CHROMOSOMAL ABERRATION AND PATHOLOGICAL ALTERATION AFTER COPPER OXYCHLORIDE FUNGICIDE INTOXICATION IN RABBITS

CHROMOSOMAL ABERRATION AND PATHOLOGICAL ALTERATION AFTER COPPER OXYCHLORIDE FUNGICIDE INTOXICATION IN RABBITS

M.F.Saad¹; A.M.Saeed¹ and A.M.H.Ahmed²

¹Department of Animal Biotechnology, Animal Production Research Institute, Dokki, Giza, Egypt

 PO Box 12816. ²Poultry Production Department, Fac. of Agri., Ain Shams Univ., Egypt.   mohamedsfahmy@yahoo. 

This study was conducted to examine the effect of Copper oxychloride fungicide is one of the most fungicide used in agriculture fields and crop storage on chromosomal aberration and some pathological alterations on rabbits. Forty five female rabbits divided into three groups (fifteen does each). The 1^st group was fed ration without fungicide (control). The 2^nd group was fed contaminated ration by Copper oxychloride (0.1184 g /Kg. BW/day as 1/20 LD₅₀) and 3^rd group was fed contaminated ration by copper oxychloride 0.2368 g /Kg. BW/day as 1/10 LD₅₀ for sixty days. Three does and nine offspring were slaughtered from each group to obtained bone marrow for cytogenetic testing and histopathological examination.

These results significant increase (P< 0.05) in total chromosomal aberrations (gap formation, chromosome break, deletion chromosome, centromeric attenuations, centric fusion, end to end associations and ring chromosome) among different groups and among does in the same group and their progeny as fungicide treatment. On the other hand we showed significant increase (P< 0.05) in the incidence of clinical signs and congestion of the portal blood vessels with severe inflammation and cellular infiltration of hepatic parenchyma mainly lymphocytes in liver, and high increase congestion of the lining epithelium of renal tubules in the form of cloudy swelling and vacuolar degeneration in kidney in 3^rd group more than the other group. The degeneration and atrasia of some ovarian follicles in ovary and increase congestion of the uterine blood vessels, focal areas of endometrial hemorrhages and hyperplasia of the endometrial lining epithelium of uterine horn associated with the increased of copper oxychloride /Kg. BW/day.

 Conclusively, these results indicated that fed contaminated ration with copper oxyhloride fungicide decreased the reproduction performance of female rabbits and make many deleterious effect on animal health.

Key words: Rabbits, Copper, fungicide, chromosomal aberration & pathological.

Copper oxychloride Cu (OH) Cl is one of agriculture fungicide which used commonly to prevent crop damage in the field and protect the harvest crops from deteriorations during storage or transport. There are many agriculture fields intensively applicated because of its low cost and good efficacy (ACP, 2007).

In animals, copper salts have moderate acute toxicity, with soluble salts being more toxic than insoluble ones. The data are limited but rats appear to be more tolerant to acute copper toxicity than other laboratory species Haywood, (1985). In sub chronic repeat dose toxicity tests, copper salts are associated with effects such as gastro-intestinal irritation and liver and kidney toxicity but no observed adverse effect level for copper sulphate of 16 mg/kg body weight/day in a 13-week study in rats (Herbert et al.,1993). Copper salts have been reported to have adverse effects on reproductive and developmental parameters like testes, seminal vesicles, uterus and ovaries have been reported at doses of et al., 1991). There are a few adequate data on chronic toxicity of copper salts. There is no evidence of direct carcinogenicity but different copper concentrations appear to have a modifying effect on tumors initiated by other agents (WHO, 2008). However, high levels of copper accumulate in the liver of rat associated with a high incidence of hepatocellular carcinoma (Masuda et al., 1992).

Chromosomal   abnormalities can result from either a variation in the chromosome number or from structural changes. These events may occur spontaneously or can be induced by environmental agents such as chemicals, radiation, and ultraviolet light. However, mutations are most likely due to mistakes that occur when the genes are copied as the cells are dividing to produce new cells (Cohen and Jon 2002).

