CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the Study

The global aquaculture industry faces significant challenges in sustainable feed production,
prompting research into alternative feed sources (Tacon & Metian, 2015). Clarias gariepinus, a
widely cultivated catfish species, offers potential for improved growth rates and market demand
(Food and Agriculture Organization of the United Nations, 2020). However, conventional fish
feed costs remain high, necessitating innovative solutions (Kumar et al., 2014). The medicinal
herb Bryophyllum pinnatum is used to treat wounds and illnesses. Bryophyllum pinnatum is an upright,
succulent, perennial shrub that grows to a height of 1.5 meters and reproduces both vegetatively from
leaf bubbils and from seeds. It is a member of the Grassulaceae family. This attractive shrub was brought
in and is already spreading like a weed around plantation crops10. Traditional uses of bryophyllum
pinnatum include treating burns, earaches, ulcers, abscesses, insect bites, diarrhea, and lithiasis. In the
southeast region of Nigeria, this plant is used to help a newborn baby's placenta drop. Every day, the
juice from the plant leaf is removed and placed to the baby's placenta after it has been gently heated.
Both the crushed leaves and the juice that was extracted are massaged on obsesses after being
combined with palm oil. Usually, it is used externally.
Known as the "miracle leaf," Bryophyllum pinnatum is extensively dispersed, particularly in the
Philippines.

Bryophyllum pinnatum's active ingredients, bufadienolides, have insecticidal, antibacterial, and

antihumorous properties16. Only a small number of these plants have their bioactive ingredients
discovered in Nigeria, as in any other underdeveloped country. However, certain plants with beneficial
bioactive qualities also contain valuable minerals and food that may be consumed by humans and
animals. The most widely cultivated fish species in Nigeria is Clarias gariepinus, which farmers choose to
cultivate above other fish species.

The high expense of imported commercial feed and the dearth of locally produced feed that is both
economically viable for farmers and nutritionally appropriate for fish make feed one of the primary
obstacles to the species' development. According to projections, at least 60% of the overall cost of
producing the species is related to fish feed.
Various local fish feeds have been developed from a variety of sources in an effort to create a more
accessible, economical, and less expensive alternative to imported commercial fish feeds.

As a result, several fish feed enterprises have grown and expanded in Nigeria, producing and marketing
a wide range of fish feed with dubious and dubious formulation, nutritional content, and production
1
methods. Without understanding the exact content, formulation, and processing of the feeds, the
majority of fish producers use this less expensive local feed. Essential nutrients like protein, lipids or fats,
ash (minerals), fiber, moisture, NFE (nitrogen free extract or carbohydrates), and vitamins should all be
present in the proper amounts in a good C. gariepinus feed. The ration should be balanced so that the
fish can eat it and grow as best they can.

1.2 Statement of the Research Problem

Diet supplementation has promise for boosting fish productivity and is a crucial component of
aquaculture management, particularly in intensive or semi-intensive fish culture. There is a dearth of
information on the nutritional value of fish feed made by various Nigerian feed firms, as well as no
trustworthy public data regarding the chemical makeup of commercial feed and feed additives.

Farmers are forced to depend only on the manufacturers' proximate feed composition due to
a lack of information on the assessment of the nutritional contents of commercial feeds. Such feed is
frequently referred to as aqua feed or local, home-made, or farm-made fish feed. The growth response
of fish to these local meals and the typical commercial feeds has not really been examined or compared
in many research.

1.3 Justification of the Research Problem

Despite Bryophyllum pinnatum’s potential benefits, limited research exists on its proximate and
mineral composition when fed to Clarias gariepinus (Olaifa et al., 2017). Understanding this
diet’s nutritional content is crucial for optimizing fish growth, health, and productivity. Practical
diets that largely rely on animal protein feedstuffs, particularly fish meal, are provided to many fish, such
as Clarias gariepinus. However, there are significant drawbacks to using animal feedstuffs, including
their high cost, the significant excess of phosphorus, the growing scarcity of fish meal supplies, growing
ecological concerns about using wild fish for aquaculture feeds, and phytoplankton bloom, which causes
eutrophication.

