DISCUSSION & CONCLUSION

6.1 INTRODUCTION

In this chapter, the results obtained during this study will be discussed. Although the
aim of this study was not to compare the normal histology of the two selected
species, but to assess them as single test organisms, it was decided to structure the
discussion in such a way that the results obtained for the two species are discussed
simultaneously in terms of the various aspects that were assessed (necropsy and
histology), to highlight both differences and similarities that were identified. This
chapter will conclude this thesis with various proposed recommendations for future
studies regarding further development of normal histology of southern African
freshwater fish species.

CHAPTER 6 6-1
 DISCUSSION & CONCLUSION

6.2 BREEDING PROCESS

As mentioned in Chapter 5, both species were bred successfully in captivity and the
required sample size of 20 sexually mature specimens per species was obtained. For
both species, the sample groups included 55% male and 45% female specimens.
Although a 50:50% ratio would be the ideal, the sample groups per sex were
sufficient to identify possible histological differences between males and females,
and to describe the gonadal histology. The specific sample size of twenty specimens
was chosen to ensure results that are representative of the species, but also
corresponds with the sample size used in field and laboratory assessments in toxicity
studies within the department, thus allowing comparison between different groups.

As mentioned above, sexually mature specimens were used for the assessments.
The macroscopic characteristics used to differentiate between males and females
and to determine sexual maturity (refer to section 3.3.1) before specimen sampling,
proved to be accurate, as all specimens were confirmed to be sexually mature during
the histological examination, identified either as mid developed (equal amounts of
early and final developmental stages present) or mature and ready to spawn
(predominantly spermatozoa and mature oocytes present).

Despite the cannibalistic behaviour demonstrated by the C. gariepinus fry, no

mortalities were recorded due to illness or infection, and all specimens appeared to
be in good health throughout the breeding process. This phenomenon was also
identified in a study by Al-Hafedh and Ali (2004) regarding the effect of feeding on
survival, cannibalism, growth and feeding conversion of C. gariepinus. It was found
that mortality was mainly caused by cannibalism, being significantly higher in fish fed
2 - 4% of body weight than with fish fed 8 - 10% of their body weight. The provision of
additional breeding tanks proved to be necessary as it enabled bigger specimens to
be separated from the rest of the fry. The C. gariepinus specimens were therefore
sampled at different ages (7-11 months) and time periods because the faster growing
specimens reached sexual maturity much earlier than expected. However, this was

CHAPTER 6 6-2
 DISCUSSION & CONCLUSION

not the case with the O. mossambicus specimens, and all fish were sampled at the
same time.

The reconstituted reverse osmosis water (RRO-water) proved to be a good breeding

medium, especially for the purpose of this study. The choice of RRO-water in
baseline studies is motivated by the need to minimise exposure to toxicants. Firstly,
metals that were identified during ICP MS analyses in the RRO-water were mostly
lower than levels detected in tap water prior to the reverse osmosis process (refer to
Chapter 4). Ammonia and zinc levels were however recorded to be higher than the
desired levels (refer to Chapter 4). Physical water quality variables remained
constant within the desired range throughout the breeding process. In addition, as
was mentioned in Chapter 4, none of the endocrine disrupting chemicals tested for
were detected (<0.5 µg/ ) in the breeding medium.

A similar procedure (the use of RRO-water) was followed in a study by Boettcher et

al. (2003). In this study, the use of reconstituted distilled water was employed to
determine baseline conditions for the effects of estrogen pollution on the reproductive
fitness of fish including baseline values for blood parameters, gonad morphology,
GSI and HSI. However, the study by Boettcher et al. (2003) used field collected
specimens which were then introduced to the reconstituted distilled water.

Considering that the desired number of healthy fish was obtained for both species
and that exposure to pollutants were minimised as far as possible, it can be
concluded that the breeding process was successful in achieving the aim of this
project.

6.3 NECROPSY

Macroscopic examination
The macroscopic examination of all fish, prior to the histological assessments,
indicated that all specimens appear to be in good health and no abnormalities were
identified regarding external features or of the visceral organs examined. Some

CHAPTER 6 6-3
 DISCUSSION & CONCLUSION

interesting differences were, however, identified in terms of the other variables

assessed during the necropsy in both species, and these will be discussed in the
following sections.

Condition factor
The condition factor varied unexpectedly between the two species. Essentially,
Fulton’s CF, calculated as stated by Carlander (1969), stipulate that a value of 1
indicates excellent fish health. However, although both species were fed equal
amounts of the same, high quality food, the mean CF for C. gariepinus (0.67) was
much lower than that calculated for O. mossambicus (1.64), and subsequently also
lower than the optimal value of 1. As measurable amounts of mesenteric fat were
only collected in C. gariepinus specimens, one would expect a higher, “healthier”, CF
for this species. As Van Sensus (1989) stated, body fat may significantly influence
CF values.

