Effect of Fipronil Exposure on Hematological Aspects of Rhesus Monkeys (Macaca mulatta): Risk and Toxicity Assessment in Agro-Workers.

Background: Fipronil (FPN) is a broad-spectrum phenylpyrazole insecticide, widely used in agriculture and veterinary medicine. Published research on FPN toxicity has established the fact that its inhalation or dermal exposure may lead to very serious clinical outcomes in non-target animals. In line to its exposure and toxicity related damage, FPN has been investigated in many invertebrates, however, its exposure-related noxiousness is less reported in higher animals. Objective: To assess the FPN-induced effects to agro-workers in the field, in the present study, we used physiological human surrogates, adult rhesus monkeys as models. Method: We exposed well habituated, chair restraint adult rhesus monkeys with a field spray concentration of FPN (0.3 mg/1 mL distilled water) through an inhalation route in the closed system. Animals were divided into control and treatment groups, each containing three animals. Inflammatory and hematological effects were determined by evaluating the kidney and liver biomarker enzymes; serum creatinine and alanine transaminase (ALT), aspartate transaminase (AST) levels respectively. Results: Our findings reveal that FPN treated monkeys show significantly increased levels of ALT (p = 0.000461), AST (p = 0.0681) and creatinine (p = 0.00656) as compared to the control group. Furthermore, significant differences of red blood cells (RBCs) (p = 0.0139) and white blood cells (WBCs) (p = 0.00642) were also observed in the treated and control group monkeys which reflect strong toxic effects on the blood cells. Conclusion: Our findings demonstrate that FPN exposure is very toxic to higher animals and causes severe damage to the liver and kidneys along with other clinical problems. The study highlights the effect and impact of passive inhalation of insecticides in intentionally carefree agro-workers and raises the concern of public awareness toward pesticides use.

Naproxen sodium nanoparticles are less toxic and gastroprotective agents than the conventional NSAID drug naproxen sodium in Balb/c mice.

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is one of the leading causes of gastric ulcers. Excellent therapeutic properties have made the use of NSAIDs widespread. Nano-drug delivery to reduce systemic toxicity through modulating drug pharmacokinetics may be a better choice. Presently, we investigated if naproxen nanoformulation (PVA capped NPRS-MgO NPs) is less toxic to be used as an alternative drug. Groups of mice were assigned to control, NPRS-treated, CNF-treated, UNF-treated, and MgO NPs-treated groups. Analyses included gross examination of gastric mucosa, calculation of ulcer and inhibition indices, determination of tissue levels of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and reduced glutathione (GSH), histological and immunohistochemical assessment of i-NOS, COX-2, and caspase-3 of stomach mucosa, q-PCR for the detection of mRNA expression of IL-1ß, IL-6, and TNF-α. Results were compared statistically at P < 0.05. Compared to NPRS-treated mice which developed multiple ulcers, had elevated MDA and ROS levels, and deceased CAT, POD, SOD, and GSH levels, significantly increased expression of IL-1ß, IL-6, and TNF-α mRNA, damaged surface epithelium with disrupted glandular architecture and leucocyte infiltration of lamina propria with a marked increase in mucosal COX-2, i-NOS, and caspase-3 expression, oral administration of coated and uncoated naproxen nanoformulations prevented the gross mucosal damage by a restoration of all biochemical, histological, and immunohistochemical alterations to near control levels. The present study demonstrates that naproxen sodium nanoformulation has a gastroprotective action and in the clinical setting can be a better alternative to conventional naproxen.

Butterfly (Order: Lepidoptera) species Richness, diversity and distribution in different localities of Battagram, Pakistan.