The Aquaculture industry is facing increasing pressure to adopt sustainable and environmentally

friendly practice. This study contribute to this goal by investing the use of natural feed additive,

Bryophyllum pinnatum as an alternative to commercial feeds considering it proximate and

mineral contents.

2
1.4 Aim and Objectives of the Study

The aim of this research study is to evaluate proximate and mineral composition of Clarias

gariepinus fed Bryophylum pinnatum diets.

Objectives of the Study are:

i. to evaluate the moisture, crude protein, crude fat, ash, fiber and Nitrogen free extracts content

of Clarias gariepinus fed Bryophyllum pinnatum supplement diets.

ii. to evaluate the calcium, phosphorous, potassium and sodium reserve of Clarias gariepinus fed

Bryophyllum pinnatum supplement diets.

3
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Aquaculture

Breeding, growing, and harvesting aquatic plants and animals in a variety of controlled water habitats is
known as aquaculture. Its goals include repopulating populations of endangered animals, preserving and
improving ecosystems, and producing food and economic items. Aquatic and freshwater aquaculture
are the two types of aquaculture. It is a crucial area of the country's economy. This is a positive step at a
time when the government is trying to diversify the economy and reduce its dependency on oil.

In addition to directly producing fish for human consumption, it has the ability to indirectly increase the
nation's food security by providing jobs for the nation's increasing number of jobless people and earning
foreign exchange through the export of fish and fish products. One of the food production sectors with
the quickest rate of growth in the world is aquaculture.

As wild fisheries reach their ecological limitations and the demand for elegant fish rises
worldwide, aquaculture production is expected to treble in the next 15 years, having doubled in the past
20. In 2012, Nigeria's annual demand for fish was 2.66 million metric tonnes, while its supply was just
1.32 million metric tonnes. 0.7 million metric tonnes are imported, compared to 0.62 million metric
tonnes produced locally. Only 200,000 metric tonnes of the world's fish supply come via aquaculture.

The current aquaculture production falls well short of its capacity of 2.5- 4.0 million metric tonnes
Nigeria is a coastal nation that shares a southern boundary with the Atlantic Ocean. It covers 923,766
square kilometers and has about 1.75 million hectares of suitable land for aquaculture production. The
aquaculture sub-sector is considering a more viable option for meeting the country's need for fish
production self-sufficiency.

The demand for aquatic food sources has significantly increased annually as a result of growing
disposable income, changing consumer preferences, and rapid population expansion in emerging
nations like Nigeria. The advent of more effective capture technology, decades of state subsidies,
increased market access, even for isolated fishing villages, and development programs to boost output
from the vulnerable open-access resource have all contributed to a significant reduction in fish supply.

As a result, there is increasing concern about the long-term viability of wild fish stocks. The
need for long-term investments is to ensure the long-term viability of aquaculture production cannot be
overstated. Design, ecosystem-based resource management, the post-harvest market, and human
resource growth are all common areas of intervention in this seemingly important yet underserved
sector.

4
With a large range of amino acids, vitamins, and minerals, fish is an essential component of many
Nigerians' diets, which are high in nutritious content. Additionally, the cost of fish products is lower in
the nation than that of beef, hog, and other animal protein sources. Fish is therefore in great demand.
About 41% of the meat in the average Nigerian diet comes from fish, yet the country must import $500
million worth of fish annually due to a lack of native availability.

2.2 Bryophyllum pinnatum

The medicinal herb Bryophyllum pinnatum is used to treat wounds and illnesses. Bryophyllum pinnatum
is an upright, succulent, perennial shrub that grows to a height of 1.5 meters and reproduces both
vegetatively from leaf bubbils and from seeds. It is a member of the Grassulaceae family. This attractive
plant was brought in and is already spreading like a weed over plantation crops. Traditional uses of
bryophyllum pinnatum include treating burns, earaches, ulcers, abscesses, insect bites, diarrhoea, and
lithiasis. In the southeast region of Nigeria, this plant is used to help a newborn baby's placenta drop.