However, in a study by Watson (2001) on both C. gariepinus and O. mossambicus

specimens collected in the Olifants River catchment (South Africa), similar trends
were identified. Mean values of 0.67 and 0.63 during winter months and 0.70 and
0.66 during summer months were recorded for C. gariepinus with higher values of
1.34 and 1.74 calculated for O. mossambicus obtained from the same river system. It
can therefore be considered, that the normal CF range for C. gariepinus is lower than
that for O. mossambicus, and possibly that of other species. As the CF incorporates
both body length and weight relationships, a possible reason for a lower CF may not
necessarily be poor health, but rather the distinctly different body shape of C.
gariepinus, being significantly more elongated than that of the cichlid species. This
raises the question whether CF is a valid health indicator in fish as it seems to be
species specific and therefore not directly comparable, especially if a value of (1) is
considered to be indicative of good health for fish in general. It seems more accurate
to establish different optimal CF values for different species. For the purpose of this
study, the baseline CF range for C. gariepinus was established to be lower than that
of O. mossambicus and most other species documented e.g. P. flavescens and C.

CHAPTER 6 6-4
 DISCUSSION & CONCLUSION

auratus (Nero et al., 2005). Future studies can possibly focus on further exploration
of this specific aspect.

Hepato-somatic index (HSI)

Bruslé and Gonzàlez (1996) state that the mean HSI value is species specific and
correlates with the amount of fat deposition (higher in species which store high
amounts of lipids) (Chiba and Honma, 1981; Oguri, 1985; Ando et al., 1993). In
Osteichthyes, the HSI is normally calculated to be between 1-2% (Bruslé and
Gonzàlez, 1996). However, intra-specific variability occurs depending on differences
in sex, season, age, and physiological condition regarding feeding, reproduction or
stress (Bruslé and Gonzàlez, 1996). According to Bruslé and Gonzàlez (1996) the
HSI is generally higher in females than in males although this was not identified in
either C. gariepinus or O. mossambicus specimens assessed. In terms of sexual
maturity, it has been shown that HSI decreases with maturation of gonads in many
teleosts e.g. P. altivelis (Aida et al., 1973) and H. hippoglossus (Haug and Gulliksen,
1988). However, experimental hormonal stimulation of estradiol induces an increase
in this value (Aida et al.,1973; Korsgaard and Mommsen, 1993). It has also been
found that HSI is highly sensitive to the nutritional status of the fish and correlates
with the quantity and quality of food (Hung et al., 1990). In addition, pollutants can
modify the HSI as shown in A. brama (Sloof et al., 1983) and it is therefore
suggested that the HSI may be a useful indicator of chemical water pollution (Bruslé
and Gonzàlez, 1996).

In this study, the mean values for both species were calculated to be within the
normal range (1-2%) although some minimum values were recorded to be lower than
1% in both sexes of both species.

Gonado-somatic index (GSI)

The GSI varied between individual specimens but was identified, on average, to be
higher in females than males. This is not unexpected, as the ovaries are noticeably
greater in size in females than the testes of equal-sized males. However, although all

CHAPTER 6 6-5
 DISCUSSION & CONCLUSION

C. gariepinus females were confirmed to be sexually mature through histological

examination, older and larger females exhibited higher GSI values. No distinct
pattern could be identified for O. mossambicus females regarding body size
measurements and GSI values. Although the GSI for the male groups of both
species were calculated to be lower than 1%, Roberts (2001) documented that the
testes of fish can approach 12% of the total body weight.

It is important to determine the GSI as part of a necropsy. Pieterse (2004) stated that
the utilisation of the GSI as a reproductive biomarker was first reported in 1927 in a
study describing the yearly variations of female P. flavescens ovaries (Meien, 1927).
According to De Vlamingh et al. (1981), there is significant evidence that exposure to
various environmental pollutants can result in gonadal changes such as decreased
GSI and/or morphological changes.

Spleno-somatic index (SSI)

Although the spleen was not included as part of the qualitative and quantitative
histological assessments in this study, the SSI for each specimen was calculated to
determine baseline values. The spleen in fish is primarily an organ of blood storage
and blood cell production (Ellis et al., 1978) and also disintegrates erythrocytes and
releases haemoglobin (Goede and Barton, 1990). Enlargement of the spleen is
considered an abnormal pathological condition (Goede and Barton, 1990) and could
be indicative of disease or immune problems (Adams et al., 1992). An increase in the
SSI can therefore indicate abnormal conditions.