Therefore, a little information is available on the effect of copper oxychloride fungicide on female’s administration on some reproductive aspects rabbits taking in consideration the changes that might occur in some relevant blood biochemical values, chromosomal changes and pathological alterations of genital organs. The objective of this study was to evaluate chromosomal aberration and pathological alteration effect in rabbits.

MATERIALS AND METHODS

Experimental work:

Forty five female rabbits divided into three groups (fifteen does each). The first group (control) was fed uncontaminated ration, the second group was fed contaminated ration with. Copper oxychloride (0.1184 g/Kg. BW/day as 1/20 LD₅₀) and third group was fed contaminated ration with copper oxychloride (0.2368g/Kg. BW/day as 1/10 LD₅₀) for sixty days. Three mothers and nine offspring were slaughtered from each group to obtain bon marrow for cytogenetic according to (Pirtskhelami et al., 2008), and testing liver, kidney, ovary and uterine horn for histopathological examination according to Cullig (1974).

Chromosomal analysis:

The animals were injected with 0.025% colchicines at dose of 0.01 ml/g intraperitoneally before slaughtering two hours to block the cells in metaphase. Animals were anesthetized with ether and processed for bone marrow sampling using the method described by Rabello-Gay and Ahmed (1980). The cells in bone marrow were flushed by pushing 2-3 times (0.9%NaCl) into the marrow cavity of femur and tibia. Cells were collected, left in (0.56% KCl) for 20 min in incubator at 37c˚ and centrifuged at 1000 rpm for 8 min. The supernatant was removed; the pellet was resuspended and fixed in 5 ml of methanol: acetic acid (3:1) for 20 min. Repeat the centrifugation and fixation two or three times. Finally, the cells were dropped on a clean wet slide and uniformly stained using a 5% Giemsa stain. The classification of aberrations was carried out as described by (Venitt and Parry 1984) and in the International System for Cytogenetic Nomenclature (ISCN 1985) From each slide 50 cells were scored under 1000x magnification to determine the frequencies of cell chromosome damaged. Aberrations were pooled as described by (Wise et al.1994). This is because deletions can only be unequivocally distinguished from achromatic lesions if the distal from centric fusion is displaced. Thus, pooling aberrations avoids artificial discrepancies between scorers due to different perceptions of the width of an achromatic lesion relative to the width of its chromatid (Wise et al.1994).  Accordingly, chromatid deletions and achromatic lesions were pooled as chromatid lesions. The frequency of chromosome aberrations was estimated on 50 metaphases.

Histopathological examination:

The organs (liver, kidney, ovary and uterine horn) were immediately removed and washed in saline solution. The organs were fixed in 10% phosphate buffered formalin. Following an overnight fixation, the specimens were dehydrated in ascending grades of alcohol, cleared in xylene and embedded in paraffin wax (58-60 C). Blocks were made and sectioned of 5 µm thickness with microtome. The tissue sections were stained with hematoxylin and eosin and observed under the light microscope.

Statistical analysis:

Analyses of variance (ANOVA) were carried out using the SAS software package (2008). Duncan^'s new multiple range tests were used to test the significance of the differences among means (Duncan, 1955).

RESULTS AND DISCUSSION

Chromosomal Investigation:

Sixty days of feeding forty five female rabbits on contaminated ration with two levels of Copper oxychloride ( 0.1184 g/Kg. BW/day as 1/20    LD₅₀ ) and (0.2368g/Kg. BW/day as 1/10 LD₅₀ ) were tested compared with the control group. The increased of the chromosomal aberrations were associated with the increased of the concentration of the copper oxychloride fungicide. We observed significant different among groups in NZW rabbits as shown in (Table 1) and the contamination with the fungicide induced significant increase (P <0.05) in total chromosomal aberrations among does and their offspring (Table 2).It may be due to accumulation of the heavy metals (copper oxychloride) in deferent tissues lead to damage of DNA as result of treatment. These results are in agreement with Alfredo Corona-Rivera et al., (2007) who observed a statistically significant increase of DNA damage in individuals exposed to 390 µg/ml of copper intake and Pirtskhelani et al., (2008), who found that oral dosage of copper oxychloride ( 1/2, 1/5, 1/10 LD50) in white mice induce significant increase (p<0,001) of chromosomal aberrations frequency (multiple fragments, lyses), genomic mutations (triploidy, tetraploidy) pathological mitosis (hollow metaphase, K-mitosis, adhesion of chromosomes) and destruction of interphase nucleuses (hollow nucleus).

 Table1: Chromosomal structural aberration as affect by copper oxychloride fungicide  (ˉx ­± se).

Values with different letters in the same row are significantly different (P<0.05).

50 cells were examined  per  animal.

The significant induction of chromosomal/ mitotic aberrations exposed to chromium compounds in present study indicated its genotoxic potential. The aberrant mitotic stages may have been the outcome of spindle poisoning that causes chromosome disturbances during mitotic cell division. Various heavy metals are known to induce chromosome breaks, fragments and micronucleus formation in plants and mammalian test systems(Knasmuller  et al., 1998), and their effects were emphasized to be the result of formation of DNA-DNA and DNA-protein cross-links.

In (Figure 1 ) we showed the most prevalent types of aberrations were gap formation (the lost area is shorter than diameter of chromatid), chromosome break (the lost  area is longer than the diameter of chromatid or equal to it), deletion chromosome (one chromatid short than its sister), centromeric attenuations (the arm of one chromatid was separated from its sister), centric fusion (the one chromosome is fusion with another chromosome by centromers), end to end associations (two chromosomes were joined at the proximal ends of the two chromatids) and ring chromosome (the ends of the two chromosome's arms were joined together) as a results to copper oxychloride fungicide treatment.

Wise et al., 1994 and Stearns et al., 1995, reported that various types of DNA damage occur in response to exposure to heavy metals (chromium

compounds) including DNA single strand breaks, DNA-protein crosslinks,

Table2: The difference responded between the does and offspring to the effect of copper oxychloride fungicide on chromosomal structural aberration (ˉx ­± se).

 Values with different letters in the same row are significantly different (P<0.05).  50 cells were examined per animal.

(a)                                                                                       (b)

(c )                                                                                 (d)

(e )                                                                                      (f)

 (Figure1): Metaphase spread of NZW rabbits fed on contaminated ration with copper oxychloride fungicide, showing (a): gap, (b): break, (c): deletion, (d): centromeric attenuation and centric fusion, (e): end to end and (f): ring.

Cr-DNA adducts, and DNA-DNA crosslinks. Sugiyama et al., 1991 and Manning et al., 1992, found the pathways for DNA damage by heavy metals in the presence of biological reducing agents appear to be highly complex. (Sugiyama et al.,1991 and Manning et al., 1992).

Histopathological changes:

The microscopic examination of the liver of rabbits fed ration contaminated with copper oxychloride (0.1184 g, Cu (OH) Cl. /kg.BW/day) as 1/20 LD50 for 60 days- revealed slight congestion of the portal blood vessels with hydropic degeneration of hepatocytes (H.D.H) as shown in Figure 2a. Also, samples in the third group (0.2368g Cu (OH)Cl  /kg.BW/day) as 1/10 LD50, showed hydropic degeneration of hepatocytes with inflammatory cellular infiltration of the hepatic parenchyma mainly lymphocytes. These results are in agreement with Haywood (1979) and Hore et al.(1997) who recorded that the signs of copper toxicity due to the most highly supplement diets in rate were early liver damage and pathological changes significantly and Singh KK. et al., (2010) who showed significant an increase in Cu concentration in the liver when recorded in fenvalerate intoxication of goats given orally 15mg Cu/kg./day for 270 days, and  Alfredo Corona-Rivera et al., (2007) who observed a statistically significant damage increase in individuals exposed to 390 µg/ml of copper.

