Coenzyme Q10 improves the quality and in vitro fertility of post-thawed buffalo (Bubalus bubalis) semen via its antioxidative effect.

This study aimed to determine the effects of Coenzyme Q10 (CoQ10) in the freezing medium on functional and oxidative stress parameters and in vitro fertilization (IVF) rate of buffalo sperm. Collected samples were relocated to the laboratory for initial evaluation, gentle dilution in extenders, cooling (4°C, 2 h), equilibration (4°C, 4 h), packaging (straws, 0.5 mL), programmable freezing, and thawing (37°C, 30 s). Statistical analysis depicted that adding CoQ10 (100 µM) in a freezing medium caused a significant augmentation in total motility (%), average path, and straight-line velocities (µm/sec) of buffalo sperm than control. Adding CoQ10 (100 µM) improved sperm progressive motility, rapid velocity, and functional parameters (%) compared to the control and 10 µM of CoQ10. Moreover, CoQ10 in a freezing medium caused a significant augmentation in seminal plasma catalase (U/mL) and glutathione reductase (GSH; nmol/109 ) at 100 µM than control and other treatments. CoQ10 inclusion (100 µM) ameliorates seminal plasma superoxide dismutase (U/mL), glutathione-S-transferase (GST; nmol/mL/min) fructose (µg/mL), and ATP (nmol/million) than control. Furthermore, CoQ10 at 100 µM improved seminal plasma glutathione peroxidase (µM) levels than control, 10 µM, and 20 µM. Lastly, hydrogen peroxide (H2 O2; nM) production was significantly lower at 100 µM than at control and 10 µM. CoQ10 (100 µM) caused a significant augmentation in the un-capacitated pattern followed by a reduction in the capacitated pattern, and apoptosis-like changes (%) than control, and other treatments, whereas viability was increased than control and other treatments. CoQ10 (100 µM) significantly improved the IVF rate in comparison with control, CoQ10 at 10 µM, and 20 µM groups. In conclusion, the addition of CoQ10 (100 µM) in the freezing medium can improve the quality and in vitro fertility of post-thawed buffalo semen via its antioxidative effect. Further studies are needed to evaluate the effect of CoQ10 on the in vivo fertility of buffalo bull semen.

The ameliorating effects of crocetin on frozen-thawed quality, and fertility via attenuating oxidative status of bubaline spermatozoa.

Current study was conducted to appraise the cryoprotective influence of crocetin on quality, oxidative status, and fertility potential of bubaline spermatozoa. Collected semen from four bulls was diluted in five aliquots with (10 µM, 5 µM, 2 µM, 1 µM, and control [0 µM] supplementation of crocetin). After gentle dilution (37 °C), cooling (4 °C, in 2 h), equilibration (4 °C, for 4 h) and packaging of samples was done in straws (polyvinyl French, 0.5 ml), and then frozen (programmable cell freezer). This study established that crocetin supplementation significantly (p < 0.05) improves CASA (Computer Assisted Sperm motion Analyzer) total motility (%), rapid velocity (%), average-path, and curved-line velocities (µm/sec, 10 µM vs. control), and progressive motility (%), straight-line velocity (µm/sec), total antioxidant capacity (TAC, µMol/l), ATP concentrations (nmol/million), and fertility potential (%) (10 µM vs. control, and 1 µM), and mitochondrial potential (%) of buffalo spermatozoa (5, and 10 µM vs. control). Crocetin supplementation significantly (p < 0.05) alleviates DNA fragmentation, seminal plasma ROS (104 RLU/20/25 million, RLU = Relative light unit) levels, and lipid peroxidation (LPO, µMol/ml) in buffalo spermatozoa (10 µM vs. control). In a nutshell, crocetin supplementation improves post-thaw quality by means of motility parameters, motion kinematics, TAC, and ATP concentrations, and fertility potential, and abolished DNA fragmentation parameters, seminal plasma ROS, and LPO concentrations of buffalo spermatozoa. The exact mechanism by which crocetin acts are not fully elucidated; however, it is probable to speculate that the reduction in ROS, and LPO recorded in this study may be related to scavenging ability of this antioxidant during cryopreservation.

Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters, and fertility potential, and alleviates oxidative stress parameters of buffalo spermatozoa.