The SSI for C. gariepinus specimens varied between individuals with no clear
differences between males and females. For O. mossambicus, the spleen was
collected for all specimens but was recorded to be below measurable limits for some
specimens, especially in females. Accurate mean values could therefore not be
calculated for this species. This small spleen size correlates with the overall smaller
size of the O. mossambicus specimens within the sample group. Although the spleen

CHAPTER 6 6-6
 DISCUSSION & CONCLUSION

was not included in the histological assessment, the SSI was calculated to establish
reference values for comparison in possible future studies.

Mesenteric fat and fat index (FI)

The determination of the amount of mesenteric fat deposits is somewhat subjective,
however, with a sample size of twenty fish, trends among, and within populations, are
readily discernable (Goede and Barton, 1990). Although not directly related to stress,
the level of fat reflects the intensity of feeding and energy deposition over the long
term and permits inference about bio-energetics (Goede and Barton, 1990).
Investigators should establish fat index criteria for the species of interest as there are
considerable differences among species (Goede and Barton, 1990). Because of
interacting variables like fish size, sex, time of year and stress level, the fat index can
not be assigned normal or abnormal values (Adams et al., 1993), but merely an index
value associated with the condition of the specific specimen. For C. gariepinus, the
fat index varied between individual specimens with a mean value of 0.7% ± 0.6 while
no measurable mesenteric fat were present in O. mossambicus specimens.

Bile colour
The colour of bile within the gallbladder is a good short-term indicator of the feeding
activity and nutritional status of fish (Love, 1980). Bile colour ranges from clear to
straw-yellow in fish that have fed within the previous couple of days to blue-green in
fish that have not eaten for a week or longer. The blue-green pigmentation is caused
by oxidation of bilirubin pigments to biliverdin (Goede and Barton, 1990). A
completely empty gall bladder indicates that the fish have probably eaten within the
last few hours (Love, 1980; Goede and Barton, 1990).

Interestingly, in this study the bile colour for C. gariepinus were identified to be straw
yellow with the exception of two specimens with a light green bile colour. In contrast,
the bile colour of all O. mossambicus specimens was identified to be light green. All
specimens were fed the day of, or before tissue sampling (depending on the time of
sampling), diverging from the theory regarding eating habits as stated above.

CHAPTER 6 6-7
 DISCUSSION & CONCLUSION

However, similar trends were identified in the study by Watson (2001), where the bile
colour of field specimens of O. mossambicus also exhibited a green colour and C.
gariepinus a yellow colour, which were attributed to the different feeding regimes of
the species in that specific study.

Haematocrit (Hct)
The haematocrit is used to measure the ratio of erythrocytes to plasma, and
effectively, measures the packed cell volume of the erythrocytes contained in the
blood (Blaxhall, 1972). According to Barnhart (1969), without the knowledge of the
normal range of haematological parameters, it is difficult, if not impossible to
differentiate between the normal and pathological status of fish. Normal
haematological values of the various blood parameters for C. gariepinus have been
presented by Hattingh (1972). He established the normal Hct to be in the region of
28.9 m /100m blood. In studies by Cyriac et al. (1989), the mean Hct for O.
mossambicus was established to be 33% ± 2.7 (pH 6.8, Temp. 28 ± 1˚C) under
laboratory conditions. However, as part of the Quantitative Health Assessment Index
for Rapid Evaluation of Fish Condition in the Field (HAI) (Adams et al., 1993), a
normal range of 30 – 45 % is considered for fish in general.

For this study, the mean Hct for C. gariepinus was calculated to be 37.1 ± 3.3 (pH
6.6, Temp 26.6˚C) and for O. mossambicus 31.3 ± 4.4 (pH 6.6 Temp. 26˚C). Both
values fall within the normal range indicated by the HAI and the values obtained for
O. mossambicus correspond with that of Cyriac (1989). Although a minimum value of
28.5% was observed for C. gariepinus, corresponding with the study of Hattingh
(1972), most values were higher than 28.5%.

Total plasma protein (TP)

According to Watson (2001), plasma proteins are essential with regard to fish
nutrition and the defence of the body against viral infections. Plasma proteins play an
important role in the physiology of fish as they regulate many functions in the blood
(Van Vuren, 1980). Low plasma protein values are often associated with starvation

CHAPTER 6 6-8
 DISCUSSION & CONCLUSION

and depletion of energy stores, a common condition of temperate-zone fish in winter

and early spring (Lockhart and Metner, 1984; Cunjak, 1988).