The histopathological changes due to copper toxicity in the kidney of rabbits were appeared in multiple areas as cloudy swelling of the lining epithelium (C.S.L.E) of some renal convoluted tubules. These microscopical changes were observed in the examined kidney of rabbits that were fed on contaminated ration with copper oxychloride  (0.1184 g Cu (OH) Cl /kg. BW/day) as 1/20 LD50 for 60 days (the second group). But in the third group fed on (0.2368g Cu (OH) Cl /kg .BW/day) in fed as 1/10 LD50 for 60 days showed congestion of the lining epithelium of renal tubules in the form of cloudy selling and vacublar degeneration (Figure 2 b). These results agreed with those observed by Haywood (1985) and Hore et al. (1997) who showed that the pathological significant changes in the kidney of rats and Abdel-Hafez (1995) who observed, in NZW rabbits, that the kidneys were soft and there were a pathological changes in its tissues.

The trace elements of the fungicide produced considerable changes of the ovarian follicular structures and the corpora lutea. The interrelationships of morphological and functional changes with increased occurrences of trace elements in various tissues necessitate further investigation.

In the present study the microscopical examination of rabbit ovaries in the third group that given (0.2368g Cu (OH) Cl /kg. BW/day) as 1/10 LD50 for 60 days revealed histological changes degeneration and atrasia of some ovarian follicles (Figure 2 c). These results are in agreement with Bires et al. (1995) who showed, in sheep that fed diet contaminated with copper intoxication, the histological changes in number of ovarian follicles and increased occurrence of primary atretic follicles and Ahmed et al. (1998) showed that copper treated rats revealed absence of mature grafian follicles and decreased number of growing follicles. Niswender et al. (1985), Wolfenson &Orlyblum (1988) and Kliment & Zithy (1989) who showed that in rabbits treated with copper small and large follicles which originate from granulose and theca cells.

 Congestion of the uterine blood vessels- focal areas of endometrial hemorrhages and hyperplasia of the endometrial lining epithelium were seen in the examined uterine horn of treated rabbits (Figure2 d). These results agreed with finding of Bires et al.(1995) who showed that in ewes fed on high level copper diets, the copper (Cu) was concentrated in uteri and changes pathological endometrium and Ahmed et al. (1998) who showed that microscopical examination of uterus of copper treated female rats – the surface epithelial cells showed focal hyperplasia- uterine glands showed degenerative and necrotizing changes of their epithelium lining.

(a)                                                                                       (b)

                        (c )                                                                                        (d)

Figure 2: (a) Liver of rabbits fed on ration contaminated with fungicide, showing extensive hydropic degeneration of hepatocytes (H.D.H) and congestion of central veins (C.V). (b) : Kidney showing cloudy swelling of the lining epithelium of renal convoluted (C.S.L.E) tubules. (c) :Ovary, showing increased number of atretic follicles. (H&E X200) and (d): Endometriun, showing focal hyperplasia of the epithelium lining. (H&EX200) arrows.

Conclusively, it was concluded that, due to toxicity of copper oxychloride and the widely using of this compound in agriculture, the use of this compound must be restricted as been as possible. Liver and kidneys of copper oxychloride exposed animals were contained with the highest copper residue levels. Copper oxychloride fungicide is the etiological factors to chromosomal aberrations, which lead to mutagenic, teratogenic and carcinogenic effect on long term contamination of human food and animal feed. It also, produces irreversible damage to some internal organs such as liver and kidney. Periodical examination should be done for meat and other meat products as well as the edible organs and their load for heavy metals which also should be evaluated according to the international guide lines as a fruitful advice to delay environmental contamination.

REFERENCES :

Abdel-hafez, H. M. (1995).Toxicological studies of copper oxychloride fungicide on rabbits. M. V. Sci. Thesis, Medical jurisprudence Toxicology department, Faculty of Veterinary Medicine, Assiut University. Egypt.

