germination and vegetative growth level is crucial. Marker assisted selection is a technique used extensively for choosing salt-tolerant genotypes from breeding populations to introduce novel genes. METHODS AND MATERIALS: The current study's main goal was to discover salt-stress resistant genes; genetic divergence and genome-wide connection by using recently designed candidate gene-based simple-sequence-repeat markers (cg-SSRs). The phenotypic connection of morphological features during the germination growth stage i.e., germination period, root length/weight and shoot length/weight, and vegetative growth stages i.e., root length/weight and shoot length/weight were tested in a group of 50 wheat genotypes. Significant difference was observed in germination rate, root length and weight among control and saline treatments. CONCLUSION: Total 30 SSR markers were utilized to test salinity resistance genes in wheat genotypes. Three (10%) of which were monomorphic, one (3.34%) showed no result, and the other 26 (86%) were polymorphic. Using 30 polymorphic markers discovered total 37 alleles. The polymorphic information content (PIC), quantifies each SSR locus capacity to discriminate between wheat, varied from 0.00 to 0.38 with an average of 0.19. Association analysis revealed that 26 primers were associated with morphological features, 03 with root length and the remaining 23 with germination. Utilizing morphological data, stress tolerance index (STI) was designed concluding that Auqab-2000, Margala-99 and Ufaq showed better resistance against salinity among other wheat genotypes. Cluster analysis demonstrated that wheat genotypes have vast genetic variability.

Crop Proteomics under Abiotic Stress: From Data to Insights.

Food security is a major challenge in the present world due to erratic weather and climatic changes. Environmental stress negatively affects plant growth and development which leads to reduced crop yields. Technological advancements have caused remarkable improvements in crop-breeding programs. Proteins have an indispensable role in developing stress resilience and tolerance in crops. Genomic and biotechnological advancements have made the process of crop improvement more accurate and targeted. Proteomic studies provide the information required for such targeted approaches. The crosstalk among cellular components is being analyzed by subcellular proteomics. Additionally, the functional diversity of proteins is being unraveled by post-translational modifications during abiotic stress. The exploration of precise cellular responses and the networking among different cellular organelles help in the prediction of signaling pathways and protein-protein interactions. High-throughput mass-spectrometry-based protein studies are now possible due to incremental advancements in mass-spectrometry techniques, sample protocols, and bioinformatic tools as well as the increasing availability of plant genome sequence information for multiple species. In this review, the key role of proteomic analysis in identifying the abiotic-stress-responsive mechanisms in various crops was summarized. The development and availability of advanced computational tools were discussed in detail. The highly variable protein responses among different crops have provided a wide avenue for molecular-marker-assisted genetic buildup studies to develop smart, high-yielding, and stress-tolerant varieties to cope with food-security challenges.

Proteomic Approaches to Uncover Salt Stress Response Mechanisms in Crops.

Salt stress is an unfavorable outcome of global climate change, adversely affecting crop growth and yield. It is the second-biggest abiotic factor damaging the morphological, physio-biochemical, and molecular processes during seed germination and plant development. Salt responses include modulation of hormonal biosynthesis, ionic homeostasis, the antioxidant defense system, and osmoprotectants to mitigate salt stress. Plants trigger salt-responsive genes, proteins, and metabolites to cope with the damaging effects of a high salt concentration. Enhancing salt tolerance among crop plants is direly needed for sustainable global agriculture. Novel protein markers, which are used for crop improvement against salt stress, are identified using proteomic techniques. As compared to single-technique approaches, the integration of genomic tools and exogenously applied chemicals offers great potential in addressing salt-stress-induced challenges. The interplay of salt-responsive proteins and genes is the missing key of salt tolerance. The development of salt-tolerant crop varieties can be achieved by integrated approaches encompassing proteomics, metabolomics, genomics, and genome-editing tools. In this review, the current information about the morphological, physiological, and molecular mechanisms of salt response/tolerance in crops is summarized. The significance of proteomic approaches to improve salt tolerance in various crops is highlighted, and an integrated omics approach to achieve global food security is discussed. Novel proteins that respond to salt stress are potential candidates for future breeding of salt tolerance.

Estimation of the burden of bacteriologically positive Tuberculosis among Adults in Kashmir: A baseline for future surveys in the Valley.

