histopathological observations in gut of clarias …...tissue preparation was carried out as...

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Histopathological observations in gut of Clarias gariepinus

juveniles exposed to aqueous extract of Piptadeniastrum

africanum bark

ADA, Fidelis Bekeh; EDET, Ekpenyong & EKPALI, Moses Esorekebere

Department of Fisheries and Aquatic Sciences,

Faculty of Agriculture and Forestry,

Cross River University of Technology, Obubra Campus,

Cross River State, Nigeria.

Correspondent author: [email protected]; [email protected], +2368068546835

ABSTRACT

The production of fish is lower than what is expected to satisfy the required 9.7 kg of fish per man per

year in Nigeria. Most of these problems of short supply of quantity and quality of fish is due to poor

aquatic environmental management. Such management practices include the negligence and wrong use of

fishing techniques. Piptadeniastrum africanum is used in villages to hunt fish while natural logs get into

water bodies as well as those introduced into water by logging activities. Aqueous bark extract of this

plant was administered to ten fish each at concentrations of 0.0 mg/L, 1.5 mg/L, 3.0 mg/L, 4.5 mg/L, 6.0

mg/L, 7.5 mg/L and 9.0 mg/L in triplicates to observe its histopathological effects on the gut of juvenile

Clarias gariepinus. Logit analysis shows that the LC50 48 hours, 72 hours and 96 hours were 9.0 mg/L,

7.5 mg/L and 6.0 mg/L respectively. The Piptadeniastrum africanum bark extract did not cause

significant variation in temperature and pH. But there was a significant decrease in dissolved oxygen

concentration in treated groups. The histomacrographs of tissues showed that there were no influence of

Piptadeniastrum africanum bark extract on the small intestine oesophagus and spleen. Histopathological

changes occurred in the liver. These changes include areas with accumulation of neutrphils, fatty changes,

and enlargement of cytoplamic vacuoles and displacement of nuclei to one side. The liver being the centre

of detoxification was affected. Mortality would have been as a result of alteration in the liver coupled

with depressed dissolved oxygen concentration resulting in inefficient cellular respiration.

Key words: Histopathology, gut, Clarias gariepinus juveniles, aqueous bark extract, Piptadeniastrum

africanum INTRODUCTION

Quality and quantity of fish supplied is influenced by several factors. These factors are divided into those

influencing capture and culture of fish. Before 1950, fish consumed in Nigeria were all from capture or

imported, because fish culture started only about this time (Dasuki, 2016). Fisheries management, if well

carried out is capable of meeting up the demand for fish, which is now higher than supply in Nigeria (Ada

et al., 2015 and Wu et al., 2010). At consumption rate of 9.7 Kg of fish per person per year, Nigeria needs

2.7 metric tons of fish to certify her population yearly. To achieve this, annual spending of about 0.6

billion US dollar is spent to import fisheries products to Nigeria (Dasuki, 2016). There is a vast expanse

of wet land capable of producing fish in Nigeria. Nigeria has a land area of 1.8 hectares that is suitable for

Aquaculture and an economic exclusive zone (EEZ) of 192,000 Km2 and 13,000 Km

2 of inland waters to

manage and produce fish for its citizens (Ita et al, 1085; Sikoki and Oyero, 1994; Dasuki, 2016 and

FAO/UNDP, 2015). Most of the areas have been depleted due to over fishing (Gordon, 2000; and Braich

International Journal of Innovative Agriculture & Biology Research 4(3):26-38, July-Sept., 2016

© SEAHI PUBLICATIONS, 2016 www.seahipaj.org ISSN:2354-2934

mailto:[email protected]


http://www.seahipaj.org/

27

and Jangu, 2015) or by outright pollution of the ecosystem (Kovinznych and Ubancikova, 1998; shlle and

oshisanya, 2013 and Okon., 2013; Svobodová et al., 1993).