Ahmed,W.M.; Desouky,H.M.; Amer,H.A.; Karima; Mhmoud,G.M. and Ahmed,Y.F. (1998). Effect of fungicide (Tri-Miltoxfort) on some reproductive aspects in female rats. Vet.Med.J., Giza, Vol.46,No.4B:833-847.

Alfredo Corona-Rivera, Patricia Urbina-Cano and Lucina Bobadilla-Morales1. (2007).  Protective  in vivo effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays J Appl Genet 48(4), pp. 389–396

Bires,j., Marcek,I., Barko,P.; Biresova,M. and Weissova,T. (1995). Accumulation of trace elements in sheep and the effects up on qualitative and quantitative ovarian changes. Vet.Human Toxicol., 37 (4) Augost.

Cohen, Jon. (2002). Sorting Out Chromosome Errors. Science, Jun. 21: 2164-2166.

Culling,C.F.A. (1974). Handbook of histological and histological techniques. Third edition Utterworth. London.

Evans,H.J. (1983). Effects on chromosomes of carcinogenic rays and chemicals. In   Chromosome Mutation and Neoplasoia. Pp. 253-279, Liss, New York.

Haddad, D.S., Al-Alousi, L.A. and Kantarjian, A.H. (1991). The effect of copper loading on pregnant rats and their offspring. Funct. Dev. Morphol. 1: 17-22.

Hamdoon,N.T. and Seddek,A.S. (1995). Chromosomal aberrations induced by aquatic weed killer (Region) in Oreochromis niloticus. Assuit Vet.Med.J. 32(64) :80-87.

Haywood,S. (1979). The effect of the sex of weaned rats on the accumulation of dietary copper in their livers. Journal of Comparative Pathology, 89(4) 481-487.

Haywood,S. (1985). Copper toxicosis and tolerance in rat. Changes in copper content of the liver and kidney. Journal of Pathology 145(2)149-158.

Hebert, C.D., Elwell, M.R., Travlos, G.S., Fitz, C.J. and Bucher, J.R. (1993). Subchronic toxicity of cupric sulfate administered in drinking water and feed to rats and mice. Fundam. Appl. Toxicol. 21: 461-475.

Hore,S. K.;S. K.;Chouhan, H. V.S.; Neelu, G.; Koley, K. M. and Gupta, N. (1997). Effect of long term exposure of (Mancozeb) on clinico-hemato-biochemical and pathological changes in rats. Indian Veterinary Journal 74:1, 26-28.

ISCN. 1985. An international system for human cytogenetic nomenclature. Cytogenet Cell Genet; 21:309-414.

Kliment,J.and Zithy,J.(1989). Morphogenesis of the genital system of New Zealand White rabbit females. 1. Developmental changes in ovarian follicles after birth. Pol′nohospodarstvo., 35:6, 555-566;7 ref.  

Knasmuller S, Gottmann E, Steinkellner H, Fomin A, Pickl C, Paschke A. 1998. Detection of genotoxic effects of heavy metal contaminated soils with plant bioassays. Mutat Res; 420:37-8.

Manning FCR, Xu J, Patierno SR. 1992. Transcriptional inhibition by carcinogenic chromate: Relationship to DNA damage. Mol Carcinog; 6:270-9.

Masuda, R., Yoshida, M.C., Sasaki, M., Dempo, K. and Mori, M. (1992). High susceptibility to hepatocellular carcinoma development in LEC rats with WHO (1982). Toxicological evaluation of certain food additives. WHO Food Additives Series, No. 17. Copper.

Niswender,g.d.; Schwall,R.H.; Fitz,T.A.; Farin,C.A. and Sawyer,H.R. (1985). Regulation of Luteal function in domestic ruminants: New concept. Recent prog. Horm. Res., 41:101-151

Permanent Crops Production, 2007. (ACP 303) 26, April 2007 URL: www.nou.edu.ng.