BACKGROUND: India has the highest burden of both Tuberculosis and MDR-Tuberculosis based on estimates reported in the Global Tuberculosis Report 2016. The estimates have been revised upwards based on the newer evidence, and the current study was done to estimate the prevalence of bacteriologically positive pulmonary Tuberculosis among the adult population and to provide baseline information for future measurements of Tuberculosis burden and trends. METHODS: A cluster-based sampling design was adopted in 10 districts of Kashmir valley in India. Assuming a prevalence of 217 per lakh population, a design effect of 2.5, a relative precision of 0.25% and the expected participation rate of 80%, a sample size of 49,716 was achieved. A total of 67 clusters were identified where each cluster had 750 subjects aged ≥15 years, and eligible individuals were questioned for pulmonary symptoms suggestive of Tuberculosis. RESULTS: Of the total 42,805 that were interviewed, 3.85% had pulmonary Tuberculosis symptoms. A total of 1539 sputum samples were collected from symptomatic and 1351 chest X- rays were done. Cartridge based nucleic acid amplification test (CBNAAT) tests were done on individuals with suspicious X-ray findings. The prevalence of bacteriologically positive pulmonary Tuberculosis was found to be 147 per 100,000 population. Females are affected more than males, and the age of female Tuberculosis patients is less than that of males. CONCLUSION: The study is the first survey of its kind providing a baseline for further research in the state. CBNAAT is going to be game-changer which surmounts the drawbacks of sputum smear microscopy.

Chemical profiling and bioactivities on the leaf extracts of Vitex neugundo.

The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and anti proliferative study of leaf extracts of V. neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier from the leaf extract of V. neugundo. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.

Chemical profiling, in vitro and in vivo bioactivities of leaf extracts of Vitex neugundo.

The Vitex negeundo is a widely used medicinal plant which has not been fully investigated in the past. We assessed the in vivo hepatoprotective and in vitro antioxidant, antibacterial, cytotoxicity and antiproliferative study of leaf extracts of V. Neugundo. The chemically profiled using HPLC, three flavonoids were quantified and GC-MS analysis revealed the presence of two new compounds those were not reported earlier. The animal study was conducted on mice treated with CCl4 using methanolic and chloroform extracts (100, 200 and 300 mg/kg b.w), with silymarin as a positive control. Hepatoprotective effects were determined by analyzing blood for liver marker enzymes, direct bilirubins and hematological parameters (RBC, WBC and platelets). The methanolic extract (300 mg/kg b.w) has shown the stronger hepatoprotective effects against abnormalities produced by CCl4. The in vivo hepatoprotective effects correlated well with the in vitro antioxidant, cytotoxicity and antiproliferative activities and with high levels of flavonoids and other organic compounds analyzed from plant extracts. The leaf extracts of this plant could be good candidates for lead compound required for the development of antioxidant/anticancer drugs.

Pharmacological and phytochemical analysis of Bergenia ciliata leaf and rhizome extracts.

Antibacterial, antifungal, antioxidant, cytotoxic, and anti-haemolytic activity of various rhizome extracts of Bergenia ciliata were evaluated in this study. The results showed inhibition of the growth of all selected bacterial and fungal strains in comparison with standard antibiotics. The antioxidant activities of Bergenia ciliata extracts were evaluated against DPPH, H2O2, ABTS, total antioxidant capacity and reducing power assays. The order of antioxidant activity of various extracts were methanol> ethanol>n-hexane> aqueous>chloroform. The cytotoxicity ( brine shrimp assay) and anti-haemolytic activities of plant extracts were also promising and varies in dose depended manner. The phytochemical analysis of rhizome extracts of Bergenia ciliata revealed presence of various secondary metabolites which might be responsible for the antimicrobial, antioxidant, cytotoxic and anti-haemolytic activities.

Synthesis, characterization and evaluation of antibacterial activity of copper oxide nanoparticles against clinical strains of Staphylococcus aureus.

Bacterial resistance is spreading globally due to excessive use of antibiotics, making it one of our times biggest challenges. To address this issue present study was conducted to evaluate the antibacterial activity of copper oxide nanoparticles against methicillin-resistant S. aureus (MRSA). Copper oxide nanoparticles were synthesized by chemical precipitation method and were characterized by UV-Visible, FT-IR spectroscopy, X-ray diffraction (XRD) and Scanning Electron Microscopy. These nanoparticles of 27nm were assessed for antibacterial activity using disc diffusion method. Our results showed superb inhibitory effects of CuO nanoparticles with increase in concentration and complete inhibition was recorded against tested strains of S. aureus at 100µl/ml and 125µl/ml concentration. The study concludes that the drugs which do not show any inhibitory effects against resistant bugs could be augmented with CuO nanoparticles to achieve the treatment goal.

Isolation and Molecular Characterization of a Model Antagonistic Pseudomonas aeruginosa Divulging In Vitro Plant Growth Promoting Characteristics.