For C. gariepinus, the mean TP value was calculated to be slightly higher (3.34 ±0.96
g/100m ) than O. mossambicus (2.01 ± 0.52 g/100m ). The mean value calculated
for C. gariepinus corresponds with the reference TP value of 3.8 ± 0.11 (Temp: 23˚C
± 1˚C) determined by Adeyemo et al. (2003) for the same species. The HAI (Adams
et al., 1993) indicates the normal range for TP to be between 3 – 6 g/100m . Mean
values for C. gariepinus were also recorded to be within this range but mean TP
values for O. mossambicus were calculated to be slightly lower than 3 g/100m .
However, the TP values for O. mossambicus correspond with haematological
reference parameters determined by Bittencourt et al. (2003) for the Nile tilapia
(Oreochromis niloticus). Bittencourt et al. (2003) established the normal range for TP
to be between 1.81 – 3.98 g/100m . However, a slightly higher reference interval of
2.9 – 6.6 g/100m was determined for hybrid tilapia (Hrubec et al., 2000). No
significant differences were identified in terms of male and female TP values (p >
0.05).

The calculation of the various aspects as discussed above (CF, HSI, SSI, GSI, FI,
and blood parameters) and the qualitative information documented as part of the
necropsy (organ and bile colour), are important to include in histological
assessments. This information serves as supporting data for histological results, as
abnormalities in macroscopic structure or deviation from the normal range of
quantitative values could be related to histological alterations that have been
identified. As toxicants can alter these indices and parameters, it is important to
determine reference values associated with normal conditions for comparison in
future histological studies.

6.4 QUALITATIVE HISTOLOGICAL ASSESSMENT

During the necropsy and macroscopic examination of the selected target organs,
distinct differences in general morphology of these organs were identified between C.

CHAPTER 6 6-9
 DISCUSSION & CONCLUSION

gariepinus and O. mossambicus in terms of size, shape and colour. However, the
qualitative histological assessment revealed fewer microscopic differences between
the same organs of the two species and most histological characteristics
corresponded with that described for other teleosts e.g. salmonids (Yasutake and
Wales, 1983). Differences that were identified will be highlighted as part of this
discussion. For the purpose of this study, macroscopic characteristics and
differences observed will be discussed together with the qualitative histological
results (as was done in Chapter 5), although these aspects were examined as part of
the preceding necropsy.

Liver
Macroscopically, the livers of both species are lobulated, but distinctly different in
appearance. The liver of O. mossambicus consists of an anterior and posterior lobe
compared to the distinct left and right lobes of C. gariepinus livers. Another clear
difference observed was the colour of the liver. The dark red colour of the liver of C.
gariepinus is generally considered to be the normal colour of fish liver, while the light
brown colour (coffee with cream) of the liver of O. mossambicus is generally
considered to be indicative of fatty livers (Adams et al., 1993). However, this colour
was also described for unexposed laboratory bred O. mossambicus livers in previous
studies (Geyer, 1989; Van Dyk, 2005).

Roberts (2001) documented that the liver in wild fish is usually reddish brown in
carnivores and light brown in herbivores but at certain times of the year it might be
yellow or even off-white. In farmed fish, where diets generally contain higher levels of
lipid, it is usually lighter in colour than in the equivalent wild specimen.

Microscopically, the livers exhibited the same histological structure with a few
exceptions. Pancreatic tissue (hepatopancreas) is a prominent feature in the liver of
O. mossambicus. At first, this feature was considered to be absent in C. gariepinus.
However, midsections of the entire liver revealed that this species do possess
hepatopancreatic tissue, but usually invade the liver parenchyma in the region of the

CHAPTER 6 6-10
 DISCUSSION & CONCLUSION

hilum region. This is not unusual, as was shown in a study by Feist et al. (2004)
where pancreatic tissue were present in flounder (Platichthys flesus) but usually
found to be absent in dab (Limanda limanda).

Another distinction between the species is the presence of a thin, connective tissue
layer that surrounds the hepatic central and portal veins of C. gariepinus. An increase
in peri-vascular connective tissue is usually considered to be pathological (Pierce et
al., 1978), but was identified to be present in the hepatic vascular systems of all C.
gariepinus specimens. However, a connective tissue layer was also found to
surround the veins of field collected C. gariepinus specimens (Van Dyk, 2003b),
although in that study it was described to be more extensive and was concluded to
be pathological in nature. Similar vascular characteristics were also identified in C.
gariepinus specimens collected in the Rietvlei and Marais dams (Rietvlei Nature
Reserve, South Africa) (unpublished field observation).