Pirtskhelami AG, Pirtskhelami NA, Gakhokidze RA, Bichilkashvili NV and Kalandiia EA. (2008).The influence of polyvitamincomplex polygene on mutagenic and cytotoxic effect of copper oxychloride in white mice.{Article in Russian, Georgian Med. News. 2008 Jun; (159): 44-47.

 Rabello G, and Ahmed A. (1980). A croniteril: in vitro cytogenetic studies in mice and rats. Mut. Res., 79: 249-255.   

Rojik, i.;Nemesok, J. and Boross, L. (1983). Morphological and biochemical studies on Liver, Kidney and gill of fishes affected by pesticides. Acta. Boil. Acad. Sci. Hung., 34:81-92.

SAS. (2008). Sas Users Guide; Statistics, ver. 6. 04, Fourth Edition, Sas Institute, Inc., Cary, Nc.

Singh KK, Gupta MK, Ram M, Singh V and Roy BK. (2010). Effect of chronic fenvalerate intoxication on tissue concentration of copper in goats and further exploration of its mechanism. Biol. Trace Elem. Res.(2010) Dec., 138 (1-3) : 163- 72. Epub. (2010) Feb.  27.

Stancliffe, T. D. and pirie (1971). The Production of superoxide radicals in the herbicide diquat FEBS Iett, 17:297-299.

Stearns DM, Kennedy LJ, Courtney KD, Giangrande PH, Phieffer LS, Wetterhahn KE. 1995. Reduction of chromium (VI) by ascorbate leads to chromium-DNA binding and strand-breaks in vitro. Biochem; 34:910-9.

Sugiyama M, Tsuzuki K, Ogura R. 1991. Effect of ascorbic acid on DNA damage, cytotoxicity, glutathione reductase, and formation of paramagnetic chromium in Chinese hamster V-79 cells treated with sodium chromate (VI). J Biol Chem; 26:3383-86.

Venitt S, Parry JM. 1984. Mutagenicity Testing: A Practical Approach, IRL Press, Oxford, Washington DC:353.

WHO (1993). World Health Organisation Guidelines for Drinking Water Quality, Volume 1 p42-43, Geneva

WHO (1999). Environmental Health Criteria 200: Copper. World Health Organization, Geneva, pp. 100-129.

WHO (2006). Guidelines for Drinking-Water Quality. Volume 1 Recommendations. First addendum to the third edition. World Health Organization, Geneva.

WHO (2008). Plan of work for the rolling revision of the WHO Guidelines for Drinking Water Quality. World Health Organization website: http://www.who.int/water_sanitation_health/gdwqrevision/en/index.html. Accessed July 2008.

Wise JP, Stearns DM, Wetterhahn, KE, Patierno SR. 1994. Cell-enhanced dissolution of carcinogenic lead chromate particles: Role of individual dissolution products in clastogenesis.Carcinog; 15:2249-54.

 Wolfenson, D. and Orlyblum (1988). Embryonic development, conception rate, ovarian function and structure in pregnant rabbits heat stressed before or during implantation. Anim. Reprod. Sci., 17: 289-270.

التغیرات الکروموسومیة و الأعراض المرضیة الناتجة عن أستخدام المبید الفطری أکسی کلورو النحاس السمی علی الأرانب