The disparity between the endogenous antioxidants concentration and free radicals in spermatozoa results in reactive oxygen species (ROS) generation. In this prospect, epigallocatechin-3-gallate (EGCG) preserves vigorous antioxidant features. Current study explored the influence of EGCG in a cryo-diluent media on microscopic parameters, oxidative stress parameters, and fertility potential of buffalo spermatozoa during cryopreservation. Concisely, collected semen from three donor bulls for four times were then evaluated for volume, motility, concentrations and then dilution in a cryo-diluent media with different concentrations of EGCG (EGCG-0 = control; EGCG-50 = 50 µM, EGCG-100 = 100 µM, EGCG-200 = 200 µM, and EGCG-300 = 300 µM) at 37 °C, cooled to 4 °C in 2 h, equilibrated for 4 h at 4 °C, and cryopreserved. At post-thawing, Computer-Assisted Sperm motion Analysis motilities (total and progressive, %) and rapid velocity (%), plasma membrane functionality, supravital plasma membrane integrity, and mitochondrial potential (%) were found higher (P < 0.05) in EGCG-200, and EGCG-300 than control, whereas average-path, straight-line, and curved-linear velocities (µm/sec), and acrosome integrity (%) were recorded higher in EGCG-300 than control. Further, comet length (µm), and tail length (µm), LPO (lipid peroxidation, µM/mL), and apoptosis-like changes (%) in spermatozoa were significantly decreased in EGCG-300 than control. Seminal plasma antioxidant enzymes activities (glutathione peroxidase, U/mL, and superoxide dismutase, U/mL) were increased with EGCG-300 than control. Moreover, EGCG-300 addition in a cryo-diluent media improves the fertility potential (%) of buffalo spermatozoa. In a nutshell, the inclusion of EGCG-300 in a cryo-diluent media enhances post-thaw microscopic parameters, and fertility potential, whereas decreases oxidative stress parameters in buffalo spermatozoa.

Supplementation of l-tryptophan (an aromatic amino acid) in tris citric acid extender enhances post-thaw progressive motility, plasmalemma, mitochondrial membrane potential, acrosome, and DNA integrities, and in vivo fertility rate of buffalo (Bubalus bubalis) bull spermatozoa.

The aromatic amino acid l-tryptophan is an essential and versatile molecule, acts by transferring an electron to free radicals and protects the plasma membrane from injuries. The aim of the present study was to investigate the effects of l-tryptophan in extender on semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during cryopreservation. Two ejaculates were collected from each bull (n = 2 ejaculates and n = 4 bulls) with artificial vagina at 42 °C followed by initial evaluation for volume, motility, concentrations and were diluted in five extenders (C = lacking l-tryptophan, D1 = 25 µ M l-tryptophan, D2 = 50 µ M l-tryptophan, D3 = 75 µ M l-tryptophan, and D4 = 100 µ M l-tryptophan) respectively, and cryopreserved. The experiment was repeated four times (n = 4 replicates). At post-dilution, sperm plasma membrane integrity (PMI, %), supravital plasma membrane integrity (SVPMI, %), hypo-resistivity (HR, %) and acrosome integrity (ACR-I, %) were significantly higher (P < 0.05) in extender supplemented with D4 than control. At post-thawing, progressive motility (PM, %), PMI, SVPMI, HR, ACR-I, and DNA-I of buffalo bull spermatozoa were significantly higher in D4 than control. Sperm in vitro longevity (%) assessed in terms of PM, SVPMI, and ACR-1 were significantly higher in D4 than control. Sperm mitochondrial membrane potential (%) was higher in treated groups than the control. The in vivo fertility rate was significantly higher in D4 than control (60.17% vs. 44.17%, P < 0.05). It is concluded that the supplementation of l-tryptophan in tris citric acid extender improves semen quality parameters, in vitro longevity and in vivo fertility rate of buffalo spermatozoa during freezing and thawing process.

Sulforaphane inclusion in a freezing medium augments post-thaw motility, functional and biochemical features, and fertility potential of buffalo (Bubalus bubalis) spermatozoa.