Clear confirmation on the presence of Kupffer cells in the livers of C. gariepinus and
O. mossambicus could not be concluded during this study. According to Roberts
(2001), functional Kupffer cells are not found in the lining of the sinusoids in fish liver.
This was demonstrated by Varichak in 1938 and has since been adequately
confirmed (Ellis et al., 1976), but descriptions of so-called Kupffer cells based solely
on morphological criteria continue to appear (Hinton and Pool, 1976).

Gills
Macroscopically, the gills of both species consist of four gill arches involved with
respiration, and a fifth, transformed in the pharyngeal bone which does not play a
role in respiration (Takashima and Hibiya, 1995). Microscopically, a distinct
difference was observed in terms of the gill epithelium of the two species. A thicker
inter-lamellar and gill arch epithelium was identified in C. gariepinus. Initially, it was
diagnosed as slight hyperplasia, but it was finally concluded to be a possible normal
feature in this species for the following reasons: (1) It correlated with observations in
field specimens of the same species (unpublished results), (2) it visually

CHAPTER 6 6-11
 DISCUSSION & CONCLUSION

corresponded with that described for the channel catfish (Grizzle and Rogers, 1976),
(3) and it was a histological characteristic observed in all twenty C. gariepinus
specimens assessed, and was not identified in O. mossambicus specimens bred
under the same conditions within the same breeding medium. However, this
observation still needs to be confirmed in future studies.

Testis
Macroscopically, the testes of C. gariepinus and O. mossambicus differ to a large
extent in terms of general appearance, as examined in sexually mature males.
However, in both species the testis is bilobed, and the histological assessments
revealed similar microscopic structure in C. gariepinus and O. mossambicus.
Interestingly, testis measurements confirmed that the left testis is constantly larger
than the right testis. This was also observed in L. dumerili in a study by Van der Horst
(1978).

Both species possess a lobular testicular organisation consisting of a central lumen,

according to the classification of Callard (1991), corresponding with that of salmonids
(Yasutake and Wales, 1983). In the two species, the males were classified to be late
spermatogenic in C. gariepinus and mid spermatogenic in O. mossambicus. This can
possibly be attributed to the fact that the O. mossambicus specimens produced a F2
generation during the breeding process, and therefore, cyclic changes differed
between the species. The presence of residual or resting primary spermatogonia
within the seminiferous lobules of C. gariepinus was also a prominent characteristic
identified in this species.

Ovary
Apart from the macroscopic differences (size and colour), the ovarian histological
structure of the two species was observed to be the same, and similar to that
described for other teleosts (Takashima and Hibiya, 1995). For both species, the
ovaries were classified to be asynchronous, meaning that a heterogeneous
population of follicles at different stages of development is present (Takashima and

CHAPTER 6 6-12
 DISCUSSION & CONCLUSION

Hibiya, 1995). The fact that post-ovulatory follicles were observed in one O.
mossambicus female can again be attributed to the spontaneous breeding behaviour
of this species in captivity and was therefore not considered to be peculiar.

Heart
During both the macroscopic and microscopic examinations, the heart ventricle of C.
gariepinus and O. mossambicus were found to be different in a number of ways, as
was illustrated in a study by Sanchez-Quintana et al. (1995) on two other fish
species. In that study, a morphological analysis of the fish heart ventricle was
executed on hake (M. merluccius), angler fish (L. piscatorius) and sea bream (P.
centrodontus). The ventricles of these species were found to be either tubular,
saccular (as was identified for C. gariepinus) or pyramidal (as was identified for O.
mossambicus) in shape. Also, it was found that in sea bream, a thin compact layer
was present in the myocardium of the ventricle (as was identified in C. gariepinus),
while the myocardium of hake and angler fish were found to be exclusively trabecular
in nature (as was identified in O. mossambicus). Apart from these structural
differences regarding the ventricle, the histological structure of the atria and bulbus
arteriosus were similar in C. gariepinus and O. mossambicus.

Kidney
In general, the kidney histology of C. gariepinus and O. mossambicus was similar to
that described for other teleosts e.g. striped bass (Groman, 1982) and salmonids
(Yasutake and Wales, 1983) but macroscopically different from that of the channel
catfish (Grizzle and Rogers, 1976) described to consist of a separate,
morphologically isolated anterior and posterior region in adult specimens. The
macroscopic shape of the kidney for both species corresponds best with that
illustrated for rainbow trout by Takashima and Hibiya (1995). Both species possess
an anterior kidney, consisting predominantly of haematopoietic tissue, and a
posterior kidney, consisting of renal corpuscles and tubules. However, in C.
gariepinus, the renal corpuscle and tubules is surrounded by a distinct matrix of

CHAPTER 6 6-13
 DISCUSSION & CONCLUSION

haematopoietic tissue in the posterior kidney, not so evident in the posterior kidney of
O. mossambicus, although observable in areas.