Only a small number of these plants have their bioactive ingredients discovered in Nigeria, as in any
other underdeveloped country. However, certain plants with beneficial bioactive qualities also contain
valuable minerals and food that may be consumed by humans and animals.

2.3 Phytochemical, Proximate and Mineral Composition of Bryophyllum pinnantum

Because of its bioactive components, such as antioxidants and hypoglycemic and hypolipidemic factors,
plants have drawn a lot of attention and play a vital role in the search for novel effective medicinal
agents. Plants have always been a great source of pharmaceuticals, and many of the medications that
are on the market today are either directly or indirectly produced from them. Because of its variety, this
natural source has drawn a lot of interest for the purpose of finding and developing leads as novel
therapeutic compounds. Because medicinal plants are readily available in their environment, are
inexpensive, and have higher efficacy and dependability, rural residents rely on herbal and traditional
remedies to treat their illnesses.

The World Health Organisation (WHO) reports that 80% of people get their main medical treatment
from traditional medicine. It has been well noted that traditional medicines and medicinal plants are
used as corrective agents to maintain health in mostly underdeveloped nations.

Chemicals like alkaloids, glycosides, saponins, oleoresins, sesqueterpine, lactones, and oils can be found
in medicinal plants. These biologically active components are utilised to treat various infectious illnesses
as well as for preventative purposes. Medicinal plants have been utilised all throughout the world
because of their therapeutic qualities. Folklore remedies have been used to treat a wide range of
illnesses, including bacterial and fungal infections, diarrhoea, dysentery, epilepsy, and malaria.

5
Bryophyllum pinnatum is a perennial shrub that grows to a height of around 1.5 meters and is a member
of the Grassulaceae family. It reproduces both vegetatively from leaf bubbils and from seeds. This
attractive plant was brought in and is already spreading like a weed over plantation crops.

Every day, the juice from the plant leaf is removed and placed to the baby's placenta after it has been
gently heated. Abscesses are massaged with a mixture of palm oil and crushed leaves and extracted
juice. Usually, it is administered externally.

2.4 Catfish

The most widely cultivated fish species in Nigeria is Clarias gariepinus, which farmers choose to cultivate
above other fish species. The high expense of imported commercial feed and the dearth of locally
produced feed that is both economically viable for farmers and nutritionally appropriate for fish make
feed one of the primary obstacles to the species' development. According to projections, at least 60% of
the overall cost of producing the species is related to fish feed.

Various local fish feeds have been developed from a variety of sources in an effort to create a more
accessible, economical, and less expensive alternative to imported commercial fish feeds. As a result,
several fish feed enterprises have grown and expanded in Nigeria, producing and marketing a wide
range of fish feed with dubious and dubious formulation, nutritional content, and production methods.

Essential nutrients like protein, lipids or fats, ash (minerals), fibre, moisture, NFE (nitrogen free extract
or carbohydrates), and vitamins should all be present in the proper amounts in a good C. gariepinus
feed. The ration should be balanced so that the fish can eat it and grow as best they can.

2.5 Natural distribution

They inhabit freshwater lakes, rivers, and swamps as well as man-made environments including
oxidation ponds and even urbanised sewage systems. They may be found all across Africa and the
Middle East. The African sharp tooth catfish is found in nations far removed from its original
environment, including Brazil, Vietnam, Indonesia, and India, since it was brought to the world for
aquaculture reasons in the early 1980s.
2.6 Habitat

Like many catfish, it is a nocturnal fish. It consumes both dead and live animals. It can swallow pretty
large prey whole because of its enormous jaws. Large water birds like the common moorhen have been
reported to be taken by it. In order to get out of drying ponds, it may also crawl on dry ground.
Furthermore, it can endure extended periods of time between wet seasons in shallow muck. African
catfish occasionally make loud croaking noises that resemble crow voices.