Sulforaphane is a natural and highly effective antioxidant safeguarding the reproductive system, and alleviate oxidative stress. This study was designed in order to elaborate L-sulforaphane effect on semen quality, biochemical parameters, and fertility of buffalo (Bubalus bubalis) spermatozoa. Semen was collected from five buffalo bull with artificial vagina (42 °C) three times and evaluated for volume, consistency (color), motility, and sperm concentration. After critical examination, semen was diluted (50 × 106 spermatozoa per ml, 37 °C) in extenders with (2 µM, 5 µM, 10 µM, and, 20 µM) or without (control) sulforaphane, cooled (from 37 to 4 °C), equilibrated (4 °C), filled (straws, 4 °C), and cryopreserved (LN2, -196 °C). Data analysis exhibited that sulforaphane addition in extender augments total motility (%, 10 µM, and 20 µM than control), progressive motility (%), and rapid velocity (%, 20 µM than control), and velocity parameters (average path velocity, µm/s, straight line velocity, µm/s and curved linear velocity, µm/s, 20 µM than control, and 2 µM). Moreover, sulforaphane augments functional features (membrane functionality, mitochondrial potential, and acrosome integrity) of buffalo sperm (20 µM than control). Sulforaphane preserves biochemical features of seminal plasma of buffalo i.e., Calcium (µM), and total antioxidant capacity (µM/L), followed by reduction in lactate dehydrogenase (IU/L), reactive oxygen species (104 RLU/20 min/ 25 million), and lipid peroxidation (µM/ml) in 20 µM than control. Lastly, sulforaphane augments fertility rate of buffalo sperm at 20 µM than control, and 2 µM. Conclusively the existing study revealed that adding L-sulforaphane (20 µM) in a freezing medium augments motilities, kinematics, functional parameters, and fertility rate of buffalo spermatozoa. Correspondingly, sperm favorable biochemical features were also augmented with sulforaphane followed by reduction in oxidative stress parameters. Further studies are highly recommended to define the particular mechanism of action of sulforaphane in augmenting buffalo post-thawed semen quality, and in vitro fertility potential.

Relaxin (a regulatory-peptide) enhances cryotolerance, fertility potential and plasma reproductive hormones of Nili Ravi buffalo (Bubalus bubalis) during low breeding season.

This study investigated the beneficial effects of relaxin on cryotolerance of buffalo spermatozoa and reproductive hormones during low breeding season. Collected semen was diluted in five aliquots with relaxin addition (0.25 mg/mL, 0.50 mg/mL, 0.75 mg/mL, 1 mg/mL, and control). After gentle dilution (37°C), cooling (4°C, 2 h), equilibration (4°C, 4 h), and packaging (straws, polyvinyl French, 0.5 mL), frozen (cell freezer), and thawed (37°C, 30 s) for analysis. Blood samples were collected at different time intervals i.e., -60, -30 and 0 min (pre-dose) and 30, 60, 90, 120 and 150 min (post-dose) from a jugular vein. This study manifest that adding relaxin (1 mg/ mL) in freezing medium ameliorates sperm motility, functionality (%), and seminal plasma total antioxidant capacity (TAC, µM/L) than control during low breeding season. Furthermore, we found that relaxin supplementation at 1 mg/mL significantly improves seminal plasma ATP concentrations (nmol/million) than control, 0.25 mg/mL, and 0.50 mg/mL, and fertility (control, and 0.75 mg/mL). Further, relaxin injection significantly improves plasma T, LH and IGF-1 levels (150 and 120 min vs. -60, and - 30), and FSH, KP, and GnRH concentrations (150 min vs. -60), during low breeding season. Taken together, this study revealed that relaxin ameliorates motility, functionality, and fertility of buffalo spermatozoa. Moreover, relaxin injection (1 mg/mL) improves essential reproductive hormones levels in buffalo signifying its importance in the field of reproductive physiology. Further studies are required to determine the exact mechanism of action of relaxin in enhancing semen quality, fertility and reproductive hormones.

Pharmacological Progress of Mitophagy Regulation.

