Egypt. J. Exp. Biol. (Zool.), 10(2): 89 – 97 (2014) © The Egyptian Society of Experimental Biology

RESEARCH ARTICLE

El-Sayed T. Rizk Shadia F. Hamada Sherif, R. AbdElGhany Amany M. Ramez

HISTOPATHOLOGICAL AND BIOCHEMICAL INVESTIGATIONS ON THE FRESHWATER SNAIL PIRENELLA CONICA (BLAINVILLE, 1829) INFECTED WITH THE DEVELOPMENTAL STAGES OF HETEROPHYES SP.

ABSTRACT: The morphology and ultrastructure alterations INTRODUCTION: of the digestive gland, as well as biochemical Gastropod snails have been reported to changes of Pirenella conica infected with be vectors of many digenean larvae Heterophyes larvae were investigated in (Choubisa, 2008). These snails mostly snails collected from irrigation canals in Port- harbour various developmental stages of Said Province during the spring-summer trematodes such as sporocysts, rediae and periods of 2012-2013. The results showed cercariae. During multiplication and growth of great histological damages in the digestive these stages, they obtain nutrients from the gland of infected snails. Meanwhile, a infected tissues like digestive gland, significant decrease (P ≤ 0.05) in total protein gonads/ovotestis, and other visceral organs of and total lipid contents was detected while the the snail host. This leads not only to the glucose concentrations exihibited a small diverse histopathological changes in the range of variation. Uric acid was significantly snails, but also to many physiological increased in both the digestive gland and the disturbances (Huffman et al., 2009). cephalopedal mass. Infected snails showed The digestive gland of gastropod snails, elevated levels (P  0.05) of nitric oxide and called the hepatopancreas is among the the antioxidant enzymes; superoxide organs, which are most frequently attacked by dismutase (SOD) and glutathione S- the trematode larvae. Because of that, transferase (GST). numerous publications have been dealing with the pathological changes in the gland caused KEY WORDS: by the parasite invasion (Bakry, 2009; Choubisa et al., 2012). To our knowledge, Digestive gland, Histopathological, Pirenella none has examined the relationship between conica. Heterophyes sp. Pirenella conica and its larval trematode Heterophyes heterophyes. The digestive gland of molluscs is involved in extracellular and intracellular digestion of food material, absorption of CORRESPONDENCE: nutrients; storage of lipids, glycogen and it El-Sayed Taha Rizk plays also a major role in detoxification (Henry et al., 1989). The snail’s digestive Zoology Department, Faculty of Science, gland (hepatopancreas) was described by Tanta University, Tanta, Egypt Brackenbury (1999). Adam et al. (1995) E-mail: [email protected] investigated the morphology and ultrastructure of the digestive gland of Bithynia siamensis goniomphalus and its Shadia Faried Hamada alteration by infection with Opisthorchis Sherif, Rmadan Abd El -Ghany viverrini, while Bakry (2009) investigated the Amany Mohamed Ramez histological alterations in the digestive glands of Biomphalaria alexandrina infected with S. Zoology Department, Faculty of Science, mansoni. Most of the morphological changes Mansoura University, Mansoura, Egypt observed in the gland were attributed to the toxic effects exerted by parasites on the host's tissues (Humiczewska, 2004) and to mechanical damages infected by parasites ARTICLE CODE: 12.01.14 present in the hepatopancreas (Choubisa et al., 2012).

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90 Egypt. J. Exp. Biol. (Zool.), 10(2): 89 – 97 (2014)