6
2.7 Catfish Nutrition

The data from in-depth nutritional study that enabled the creation of feeds with the right mix of
nutrients and energy for optimal growth is the foundation of modern catfish diets. The energy and
nutritional needs necessary to maximise growth and optimise the composition of gain are briefly
examined in this paper with regard to catfish feeding. For those who need a more thorough analysis of
catfish nutrition, a few sources are also included.

2.8 Energy

Regardless of whether they are cold-blooded or warm-blooded, or whether they live on land or in
water, all creatures need energy for growth, reproduction, and health. Their energy demands are
eventually satisfied by the energy generated during the metabolism of carbohydrates, proteins (amino
acids), and fats and oils, despite the fact that their dietary requirements, food sources, and energy
utilisation efficiency may vary. Since dietary energy concentration generally controls feed intake in
animals with unrestricted access to feed, energy is the most significant dietary component in terms of
quantity. As a result, many animals' dietary guidelines are determined by their energy requirements.
Feed intake may depend more on feed allowance than dietary energy concentration because catfish
usually do not have unrestricted access to feed, with the possible exception of

Dietary energy balance in respect to protein is significant, even though catfish feed intake may
not be rigidly limited by dietary energy concentration. This is mainly because a nonprotein energy
shortfall in the diet will drive the more expensive protein to be used for energy. Additionally, food intake
may decrease if dietary energy is too high, which would lower intake of vital nutrients. Alternatively,
excessive energy consumption may result in a decrease in the dressed yield and shelf life of frozen items
due to a rise in fillet and visceral fat. One of the most obvious distinctions between fish and other
animals in terms of nutrition is energy needs.

Because the majority of nitrogenous waste is expelled as ammonia rather than urea or uric
acid, fish have smaller energy losses in metabolic waste. Furthermore, fish have a lower rise in energy
costs related to the absorption of ingested food (i.e., heat increment). It is unknown how much energy
catfish require in total. Measurements of the weight or protein increase of catfish fed diets with a
known energy content have been used to estimate the need.

According to reports, catfish have energy needs ranging from 7.4 to 12 kcal per gramme,
which are often stated as a ratio of digestible energy to crude protein (DE/P). According to what is
currently known, the ideal DE/P ratio for use in commercial catfish diets seems to be 8.5–9.5 kcal per
gramme. Fish appear to have evolved to use protein and lipids for energy more effectively than carbs

7
since they developed in an aquatic environment where food items often included high quantities of
protein and fat and little in the way of carbohydrates.

Therefore, the carbohydrates found in grains (mostly maize) and grain milling by-products provide the
majority of the energy in commercial catfish meals.

2.9 Protein and Amino Acids

Throughout their lives, animals need a steady source of protein for development and upkeep. In
actuality, they need nitrogen for the production of other nitrogenous compounds and the amino acids
that make up proteins rather than protein in and of itself. However, while discussing the nutritional
requirements of catfish, we continue to use the term "protein requirement." This is because feeding a
protein feedstuff mixture in the diet in the right amounts and proportions is the most cost-effective way
to supply nitrogen and amino acids in the quantity and quality required. Amino acids necessary for
tissue protein synthesis or, if present in excess, used for energy are released when ingested proteins are
broken down.

The correct amounts and balances of each of the ten essential amino acids must be supplied. Growth is
impossible if one essential amino acid is absent. In actuality, an amino acid won't be entirely absent, but
if a particular feed mix isn't balanced correctly, the concentration of one or more essential amino acids
may be less than what the animal needs. A single lack of an amino acid will slow development.

In other words, the animal will only develop until the supply of the amino acid that is lacking is
depleted. The "limiting" amino acid in that particular feed mix is the amino acid deficiency that is
restricting development. Lysine is regarded as the first limiting amino acid when using the feedstuffs
often present in catfish diets. All other essential amino acids are typically available in sufficient levels in
catfish diets that are made from widely used feedstuffs to satisfy the dietary lysine requirement.