With the advancement in novel drug discovery, biologically active compounds are considered pharmacological tools to understand complex biological mechanisms and the identification of potent therapeutic agents. Mitochondria boast a central role in different integral biological processes and mitochondrial dysfunction is associated with multiple pathologies. It is, therefore, prudent to target mitochondrial quality control mechanisms by using pharmacological approaches. However, there is a scarcity of biologically active molecules, which can interact with mitochondria directly. Currently, the chemical compounds used to induce mitophagy include oligomycin and antimycin A for impaired respiration and acute dissipation of mitochondrial membrane potential by using CCCP/FCCP, the mitochondrial uncouplers. These chemical probes alter the homeostasis of the mitochondria and limit our understanding of the energy regulatory mechanisms. Efforts are underway to find molecules that can bring about selective removal of defective mitochondria without compromising normal mitochondrial respiration. In this report, we have tried to summarize and status of the recently reported modulators of mitophagy.

Polymorphs of Substituted p-Toluenesulfonanilide: Synthesis, Single-Crystal Analysis, Hirshfeld Surface Exploration, and Theoretical Investigation.

Polymorphism is an exciting feature of chemical systems where a compound can exist in different crystal forms. The present investigation is focused on the two polymorphic forms, triclinic (MSBT) and monoclinic (MSBM), of ethyl 3-iodo-4-((4-methylphenyl)sulfonamido)benzoate prepared from ethyl 4-amino-3-iodobenzoate. The prepared polymorphs were unambiguously confirmed by single-crystal X-ray diffraction (SC-XRD) analysis. According to the SC-XRD results, the molecular configurations of both structures are stabilized by intramolecular N-H···I and C-H···O bonding. The crystal packing of MSBT is different as compared to the crystal packing of MSBM because MSBT is crystallized in the triclinic crystal system with the space group P1̅, whereas MSBM is crystallized in the monoclinic crystal system with the space group P21/c. The molecules of MSBT are interlinked in the form of dimers through N-H···O bonding to form R22(8) loops, while the MSBM molecules are connected with each other in the form of an infinite chain through C-H···O bonding. The crystal packing of both compounds is further stabilized by off-set π···π stacking interactions between phenyl rings, which is found stronger in MSBM as compared to in MSBT. Moreover, Hirshfeld surface exploration of the polymorphs was carried out, and the results were compared with the closely related literature structure. Accordingly, the supramolecular assembly of these polymorphs is mainly stabilized by noncovalent interactions or intermolecular interactions. Furthermore, a density functional theory (DFT) study was also carried out, which provided good support for the SC-XRD and Hirshfeld studies, suggesting the formation of both intramolecular and intermolecular interactions for both compounds.

Improving the quality and in vitro fertilization rate of frozen-thawed semen of buffalo (Bubalus bubalis) bulls with the inclusion of vitamin B12 in the cryopreservation medium.

This study was designed to evaluate the effects of vitamin B12 in cryopreservation medium on frozen-thawed semen of buffalo (Bubalus Bubalis) bulls. Semen from five bulls (fertility-proven) were diluted in five aliquots not supplemented (control), or supplemented with 1, 2, 4, or 5 mg/mL of vitamin B12 and evaluated using the Computer Assisted Sperm motion Analysis, antioxidant enzymes, lipid peroxidation (LPO), reactive oxygen species (ROS), ATP concentrations, and in vitro fertilization rate (%). Sperm progressive motility, rapid velocity (%), mitochondrial potential, and acrosome integrity were greater (P < 0.05) with supplementation of 4, and 5 mg/mL vitamin B12 than the control sample. Similarly, compared with the control, samples with 5 mg/mL vitamin B12 supplementation had markedly greater average-path, straight-line, and curved-line velocities (µm/sec). Semen samples supplemented with 2, 4 and 5 mg/mL vitamin B12 had greater concentrations of GPx (U/mL) and SOD (U/mL), whereas LPO (µM/mL) was less (P < 0.05) compared with the control sample. Seminal plasma ROS concentrations (104/25 × 106) were less in the 5 mg/mL vitamin B12 supplemented than control sample. Semen samples supplemented with 5 mg/mL of vitamin B12 had greater concentrations of ATP than control and the 1 mg/mL vitamin B12 supplemented sample. Semen samples supplemented with 5 mg/mL of vitamin B12 had greater plasmalemma and DNA integrities (%) than the control sample. In summary, vitamin B12 supplementation augments semen quality, as evidenced by values for CASA variables, antioxidant enzymes, and ATP concentrations, which may occur as a consequence of inhibition in LPO and ROS production by buffalo spermatozoa.