Pollution of water is caused by both natural and anthropogenic activities. Anthropogenic sources of

pollution are from house hold waste, industrial effluents, agro-chemicals and mineral exploitations.

Timbering, apart from destroying water shade, does cause pollution in aquatic habitat. It is known that

immersion of timber product from a tree called small leaf (Piptadeniastrum africanum) leads to fish

mortality in water. Because of this, many local fishers use the plant product to hunt and kill fish in rivers

and streams. The modalities for killing the fish by this plant are not understood.

Clarias gariepinus juveniles were exposed to the aqueous bark extract of Piptadeniastrum africanum to

study its effects on the gut of the fish. This fish was selected for this experiment because of its ecological

and economic values. Nasar et al. in FAO (2002) state that it is to aquatic organisms’ research what

guinea pig is to terrestrial organisms in the laboratory. Not only that, the fish is widely cultured in Nigeria

and as Offem et al. (2010) and Awachie. and Ezenwaji (1998) revealed, is the third most cultured in the

world. It can easily be manipulated in captivity and it is one of the likely candidates to bridge the gap

between fish demand and supply given appropriate management and cultural practices.

MATERIALS AND METHODS

Collection of experimental fish

Apparently healthy looking Clarias gariepinus juveniles with a mean weight of 2.35 0.18g and mean

total length of 4.45 0.31cm were collected from a single population in CRUTECH fish farm Obubra.

They were transported in 50L Jerry cans by wheel barrow to the fisheries laboratory and acclimated for

twenty days before experimentation. They were fed twice a day with a commercial feed (COPPEN) at 2

% body weight at 0800 and 1600 h, respectively. All the fish were starved for 24 hours prior the

commencement and during the experiment to minimize the contamination of the test media.

Collection and preparation of Piptadeniastrum africanum

Piptadeniastrum africanum bark was obtained from Cross River National Park, Okwangwo Range –

Butatong Station Botanical Garden, Boki Local Government Area of Cross River State, Nigeria. The

fresh bark of Piptadeniastrum africanum was grinded to powder and then dissolve in water to obtain the

concentration.

Acute toxicity test

A preliminary range finding test was conducted for 24 hours to determine the toxic level of

Piptadeniastrum africanum using a standard method or procedure of American Public Health

Association, APHA (1981). The behavioural and biological characteristics of the test fish were monitored

and recorded.

Eighteen (35 cm x 26 cm x 26 cm) transparent plastic tanks of 60 litres capacity each were filled with 20

litres of stream water in which 10 Clarias gariepinus juveniles were batch – weighed with a top-loading

meter balance (SCAUT PRO. OHAUS) and distributed randomly in triplicate per treatment. There was no

change of water or feeding throughout the test. The concentrations of 0 mg/L, 1.5 mg/L, 3.5 mg/L, 4.5

mg/L, 6.0 mg/L, 75 mg/L and 9.0 mg/L of Piptadeniastrum africanum aqueous bark extract were used.

Fish mortality was monitored and recorded hourly for the first four hours. The inability for the fish to

respond to external stimuli was seen as an index of death. A part from the monitoring and recording of

fish mortality, the fish behaviours such as erratic swimming, air gulping and loss of reflex, discoloration

and moulting was also monitored. LC50 of Piptadeniastrum africanum was estimated by probit analysis

(Herwig 1979, USEPA 2000).

Ada et al. ….. Int. J. Innovative Agric. & Bio. Res. 4 (3):26-38, 2016

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Histopathological analysis of the guts

At the end of the experiment, one fish per replicate was degutted using a dissecting kit. Tissues such as

the gullet, stomach, small and large intestine, and liver were removed were stored in 10% formalin for

three days. Tissue preparation was carried out as described by Baskar (2014).