The use of microbial technologies in agriculture is currently expanding quite rapidly with the identification of new bacterial strains, which are more effective in promoting plant growth. In the present study 18 strains of Pseudomonas were isolated from soil sample of Balochistan coastline. Among isolated Pseudomonas strains four designated as SP19, SP22, PS24, and SP25 exhibited biocontrol activities against phytopathogenic fungi, that is, Rhizopus microsporus, Fusarium oxysporum, Aspergillus niger, Alternaria alternata, and Penicillium digitatum; PS24 identified as Pseudomonas aeruginosa by 16srRNA gene bank accession number EU081518 was selected on the basis of its antifungal activity to explore its potential as plant growth promotion. PS24 showed multiple plant growth promoting attributes such as phosphate solubilization activity, indole acetic acid (IAA), siderophore, and HCN production. In order to determine the basis for antifungal properties, antibiotics were extracted from King B broth of PS24 and analyzed by TLC. Pyrrolnitrin antibiotic was detected in the culture of strain PS24. PS24 exhibited antifungal activities found to be positive for hydrogen cyanide synthase Hcn BC gene. Sequencing of gene of Hcn BC gene of strain PS24 revealed 99% homology with the Pseudomonas aeruginosa strain PA01. The sequence of PS24 had been submitted in gene bank accession number KR605499. Ps. aeruginosa PS24 with its multifunctional biocontrol possessions can be used to bioprotect the crop plants from phytopathogens.

Isolation and characterization of antibiotic producing bacterial strains from red soil of Himalayan region of Pakistan.

The emergence of multi drug resistant microbial pathogens has become a global health challenge and set a dire requirement of searching new effective antimicrobials. Soil is an ultimate reservoir of biologically active micro flora, which harbors trillions of microbial strains producing compounds of commercial interest. Hence aim of the present study was an attempt to isolate and identify the antibiotic producing microbial strains from the red soil of Himalayan an unexplored region of Pakistan. In this study from 10 different soil samples only one bacterial strain was isolated capable of antimicrobial activity. Strain was identified by biochemical characteristics and final identification was done by API 20 NE kit which showed 99% homology with P. aeruginosa. Hence the strain was identified as P. aeruginosa S2. Antibacterial and antifungal activity of the P. aeruginosa S2 showed that Staphylococcus aureus was extremely sensitive to it with a zone of inhibition of 42mm. Staphylococcus epidermidis, Enterobacter aerogenes, Aspergillus fumigatus and Candida albicans were also inhibited by the isolated strain. Effect of Glycerol, Copper sulphate (CuSo4), Sodium sulphate (Na2SO4) and Glycerol on antibiotic production was also evaluated by supplementing growth media with these chemicals. Pseudomonas aeruginosa was grown in bulk quantity using solid state fermentation and crude extract was prepared using organic solvents and subjected to silica gel column chromatography for purification of active compound. Purified compound showed antibacterial against human pathogens. The unexplored Kashmir Himalayas are of great significance because of its richness in biodiversity and need to be explored for isolation and characterization of native microbes for biologically active secondary metabolites. This untouched region may be considered as hub of new antimicrobials and may have applications in natural product-based drug discovery.

Biosynthesis, characterization and antimicrobial action of silver nanoparticles from root bark extract of Berberislycium Royle.

Various biological methods are being recognized for the fabrication of silver nanoparticles, which are used in several fields. The phytosynthesis of nanoparticles came out as a cost effective and enviro-friendly approach. When root bark extract of Berberis lycium was treated with silver ions, they reduced to silver nanoparticles, which were spherical, crystalline, size ranged from 10-100nm and capped by biomolecules. Synthesized silver nanoparticles were characterized by UV-visible spectroscopy, Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDX), Transmission Electron Microscopy (TEM), X-Ray Diffraction (XRD) and Fourier Transform Infra Red Spectroscopy (FTIR). The plant mediated synthesized silver nanoparticles showed pronounced antimicrobial activities against both Gram negative bacteria (Escherichia coli, Klebseilla pneumoniae, Pseudomonas aeruginosa) and Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis). The plant mediated process proved to be non-toxic and low cost contender as reducing agent for synthesizing stable silver nanoparticles.

Proteomics analysis of sensitive and tolerant barley genotypes under drought stress.

Drought is a severe environmental constraint to plant productivity and an important factor limiting barley yield. To investigate the initial response of barley to drought stress, changes in protein profile were analyzed using a proteomics technique. Three-day-old barley seedlings of sensitive genotype 004186 and tolerant genotype 004223 were given two treatments, one with 20 % polyethylene glycol and the second with drought induced by withholding water. After 3 days of treatments, proteins were extracted from shoots and separated by 2-dimensional polyacrylamide gel electrophoresis. Metabolism related proteins were decreased in the sensitive genotype under drought; however, they were increased in the tolerant genotype. Photosynthetic related proteins were decreased and increased among the three sensitive and three tolerant genotypes, respectively. In addition, amino acid synthesis and degradation related proteins were increased and decreased among the three tolerant genotypes. These results suggest that chloroplastic metabolism and energy related proteins might play a significant role in the adaptation process of barley seedlings under drought stress.