On the other hand, the digestive gland Since information, about one snail of molluscs is involved in extracellular and host/larval trematode system cannot be intracellular digestion of food material, applied to other systems; the aim of this study absorption of nutrients; storage of lipids, is to screen the physiological alterations (total glycogen and plays a major role in proteins, total lipids, glucose, uric acid, nitric detoxification (Henry et al., 1989). The snail’s oxide level, and antioxidant capacity) in digestive gland (hepatopancreas) was Pirenell conica in response to the infection described by Brackenbury (1999). Adam et al. with Heterophyes larvae. Besides, (1995) investigated the morphology and morphological and ultrastructural alterations ultrastructure of the digestive gland of occurring mainly in the digestive-gland tissue Bithynia siamensis goniomphalus and its of the snail are demonstrated on the light and alteration by infection with Opisthorchis transmission electron microscope levels. viverrini, while Bakry (2009) investigated the histological alterations in the digestive glands MATERIAL AND METHODS: of Biomphalaria alexandrina infected with S. A total of 1250 freshwater snail mansoni. Pirenella conica; the intermediate host of Most of the morphological changes Heterophyes spp., were collected from observed in the gland were attributed to the irrigation canals in Port-Said Province using a toxic effects exerted by parasites on the hand wire mesh-scoop (Abou El-Azm, 1991) host's tissues (Humiczewska, 2004) and the during spring-summer periods of 2012-2013. mechanical damages caused by parasites They were brought to the laboratory and present in the hepatopancreas (Choubisa et maintained in separate aquaria containing al., 2012). fresh water and aquatic plants. The snails On the other hand, the larval trematode were successively examined for natural stages caused series of physiological trematode infection by exposure to artificial alterations as a result of living in their light at 28oC for cercarial shedding. Shells of intermediate hosts. The parasite larvae the snails were removed, and the soft tissues depend on the host for their survival, resulting were separated into the cephalopedal mass in a close physiological and metabolic and digestive gland. The fresh tissues were relationship between both parasite and host weighed, divided into two groups and (Pinheiro et al., 2009). maintained at −10°C until use. Small pieces of Many authors have reported various the digestive gland were immediately fixed in metabolic alterations in different parasite– 10% neutral formalin for 24 hours. host models. Shetty et al. (1992) noted a Biochemical determinations: significant reduction in the concentration of Total protein concentration was various lipids of the digestive gland-gonad estimated in the cephalopedal mass and complex of B. glabrata infected with E. digestive gland homogenates according to caproni. Humiczewska and Rajski (2005) Henry (1964) and expressed as milligram of stated that digestive gland lipid level of total protein per gram of wet tissue. Lymnaea truncatula infected with Fasciola The total lipids concentration in the hepatica was found to be almost halved. cephalopedal mass and in the digestive gland Pinheiro et al. (2009) stated that the intense homogenates was determined according to protein degradation results in an extensive ZÖllner and Kirsch (1962) and expressed as increase of the uric acid contents. Tunholi et milligram of total Lipids per gram of wet al. (2011) reported significant decrease in the tissue. concentrations of total proteins in the infected Biomphalaria glabrata snails, as well as an Glucose concentration was estimated increase in the nitrogenous products of according to Trinder (1969) and expressed as excretion. Pinheiro et al. (2001) reported milligram of glucose per gram of wet tissue. alterations in protein content and enzyme Glutathione-S-transferase activity was activities in mollusks tissues in response to spectrophotometrically measured using 1- larval trematodes infection. chloro-2,4-dinitrobenzene (CDNB) and Moreover, Barcia and Ramos-Martinez glutathione as described by Habig et al. (2008) pointed out that Nitric oxide production (1974) and expressed as µmol/min/g tissue. in molluscan haemocytes is elevated following Superoxide dismutase (SOD) was exposure to parasites or pathogens. Activities assayed in tissue extract by the procedure of of detoxifying and antioxidant enzymes are Nishikimi et al. (1972) and expressed as U/g affected by various invasions (Bender et al., tissue. 2007; Pinheiro et al., 2009). In addition, Uric acid concentration was estimated Vorontsova et al. (2010) found that the according to Barham and Trinder (1972) and activities of glutathione-S-transferase expressed as mol/g tissue. decrease, while the superoxide dismutase activity increases in the body homogenate of Nitric oxide (NO) concentration was the pond snail Lymnaea stagnalis. estimated according to the method of

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Rizk et al., Histopathological and Biochemical Investigations on Pirenella Conica Infected with Heterophyes Sp. 91

