INTRODUCTION
The
liver, gall-bladder, pancreas and swim bladder are the accessory organs of the
digestive tract and each of the parts plays a vital role to the overall growth
and development of fish (Ali, 2015). The fish liver is a key organ which
controls many functions and plays an important role in fish physiology, both in
anabolism as in catabolism (Claudemir, Renata, Maria, Carlos & Irene,
2014). Fertilizers have considerably contributed to increase in food production
and food security, hence over the years, different type of fertilizers are used
to improve crop production. Fertilizer application whether organic or inorganic
has over the years been essential for increase in agricultural output. They
play an essential role in modern agricultural production, however, when wrongly
used can cause imbalance within the environment (Gupta & Kumari, 2021).
Study by Han, An, Hwang, Kim & Pank (2016) and Rosal, Solanic, Agan,
Mondea, Villa & Sanchez (2021) shows that inorganic fertilizers are
relatively inexpensive, have high nutrient contents, and are rapidly taken up
by plants however, the use of excess inorganic fertilizer can result in number
of problems such as histological and histochemistry changes in fish anatomy. The
effects of Urea fertilizer on fish have been reported by Asuquo, Essien-Ibok
& Abiaobo, (2016); Ajima, et al., (2017); Gupta & Kumari, (2021);
Rosal, et al., (2021). Studies have been documented on the liver of fish
(Ikpegbu, Nlebedum & Ibe, 2014). However, despite decades of agricultural use
of Urea fertilizer and widespread detection of this chemical in freshwater, no
data is available on the effect of sub lethal concentrations of Urea fertilizer
on the histology and histochemistry of the liver of African catfish (C. gariepinus) exposed to sub-lethal
concentrations of Urea fertilizer. Hence,
this study seeks to investigate changes in the histology and
histochemistry of age-related African catfish (Clarias gariepinus) liver
exposed to sub-lethal concentrations of Urea fertilizer.
MATERIALS AND METHODS
The
study on histology and histochemistry of the liver of age-related African
catfish exposed to sub-lethal concentrations of Urea fertilizer was
investigated in the Applied Hydrobiology and fisheries Laboratory, Department
of Zoology, University of Jos, North Central, Nigeria. One hundred each of
apparently healthy C.gariepinus
fingerlings, juveniles and adults mixed sex with mean weight 4.22±0.15;
22.67±1.10 and 643.96±48.75g respectively, mean total length 8.77±0.25;
12.80±0.21 and 38.93±0.39cm respectively and mean standard length 7.60±0.21; 10.63±0.24
and 24.93±0.45cm respectively were purchased from a reputable fish farm in Jos,
Plateau State, North Central Nigeria. The fish were transported in an aerated
plastic containers to post graduate Hydrobiology and fisheries laboratory,
Department of Zoology, University of Jos, Nigeria. The fish were acclimated for
two weeks in plastic containers separately according to age groups. During the
acclimation period, fish were fed with commercial pelleted feed (skretting;
crude protein- 42%, crude fat- 10%, crude fibre- 3.2%, ash- 7%).
Source and types of
Urea fertilizer;
three different types of Urea fertilizer: Indorama Urea - N46%; Dangote Urea -
N46% and Notore Urea - N46%were procured from Plateau Agricultural Development
Program (PADP).
Source and types of
fish feeds;
two types of foreign fish feeds: Copen and Skretting were procured from
reliable marketers.
Solubility test: Five grams of the
different types of Urea fertilizers; A B C were dissolved in five liters of
water and stirred with a glass rod. Each of the three Urea types procured above
dissolved within two minutes.
Stability test for
fish feeds:
Five grams of feed procured; A and B were dropped in five liters of water and
observed for thirty minutes to
determine which one is more stable in water for use in feeding the experimental
fish throughout the research period.
Preparation of stock
solution:
The chosen Urea fertilizer was weighed into different sub lethal concentrations
as in Ajima et al. (2017); 0.75g/L, 1.50g/L and 3.00g/L. One kilogram each of
the test feed (skretting) according to
age of fish was poured into each 0.75g/L, 1.50g/L and 3.00g/L concentrations of
Urea fertilizer and enable the feed to absorb the Urea fertilizer solution for
30 minutes, stirred with a glass rod to obtain a homogenous mixture then sieved
out and allowed to dry under laboratory
condition. Each of the dried feed was
labelled (0.75g/L, 1.5g/l and 3.00g/l) accordingly.
Experimental Design: The experiments
were conducted in twenty-four (24) plastic bathes according to age. A total of
three test concentrations; 0.75g/L, 1.5g/l and 3.00g/l of fish feed each for C. gariepinus fingerlings, Juveniles and
adults and control each duplicate replicate were labelled A1, A2,
B1, B2, C1, C2, F1, F2
each for the three age groups. The 24 containers were grouped into eight
according to age of fish labelled and randomly distributed on a platform in the
laboratory.
