الفهرس 
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Abstract
The present work was carried out to estimate the effect of two different types of insecticide (phoxim and flumethrin ) on fertility in mature male rat and also their effect on liver and kidney function via estimation of hematological and biochemical changes, gene expression, oxidative stress markers in blood and estimate presence or absence of histopathological changes in white albino rats and the potential protective role of (spirulina and Nano zinc oxide) against phoxim and flumethrin reproductive toxicity in adult male albino rats.
1. Drugs:
1.1. PhoximEC (Sebacil 50%):
Phoxim is a promising new broad-spectrum insecticide and the strongest organophosphorus currently being developed. Phoxim has demonstrated exceptional potency against a number of agriculturally and farm pests .In our investigation it we used it to investigate its effect on reproductive performance and hepatorenal toxicity on albino male rat with a dose 30 mg/kg B.wt (1/20 of LD50).
1.2. FlumethrinEC (bayticol 1%):
Flumethrin is a type II synthetic pyrethroid used as an ectoparasiticide, and in veterinary management it is applied topically on cattle, goats and sheep as a 1% weight/volume pour-on for the control of ticks, lice, mites and nuisance insects. In our investigation it we used it to investigate its effect on reproductive performance and hepatorenal toxicity on albino male rat with a dose 10mg/kg B.wt (1/20 LD50).
1.3. Spirulina platensis:
Spirulina (Arthrospira) is a type of blue-green microalgae, multicellular, filamentous. It is categorized as a microalga in the Cyanophyceae family. In our investigation we used it to ameliorate the toxic effect of phoxim and flumethrin on albino male rat with a dose 300 mg/kg B.wt.
1.4.Nano zinc oxide:
Zinc oxide nanoparticles (ZnO-NPs) are used in several applications, including sunscreens , food additives, manufacturing of rubber, pharmaceuticals and electrical materials. For its preparation we use planetary ball milling machine to obtain particles with a diameter ranges of 200 nm by SEM. In our investigation we used it to protect against the toxic effect of phoxim and flumethrin on albino male rat with a dose 15mg/kg B.wt [1/20 LD50].
2. Experimental design :
The study was carried out on (45) mature male albino rats with a mean weight of 180–200g. Prior to the start of the experiment, the animals were housed in individual cages in groups of five rats per cage and after 2 weeks of acclimatization, the drugs were administered orally using an intra-gastric gavage tube, once daily for 56 consecutive days as following:G1 group: was served as control group fed only rat pellets and water.G2 group: was received phoxim at a dose of 30 mg/kg b.wt.G3 group: was received Spirulina at dose 300 gm/kg b.wt and after one hour, phoxim was given at dose 30 mg/kg b.wt.G4 group: w a s given Nano zinc oxide at a dose 15 mg/kg b.wt and after one hour, phoxim was given at dose 30mg/kg b.wt. G5 group: given spirulina at dose 300 mg/kg b.wt. G6 group: was received Nano zinc oxide at dose 15mg/kg b.wt . G7 group: was received flumethrin at dose 10 mg/kg b.wt.G8 group: was given spirulina at dose 300 gm./kg b.wt and after one hour, flumethrin was given at dose 10 mg/kg b.wt.G9 group: was received Nano zinc oxide at dose 15 mg/kg b.wt and after one hour, given flumethrin at dose 10 mg/kg b.wt.
• Blood sampling:
At the end of our experiment, the blood samples were collected in heparinized mirohaematocrite tubes to obtain plasma and in non- heparinized mirohaematocrite tubes to obtain serum from retro-orbital venous plexus blood collection.
• Animal sacrifice:
After 56 days of treatment, Rats were weighted and anesthetized by using ketamine 90 mg/kg of body weight and 5 mg/kg xylazine.
• Results:
• Effect on body weight of rats:
At the end of our experiment , the animals were weighted .Our investigation revealed that there is significant DROP in body weight in phoxim and flumethrin treated groups as compared to control group . There is significant increase in body weight in spirulina treated group, on the other hand ,ZnO Nps treated groups caused decrease in the body weight.
• Hematological parameters:
• Blood picture:
The result revealed a significant reduction (p<0.05) in haemoglobin content , RBCs count and PCV level in flumethrin treated rats (G7) when compared to rats of control group(G1) as well as ZnO Nps (G6) or Spirulina platensis (G5) treated groups alone or as a protective prior to insecticide administration . Haemoglobin content and PCV were significantly reduced in phoxim treated rats (G2) .
Differential leucocytic count:
Our investigation revealed that there is significant elevation in neutrophil count in phoxim / nano zinc oxide treated group( G4 ) and spirulina treated group (G3) ,respectively, wherever occur increase in monocyte count in flumethrin plus Spirulina(G8) and flumethrin plus Zno Nps (G9)treated groups and reduction in monocyte count in phoxim plus nano zinc treated group(G4).
• Biochemical parameters:
• Liver and kidney function
There is a significant marked elevation in levels of ALT, AST and Urea in phoxim (G2) and flumethrin (G7) treated groups in comparison to other treated groups which indicates hepatorenal inflammatory changes and sever tissue damage which is alleviated in the groups in which spirulina (G3,G8) and nano zinc oxide (G4,G9) are co-administrated with this toxins, that revealed phoxim and flumethrin show sever hepatic and renal damage on the other hand ameliorating effects of nano zinc oxide and spirulina against oxidative stress and hepatorenal damage of flumethrin and phoxim.