Butterflies are the beautiful creatures and need to be conserved. The present survey was conducted to explore the biodiversity of butterflies of District Battagram Khyber Pakhtunkhwa Pakistan, from March to September 2021. During this study the butterflies were collected from 2 Tehsil including 12 localities using line transect method. A total of 572 specimens were collected from all localities. Species identified were belonging to 3 families and 7 genera. The species were Cynthia cardui. Danaus chrysippus, Junonia orithya, Papilio demoleus, Papilio polytes, Colias croceus, Pieris ajaka, Pontia daplidice and Pieris napi. In the recorded 9 species Papilo demoleus was the most common species of the district Battagram and the most rear specie was Pieris ajaka during this study. The current study is new detailed work on the butterflies from district Battagram . It is concluded from the present study that district Battagram is rich in flora and provide a much suitable environment and place for biodiversity to insects. As this study is the first survey of butterflies population in the district and recorded rich diversity, so more explorative work is needed for its population estimation and specie abundance.

Intraperitoneal kisspeptin-10 administration ameliorates sodium arsenite-induced reproductive toxicity in adult male mice.

The current study investigated the protective ameliorative effect of intraperitoneally administered kisspeptin-10 (50 nmol/day) against reproductive toxicity in adult male mice challenged with 35 days of exposure to sodium arsenite in drinking water. Mice were divided into tap water control, sodium arsenite-alone (4 ppm and 10 ppm), kisspeptin-alone (intermittent and continuous) and combined (sodium arsenite +kisspeptin-10 intermittent and continuous) treatment groups. Results revealed protective effect of both intermittent and continuous kisspeptin doses on reproductive organs against sodium arsenite-induced toxicity. This was indicated by an increase (p < 0.001) in the activity of antioxidant enzymes and a decrease (p < 0.001) in the levels of oxidative stress biomarkers. Concomitant significant increase was noticeable in the relative organ weight (p < 0.01), and serum testosterone and seminal fructose (p < 0.001), and a significant improvement in sperm parameters was also observed. A significant downregulation of lactate dehydrogenase concentration demonstrated further the protective effect of kisspeptin against tissue damage. Histologically, both treatment regimens of kisspeptin combined with sodium arsenite exposure prevented massive germ cell loss and tissue damage, a condition prominent in sodium arsenite-alone-treated mice. The study demonstrates for the first time kisspeptin's potential to mitigate the biochemical and histotoxic effects of arsenic on male reproductive system.

Dose-Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study.

BACKGROUND: The discovery of kisspeptin signaling as a key regulator of gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone, and spermatogenesis are studied in detail. OBJECTIVE: The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. METHODS: We administered a range of kisspeptin-10 doses (1 µg, 1 ηg, and 10 ρg) intraperitoneally to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. RESULTS: At the end of the treatment, plasma concentrations of testosterone were measured by competitive binding radioimmunoassay, and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1µg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregularly shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration, and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. CONCLUSION: It is concluded that chronic intermittent administration of kisspeptin-10 has a dose-dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.

In vitro administration of sodium arsenite in mouse prepubertal testis induces germ cell loss and apoptosis.

High levels of arsenic contamination in drinking water pose serious health risks in numerous countries. The documentation reporting arsenic toxicity on reproduction and development is increasing, with evidence of arsenic inducing fertility and developmental issues. Nonetheless, the impact of arsenic exposure on the development of the male reproductive system is not fully elucidated. In the present study, we have investigated the direct effects of arsenic on prepubertal mouse testis using an in vitro testicular organ culture system. Culture medium was supplemented with a range of concentrations of sodium arsenite, examining effects of low (0.5 and 1 µM) and high (10, 50, 100 µM) concentrations, in cultures of post-natal day 5 CD1 mouse testis. In vitro exposure of low arsenic concentrations (0.5 or 1 µM) for 6 days did not cause any change in the testicular morphology, germ cells density, or apoptotic marker cleaved caspase 3 (CC3) expression. In contrast, exposure of prepubertal testis to high arsenic concentrations (10, 50 or 100 µM) induced drastic changes: severe destruction of testicular morphology, with loss of seminiferous tubule integrity; a dose-dependent decrease in germ cell density, and a hundred-fold increase in CC3 expression after 50 µM arsenic exposure. In conclusion, high arsenic treatment induced a dose-dependent induction of apoptosis and germ cell loss in prepubertal mouse testis.