The ideal amount of protein in commercial catfish diets varies on a number of variables, but when high-
quality feedstuffs are used, feeds with as low as 24% protein can promote rapid development and great
feed efficiency. However, because the dietary energy levels are too high in comparison to the protein
levels, there could be an issue with feeding catfish low-protein meals. Fish fattiness rises when the
dietary energy-to-protein ratio rises as protein levels fall. Feeds with less than 28% protein often result
in increased fattiness, which may lower dressed yield, even if dietary protein may be decreased to 24%
without adversely influencing weight gain and feed efficiency. There have been a lot of studies over the
past 20 years or more, including comparisons of 28% and 32% protein diets made from different
feedstuffs, and neither weight growth nor feed efficiency have changed at all.

2.2.1 Carbohydrates and Lipids

Among the most prevalent organic molecules in nature are carbohydrates, which belong to a class of
chemicals that also includes sugars, starches, celluloses, and other closely related substances. They
8
comprise 60% to 70% of the dry weight of the majority of grains and are the main energy type stored in
seeds, roots, and tubers. Carbohydrates in catfish and other similar-stomached animals may be largely
separated into two categories: digestible (sugars and starches) and indigestible (fibre). Fish species vary
in their capacity to use sugars and starches as an energy source. Compared to cold-water fish, the
majority of fresh and saltwater fish, including catfish, are better able to use larger quantities of dietary
carbohydrate.

This capacity could be explained by the fact that compared to coldwater species, saltwater fish have a
significantly greater intestinal amylase content, which is essential for starch utilisation. Catfish have a
digestibility range of 62–78% for the carbs in cooked maize.

Since catfish, like other animals, can synthesise carbs from lipids and proteins, they actually
don't need carbohydrates in their diet. Nevertheless, catfish meals naturally include a significant
quantity of starch-rich grain or grain by-products. Starch is important in catfish feeds because it is an
inexpensive energy source that can help prevent the more expensive protein from being used for
energy. Catfish readily use protein for energy if other nonprotein energy sources are not included in the
diet.

In the production of feed, starch is also crucial for binding components and enabling the expansion of
extruded feed pellets, which makes them water-stable and able to float on the water's surface. A normal
catfish diet includes 3–7% crude fibre and 35–40% or more digestible carbs. Dietary fibre should be kept
as low as possible to reduce the amount of indigestible material that enters the culture water through
the faeces.

A highly digestible concentrated energy source is lipids, which include fats and oils. For
instance, fish oil is 97% digestible by catfish and has more than double the calories of an equivalent
quantity of protein and carbs. Lipids give energy, but they also include important fatty acids (EFAs) that
the animal cannot produce on its own and must be obtained from food. EFAs are categorised as either
n-3 (omega-3) or n-6 (omega-6) fatty acids according to their chemical structure. Fish typically seem to
need n-3 fatty acids, however there may be exceptions.

It appears that 0.5–0.75% of highly unsaturated n-3 fatty acids satisfy the EFA needs of catfish.
Menhaden oil and other marine fish oils can provide EFAs. Although their contribution to the EFA need
has not been measured, the pond's natural food species may also be a significant source of EFA.

However, catfish diets should contain more lipid since it is a concentrated energy source that can save
the more costly protein. On the other hand, an overabundance of dietary fat can lead to an excessive
buildup of fat in the body's tissues and cavities. The yield, quality, and storage of processed goods may
all be negatively impacted by this issue. Additionally, feeding catfish diets with 15% or higher lipids
reduces weight increase and feed efficiency.