3.5. Water quality Water quality parameter monitoring was done prior to the experiment, during the experiment and after the

experiment. The parameters recorded include: pH, turbidity, Dissolved oxygen (DO) concentration and

temperature. pH was determined using a digital meter (MALLET TOLEDO 320). Dissolved Oxygen

Concentration was measured using a digital dissolved oxygen meter (WTW PH 90) (Ada et al., 2015 and

Jenway 1997) once a day at 8:30 am. Temperature was measured using mercury in glass thermometer,

which will placed or inserted in the medium inside the aquarium until reading will be taken or made.

Water quality parameters were analyzed using computerized, analysis of variance (ANOVA) (Ayotunde

et al., 2010).


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Table 1. Showing biological and behavioural responses in Clarias gariepinus exposed to Piptadeniastrum africanum aqueous bark extract

during a definitive experiment.

6 HOURS 12 HOURS 24 HOURS 36 HOURS 48 HOURS 72 HOURS 96 HOURS

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

0.0

mg

/L

3.0

mg

/l

30

.0 m

g/L

30

0.0

mg

/L

30

00

.0 m

g/L

30

00

0.0

mg

/L

Loss of

reflex

N N N N Y Y N N Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y

Molting N N N Y Y Y N N Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y

Discolorati

on

N N N N N N N N Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y

Air

gulping

N N N Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y

Erratic

swimming

N N N Y Y Y N N N Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y

Haemorrh

age

N N N N N N N N N Y Y Y N N N Y N N N N N N N N N N N N N N N N N N N N N N N Y Y Y

Swimming

upside

down

N N N N N Y N N Y Y Y Y N N N Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y N Y Y Y Y Y


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Figure 1 is showing the LC50 24 hrs, 48 hrs, 72 hrs and 96 hrs of Clarias gariepinus juveniles exposed to

Piptadeniastrum africanum in a definitive test experiment. The LC50 96 hours was 6.0 mg/L; 72 hours

was 7.5 mg/L; 48 hours was 9.0 mg/L and none of these concentrations killed 50 % of the fish within one

day. Key to histological slides: Histological changes in liver of Clarias gariepinus juvenile exposed to

Piptademastrum bark extract. Cv = central vein; s = sinusoid; H = hepatpcytefc = fatty changes; an =

accumulation of neutrophils; V = vacuole.

0.0 mg/L 1.5 mg/L 3.0 mg/L

4.5 mg/L 6.0 mg/L

7.5 mg/L

1 2

3

4 5 6

cv

s

cv

cv V

V


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Plate A1 to A6 are X10 and A7 is X40. At 0.0 mg/L, Normal hepatic tissue, showing normal hepatocytes

with no architectural defect; at 1.5 mg/L, Normal appearance of the general tissue structure of the liver

section. Also seen are hepatocytes and sinusoids without any form of abnormality. No inflammation is

seen. At 3.0 mg/L Section shows normal appearance of the liver with no fibrosis of the cells. The

hepatocyes appears with normal size with no noticeable inflammation. At 4.5 mg/L, liver section with

areas of accumulation of neutrophils, area of fatty changes At 6.0 mg/L shows liver section with some

prominent fatty changes resulting into large cytoplasmic vacuoles within some hepatocytes displacing the

nucleus to one side.At 7.5 mg/L Liver section shows numerous hepatocytes with fatty changes with the

central vein centrally located. At 9.0 mg/L, section shows liver tissue with prominent central vein and

hepatocytes mostly exhibiting fatty changes with the displacement of the nuclei and enlargement of the

cytoplasm.

9.0 mg/L

7

fc

an

6

4

5

1 2 3

0.0 mg/L

1.5 mg/L

3.0 mg/L

4.5 mg/L

6.0 mg/L 7.5mg/L


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Plate B showing Histological changes in eosophagus of Clarias gariepinus juvenile exposed to

Piptadeniastrum africanum bark extract All sections at X40. Normal oesophagus with no pathological

lesions observed at concentration of 0.0 mg/L. At 1.5 mg/L, oesophageal tissue was with no visible

abnormality. Dense regular connective tissue with finely arranged stratified squamous epithelia, general

tissue structure of the tissue not distorted at 3.0 mg/L. Connective which contains oesophageal glands that

secrete mucus to help ease the passage of swallowed food. At 6.0 mg/L, tissue with finely arranged

stratified squamous epithelial cell with no visible abnormality seen and at 7.5 and 9.0 mg/L, there were

dense regular connective tissues with no noticeable inflammation.