Montgomery and Dymock (1961) and and 43.36 mg/g, respectively (Table 1). In expressed as µmol/g tissue. the digestive gland homogenate of infected Statistical analysis: snails, protein content was significantly reduced by 45.7%. A similar reduction in The results obtained in the present work were protein concentration was observed in the evaluated by One Way ANOVA (analysis of variance) cephalopedal mass. test and post comparison was carried out with Duncan test. The results were expressed as means ± In comparison with uninfected snails, standard error (X±SE). The values of p≤0.05 were the data indicated a significant decrease in considered statistically significant. lipid levels (reached about 74%) in the infected snails. The total lipids in the Light microscopy: cephalopedal mass and the digestive gland For light microscopy, the specimens of non-infected snails were 394.71 and were dehydrated in a graded series of 624.98 mg/g, respectively, while in the ethanol, cleared i n xylene for 20 minutes (two infected ones the total lipids were 118.35 changes) then impregnated in paraffin wax and 137.34 mg/g of the cephalopedal mass o (three changes) at 60 C for two hours and and the digestive gland, respectively (Table embedded in paraffin wax. Sections 4-5 µm 1). thick were cut using microtome, mounted on It was illustrated that the glucose glass slide and stained with hematoxylin- content in cephalopedal mass (CP) and eosin. digestive gland gonad complex (DGG) in Transmission electron microscopy: infected snails didn’t show significant Specimens of the two groups were variation when compared with the values of fixed by immersion for 3 hr in 2.5% the uninfected control ones (Table 1). glutraldehyde buffered in 0.1 M sodium Tabl e 1 also shows uric acid o cacodylate buffer with pH 7.2 at 4 C and concentration in cephalopedal mass (CP) and o rinsed overnight in the buffer at 4 C. Then, digestive gland gonad complex (DGG) in both followed by post-osmofixation in 1% cold control and infected snails. The snails osmium tetroxide buffered in 0.1 M sodium infected with the larval trematodes exhibited cacodylate at pH 7.2 for 3 hr. Sections were an increase in uric acid concentrations from stained for 60 minutes in 1% uranyl acetate in 4.13 to 11.38 mg/g in the cephalopedal mass 25% ethyl alcohol. The tissues were then with a percentage of change reaching 64% dehydrated and rinsed three times in 30% and from 8.08 to 13 mg/g in the digestive ethyl alcohol and taken up for further staining gland with a change percentage reaching in lead citrate. Examination of the ultra-thin 38% compared with the control snails. sections and selected areas were The snails infected with the larval photographed using JEOL 100 CX electron stages of Heterophyes showed elevated microscope, in Electron Microscope Unit at levels of nitric oxide compared to uninfected the Faculty of Science, Alexandria University, snails (Table 1). The rise in the nitric oxide Egypt. levels increased from 22.53 to 58.31 µmol/g in the cephalopedal mass with a change RESULTS: percentage of 61% and from 25.87 to 76.26 The total protein contents in the µmol/g in the digestive gland with a change cephalopedal mass and digestive gland percentage of 66%. homogenates of uninfected snails were 64.67 Table 1. Total protein, lipid, glucose, uric acid and nitric oxide concentration in the cephalopedal mass (CP) and the digestive gland gonad complex (DGG) of uninfected (control) and infected Pirenella conica. CP DGG Groups Anova Uninfected (Control) Infected Uninfected (Control) Infected

Protein concentration Mean ± SE 64.67 ± 0.2 35.89 ± 1.6 43.36 ± 0.7 23.52 ± 0.1 (mg/g tissue) % of change 44.5* 45.7**

Lipids concentration Mean ± SE 394.71 ± 35.3 118.35 ± 6 624.98 ± 28.5 137.34 ± 9 (mg/g tissue) % of change 70* 78**

Glucose concentration Mean±SE 158 ± 38 123 ± 4.5 167 ± 34 109 ± 4.2 P≤0.05 (mg/g tissue) % of change 22.3* 34.8**

Uric acid Concentration Mean ± SE 4.13 ± 0.1 11.38 ± 1 8.08 ± 0.6 13 ± 0.8 (mg/g tissue) % of change 63.7* 37.8**