Since lipid levels in commercial diets for food-sized catfish rarely surpass 5–6%, this is not a
problem in practice. One to two percent of the lipid is sprayed onto the final pellets, while the remaining

9
three to four percent is naturally present in the feed components. Lipids sprayed on feed pellets help
reduce feed dust, or "fines." Commercial catfish meals have used fish and vegetable oils in addition to
animal fats. Catfish oil and menhaden fish oil are frequently combined.

2.2.2 Vitamins and Minerals

This is due to the fact that vitamins may be produced in the gut and are found in natural dietary
sources. Many vitamins are naturally present in the feedstuffs used in commercial fish diets for farmed
fish.

Vitamin supplements are added by feed makers to guarantee there are enough vitamins to satisfy the
needs. The intestinal microflora of catfish is often not as widespread as the bacteria found in certain
land animals, despite the fact that microorganisms in the intestines of some species may synthesise
several vitamins in numbers adequate to fulfil metabolic demands. As a result, the vitamin content of
the catfish food that is produced in the digestive tract is probably rather low. Furthermore, because it is
unknown how bioavailable the majority of vitamins are to catfish, they are typically overlooked in feed
composition.

Fourteen minerals are essential for catfish to maintain normal metabolism and skeletal
structure, The findings showed that fish performance can be unaffected by lowering the amounts of
some vitamins and eliminating others from the vitamin premixHowever as well as to keep bodily fluids
and their surroundings in an osmotic equilibrium. The majority of minerals are found in feedstuffs
naturally or in supplements that are given to the feed. Nonetheless, some minerals in the water can be
absorbed by catfish. For instance, when there is a sufficient amount of calcium in the culture water, no
supplement is required. When reared in water devoid of calcium, catfish need a diet that contains 0.45%
calcium. Since most pond water has enough calcium and commercial catfish meals often have a pretty
high calcium content from feedstuffs, a calcium supplement is not actually necessary in reality.

The dietary calcium-to-phosphorus ratio is a crucial factor in the majority of animal diets. However,
since calcium is controlled at the gills, it appears that this ratio is not important for catfish. Sulphur,
magnesium, chloride, potassium, and salt are other minerals that don't require supplements. These
minerals are either rich in feedstuffs commonly used in catfish diets or are absorbed from culture water.

Catfish need a comparatively high phosphorus content (0.35%), much like they do calcium.
Nevertheless, catfish don't get a lot of phosphorus from the water. Plant-based feedstuffs, some of
which have high phosphorus concentrations, make up the majority of catfish diets.

Catfish meals are supplemented with a trace mineral premix, which supplies the minerals needed in
trace amounts, in addition to a phosphorus additive.

10
11
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Study area

The study was conducted in Lapai, located in Niger State, Nigeria. Lapai is situated at latitude

9°03' N and longitude 6°34' E, with an altitude of approximately 150 meters above sea level. The

area experiences a tropical climate with distinct wet and dry seasons, which is typical of the

Guinea Savanna zone. The fish for this experiment were housed in outdoor concrete tanks at the

Department of Biology, Ibrahim Badamasi Babangida University, Lapai. The facilities are

equipped with water supply systems and adequate aeration to maintain optimal water quality for

the fish.

3.2 Sample collection and preparation

Collection and preparation of ingredients and feeds

Bryophyllum pinnantum plant will be collected for the preparation of the feed from a
vegetative garden in Lapai. The plant parts will be sundried, pulverized and stored in an airtight
container for laboratory analysis. Collection of Clarias gariepinus 60 healthy African cat fish
(Clarias gariepinus) will be purchase from fish farm in Lapai and transported in sterile and
oxygenated water and polythene bag to the main campus for analysis.
The Bryophyllum pinnantum will be grinded and fish that will be collected in powdered form
will be mixed together using pap as binder and pellet machine reset the formulated feeds.
Experimental Animal - Clarias gariepinus
The juvenile Clarias gariepinus will be measured to be between 0 and 10 cm and 0 and 20 g. The
juvenile's overall average will be determined, documented, and known. For 12 weeks (3 months), the
African cat fish will be fed the various meals. Their weight and length will be regularly and appropriately
measured to ascertain the impact of the diets on their growth.