Experimental
Procedures:
Ten (10) apparently healthy acclimated fish were exposed to each of the
aforementioned test concentrations in the 24 plastic containers; C. gariepinus age groups and filled with
dechlorinated water. The test fish were fed three times a day for 62 days
within which behavioral changes were observed. The test fish were not fed 24
hours a day prior to commencement of the experiment.
Determination of
water quality parameters: Water quality parameters were determined bi-weekly throughout
the exposure period using the methods of APHA. (2005).The water quality
parameters monitored were; temperature, free carbon dioxide, dissolved oxygen,
hydrogen ions (pH), alkalinity, nitrite, ammonia and nitrate.
Histological Processing of Liver Tissues of C. gariepinus age group
Liver tissues were
histologically processed according to method by Drury and Wallington (1980). Liver
tissues of 5mm thickness were obtained by trimming the tissues using sharp
razor blade. Thereafter, trimmed liver tissues were histologically processed.
Briefly, trimmed tissues were dehydrated in graded concentrations of alcohol;
70% for 1 hour, 90% thrice for 1hour and two times in absolute alcohol (100%)
for 1 hour each. This was succeeded by clearing of dehydrated tissues in xylene
for 2 hours each and then embedded in paraffin wax at 60°C. The
waxed tissue blocks were then sectioned at 5μm thickness using rotary
microtome (Leica, USA). Sections obtained were mounted on clear albuminized
slides subsequent to floating on a warm water bath and then dried in an oven
and stained with haematoxylin and eosin (H & E). The stained tissues were
later viewed under light microscope (Olympus, China) for variation in
parenchymal histoarchitectural changes in different groups( Ikpegbu Nlebedum,,
Nnadozie & Agbakwuru, 2012;
Thayappan, Maghil, Annadurai & Narayanasamy, 2014; Pollyanna, Debora,,
Analucia, Sirlene, Jerusa, Alex & Jeneri,
2015)..
Histochemistry
of the Liver of C. gariepinusage
groups
The
periodic acid Schiff (PAS) technique was carried out using procedure of
Blachall and Daisley (1973)and Omirinde,
Olukole and Oke (2021). Waxed sections of various segments of liver from the
different cat fish groups were dewaxed in xylene for 5 minutes and rinsed
consecutively in 100%, 96% and 70% alcohol for 1minute each. This was followed
by placing rinsed tissues in distilled water and subsequently treating with
undiluted periodic acid for 10 minutes. The treated tissues were washed in
eight changes of distilled water, exposed to Schiff's solution for 1hour and
washed in running tap water for 10 minutes. The nuclei were distinctly stained
with Lilly Mayer’s heamatoxylin for 1 min and further differentiation was
avoided. Bluing of the tissues was done under tap water for 10 minutes. This
was followed by tissue dehydration in 96% and 100% alcohol, cleared in xylene
and mounted in Entellan. When viewed under microscope, parts of the tissue that
were positive for glycogen stained magenta while the nuclei stained bluish. Furthermore, the respective photomicrographs of the PAS‑stained
slides were quantified using Image J software (NIH, Bethesda, MD, USA).
Statistical Analysis
Two-way analysis of variance (ANOVA) was used
to evaluate significant differences across groups and the values of P<0.05
was considered significant. A Turkey post hoc test was further used to evaluate
the significant differences between and within groups. Statistical package for
social sciences (SPSS) version 17.
RESULTS
The hepatic histo-morphology of the control
groups of C. gariepinus fingerlings,
juvenile and adult is devoid of lesion and characterized by distinct central
vein (cv), sinusoidal spaces (red arrow), intact hepatocytes with regular
outline, roundish nucleus and substantial cytoplasm (Plates. 1A, 2A & 3A).
Similarly, the liver of all the age category of fish exposed to 0.75 g/L of
Urea fertilizers shared similar appearances with their respective controls (Plates
1B, 2B & 3B). However, 1.5 and 3.0 g/L concentrations of Urea fertilizers
precipitated moderate to severe histopathological lesions (central veinous
congestions, cellular infiltration, hepatocellular degeneration and necrosis)
in their respective liver parenchyma (Plates 1, 2 & 3 C-D).
The
periodic acid–Schiff (PAS) positive areas were mainly
demonstrated within the cytoplasmic hepatocyte and in the boundary area between
hepatocytes across all the different age-groups of C. gariepinus exposed to concentrated grades of Urea fertilizer (Plates
4-6). The PAS intensity values in the C. gariepinus fingerlings and adult liver decreased significantly
(p<0.05) with the increasing grades of Urea fertilizer concentration (Table
1). However, there was no significant difference (p>0.05) in the PAS
intensity of the liver of C. gariepinus juvenile
exposed to different grades of Urea fertilizer concentration compared to the
control. The trend in liver PAS intensity variation across the age groups of C. gariepinus exposed to Urea fertilizer
initially appeared to significantly increased (p<0.05) from C. gariepinus fingerlings to juvenile
and then significantly reduced in the C.
gariepinus adult (Table 1).