Comparative phylogenetic analysis of Schizothorax plagiostomus and Schizothorax esocinus with other members of subfamilies of cyprinidae on the basis of complete mitochondrial genome and 12S, 16S ribosomal RNA from Northren areas of Pakistan.

We assessed the relationship of Schizothoracinae species with other subfamilies Alburninae, Xenocyprinae, Cultrinae and Squaliobarbinae of family Cyprinidae by creating the phylogenetic trees using complete mitogenome and 12S and 16S RNA. Our representative species show the great affiliation with other but separated from a group composed of Metzia mesembrinum, Metzia longinasus, Metzia lineata and Metzia formosae of subfamily Alburninae while other subfamilies formed distinct group. The members of subfamily Schizothoracinae shows separate line of evolution from subfamily Barbinae.

Supplementation of green tea extract (GTE) in extender improves structural and functional characteristics, total antioxidant capacity and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa.

The aim of this study was to elucidate the beneficial effects of green tea extract (GTE) in tris citric acid extender on post thaw structural and functional characteristics, DNA fragmentation (%), total antioxidant capacity (TAC, µM/L), lipid peroxidation (LPO, µM/mL) levels and in vivo fertility of buffalo (Bubalus bubalis) bull spermatozoa. GTE is a acknowledged natural antioxidant, act as a free radical scavenger and protects spermatozoa against oxidative stress. Three mature and donor buffalo bulls were used in this experiment. Two ejaculates were collected per bull on each collection day, followed by initial evaluation for consistency (colour), volume (mL), progressive motility and concentration (x109) and were diluted in five extenders @ 50 x 106/ mL (C = control, no GTE; T1 = treatment 1, GTE 0.1%; T2 = treatment 2, GTE 0.5%; T3 = treatment 3, GTE 0.75% and T4 = treatment 4, GTE1.0%). The experiment was repeated thrice. Data analysis showed that sperm progressive motility (%), plasma membrane integrity (%), supravital plasma membrane integrity (%), viable sperm with intact acrosome (%) and TAC were significantly higher (P < 0.05) in extender supplemented with T4 as compared to control. Furthermore, sperm DNA fragmentation and occurrance of LPO in buffalo bull spermatozoa were significantly lowered in T4 than control. In vitro longevity (%) of spermatozoa was significantly higher in T4 compared to control during 45 and 90 min of incubation at 37 °C. In vivo fertility rate of buffalo bull spermatozoa was significantly higher in T4 compared to control (64.96 vs. 48.40%, P < 0.05). It is concluded that supplementation of tris citric acid extender with 1.0% GTE improved structural and functional characteristics, TAC, in vitro longevity (%) and in vivo fertility, whereas decreased DNA fragmentation and LPO occurrence in buffalo bull spermatozoa after freezing and thawing protocol.

Bioaccumulation of Some Heavy Metals: Analysis and Comparison of Cyprinus carpio and Labeo rohita from Sardaryab, Khyber Pakhtunkhwa.

We examined and compared heavy metals bioaccumulation in Cyprinus carpio and Labeo rohita netted from Sardaryab, a tributary of River Kabul. By using atomic absorption spectrometry we assessed different organs including livers, gills, and muscles. Metals studied were chromium, iron, zinc, lead, and copper. Livers of both species showed higher concentrations of metals while muscles showed the least amount. Chromium and iron were the highly concentrated metals in the gills and livers of both species. A quantity of 0.154 ± 0.011, 0.199 ± 0.0079, and 0.024 ± 0.008 µg/g of chromium was found in the gills, livers, and muscles of Cyprinus carpio, respectively. Similarly, the gills, liver, and muscles of Labeo rohita contained 0.133 ± 0.008, 0.165 ± 0.01, and 0.019 ± 0.006 µg/g of Cr, respectively. Iron was highest in carp in the range of 0.086 ± 0.01 in gills and 0.067 ± 0.011 µg/g in muscles, comparatively. All the studied metals were found within the US recommended daily dietary allowances (RDA) limits; hence no immediate risk in their consumption for human was found. The data showed that Cyprinus carpio being omnivorous and bottom feeder stored higher concentrations of metals as compared to Labeo rohita.