Nitric oxide Concentration Mean ± SE 22.53 ± 1.2 58.31 ± 1.9 25.87 ± 1.2 76.26 ± 2.1 (µmol/g tissue) % of change 61.3* 66** Percentage of change, (n = six for each group).*: percentage of change in the cephalopedal mass (CP) compared to control group, **:% of change in the digestive gland gonad complex (DGG) compared to control group. Significant change at p ≤ 0.05 compared to control group. ISSN: 2090 - 0511 On Line ISSN: 2090 - 0503 http://www.egyseb.org

92 Egypt. J. Exp. Biol. (Zool.), 10(2): 89 – 97 (2014)

Table 2 shows GST and SOD activities in µmol/min/g in the (CP) and the (DGG), the cephalopedal mass (CP) and the digestive respectively while in the infected snails the GST gland gonad complex (DGG) in both uninfected activities were 3.2 and 3.81 µmol/min/g, and infected snails. It is evident that the GST respectively. On the other hand, the SOD and SOD activities showed a significant activities was elevated from 7.9 to 15.7 U/g in increase in infected snails when compared with the cephalopedal mass and from 8.05 to 18.2 the uninfected ones. The levels of GST activity U/g in the digestive gland of uninfected and in the uninfected snails were 1.76 and 2.29 infected snails, respectively. Table 2. Glutathione S transferase (GST) and Superoxide dismutase (SOD) activities in the cephalopedal mass (CP) and in the digestive gland gonad complex (DGG) in both uninfected (Control) and infected snails CP DGG Groups Anova Uninfected (Control) Infected Uninfected (Control) Infected GST Concentration Mean ± SE 1.76 ± 0.1 3.2 ± 0.1 2.29 ± 0.1 3.81 ± 0.2 (µmol/min/g tissue) % of change 55* 60** P ≤ 0.05 SOD Concentration Mean ± SE 7.9 ± 0.4 15.7 ± 1.1 8.05 ± 0.3 18.2 ± 0.5 (U/g tissue) % of change 49.6* 55.7** Percentage of change, (n = six for each group).*: percentage of change in the cephalopedal mass (CP) compared to control group, **:% of change in the digestive gland gonad complex (DGG) compared to control group. Significant change at p ≤ 0.05 compared to control group. Histological and ultrastructural alterations: Healthy digestive glands could easily identify due to their dark orange coloration. The gland is composed of numerous oval tubules having lumen surrounded by single layer of epithelial cells. It shows two types of cells: digestive and secretory cells. Tubules are generally connected to each other by fine connective tissues. The whole mass of digestive gland enclosed in the outermost membrane, the tunica propria (Figs 1-3).

Fig. (3). Enlarged view of the (DGT) showing the digestive cells (DC), the secretory cells (SC) and secretory bodies (b). The digestive cells are characterized by their high degree of vacuolation. Apically, a dense microvillar borders visible. Beyond the microvilli, small electron-dense particles are seen. Toward the basal part of the cell, the diameter of vacuoles increases and the vacuoles contain flocky to granular material. The nucleus is oval without a distinct nucleolus. Mitochondria are distributed all over the cell (Figs 4-6).

Figs (1, 2). Light micrograph of the digestive gland of uninfected snails showing the digestive tubules (DGT) connected with a connective tissue (CT) and surrounded by the tunica propria (tp).

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Rizk et al., Histopathological and Biochemical Investigations on Pirenella Conica Infected with Heterophyes Sp. 93