The different renal tubules were not always identifiable, but the second segment of
the proximal tubule and the distal tubule made up most of the renal structure of the
posterior kidney in both species. The initial neck segment was not identified in kidney
samples. However, to conclude that this segment is not present in these species will
be immature and needs further investigation. A possible reason for the predominant
occurrence of certain tubule segments compared to others segments, might be
attributed to the section orientation of the tissue sample.

Another distinct feature identified in most specimens was hyaline droplet

degeneration. However, this phenomenon is also listed as a histological alteration in
the quantitative histological assessment protocol, and will therefore be further
discussed in the following section.

6.5 QUANTITATIVE HISTOLOGICAL ASSESSMENT

As mentioned in Chapter 1, various factors can influence the histological integrity of a

fish specimen, including age, season, sex, sexual maturity, nutrition, physical water
quality, and chemical and/or toxicant exposure. Hence histological alterations are
expected in target organs of any fish specimen. However, by maintaining controlled
conditions and minimising exposure to harmful chemicals and/or toxicants, the
presence of toxicant induced alterations is significantly reduced.

Various histological alterations were identified in the selected target organs during
the histological assessment and those were subsequently quantified by means of the
quantitative histological assessment protocol (Bernet et al., 1999). The aim of the
quantitative assessment was to determine baseline reference values and intervals for
the selected target organs in terms of the specific protocol used in histological
assessments within the department, and simultaneously assess the health of the
sample groups. However, in a baseline study, where reference intervals want to be

CHAPTER 6 6-14
 DISCUSSION & CONCLUSION

determined, it is important to consider the number of specimens in which the

alterations occurred to subsequently define the occurrence as either typical or non-
typical.

For the C. gariepinus sample group, alterations were identified in the liver, gills,
testes and kidney. For the liver, these included cord disarray (1/20), intra- (19/20)
and inter- (1/20) cellular deposits, the presence of mono-nuclear leucocytes (1/20),
focal necrosis (5/20), cellular swelling (4/20) and nuclear activity (3/20). Alterations
identified in the gills included intercellular oedema (6/20) and plasma alterations
(1/20). In the testes, vacuolation within spermatocyte cysts were identified (1/11) and
in the kidney hyaline droplet degeneration (19/20) and thickening of the Bowman’s
capsule (1/20) were observed.

In the O. mossambicus sample group, alterations were identified in the liver, ovary
and kidney. For the liver, these included cord disarray (1/20), intra- (20/20) and inter-
(11/20) cellular deposits and nuclear activity (4/20). In the ovaries, intercellular
deposits were identified (3/9), and in the kidney, hyaline droplet degeneration (19/20)
and focal necrosis (1/20) were observed.

The specific root causes of these alterations can be speculated and can be attributed
to any of the factors listed in the first paragraph of this section. However, in this
study, all these factors were documented and hence these alterations can be
associated with the combined measurements of variables and parameters as
recorded throughout the breeding process including water quality. Nevertheless,
although not equally prominent, all alterations were included in the calculation of the
various indices to establish baseline intervals. However, those alterations identified in
the majority of the specimens (>50%) (hyaline droplet degeneration, and intra- and
intercellular deposits) requires further discussion.

Hyaline droplet degeneration in the kidney

According to Takashima and Hibiya (1995), hyaline droplet degeneration of the
epithelial cells is one of the typical changes that occur in the renal tubules. Coarse

CHAPTER 6 6-15
 DISCUSSION & CONCLUSION

eosinophilic granules appear in the cytoplasm. The size of the granules is not
uniform, but varies from rather large to small granules. In extreme cases, excess
accumulation of granules may lead to necrosis, which is evidenced by pyknosis and
vacuolation of the cytoplasm (Takashima and Hibiya, 1995). The granules may be
produced within the cell itself, or formed by the reabsorption of excess amounts of
proteineous substances filtered through the glomerulus. However, in addition to the
pathological condition described above, eosinophilic hyaline granules may also
appear in the epithelial cells of the renal tubule under normal conditions, but in this
case, granules and nuclear or nucleolar changes are not evident (Takashima and
Hibiya, 1995). Hyaline droplet degeneration was also identified in reference groups in
a study by Bernet et al. (2004). As nuclear changes were not identified in epithelial
cells of renal tubules in this study, this phenomenon can be considered to be that
associated with normal conditions.