In vitro application of sodium arsenite to mice testicular and epididymal organ cultures induces oxidative, biochemical, hormonal, and genotoxic stress.

Arsenic poisoning is well-known for its innumerable toxic and carcinogenic effects. In vivo data on reproductive toxicity are also known but in vitro data are scant. Presently, we evaluated the in vitro toxic effects of sodium arsenite (NaAsO2) on adult mice testes and epididymal tissues using organ cultures. Testicular and epididymal fragments were incubated at 37°C and 33°C, respectively, with 1, 10, 50, and 100 µM concentrations of NaAsO2. Cultures were allowed to incubate for 2 and 24 h. Levels of oxidative stress markers, the reactive oxygen species (ROS) and thiobarbituric acid reactive substance assay (TBARS), antioxidant enzymes, testosterone concentrations, and the extent of sperm DNA damage, were estimated. Results were analyzed statistically at p < 0.05. Results demonstrated both time- and dose-dependent alterations whereby, following 24-h incubation with NaAsO2, substantial increases were noticeable in ROS and TBARS levels and sperm DNA damage (p < 0.001), while decreases (p < 0.001) occurred in catalase, peroxidase, and superoxide dismutase levels at 10, 50, and 100 µM concentrations. Incubations for 2 h revealed similar but relatively less toxic effects. Testosterone concentrations decreased significantly only after 24 h of incubation with 50 (1.95 vs. 2.93 ng g-1; p < 0.01) and 100 µM (1.32 vs. 2.93 ng g-1; p < 0.001) NaAsO2 concentrations. The study concluded that exposure of testicular and epididymal tissue fragments to arsenic under in vitro conditions induces rapid and immediate metabolic and genotoxic damage at higher concentrations.

Short-term exposure of Balb/c mice to buprofezin insecticide induces biochemical, enzymatic, histopathologic and genotoxic damage in liver and kidney tissues.

Buprofezin is a type-1 chitin synthesis inhibitor insecticide used to control hemipteran insects. It is generally considered safe for humans, but its persistent nature may become a health hazard if long-term exposure takes place. Adverse effects on mammals are remaining to be explored. The present study investigated buprofezin toxicity on liver and kidney tissues of Balb/c mice treated intraperitoneally with 4.0, 6.0 and 8.0 µg/kg b.w doses respectively for 24 h. Statistical analyses demonstrated increased activities (p < 0.05) of serum alanine aminotransferase, aspartate aminotransferase, creatinine and urea, ROS and TBARS (thiobarbutaric acid) in liver and kidney tissues. Concomitant significant decrease occurred in tissue total protein, antioxidants enzymes, the superoxide dismutase, catalase and peroxidase and non-enzymatic reduced glutathione. Significantly altered histomorphology of liver and kidney tissues revealed excessive tissue damage. Congestion, hepatocyte necrosis, decreases sinusoidal damage in liver, while in kidneys, glomerular shrinkage, capillary damage, widened Bowman's space and lumens of tubules and collecting ducts and necrosis of tubular epithelial cells were evident. TUNEL assay confirmed apoptosis, the Comet assay demonstrated DNA damage by an increase in the head length, tail length, comet length, tail moment and olive tail moment. The study concludes that buprofezin is highly toxic for mammalian tissues and warrants further biochemical, molecular and cellular studies.

Short and long term modulation of tissue minerals concentrations following oral administration of black cumin (Nigella sativa L.) seed oil to laboratory rats.