Figs (4-6). A view of the digestive gland of uninfected snails. The digestive cells are characterized by several digestive vacuoles (DV), normal endoplasmic reticulum (RER), mitochondria (M), the microvillar border (MV) at the apical region and electron dense particles (EDP) beneath the microvilli. Figs (7-9). Secretory cell of a control snail. Note The secretory cells are located in groups of the triangular shape of the cell with a basal three to four cells. These cells are triangular with a nucleus (N), a secretory vacuole (SV), nucleus broad cell base. Secretory cells are recognizable by a (N), mitochondria (M), rough endoplasmic reticulum (RER), lysosomes (L), and golgi single secretory vacuoles and large nuclei situated at complex (GC). the base of the cell. The nuclei showed condensed chromatin patches. These cells are characterized by Infected digestive glands appeared the extended endoplasmic reticulum (RER) and large brown or grey in colour, swollen and friable. number of mitochondria, The RER is often located The infected tissue showed mechanical and near mitochondria (Figs 7-9). physiological damages. Mechanical damage was observed in the form of degenerating changes such as rupture of the tunica propria, irregular shape of digestive tubules and consequently enlargement of inter-tubular areas, as well as complete or partial destruction of tubules, (Figs 10-13). Physiological damage was observed in the form of loss of tissue integrity and necrosis of epithelial cells in heavily infected digestive gland (Fig. 13). The sporocysts and cercariae permeated the structure and were apparent to the naked eye. They found under the tunica proporia. The variety of histopathological changes in digestive glands induced by larval digenean trematode parasites was found to be dependent upon the severity of infection.

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94 Egypt. J. Exp. Biol. (Zool.), 10(2): 89 – 97 (2014)

Figs (10-13). Light micrographs of the infected digestive gland showing the damaged tubules (ADGT) surrounded by the larval stages of the parasite mainly cercariae (C) and sporocysts (SP). In digestive cells of infected animals, the vacuoles size increases and their contents become more in homogeneous. The microvilli decreased dramatically in number. Fragmentation of the endoplasmic reticulum, deformities of the mitochondria and autolysis of the cell were observed due to the ruptured vacuoles and lysosomes. The early stages of the developing sporocysts start to appear resulting in the increased number of the lysosomes. The mature sporocysts appeared in advanced cases of infection (Figs 14-17).

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Rizk et al., Histopathological and Biochemical Investigations on Pirenella Conica Infected with Heterophyes Sp. 95

The secretory cells of infected animals lost their shape and formed irregular cytoplasmic protrusions reaching the lumen. The number of mitochondria decreased and the endoplasmic reticulum became degranulated and formed a whorl-like structure. (Figs 18-20).

Figs (18-20). Ultrastructural alterations within the secretory cell in the digestive gland of infected animal. Note the autolysis and the irregular cytoplasmic protrusions (CP) towards the lumen (Lu), degenerated mitochondria (DM) and lysosomes (L). The RER become degranulated and forms a whorl-like (wh) structure.

DISCUSSION: It seems that there is an agreement between many authors in considering the intermediate host snails to show multiple Figs (14-17). Ultrastructural alterations within defensive behaviours in response to the digestive cells of an infected animal showing the enlarged digestive vacuoles (DV) with ruptured stress associated with the infection with larval vacuole membranes (arrows), fragmented trematodes. A number of complex interactions endoplasmic reticulum (RER), deformed exist between Heterophyes larvae and their mitochondria (M) and golgi complexs (GC) and molluscan intermediate host (Bayne, 2009; ruptured lysosomes (L). Note the increased no. Pinheiro et al., 2009). of lysosomes (L) and increased lipid droplets (LD) and sporocysts (SP) at the sites of The present study showed a considerable infection. decrease in the total protein content. These ISSN: 2090 - 0511 On Line ISSN: 2090 - 0503 http://www.egyseb.org