Intracellular deposits in the liver

Hinton and Laurén (1990) stated that cytoplasmic inclusions (deposits) are common
biomarkers of metal exposure in mammals but have not been commonly reported in
fish. Leland (1983) reported the presence of electron dense deposits in hepatocytes
of trout exposed to both copper and zinc. Sorenson (1976) found similar cytoplasmic
and nuclear deposits in sunfish (L. cyanellus) exposed to high concentrations of
arsenic. Van Dyk (2005) also reported hyaline deposits in hepatocytes of O.
mossambicus exposed to cadmium and zinc. Although not as severe as the findings
by Van Dyk (2003), the intracellular deposits identified in C. gariepinus and O.
mossambicus stained PAS positive indicating possible hyaline droplet degeneration.

This presence of various sized, eosinophilic deposits was identified in areas of most
livers assessed. The term hyaline, a generic term used by histologists for dense,
eosinophilic inclusions, describes only the physical appearance as seen by light
microscopy and do not relate to chemical composition (Cheville, 1994). According to
Cheville (1994), protein inclusion bodies are common in metal toxicities possibly
explaining the PAS positive reaction. These deposits can therefore be considered to

CHAPTER 6 6-16
 DISCUSSION & CONCLUSION

be a possible result of exposure to the low levels of zinc detected in the reconstituted
reverse osmosis water during the breeding process.

Inter cellular deposits in the liver

These structures were only prominent in some O. mossambicus livers and mostly
visible in the vicinity of the central veins. This phenomenon was also identified by
Grant (2004). These structures do not stain PAS positive and do not resemble a
constituent of melano-macrophage centres identified in both control and exposed
livers of O. mossambicus by Grant (2004) and Van Dyk (2005). However, in the
study by Grant (2004), histo-chemical demonstration of these structures proved
negative with the Rubeanic Acid method for the demonstration of copper deposits but
positive with the Mallory and Parker’s haematoxylin method for copper deposits. As
no levels of copper were detected during the metal analyses, the exact cause of
these deposits is not clear.

Histological indices
Index values were subsequently calculated for the various target organs. In terms of
the reaction indices, the results showed that in both species, regressive changes
were most prominent of the five reaction patterns as classified by Bernet et al.
(1999). The organ indices of both species indicated that the liver showed most of
these regressive histological alterations. This is not unexpected, as the liver is
regarded as the detoxification organ in the body and by metabolising and/or storing
toxicants, structural alterations will result.

Although the reaction and organ indices indicated that different alterations, or similar
alterations of different severity, were identified in C. gariepinus and O. mossambicus
respectively, the total organ indices for the two sample groups were calculated to be
in close range with a difference of only 1.1 index units. This result indicated that the
overall health condition of both species was similar. This is not unexpected as both
these groups were bred under the same breeding conditions.

CHAPTER 6 6-17
 DISCUSSION & CONCLUSION

These quantitative histological results will provide reference material in future studies
where the same protocol will be applied to the same species. However, it must be
mentioned that the total organ indices can not be compared between groups if the
same organs are not assessed. Organ indices can, however, be compared between
groups, or between individuals within the same group. When the results obtained
were compared to control groups of other species in studies where this assessment
protocol has been employed (refer to Table 5.7 and 5.14), the corresponding organ
indices for C. gariepinus and O. mossambicus were either lower, or within the range
of normal values determined for the species in those studies. Hence, the values
calculated in this study, correspond with those considered to be normal for other
species.

The same quantitative protocol has also been applied in current histology related
toxicity studies at the University of Johannesburg on both C. gariepinus and O.
mossambicus specimens and the results showed higher index values (unpublished
results) than the reference values calculated in this study for the same species. For
the baseline study, it is recognised that the values calculated are associated with the
specific breeding conditions recorded throughout the breeding process and that
specimens obtained from different systems may show different results. However,
these breeding conditions correspond with standard protocols used in toxicity testing
within the department, and therefore these results can serve as baseline values for
those studies.

6.6 CONCLUSION

As mentioned before, Yonkos et al. (2000) stated in an article pertaining to the

fathead minnow, that identification of tissue lesions requires a baseline appreciation
of normal tissue conditions, and while histological atlases exist for several
commercially valuable species (e.g. striped bass, salmonids, the channel catfish),
such a resource has been unavailable for cyprinids. Hence these authors generated
an atlas of the normal microanatomy of the fathead minnow. Similarly, this project
originated from a need for baseline histological data of the two species most used in

CHAPTER 6 6-18
 DISCUSSION & CONCLUSION

histological research at the University of Johannesburg, Clarias gariepinus and

Oreochromis mossambicus. Although control groups usually serve as reference in
toxicity studies, accurate histological baseline data requires the history of these
control specimens to be known (e.g. prior exposure to toxicants throughout its
lifespan).