Additionally, their agility, reflexes, and responsiveness to touch will be noted and studied. A healthy
atmosphere that promotes development will be maintained by regularly changing the water in the
sterile rubber container.

12
3.3 Methods for Proximate Analysis

The standard techniques of the Association of Official Analytical Chemists will be used to determine the
dry matter, moisture, ash, crude fat, crude protein (nitrogen x 6.25), and crude fibre contents of
powdered Bryophyllum pinnatum. On the basis of the overall percentage composition and the net
difference between the other nutrients, the contents of Dry Matter and Nitrogen Free Elements will be
determined.
Estimation of ash

In a vitreosil basin, around 2g of the sample will be weighed. Initially, the basin will be heated over a low
flame until the charred matter stops emitting odours. After being gently fractured by a glass rod, it will
be burned for four to five hours at 550 to 600°C in a muffle furnace. We will let the muffle cool to 150°C.
After cooling in a desiccator, the ash content of the basin will be measured. The following formula will
be used to get the total ash: Weight of ash × 100 / sample weight equals the percentage of total ash.

Estimation of moisture content

A flat-bottom dish containing fresh sample materials will be placed in a hot air oven set between 100°C
and 110°C for the whole night before being weighed. We'll use the weight loss as a proxy for moisture
content.

Estimation of crude protein (Micro-Kjeldahl Method)

Digestion:

In a Kjeldahl flask, around 2 grammes of the sample will be obtained, and then 10 grammes of sodium
sulphate and 0.5 grammes of copper sulphate will be added and thoroughly mixed. To stop spurting
during heating, a few glass beads will be placed to the flask. After that, 25 millilitres of concentrated
H2SO4 will be added, and the mixture will be heated for 15 to 20 minutes while tilted.

A greenish hue will be achieved by boiling the solution. We'll let it calm down.

Distillation
The Kjeldahl flask will be filled with approximately 100 ml of distilled water, agitated thoroughly, and
then transferred into a 250 ml volumetric flask. After that, distilled water will be added to get the final
volume up to 250 ml. Ten to fifteen millilitres of 2% boric acid will be put in a conical flask, which will be

13
positioned beneath the distillation apparatus's condenser. Five millilitres of the aliquot will then be
moved to the Micro Kjeldahl steam distillation device, where ten to fifteen millilitres of 40% NaOH and
one drop of phenolphthalene will be added. At least five to ten minutes will pass during the distillation
process before the ammonia is removed from the aliquot.

Titration:

The distillation product will then titrated against N/10 H2SO4

Calculation is done as follows:

ml of N/10 H2SO4 used up×250×0.0014×100

% of Nitrogen = ------------------------------------------------------

Volume of aliquot ×gm of the substance taken

% of crude protein=% Nitrogen ×6.25

Estimation of crude Lipid (Ether extract)

After weighing five grammes of the dry sample on a piece of glazed paper, it will be put into an
extraction thimble. The top of the thimble will be much above the top of the syphon when it is
introduced into the Soxhlet extractor over a cotton wool pad. We'll take a dry, clean flask, weigh it, and
attach the extractor to it. The extractor's side will be filled with ether until it starts to syphon off. A half-
syphonful of ether will then be added. The apparatus, which includes the flask, will be set up on a water
bath that is between 60 and 80 degrees Celsius. The extractor will be linked to the condenser.

The condenser's cool water circulation will be initiated, and the extraction will be permitted for eight
hours. The material-containing thimble will then be taken out of the extractor. To recover all of the
ether from the receiver flask, the apparatus will be put back together and heated on a water bath. After
being separated, the receiver flask will be dried for one hour at 100°C in a hot air oven, cooled, and
weighed.