BACKGROUND: Nigella sativa, or commonly called black cumin is a small herb of family Ranunculaceae is a well-known medicinal plant but its effects on tissue mineral concentrations of animal bodies is unknown. PURPOSE: To study the effect of oral administration of fixed oil of black cumin seeds on tissues mineral content using laboratory rats as experimental model. STUDY DESIGN: Experimental animals were exposed to two oral doses of seed oil (60 and 120 ml kg-1 body weight). Short- and long term experiments lasted 24 h and 60 days respectively, with three replicates each. METHODS: Oil extracted from black cumin seeds was subjected to GC-MS to identify chemical components. Following the wet digestion in nitric acid, samples of whole blood and organs of rats were subjected to atomic absorption spectrophotometry for determination of elements concentrations. Data were compared statistically at p < .05. RESULTS: Compared to control, Cr, Mn, Ni, Cu, Zn showed decrease, whereas Co, Na, Mg and K demonstrated increase, but Ca showed both increase and decrease in most of the tissues upon short term exposure to low and high doses of black cumin oil. During long term exposure, Cr, Fe, Mn, Cu exhibited decrease; Co, Na, Mg and Ca concentrations demonstrated an upregulation, whereas Ni and Zn showed increase and decrease in most of the tissues. Comparison of short term with long term experiments at low dose revealed increases in Fe, Zn, Cu, Mg, K and Ca, a decrease in Cr, Mn, Ni and Cu in most tissues, but both increase and decrease in Na. At high dose, an increase occurred in Fe, Ni, Zn, K, Ca, Mg, a decrease in Cr, while both increase and decrease in Cu, Co and Na concentrations. CONCLUSION: Our study demonstrates that oral administration of black cumin seeds oil to laboratory rats significantly alters tissue trace elements and electrolytes concentrations. The study appears beneficial but indicates modulatory role of black cumin oil as regards mineral metabolism with far reaching implications in health and disease.

Vitamin C attenuates biochemical and genotoxic damage in common carp (Cyprinus carpio) upon joint exposure to combined toxic doses of fipronil and buprofezin insecticides.

In the present study, potential protective role of Vitamin C (l-ascorbic acid) was investigated in aquaria acclimated common carp (Cyprinus carpio) following exposure for 96 h to combined toxic doses of fipronil (FP) and buprofezin (BPFN) insecticides in combination (FP: 200 µg/L; 4.57 × 10-7 mol/L and BPFN: 50 mg/L; 1.64 × 10-4 mol/L). At end of 96 h exposure, fish were supplemented with low (25 mg/L) and high (50 mg/L) doses of Vitamin C, added once daily to aquaria water for continuous three weeks. Appropriate control groups were run in parallel. Fish behavior was monitored throughout for signs of toxicity. At completion of experiments, liver, kidney, brain and gills were excised for toxicity assessment and possible remediation by the Vitamin C through biochemical determination of reactive oxygen species (ROS), thiobarbituric acid reactive substances or TBARS, reduced glutathione (GSH) and total protein content, levels of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD), and the Comet assay. Hepatosomatic index (HSI), condition factor (CF), survival rate (SR), and combination index (CI) were also determined. Data were compared statistically at p < 0.05. Results showed significant behavioral and biochemical alterations, and DNA damage in the fish group exposed to FP and BPFN in combination. In fish groups supplemented with Vitamin C following FP and BPFN treatment, significant alleviation in tissue damage and toxic effects was represented by substantial decreases in ROS and TBARS production (p < 0.001), along with a concomitant significant increase in the survival rate, GSH and total protein content, HSI, CF, and activities of SOD, CAT and POD enzymes (p < 0.001). Mean tail length of comet and percent tail DNA decreased significantly (p < 0.001), which indicated amelioration of DNA damage. The study concludes that Vitamin C is an effective remedial treatment against FP and BPFN-induced damage in exposed fish.

Vocalizations of adult male Asian koels (Eudynamys scolopacea) in the breeding season.