Characterization of ascorbate peroxidase in soybean under flooding and drought stresses.

Flooding and drought are the two different forms of water stress that adversely affect the growth and development of soybean plant in particular at early stage. Ascorbate peroxidase (APX) is a known antioxidant enzyme that plays key role in abiotic stresses. To investigate the changes in APX in soybean under drought and flooding stresses, western blotting, enzyme activity assay and biophoton emission techniques were used. Flooding stress was imposed by adding excess amount of water in the sand and drought by withholding water supply. Under flooding stress, a decrease in APX was detected with time. Completely opposite trend was evident in hypocotyl and root of plants exposed to drought. Western blotting and APX activity results are complementary to each other. Biophoton emissions further confirmed the increasing and decreasing trend of APX under drought and flooding stress, respectively.

Assessment of reproductive parameters in female Dwarf goat (Capra hircus) on the basis of progesterone profiles.

A study was undertaken to look into the reproductive performance of female Dwarf goats reared under traditional conditions at NIAB Farm, Faisalabad, Pakistan. The serum progesterone profile was used to monitor various reproductive parameters (length of postpartum period, resumption of cyclicity, gestation period, prepartum period, parturition) in two lots of goats. Litter size, birth weight of kids and kidding interval were also observed. Most of the animals conceived within 15-59 days of postpartum period. All the does conceived at first or second estrus. During gestation period, higher levels of progesterone were maintained with wide variations falling in the range of 3-13 ng ml(-1). However, a few days before parturition a decline was noticed at 6+/-0.9 days and it reached to the basal level of 0.1 ng ml(-1) after the completion of parturition process. The length of gestation period was found to be 145.8+/-5 days in the first lot and 145.2+/-4 days in the second lot. A very short kidding interval (203.7+/-46 days) and considerably bigger litter size (1.8+/-0.8) was observed. All the parturitions were normal and a considerable weight gain (8.2+/-0.3 kg) of mothers was recorded during pregnancy. The initial birth weight of kids was averaged as 2.1+/-0.5 kg in the first and 1.6+/-0.2 kg in the second lot. It was concluded that Dwarf goat has short gestation length, postpartum period and kidding interval along with multiple births being common. Due to these factors, its reproductive efficiency can be exploited for efficient goat meat production.

Manipulation of estrous cycle in dwarf goat (Capra hircus) using estrumate under different management conditions.

A study was conducted to elucidate the effectiveness of exogenous estrumate (prostaglandin F(2alpha)) treatment as a synchronizing agent for Dwarf goats and to establish the progesterone levels at different reproductive stages under two different environmental and nutritional conditions. Female Dwarf goats of various ages were raised, 10 each at two sites, viz. the Bio-Saline Research Substation (BSRS), Lahore and at the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad. The animals at the NIAB farm were reared on non-saline soil under normal grazing conditions, while the other animal lot was raised on salt-affected lands at the BSRS, Lahore and was fed on non-conventional fodders produced by salt-affected lands. The animals received two intramuscular doses (0.5 ml each) of estrumate (125 microg/ml) at 10 days interval. Nineteen of the 20 animals exhibited estrus after 56-72 h of the second injection of estrumate. The mean progesterone concentration in the NIAB and BSRS lots was 2.8 and 2.4 ng/ml, respectively, at the time of second injection and declined to the basal level (estrus) within 48 h. A gradual increase in the progesterone level occurred during the metestrus, reaching maximum during the luteal phase when it was 6.3 and 6.7 ng/ml in NIAB-lot and BSRS-lot, respectively. During the proestrous phase, the progesterone level decreased to the basal level (0.1 ng/ml) at the onset of the next estrus after 22 days. The length of the induced or natural estrous cycle (20+/-1 days) was not affected by the estrumate treatment, nor by the environmental and nutritional conditions. Breeding was allowed after the natural estrous cycle at the onset of third estrus and a high fertility rate (95%) was achieved. The progesterone concentrations remained at higher level (4.5-9.4 ng/ml) throughout the gestation period, declined gradually in the prepartum period, and dropped to the basal level at the completion of parturition. The results suggested that estrumate is an efficient synchronizing agent for the Dwarf goats kept under different environmental-nutritive conditions and that the progesterone profile is a useful indicator to assess the reproductive status of the goats.