Plate 1. Photomicrographs of
the Liver of Clarias gariepinus Fingerlings
Exposed to Graded Concentrations of Urea Fertilizer. A. Control: The
liver histo-architecture is devoid of lesion and typified by distinct central
vein (cv), sinusoidal spaces (red arrow), intact hepatocytes with regular
outline, roundish nucleus and substantial cytoplasm (black arrow). B. 0.75
g/L of UF: Has no visible lesion C. 1.5 g/L of UF: Moderate central
venous congestions (black arrow), diffuse hepatocellular degeneration (red
arrow) and moderate cellular infiltration (white arrow). D. 3.0 mg/L of UF:
Marked hepatocellular degeneration (black arrow). Magnification: X400; Stain:
Haematoxylin and Eosin.
Plate
2. Photomicrographs of the Liver of ClariasgariepinusJuvenile Exposed to Graded Concentrations of Urea
Fertilizer. A. Control: Normal histological appearance (distinct central vein
(cv) and roundish nuclei (black arrow) within cytoplasm. B. 0.75 g/L
of UF (Urea Fertilizer): No lesion observed.
C. 1.5 g/L of UF: Moderate central
venous congestions (cv) and diffuse hepatocellular necrosis (black arrow). D.
3.0 g/L of UF: Diffuse hepatocellular
necrosis (black arrow). Magnification: X400; Stain: Haematoxylin
and Eosin
Plate
3.
Photomicrographs of the Liver of Clariasgariepinus Adult Exposed to Graded Concentrations of Urea Fertilizer. A.
Control: The liver histo-architecture appeared normal and characterized by
intact hepatocytes with regular outline, roundish nucleus, substantial
cytoplasm (black arrow) and distinct portal vessel (pv). B. 0.75 g/L of Urea
Fertilizer (UF): Share similar histo-architecture with the control. C.
1.5 g/L of UF: Moderate central venous (cv) and sinusoidal congestions
(white arrow) as well as peri-portal hepatocellular degeneration (black arrow).
D. 3.0 g/L of (UF): Severe hepatic degeneration (white arrow) and marked
hepatocellular infiltration (black arrow). Magnification: X400; Stain:
Haematoxylin and Eosin.
Plate 4. PAS staining of the
Liver of Clarias gariepinus Fingerlings Exposed to Different
Concentrated Grades of Urea Fertilizer. A. Control B. 0.75 g/L C. 1.5 g/L D.
3.0 g/L. Also, note the demonstration of periodic
acid–Schiff (PAS) positive areas within the cytoplasmic hepatocyte and in the
boundary area between hepatocytes (white arrow). Magnification: X400.
Plate 5. PAS
Staining of the Liver of Clarias gariepinus Juvenile Exposed to
Different Concentrated Grades of Urea Fertilizer. A. Control B. 0.75 g/L C. 1.5
g/L D. 3.0 g/L. Note the demonstration of PAS positive areas within the
perivascular tissue in A, B & D (white arrow) as well as in the boundary
area between hepatocytes in C (black arrow).Magnification: X400.
Plate 6. The PAS Staining of
the Liver of Clarias gariepinus Adult Exposed to Different Concentrated
Grades of Urea Fertilizer. A. Control B. 0.75 g/L C. 1.5 g/L D. 3.0 g/L. Note the demonstration of PAS positive areas within the
perivascular tissue and between hepatocytes in A (white arrow and arrowhead),
within cytoplasmic hepatocytes in B & C (white arrow) and inter-hepatocytes
in D (white arrow). Magnification: X400.
Table 1: PAS Staining Intensity in the Liver of
the Different Age Groups of Clarias gariepinus Exposed to Concentrated
Grades of Urea Fertilizer.
|
|
|
Concentration of
|
Urea fertilizer(g/L)
|
|
|
|
Organ PAS
Intensity (%Area)
|
Age
grouping
|
Control
|
0.75
|
1.50
|
3.00
|
|
Liver
|
Fingerling
|
72.92 ± 2.71a#
|
58.43 ± 3.00b#
|
46.69 ± 1.22 c##
|
47.64 ± 1.15c##
|
|
|
Juvenile
|
70.83 ± 3.97a#
|
70.60 ± 2.50a##
|
66.24 ± 1.15a###
|
68.68 ± 2.03a###
|
|
|
Adult
|
67.04 ± 2.74a#
|
60.99 ± 1.99b#
|
38.73 ± 0.55c#
|
40.97 ± 1.83c#
|
PAS –
Periodic Acid Schiff - Values with the different alphabet superscripts
(a,b,c,d) in the same row (comparison along each age group) are significantly
different
-
Values with the different number of harsh tag (#) in the same row (comparison
across different age groups) are significantly different.