• Oxidative stress markers (TAC):
occur marked reduction in TAC in phoxim treated group(G2) and flumethrine treated group(G7) in comparison to control group and to their compination with spirulina (G3,G8) and nano zinc groups(G4,G9)and that indicates increased level of free radicals and harmful substances in the body due to reduction in antioxidant enzymatic activity.
• Relative reproductive organ weightper 100 gm b.wt. :
The weight measures of reproductive organs significant reduction occur in reproductive organ weight (testis ,epididymis ,seminal vesicle and prostate gland ) in phoxim(G2) and flumethrine treated group(G7) in comparison to control group(G1 ) ,on the other hand there is significant elevation occur to spirulina platensis treated group(G5) in reproductive organ weights , also pretreatment of rats with spirulina platensis (G3,G8) and ZNO NPs (G4,G9 ) and prior to toxin administration showing alleviation in toxin effect on these organ weights . Spirulina platensis and ZNO-NPs do not significantly enhance testis and other reproductive organ weights in comparison to control group but they compete the impact of organ weight reduction caused by phoxim and flumethrine when they are co-administrated with these toxins.
• Hormonal changes related to: Fertility (FSH, LH, Testosterone):
There is significant reproductive hormonal disturbance in the form of reduction of Testosterone, FSH and LH in both phoxim (G2) and flumethrine treated group(G7) in comparison to control group (G1 )and we notice that this reduction is alleviated in groups at which phoxim and flumethrin are pretreated with spirulina platensis(G3,G8) and ZNO NPs (G4,G9 ) which give an indication about their validity as reproductive performance enhancer.
• Semen picture in male rats:
The result showed a dramatic DROP in sperm count, decreased individual motility as well as life to dead sperm ratio and also increased level of total abnormalities occur in phoxim (G2) and flumethrin (G7) treated groups in comparison to control group (G1 ), On the other hand co-administrated groups by spirulina platensis (G3,G8) showing normal semen picture associated with normal individual motility with non significant change in total abnormalities and elevation of sperm count than normal. In addition to that co-administrated groups by ZnO-NPs treated group (G4,G9 ) showed marked protection against toxic effect of flumethrin and phoxim which appeared on the form of increased on spermatic count and individual motility with reduction of dead to live ratio and so increased in total abnormalities.
• Gene expression :
• Testicular tissue gene expression findings:
In the current study, phoxim (G2) and flumethrin (G7) treated groups shows significant down regulation of the testicular mRNA expression levels of CYP11A1 StAR and HSD-3b which is represented by testicular and accessory glands. It was determined that all groups treated with spirulina platensis (G3,G8) and ZNO NPs (G4,G9 ) counteracted the negative effects of both toxins and significantly increased the testicular mRNA expression levels of the subjects studied .However, nano zinc oxide was discovered to surpass spirulina in testicular protection because it significantly upregulated the studied genes when compared to spirulina
• Liver and kidney gene expression findings:
Our investigation revealed that both phoxim(G2) and flumethrin (G7) treated groups resulted in significant upregulation of TNF- and IL-1 mRNA expression levels in the liver and kidney, on the other hand all treatments(spirulina platensis (G3,G8) and ZNO NPs (G4,G9 ) treated groups ) were effective for mitigating the negative effects of both phoxim and flumethrin by lowering the mRNA expression levels of the studied genes Interestingly, these findings support the potential role of nano zinc and spirulina as protective agents against phoxim and flumethrin hepatorenal toxicity.
• Histopathological examination
• Kidney:
Our investigations revealed that the phoxim(G2) and flumethrin (G7) treated group ’s renal tissue showed degeneration and atrophy of renal corpuscles. The renal tubules were severely degenerated, with cystic dilatation. The renal blood vessels appeared to be extremely congested, On the other hand, spirulina platensis (G3,G8) and ZNO NPs (G4,G9 ) co-administered groups appeared normal with normal renal corpuscles and normal renal tubules (arrowhead).
• Liver:
Our result revealed that the hepatic tissue of Phoxim-treated group (G2) showed severe vacuolar degeneration in the hepatocytes (arrow) around the portal area with marked congestion of the portal blood vessels and the central vein and flumethrine-treated group (G7) the hepatic tissue of showing congested central vein and portal blood vessels . The hepatocytes especially around the portal area suffered from severe vacuolar degeneration (arrow), on contrary the hepatocytes adjacent to central vein appeared normal (arrowhead).In comparison to the hepatic tissue of spirulina platensis (G3,G8) and ZNO NPs (G4,G9 ) treated groups co-administered groups appeared normal with normal hepatocytes (arrowhead), normal central vein.
● Testes and accessory genital glands:
Our result revealed that testis and accessories in phoxim(G2) and flumethrin (G7) treated groups suffering from degenerative changes and atrophy of testicular tissue. Co-administrated groups with Spirulina(G3,G8) appear with normal sections in both seminefrous tubules, prostate, epididymis and seminal vesicle, also co- administrated group with Nano zinc oxide (G4,G9 ) show protective effect to testicular tissue and accessory gland ,even if it ameliorates the impact of toxins on testis but with less degree compared to Spirulina platensis treated groups.