Defining the vocal repertoire provides a basis for understanding the role of acoustic signals in sexual and social interactions of an animal. The Asian koel (Eudynamys scolopacea) is a migratory bird which spends its summer breeding season in the plains of Pakistan. The bird is typically wary and secretive but produces loud and distinct calls, making it easily detected when unseen. Like the other birds in the wild, presumably Asian koels use their calls for social cohesion and coordination of different behaviors. To date, the description of vocal repertoire of the male Asian koel has been lacking. Presently we analyzed and described for the first time the vocalizations of the adult male Asian koel, recorded in two consecutive breeding seasons. Using 10 call parameters, we categorized the vocalization type into six different categories on the basis of spectrogram and statistical analyses, namely the; "type 1 cooee call", "type 2 cooee call", "type 1 coegh call", "type 2 coegh call", "wurroo call" and "coe call". These names were assigned not on the basis of functional analysis and were therefore onomatopoeic. Stepwise cross validated discriminant function analysis classified the vocalization correctly (100%) into the predicted vocal categories that we initially classified on the basis of spectrographic examination. Our findings enrich the biological knowledge about vocalizations of the adult male Asian koel and provide a foundation for future acoustic monitoring of the species, as well as for comparative studies with vocalizations of other bird species of the cuckoo family. Further studies on the vocalizations of the Asian koel are required to unravel their functions in sexual selection and individual recognition.

Effect of kisspeptin-10, LH and hCG on serum testosterone concentrations in stallions, donkeys and mules.

This study was conducted to determine the response of serum testosterone (T) in male equines (stallions, donkeys and mules) after administering intravenous doses of kisspeptin-10 (KP-10), human chorionic gonadotropin (hCG) and luteinizing hormone (LH) and saline as a control. The animals were divided into four groups of three each: Group I, 3 ml of 0.95% saline; Group II, 50 µg KP-10; Group III, 2500 IU hCG and group IV, 400 µg LH. The administration of KP-10 and hCG to stallions resulted in a significant increase in serum T concentration at 240 min; whereas it was significantly higher at 30, 60, 120, and 240 min with LH treatment as compared to pre-dose concentrations. Both KP-10 and hCG significantly elevated the T concentrations in donkeys at 120 and 240 min, respectively; whereas it was significantly higher at 60, 120, and 240 min with LH treatment as compared to pre-dose concentration. Both KP-10 and LH elevated T in donkeys at 240 min as compared to the control and hCG concentrations. After 120 and 240 min, T concentrations in mules were higher (p < 0.05) with administration of KP-10, hCG and LH as compared to the control. In conclusion, the administration of KP-10, hCG and LH elevate the serum T concentration in normal male equines. It is suggested that KP-10 may be useful in situations where an increase in T is desired. Further work is required to determine the effect of KP-10 on T in male equids with reproductive abnormalities before it can be used in clinical situations.

Serum APOE, leptin, CFH and HTRA1 levels in Pakistani age related macular degeneration patients.

OBJECTIVE: To determine the association between serum levels of apolipoprotein E, leptin, complimentary factor H and high temperature requirement A-1 in patients with age-related macular degeneration. METHODS: This case-control study was conducted at the Quaid-i-Azam University, Islamabad, Pakistan, from May to October 2013, and comprised patients with age-related macular degeneration and matching controls. The confirmation of age-related macular degeneration was carried out through slit lamp examination, fundoscopy and ocular coherence tomography. The selected subjects were not suffering with any other systemic or ophthalmic complication(s). Serum apolipoprotein E, leptin, complimentary factor H and high temperature requirement A-1 were estimated in serum samples of all subjects. SPSS 18 was used for data analysis. RESULTS: Of the 190 participants, 90(47.4%) were patients with age-related macular degeneration and 100(52.6%) were controls. Significantly elevated serum apolipoprotein E (p<0.0024) and high temperature requirement A-1 (p<0.0001) levels were observed in the patients, while serum leptin (p<0.008) and complimentary factor H (p<0.0001) levels were significantly reduced. Logistic regression showed that lower leptin (p<0.026) and elevated high temperature requirement A-1 (p<0.0001) were the relevant risk factors. CONCLUSIONS: Serum apolipoprotein E, leptin, complimentary factor H and high temperature requirement A-1 levels were altered in age-related macular degeneration patients.