96 Egypt. J. Exp. Biol. (Zool.), 10(2): 89 – 97 (2014) abnormalities agree with the data of Pinheiro et using different snails have highlighted larval al. (2009) who stated that the total protein was trematodes ability to raise NO output. Brunet reduced by 80.01% and 76.42% in the (2001) found that NO limits parasite hemolymph and digestive gland respectively of development not only in vertebrate definitive but the intermediate snail host Lymnaea columella also in invertebrates intermediate hosts. infected with the digenean Echinostoma The mechanical damages of the digestive paraensei larvae. Schnell et al. (1985) have tubules appear to be the cumulative effect of also reported reduction in proteins and amino the larval movement, feeding, growth and their acids. It seems that the high consumption of multiplication. The physiological changes as carbohydrates as an energy source by the larva autolysis and necrosis are possibly the result of depletes the snail glycogen deposits. The host the release of proteolytic enzymes from the must utilize proteins as an alternative substrate ruptured digestive cells and enzymatic secretion source to obtain energy for its metabolism. and metabolites from trematode larvae. This is Pirenella lipid levels might be expected to supported from previous reports of certain snail decrease with infection as has been observed species. It has been discussed in earlier studies by Humiczewska and Rajski (2005) in Fasciola that redial stages cause more mechanical and hepatica-infected Lymnaea snails relation. They physiological damage when compared to stated that the loss of lipids in the digestive sporocysts because of their mouth and gland of the infected snails pointed at locomotory organs while their pharyngeal mobilisation of lipid energy reserves to glands and gut contribute for the physiological compensate for the deficiency of carbohydrates, damages. It has also been observed that rediae used by these parasites. engulf the host’s digestive cells and utilize the Infection by trematodes can interfere with hydrolases for their extra cellular digestion the endocrine system of the snail host (de Jong- (Choubisa, 2008). Brink et al., 1988), and this may explain the Choubisa et al. (2012) reported that balance in the glucose concentration observed. rediae engulf the host’s digestive cells and Thompson and Lee (1986) stated that the utilize the hydrolases for their extra cellular glucose concentration in the hemolymph of digestion. Besides, other contributory factors snails is very precisely regulated by can be parasitic secretions and excretory homeostatic mechanisms, which maintain the products that produce toxic effects. glucose level in a small range of variation. Mechanical pressure caused by the The uric acid content had increased by infiltration of rediae and the movement of 63.7% in the cephalopedal mass and by 37.8% cercariae leads to shrinkage of cells, and in digestive glands of infected snails. Tunholi et eventually to a decrease in their number and of al. (2011) observed similar increases in the uric the gland tubules. Porter et al. (1967) reported acid concentrations in the haemolymph of B. the destruction of the digestive gland of glabrata that infected with E. paraensei Oxytrema siliqua by the extrinsic mechanical miracidia. Probably the increased concentration pressure caused by Nanophyetitssa lmincola. of nitrogenous products results from the Porter (1970) reported a similar situation in increased catabolism of proteins to meet the Flumenico lavirens infected with infected snail’s energetic requirements. Plagioporusvirens. Both of whom referred the The parasitic infection caused significant digestive gland destruction to the accumulation increase in NO production. Previous studies of waste products.