7

9.0 mg/L


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Plate C is showing Histopathological changes in small intestine of Clarias gariepinus juveniles exposed

to aqueous bark extract of Piptadeniastrum africanum. 0.0mg L Section shows normal intestine no

pathological lesion observed 1.5 mg/L Section shows normal appearance of the general tissue of an

intestinal tissue, no abnormality is seen on the villi, lamina proptia, goblet cells and on all other

component of the tissue. 3.0 mg/L Section shows a section of intestine with no damage to muscularis with

prominent goblet cells. 4.5 mg/L Section shows normal appearance of the general tissue structure of an

intestinal tissue, with normal appearance of the villi with slight noticeable damage to the muscularis

6.0mg/L Section of an intestinal tissue showing regular appearance of the general tissue with no

noticeable pathology. 7.5 mg/L Section of an intestinal tissue shows regular appearance of the intestinal

tissue, erosion of the mucosa lining 9.0 mg/L Section of an intestinal tissue shows normal villi, goblet

cell, muscularis, and mucosa lining.

6

7

5

4

3 2 1 0.0 mg/L 1.5 mg/L

3.0 mg/L

4.5 mg/L 6.0 mg/L 7.5 mg/L

9.0 mg/L

9.0 mg/L


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Plate D showing Histopathological changes in spleen of Clarias gariepinus exposed to aqueous bark

extract of Piptadeniastrum africanum. At 0.0 mg/L section had no pathological lesion observed. At 1.5

mg/L and 3.0 mg/L, there was spleenic tissue with normal appearance of the general tissue structure.

There is no visible treatment related lesion on the tissue No apoptotic debris was seen. At 4.5, mg/L

spleen shows no damages of cellularity. At 6.0 mg/L section shows no damage to cells. Germinal center

development within the lymphoid follicles appears regular. At 7.5mg/L section shows spleenic tissue with

no visible abnormality in the cell or the nuclei. Both the red pulp and the white pulp were not damaged

Figure 2 shows the significant variations (α < 0.05) that occurred in dissolved oxygen concentrations in

water containing Clarias gariepinus juveniles exposed to bark extract of Piptadeniastrum

africanum.Means that bear the same letters are the same while those with different letter are different.

7.5 mg/L

7.5 mg/L

6

3

3.0 mg/L

5

6.0 mg/L

4

4.5 mg/L

2 1.5 mg/L

1 0.0 mg/L


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Figure 3 shows the insignificant variations (α < 0.05) in temperatures of water containing

juveniles of Clarias gariepinus exposed to different concentrations of Piptadeniastrum

africanum bark extract.

Figure 4 shows the influence of Piptadeniastrum africanum on water pH of water containing Clarias

gariepinus juveniles. There were no significant changes between the treatments (α < 0.05)

DISCUSSION In the work of Ateulack et al. (2015), Piptadeniastrum africanum showed protective and healing effects

against stomach ulcers in Laboratory rats. These workers feed laboratory rats with 125mg/kg, 250mg/kg

and 500 mg/kg of food. The rats were allowed to eat freely (ad libitum). These workers found that in rats

where ulcer was induced, photomicrograph showed haemorhagic erosion, discontinuity in the lining of

epithelial cells as well as damage to submucosa. These are deviation from normal mucosa having small

trophic gland, mild hyperplasia with no edema. The ability of Piptadeniastrum to inhibit gastric ulcer lies

in the fact that it reduces acid secretion or improves acid neutralization in the gut (Deore et al., 2011).