Synthesis, pH dependent photometric and electrochemical investigation, redox mechanism and biological applications of novel Schiff base and its metallic derivatives.

A novel Schiff base, 1-((2, 4-dimethylphenylimino)methyl)naphthalen-2-ol abbreviated as (HL) and its four metallic complexes were synthesized and confirmed by 1H and 13C NMR, FTIR, TGA and UV-Visible spectroscopy. Schiff base was also characterized by X-ray analysis. The photometric and electrochemical responses of all the synthesized compounds were investigated in a wide pH range. Structures of the compounds were optimized computationally for the evaluation of different physico-chemical parameters. On the basis of electrochemical results the redox mechanistic pathways of the compounds were proposed. The cytotoxicity analysis on Hela cells revealed that HL and its complexes inhibit cell growth as revealed from their IC50 values (HL):106.7µM, (L2VO): 40.66µM, (L2Sn): 5.92µM, (L2Zn): 42.82 and (L2Co): 107.68µM. The compounds were tested for anti-diabetic, triglyceride, cholesterol, anti-microbial, anti-fungal and enzyme inhibition activities. The results revealed that HL and its complexes are promising new therapeutic options as these compounds exhibit strong activity against cancer cells, diabetics, fungal and microbial inhibition.

Chicken egg yolk plasma in tris-citric acid extender improves the quality and fertility of cryopreserved water buffalo (Bubalus bubalis) spermatozoa.

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (UV)-C-irradiated chicken egg yolk plasma (EYP; v:v; 10%, P1; 15%, P2; 20%, P3) or 20% (v:v) of whole chicken egg yolk (WCEY) in tris-citric acid (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C-irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (µm/s) and straight line velocity (µm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C-irradiated chicken EYP (20%) in TCA extender for cryopreservation of water buffalo spermatozoa.

Cryoprotection synergism between glycerol and dimethyl sulfoxide improves the mitochondrial transmembrane potential, plasmalemma, acrosomal and DNA integrities, and in vivo fertility of water buffalo (Bubalus bubalis) spermatozoa.

The objective of the study was to devise a cryoprotection synergism between glycerol and dimethyl sulfoxide (DMSO) for water buffalo spermatozoa. Additionally, the effect of best evolved concentrations of glycerol and DMSO in extender was assessed on in vivo fertility of buffalo spermatozoa. Ejaculates (n = 30) were equally distributed into five aliquots; first aliquot was diluted at 37 °C in extender having 7 % glycerol (control); second aliquot was diluted at 37 °C as well as at 4 °C in extender having 3.5 % DMSO (Group 1); third aliquot was diluted at 37 °C in extender having 3.5 % glycerol and then at 4 °C in extender having 3.5 % DMSO (Group 2); fourth aliquot was diluted at 37 °C in extender having 3.5 % DMSO and then at 4 °C in extender having 3.5 % glycerol (Group 3); fifth aliquot was diluted in extenders having 1.75 % glycerol and 1.75 % DMSO at 37 as well as at 4 °C (Group 4). At post thawing, sperm progressive motility (%), rapid velocity (%), average path velocity (µm/s), curved line velocity (µm/s), in vitro longevity (%), structural and functional integrity of plasmalemma (%), mitochondrial transmembrane potential (%) and viable sperm with intact acrosome (%) were higher (P < 0.05) in Group 4 compared to other treatment groups and control. Regarding sperm DNA integrity (%); it was higher (P < 0.05) in Group 4 compared to Group 1, 3 and control. The in vivo fertility (%) of buffalo spermatozoa was significantly higher with Group 4 compared to control (69.45 vs. 59.81). In conclusion, synergism exists between glycerol and DMSO (Group 4) in improving the quality and in vivo fertility of cryopreserved water buffalo spermatozoa.