¹محمد فهمی سعد , ¹أیمن مصطفی سعید ، ²أیمن محمد حسن

¹قسم التکنولوجیا الحیویة بمعهد بحوث الأنتاج الحیوانی

²قسم تربیة الدواجن بکلیة الزراعة جامعة عین شمس

أجرى هـذا البحـث لدراسـة تأثیر تلوث مواد العلف المختلفة (الأعلاف الخضراء والمصنعة)  بالمبیدات الفطریة مثل (أوکسى کلورو النحاس) على حیوانات المزرعة حیث استخدم فى هذه الدراسة عدد 45 أنثى ناضجة من الأرانب النیوزیلاندى الأبیض فى عمر 5-6 شهور بمتوسط وزن 3 کجم وقد تم تقسیمها الى 3 مجموعات الأولى منها ضابطة وهذه غذیت على علیقة مصنعة خالیة تماما من أى تلوث بالمبید ، والمجموعة الثانیة غذیت على علیقة ملوثة بالمبید بواقع 0.1184 جم من المبید/ /کجم وزن حى للحیوان / یومیا (1/20 من الجرعة النصف ممیتة)، أما المجموعة الثالثة فقد غذیت على علیقة ملوثة بالمبید بواقع  0.2368جم / کجم وزن حى للحیوان/  یومیا أى حوالى1(/10 من الجرعة النصف ممیتة) وکانت نسبة ترکیز عنصر النحـاس فى هذا المبید الفطرى حوالى 50%  وقد استمرت التغذیة لمدة 60 یوما0

وبعد انتهـاء مدة التغذیة  تم ذبح 3 أمهات من کل مجموعة وکـذا ذبح عدد 3 أبنـاء لکـل أم أى أن العدد الکلى للحیوانات المذبوحة 27 حیوان  لدراسة التغیرات السیتوجینیة والهستوباثولوجیة .

أولا : الدراسة السیتوجینیة :

تم فحص 450 طور استوائى للأمهات و 1350 طور استوائى للنسل فـأوضحت الدراسة أن نسبة الاختلالات فى التراکیب الکروموسومیة کانت 2.71% ، 8.75% ، 12.17% فى المجموعات الثلاثة على الترتیب کما أوضحت الدراسة أن نسبة الاختلالات الکروموسومیة فى النسل کانت 6.42%.

ممـا سبق یتضح أن هناک زیادة معنویة فى نسبة الاختلالات الکروموسومیة نتیجة التعرض للترکیزات المختلفة من المبید الفطرى بین المجامیع وأن هناک تناسبا طردیا بین ترکیز المبید المستخدم وبین نسبة الاختلالات الکروموسومیة للأفراد.

ثانیا :الدراسة الهستوباثولوجیة:

تم أخذ عینات لکل من الأعضاء (الکبد – الکلیة – المبیض – الرحـم ) للدراسة الهستوباثولوجیة حیث أنه من خلال الفحص الهسـتوباثولوجى تلاحظ وجـود اصـابة ســائدة بالخــلایا الکبدیة ومعظــمها بؤرى ویصاحبـها أحیانا تفاعل خلوى للخـلایا وحیدة النواة وذلک فى المجموعة الثانیة . کماشـوهد أیضا فى المجموعة الثالثة زیادة فى تلیف الخلایا الکبدیة وزیادة فى التهاب بعض المناطق مصحوبا بنزیف دموى فى نسیج الکلیة وکانت خلایا الأنابیب الکلویة منتفخة وبها حبیبات واحتقان فى المنطقة المبطنة لجدر الأنابیب الکلویة وذلک فى کل من الجموعتین الثانیة والثالثة , کمـا لوحظ عند فحص المبیض أن هناک زیادة معنویة فى ضـمور فى الحویصلات المبیضیة الأولیة والثانویة وذلک فى الجموعتین الثانیة والثالثة کما شـوهد أیضا فى نفس المجموعتین زیادة معنویة فى احتقان الأوردة الدمویة المغذیة للرحم مع وجود التهابات فى النسیج الحمى وتضخم الغدد الرحمیة وغزو لخلایا مستدیرة للطبقة المبطنة للرحم.

التوصیة: یوصى البحث مراعاة عدم تعرض حیوانات المزرعة لأى تلوث من المبیدات المستخدمة ســواء فى الانتاج الزراعى أو الانتاج الحیوانى مع أخذ الاحتیاطات الکافیة لتجنب هذه الملوثات کما یوصى البحث العمل على استخدام البدائل العضویة فى مکافحة الفطریات فى الانتاج الزراعى. کما أنه یوصى بعدم أکل بعض الأعضاء الداخلیة للذبیحة واستبعادها مثل الکبد والکلیة .