Acid neutralization ability of Piptideniastrum extract has been attributed to the presence of alkaloids

known to be involved in acidity reduction (Ateulack et al. 2015). Similarly, livingstrong.com (2016), said

‘it may come as a surprise that hot pepper, rather than harming actually play beneficial role in helping to

protect the stomach’. Pepper may cause dyspepsia (stomach discomfort and increased acid reflux), but

does not cause any histological damage to the gut tissues. The presence of hot substances like pepper

(Capcicum) and small leaf (Piptadeniastrum) cause blood vessel dilation, leading to increased supply of


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blood , thus bicarbonate to neutralize acid. The damage of gastric mucosa is also related to the increase in

neutrophil infiltration into ulcerated tissues. These neutrophils inhibit ulcer healing mediated lipid

peroxidation through the release of cytotoxic factors such as superoxide and hydrogen peroxide. Thus the

removal of the infiltration of neutrophils by anti-inflammatory activity of the extract could improve a

recovery mechanism. Nitrogen II Oxide (NO) is important in maintaining of integrity of the mucosal

epithelium due to its enhancement in the production of mucus. No acid is also known for production of

growth factors that are responsible for epithelial cell multiplication. The ulcer preventive and protective

activity demonstrated in the present study provides a strong support for the traditional use of this plant in

the treatment of gastric ulcer. Further studies are required to confirm the exact mechanism underlining the

ulcer healing and protecting property of the extracts and identify the chemical constituents responsible for

it.

The none involvement of Piptadeniastrum africanum aqueous bark extract in using fish gut directly to kill

it means involvement of some other organs should be suspected. However, some histopathological

observations were seen in the liver, which is an associated organ of the gut. Ada et al. (in press)

investigated this extract on blood of Clarias gariepinus juveniles and also reported that it did not have

negative haematological changes on the fish. Since this extract presents ‘pepperish’ sensation, coupled

with its influence on dissolve oxygen concentration, it may be reasonable to suggest that fish death is

caused by deficiency in oxygen absorption through the gills and pathology of the liver. The liver as gut’s

associated organ was badly affected when exposed to bark extract of Piptadeniastrum africanum. The

liver is the central organ responsible for detoxification. The first step in the process of detoxification is

absorption of the toxin and then neutralization. If the poison is absorbed faster than detoxification

process, the result is the damaging effect as seen in plate A, where there were neutrophil accumulation,

fatty changes, enlargement of cytoplasmic vacuole, displacement of nuclei to one side at concentration of

4.5 to 9.0 mg/L (Velmurugan et al., 2009; Bosi et al., 2005; Samanta et al., 2016). As it was observed in

this work, oxygen concentration was significantly lower in the treated groups. Ayoola (2008) also observe

reduction in dissolved oxygen concentration in water containing Oreocrhomis niloticus exposed to

Glyphosate. But the mechanism of oxygen depletion is also not clear. On the other hand, Baskar (2014)

explained that ‘fish gills is compose of a large part of fish body that contacts the external environment and

play an important role in the gas and ion exchange between the organism and environment’. The gills and

the skin are continuously exposed to water and are the principal sites of activity of numerous toxic

substances. The gill surface, when stretched out is more than half of the entire body surface area. The

internal environment of fish is separated from the external environment by only a few microns of delicate

gill epithelium. Gill function is very sensitive to environmental contamination. The gills are therefore,

considered the most vulnerable organ to external pollutants owing to their direct contact with water

because of their lamellar epithelial penetration and movement of substances into blood circulation.

However, sub lethal concentrations of such chemicals are capable of causing pathological changes in

tissues of organisms. In a feature experiment, sub-lethal concentration of this extract be administered to

the fish for observations.

ACKNOWLEDGEMENT We are grateful to Mr igri, Igri I. of the Central Laboratory of the Faculty of Agriculture, University of

Calabar, Nigeria for carrying out histopatholgical processing of slides for this work.

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