Exposure to sub-acute doses of fipronil and buprofezin in combination or alone induces biochemical, hematological, histopathological and genotoxic damage in common carp (Cyprinus carpio L.).

Use of pesticides or insecticides can be highly toxic to aquatic life forms due to leaching and agricultural runoff, rains or flood. Fipronil (FP) is a GABA receptor inhibitor, while buprofezin (BPFN) is an insect growth regulator. Presently, we exposed groups of aquaria acclimated carp fish (Cyprinus carpio) for 96h to sub-lethal concentrations of fipronil (400µgL(-1); 9.15×10(-7)molL(-1)) and buprofezin (BPFN, 100mgL(-1); 1.072×10(-6)molL(-1)) singly or in combination. The extent of damage was assessed at biochemical, hematological, molecular biological and histopathological level. Results obtained in treated fish were compared statistically with those of control non-treated fish and also among treatment groups. Significance level was p<0.05. Compared to control, serum total protein and globulin concentrations decreased significantly (p<0.0001) in fish treated with FP; while albumin concentration remained unaltered with all treatments. Glucose concentration decreased significantly (p<0.002) in fish treated with FP. In contrast, combined FP+BPFN treatment and BPFN treatment caused insignificant elevation of glucose concentration. Hematological assessment demonstrated significant decrease in red blood cell and thrombocyte counts, hemoglobin concentration and hematocrit percent; while white blood cell count showed an increase in all treatment groups (p<0.0001). Blood smears from pesticide treated fish revealed aberrant erythrocyte morphologies which included necrosis, micronuclear formation and hyperchromatosis. DNA laddering assay carried out on whole blood demonstrated excessive smear formation in combined FP+BPFN and BPFN treatment groups but no smear formation was noticeable in FP treated fish. Compared to control, whole blood DNA content increased significantly in the combined FP+BPFN and BPFN treatment groups (p<0.001 and p<0.009). With all treatments histopathological changes observed in the gills were: epithelial uplifting and necrosis of lamellae, lamellar atrophy, disruption of cartilaginous core, fusion and disorganization of lamellae and telangiectasia. In liver these were: karyorrhexis, hepatocellular hypertrophy, nuclear hypertrophy, melanomacrophage aggregates and central vein contraction, while in the kidney: deterioration of glomerulus and dilatation of Bowman's space, dilatation of renal tubules, thyroidisation, altered tubular lumen, nuclear hypertrophy, cellular atrophy, and cellular necrosis were the outcome. Our study revealed that FP and BPFN produce highly toxic effects on fish when given in combination or singly. To our knowledge, this is the first report on toxicity caused by FP and BPFN in single and combined state.

Association of gene polymorphism with serum levels of inflammatory and angiogenic factors in Pakistani patients with age-related macular degeneration.