Culturing a reference group under controlled conditions (as was done in this study),
enables the water quality to be monitored throughout the fish’s lifespan and
subsequently the determination of normal reference intervals for the different
parameters usually examined during histological assessments in toxicity studies.
However, it is acknowledged that the confirmation of reference values and intervals
representing normal conditions in fish requires the collaboration of the results of
various qualitative and quantitative baseline studies, as different factors can influence
the histological integrity of a fish specimen including age, season, sex, sexual
maturity, nutrition, physical water quality and chemical and/or toxicant exposure. A
single study cannot incorporate all the possible different influences these variables
will have on the histological structure of target organs. However, separate, smaller,
more specific baseline studies, allow the systematic determination of these reference
intervals for specific variable conditions at a time.

The aim of this baseline study was to establish such reference material, both
qualitative and quantitative, for two southern African freshwater fish species in the
form of descriptive histology (qualitative assessment) and the associated reference
values and intervals for related quantitative aspects, including somatic indices,
condition factor, selected blood parameters and quantitative histological results
(quantitative assessment).

Breeding these baseline specimens in reconstituted reverse osmosis water

(according to the criteria as stated in Chapter 3, section 3.2), proved to be successful
in minimising exposure to pollutants, and produced the desired number of healthy,
sexually mature specimens per sample group. It also allowed the history of the fish to

CHAPTER 6 6-19
 DISCUSSION & CONCLUSION

be known and documented, distinguishing these specimens from other control

groups which are usually collected in the field and then acclimatised within an
aquarium setup. The hypothesis for this study as stated in Chapter 1 could therefore
be accepted. Only selected target organs (liver, gills, gonads, heart and kidney) were
included in this study due to time constraints. However, the determination of
reference material for these specific organs was regarded as a priority as they are
included in histological assessments in toxicity studies within the department.

So, to conclude, the results of both the qualitative and quantitative histological
assessments (including the necropsy) produced the necessary baseline data for use
and incorporation in future toxicity studies, complying with the aim of this study as
stipulated in Chapter 1. Furthermore, the results provide: (1) knowledge and a better
understanding of freshwater fish histology of selected target organs of two indicator
species; (2) contributes to a limited database regarding normal fish histology in South
Africa; (3) provides reference material for target organ histopathological analyses
with the application of a standardised assessment protocol which includes specific
criteria to ensure proper and accurate histopathological diagnoses; (4) will eventually
contribute to the creation of a comprehensive freshwater fish histology atlas for
southern African species; (5) provides baseline values and intervals for quantitative
parameters measured as part of histological assessments (somatic indices, selected
blood parameters and condition factor); (6) and provides a working document, to be
used in, and supplemented by the results of, future histological assessments to assist
in basic fish histology training within the department.

Various additional related aspects could form part of future studies for the further
advancement of histology and histopathology of freshwater fish in South Africa.
These are stated in the following section.

6.7 FUTURE RECOMMENDATIONS

This project was the results of recommendations from previous histological studies
involving Clarias gariepinus and Oreochromis mossambicus, where a need for the

CHAPTER 6 6-20
 DISCUSSION & CONCLUSION

normal histology of these two species were emphasised. However, as mentioned in

Chapter 1, the range of normal histological characteristics is usually confirmed with a
collaboration of various histological assessments of which this project was a first
step. Subsequently, various future aspects can be recommended to further the
development of a firm and comprehensive histological database for these species:

(1) Apart from the five target organs assessed in this study, various other
target organs of the two selected species could also be studied to add
to a growing database of normal histology (e.g skin, spleen, muscle,
brain, thyroid, intestine, pancreas and blood).

(2) Age and seasonal histological differences must be examined for these
species, especially in terms of gonadal development.

(3) Ultrastructural data and morphological characteristics needs to be

incorporated to better understand the sub-cellular morphology of
specific target organs.

(4) Other freshwater fish species, endemic to South Africa, could be

considered in future studies by applying the same protocol as this
study, to also include histological characteristics of more sensitive
species to the histological database.

(5) Results from laboratory baseline specimens needs to be compared to

that obtained from field specimens collected from so-called “pristine”
aquatic systems. However, the existence of such systems is
questionable.

(6) The incorporation of expertise in bacteriology, virology, and

parasitology in histological assessments is essential for future
histopathological research, to allow the researcher to be able to
distinguish between pollution associated and infection associated organ
lesions.

CHAPTER 6 6-21
 DISCUSSION & CONCLUSION

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