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Oswaldo Cruz, 96(8): 1161–1164. دراﺳﺎت ھﺴﺘﻮﺑﺎﺛﻮﻟﻮﺟﯿﺔ وﺑﯿﻮﻛﯿﻤﯿﺎﺋﯿﺔ ﻋﻠﻰ ﻗﻮﻗﻊ اﻟﻤﯿﺎه اﻟﻌﺬﺑﺔ ﺑﯿﺮﻳﻨﯿﻠﻼﻛﻮﻧﻜﺎ اﻟﻤﺼﺎب ﺑﺎﻷطﻮار اﻟﯿﺮﻗﺎﻧﯿﺔ ﻟﻄﻔﯿﻞ ھﯿﺘﯿﺮوﻓﯿﺲ اﻟﺴﯿﺪ طﻪ رزق*، ﺷﺎدﻳﺔ ﻓﺮﻳﺪ ﺣﻤﺎدة**، ﺷﺮﻳﻒ رﻣﻀﺎن ﻋﺒﺪ اﻟﻐﻨﻰ**، أﻣﺎﻧﻲ ﻣﺤﻤﺪ راﻣﺰ** * ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻛﻠﯿﺔ اﻟﻌﻠﻮم، ﺟﺎﻣﻌﺔ طﻨﻄﺎ، ﻣﺼﺮ ** ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻛﻠﯿﺔ اﻟﻌﻠﻮم، ﺟﺎﻣﻌﺔ اﻟﻤﻨﺼﻮرة، ﻣﺼﺮ ﺗﻢ ﻓﻲ ھﺬه اﻟﺪراﺳﺔ دراﺳﺔ اﻟﺘﻐﯿﺮات اﻟﻈﺎھﺮﻳﺔ اﻟﯿﻮرﻳﻚ ﻓﻲ ﻛﻞ ﻣﻦ اﻟﻐﺪة اﻟﮫﻀﻤﯿﺔ واﻟﻜﺘﻠﺔ اﻟﺮأﺳﻘﺪﻣﯿﺔ. واﻟﺘﺮاﻛﯿﺐ اﻟﺪﻗﯿﻘﺔ ﻟﻠﻐﺪة اﻟﮫﻀﻤﯿﺔ وﻛﺬﻟﻚ اﻟﺘﻐﯿﺮات أظﮫﺮت اﻟﻘﻮاﻗﻊ اﻟﻤﺼﺎﺑﺔ زﻳﺎدة ﻓﻰ ﻣﺤﺘﻮى ﻛﻞ ﻣﻦ أﻛﺴﯿﺪ اﻟﺒﯿﻮﻛﯿﻤﯿﺎﺋﯿﺔ ﻟﻘﻮاﻗﻊ ﺑﯿﺮﻳﻨﯿﻠﻼ ﻛﻮﻧﻜﺎ اﻟﻤﺼﺎﺑﺔ ﺑﯿﺮﻗﺎت طﻔﯿﻞ اﻟﻨﯿﺘﺮﻳﻚ وﻛﺬﻟﻚ زﻳﺎدة ﻓﻲ ﺗﺮﻛﯿﺰ اﻹﻧﺰﻳﻤﺎت اﻟﻤﻀﺎدة ﻟﻸﻛﺴﺪة اﻟﮫﯿﺘﯿﺮوﻓﯿﺲ واﻟﺘﻲ ﺗﻢ ﺗﺠﻤﯿﻌﮫﺎ ﻣﻦ ﻣﺤﺎﻓﻈﺔ ﺑﻮرﺳﻌﯿﺪ (ﺟﻠﻮﺗﺎﺛﯿﻮن ﺗﺮاﻧﺴﯿﻔﯿﺮﻳﺰ وﺳﻮﺑﺮ أﻛﺴﯿﺪ دﻳﺴﻤﯿﻮﺗﯿﺰ). (ﻗﺮﻳﺔ ﺷﻄﺎ ﻣﻠﺢ) ﺧﻼل ﻓﺼﻞ اﻟﺮﺑﯿﻊ إﻟﻰ ﻓﺼﻞ اﻟﺼﯿﻒ ﻋﺎم 2012-2013. أظﮫﺮت اﻟﻨﺘﺎﺋﺞ ﺣﺪوث ﺗﺪﻣﯿﺮ واﺿﺢ ﻟﻠﻐﺪة اﻟﮫﺎﺿﻤﺔ ﻟﻠﻘﻮاﻗﻊ اﻟﻤﺼﺎﺑﺔ. و ﻛﺬﻟﻚ أوﺿﺤﺖ اﻟﻨﺘﺎﺋﺞ ﻧﻘﺺ ذو اﻟﻤﺤﻜﻤﻮن: دﻻﻟﺔ إﺣﺼﺎﺋﯿﺔ (P≤0.05) ﻓﻲ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻰ وﻛﺬﻟﻚ اﻟﺪھﻮن، ﺑﯿﻨﻤﺎ ﻛﺎن ھﻨﺎك ﺗﻐﯿﺮات طﻔﯿﻔﺔ ﻓﻲ ﺗﺮﻛﯿﺰ اﻟﺠﻠﻮﻛﻮز. أ.د. ﻣﺤﻤﺪ ﺣﺴﻦ ﻣﻨﺎ ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻋﻠﻮم طﻨﻄﺎ وأﻳﻀﺎ ﻛﺎن ھﻨﺎك زﻳﺎدة ذات دﻻﻟﺔ إﺣﺼﺎﺋﯿﺔ ﻓﻲ ﺗﺮﻛﯿﺰ ﺣﻤﺾ أ.د. ﻋﺒﺪ اﻟﺮﺣﻤﻦ ﺑﺸﻄﺮ ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﯿﻮان، ﻋﻠﻮم اﻟﻘﺎھﺮة

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