PURPOSE: To study the association of serum levels of inflammatory mediators and angiogenic factors with genetic polymorphism in Pakistani age-related macular degeneration (AMD) patients. METHODS: This was a cross-sectional and case-control study that included 90 AMD patients diagnosed through slit-lamp examination, fundoscopy, and ocular coherence tomography. For reference and comparison purposes, 100 healthy age-matched subjects (controls) were also recruited. IL-6, IL-8, VEGF, and CRP levels were estimated in the serum samples of patients and control subjects. Using restriction fragment length polymorphism, single nucleotide polymorphisms were studied in IL-6 (rs1800795, rs1800796, rs1800797), IL-8 (rs4073, rs2227306, rs2227543), VEGF (rs3025039, rs699947), and CRP genes (rs1205, rs1130864). Since the data were obtained from a sample population, the Box-Cox transformation algorithm was applied to reduce heterogeneity of error. Multivariate analyses of variance (M-ANOVA) were applied on the transformed data to investigate the association of serum levels of IL-6, IL-8, VEGF, and CRP with AMD. Genotype and allele frequencies were compared through χ(2) tests applying Hardy-Weinberg equilibrium. The serum concentrations of IL-6 and IL-8, VEGF, and CRP between homozygotes and heterozygotes were compared through one-way ANOVA. Significance level was p<0.05. RESULTS: Compared to control subjects, serum IL-6 (p<0.0001), IL-8 (p<0.0001), VEGF (p<0.0001), and CRP (p<0.0001) levels were significantly elevated in the AMD patients. For rs1800795, patients with the GG genotype showed significantly raised levels of IL-6 compared to those with GC and CC genotypes (p<0.0001). Serum IL-8 levels were significantly higher in patients with the GG genotype compared to the GC and CC genotypes for the single nucleotide polymorphism (SNP) rs2227543 (p<0.002). Similarly, significantly higher VEGF levels were detected for genotype TT for rs3025039 SNP (p<0.038). However, no significant alteration in serum CRP levels was detected in hetero- or homozygotes for rs1205 and rs1130864 SNPs. CONCLUSIONS: Serum IL-6, IL-8, and VEGF levels are substantially increased in AMD, and the levels coincide with polymorphism in the respective gene. No such relationship appears to exist with regard to SNPs of CRP.

Assessment of heavy metals and metalloids in tissues of two frog species: Rana tigrina and Euphlyctis cyanophlyctis from industrial city Sialkot, Pakistan.

In the present study, we investigated the concentrations of Ni, Fe, Pb, Cu, Co, Zn, Cd, Mn, and Cr in selected body tissues (liver, stomach, kidney, heart, lungs, and skeletal muscles) of two frog species: Rana tigrina and Euphlyctis cyanophlyctis captured from industrial wastewater of Sialkot city known worldwide for its tanning industry. The both frog species had darker appearance, distinctively different wet body weight, and snout-vent length. The results revealed that the heavy metal concentrations were high in the samples collected from industrial sites as compared to non-industrial sites. The different tissues of R. tigrina and E. cyanophlyctis exhibited little significant differences from two sites. The concentrations of heavy metals were more in tissues of R. tigrina as compared to E. cyanophlyctis. Mean concentration of Cd, Fe, Ni, Mn, Cu, and Cr was comparatively greater in R. tigrina, whereas Pb and Co were higher in E. cyanophlyctis. The concentration of Cu and Cd in the liver and kidney were relatively more in both species as compared to other organs. Further, the results indicated that frogs collected from industrial sites showed decreased body length and weight, and greater metal accumulation. The results will help the authorities for the conservation of these frog species which are under the influence of heavy metal contamination.

Assessment of serum lipids in patients with age related macular degeneration from Pakistan.

OBJECTIVE: To determine serum lipids in patients with age related macular degeneration from Pakistani population. METHODS: The study was a cross sectional, randomized and case-control. Selected subjects ages were > or = 50 years and were normotensive, non-diabetic with no family history of any such disease and no complication of posterior ocular chamber other than age related macular degeneration (AMD). Controls were age matched healthy individuals with no symptoms of AMD. Diagnosis of AMD was done through conventional diagnostic techniques by professional ophthalmologists. Serum samples were analyzed for total cholesterol, triglycerides, LDL and HDL using commercially available kits. Data were compared with Student's t-test. Pearson correlation was calculated for relationship between different parameters. P < 0.05 was considered significant. RESULTS: Compared to controls, AMD patients had significantly greater total cholesterol concentration (p < 0.041), and power HDL/LDL ratio (p < 0.038), while serum triglycerides, HDL and LDL were non-significantly different from control subjects. Total cholesterol in AMD patients was significantly correlated with TG, LDL and HDL (p < 0.0001). CONCLUSION: The study indicates that high cholesterol might be a predictor of AMD and can be a diagnostic parameter.