DISCUSSION
The liver is the major metabolic centre in
all vertebrates and any damage to this organ would subsequently lead to many
physiological disturbances which will cause mortality in fish (Mishra & Poddar,
2016; Madhulatha & Rajyasree, 2019).
|The result of the current study revealed that C. gariepinus age groups exposed to graded concentrations of Urea
fertilizer manifest to some extent histological alterations in the liver while
those exposed to normal/control tanks shows normal structure. These
histological alterations in the liver of C.
gariepinus age groups exposed to graded concentrations of Urea fertilizer
were in line with the work of Scham, Hanan, Fawzia and Midhah (2014). The
alterations in the liver of fish under study may be attributed to the intake of
food substances imbibed by different concentrations of Urea fertilizer.
Findings revealed that the normal liver of C.
gariepinus age groups and C.
gariepinus age groups exposed to 0.75gL concentration of Urea fertilizer
exhibited normal structure, devoid of lesion and were characterized by distinct
central vein, sinusoidal spaces, undistorted hepatocyte, roundish nucleus and
substantial cytoplasm. Similar finding was observed in the liver of Channa
striatus (Madhulatha & Rajyasree, 2019); Gupta and Kumari (2021).
Histological study in the present study revealed that the liver of C. gariepinus exposed to 1.5g/l and
3.0g/l concentrations of Urea fertilizer showed varied degree of ventral
congestions, diffuse hepatocellular degeneration, moderate cellular
infiltration, marked hepatocellular degeneration, severe hepatic degeneration,
necrosis vacuoles degeneration and necrosis of hepatic cells. Simillar finding
was reported by Ullah, et al. (2015) who observed significant changes such as
disintegration of hepatic mass and necrosis in the liver of Labeo rohita exposed to cypermethrin.
The liver of C. gariepinus exposed to higher
concentrations of Urea fertilizer in the present study showed hyperplastic
hepatic and necrosis of hepatic cells. Similar observations were recorded by
Kalaiyarasi et al. (2017). The necrosis of the liver of C. gariepinus age groups exposed to higher concentrations of Urea
fertilizer in the present study could be due to the extra work load on
hepatocyte during detoxification of the Urea fertilizer.
Histochemistry
studies on the effect of sub-lethal concentrations of Urea fertilizer: 0.75,
1.5, 3.0 g/L and control on the liver of C.
gariepinus fingerlings revealed a PAS positive within the cytoplasmic
hepatocyte and the boundary area between hepatocyte. Similar finding was
reported on Cyprinus carpio (Khalid,
et al.2020). The presence of PAS positive cells in tissues often indicates the
presence of neutral mucin. The presence of PAS+ within the cytoplasmic
hepatocyte of the liver of C. gariepinus
Juvenile exposed to graded concentrations of Urea fertilizer also indicate the
presence of neutral mucin. The liver of
C. gariepinus juvenile exposed to graded concentrations of Urea fertilizer
shows PAS positive area within the perivascular tissues as well as bonding
areas between hepatocytes while that of the C.
gariepinus adult exposed to all the concentrations including the control
recorded PAS positive within the perivascular tissue and between hepatocytes,
within cytoplasmic hepatocytes. This is contrary to findings by Olivera-Ribeiro
& Fanta (2000); Johnson, et al. (2005). The histochemistry of the liver of C. gariepinus fingerlings and adults
shows that PAS values decreased with increasing grades of Urea concentration.
Findings on histochemistry study revealed that PAS intensity variations of the C. gariepinus liver exposed to graded
concentrations revealed an increased from fingerlings to Juveniles but reduced
as the C. gariepinus increased in age
thereafter.
CONCLUSION
In conclusion, the hepatic histo-morphology
of C. gariepinus fingerlings,
juvenile and adult in all the control groups and those exposed to 0.75 g/L were
devoid of lesion. However, all the C.
gariepinus age groups exposed to 1.5 and 3.0 g/L concentrations of Urea
fertilizers precipitated moderate to severe histopathological lesions in their
respective liver parenchyma. The liver PAS intensity variation across the age
groups of C. gariepinus exposed to
Urea fertilizer initially appeared to significantly increased (p<0.05) from C. gariepinus fingerlings to juvenile and
then significantly reduced in the C.
gariepinus adult. The periodic acid–Schiff (PAS)
positive areas were mainly demonstrated within the cytoplasmic hepatocyte and
in the boundary area between hepatocytes across all the different age-groups of
C. gariepinus exposed to concentrated
grades of Urea fertilizer.
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