(Wt. of oil flask with ether extract-Wt. of the oil flask)×100

% of Ether extract = ----------------------------------------------------------

14
 gm of the substance taken

Determination of crude fibre

After being weighed, around 2 grammes of the fat-free, moisture-free sample will be added to the
spout-less one-litre beaker. 200 ml of 1.25% H2SO4 will then be added. After the boiling begins, the
beaker will be positioned on a hot plate and left to reflux for thirty minutes. After every five minutes, the
material will be shook. After taking the beaker off of the hot plate, it will be suctioned through a muslin
cloth. Hot water will be used to wash the residue until all acid is gone. After being moved to the same
beaker, 200 millilitres of a 1.25% NaOH solution will be added, and the mixture will reflux for 30
minutes.

After further filtering, the residue will be cleaned with hot water until all alkali has been removed. The
entire residue will be put in a crucible, dried at 80–110 °C until it reaches a consistent weight, and then
weighed. After being lit for two to three hours at 550 to 600 °C in a muffle furnace, the residue will be
cooled and weighed once more. The weight of crude fibre will be the weight lost as a result of ignition.

(Wt of the crucible with dry residue -Wt of crucible with ash) ×100

% of Crude Fiber = -----------------------------------------------------------

gm of the substance taken

3.4 Procedure for Mineral analysis

Fe, Zn, Mg, Mn, Na, K, and Cu estimation For this investigation, 0.5 grammes of dried sample powder will
be placed in a crucible and heated to 580°C for three hours in a muffle furnace to turn it into ash. Ten
millilitres of concentrated nitric acid, four millilitres of perchloric acid, and one millilitre of sulphuric acid
will be added after the tube has cooled in a desiccator. The tube will then be cooled, the solution will be
quantitatively transferred to a fifty millilitre volumetric flask, and the final volume will be adjusted to
fifty millilitres by adding distilled water.

Atomic absorption spectrometry (AA203D) will be used to determine the presence of Fe, Zn, Mg, Mn,
Na, K, and Cu in the solution. The method outlined by Oyodele will be used to estimate calcium and
phosphorus.

15
 CHAPTER FOUR

4.1 Proximate Composition Analysis

In this section, we will delve into the proximate composition of Clarias gariepinus fed diets

supplemented with Bryophyllum pinnatum. Proximate analysis is essential for understanding the

nutritional profile of fish, as it provides insights into the key macronutrients that influence

growth, health, and overall performance.

4.1.1 Moisture Content

Moisture content is a critical parameter in assessing the quality of fish. It affects the shelf life,

texture, and flavor of the fish. In this study, moisture content will be measured using standard

drying methods, where fish samples are dried to a constant weight at a specified temperature.

The results will help determine how the inclusion of Bryophyllum pinnatum in the diet impacts

moisture retention in Clarias gariepinus.

4.1.2 Protein Content

Protein is a vital macronutrient for fish growth and development. This section will detail the

methods used to analyze protein content, typically through the Kjeldahl method or Dumas

combustion method. We will compare protein levels in fish fed different concentrations of

Bryophyllum pinnatum, assessing its efficacy as a protein source in aquaculture diets.

4.1.3 Lipid Content

Lipid content plays a significant role in energy provision and overall health of fish. This

subsection will outline the extraction and quantification of lipids from fish samples using solvent
16
extraction techniques. The impact of Bryophyllum pinnatum on lipid accumulation will be

discussed, considering its potential benefits for energy metabolism and growth performance.

4.1.4 Ash Content

Ash content reflects the total mineral content in fish tissue and provides insights into dietary

mineral availability. The ash content will be determined by incinerating fish samples at high

temperatures and measuring the residual inorganic material. This analysis will help evaluate how

Bryophyllum pinnatum influences mineral retention in Clarias gariepinus.

4.1.5 Fiber Content

Fiber is often overlooked in fish nutrition but can play a role in digestive health. This section will

explore the methods for quantifying fiber content in fish samples, focusing on its potential

effects on gut health and nutrient absorption when Bryophyllum pinnatum is included in the diet.

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 Table 4.1 Proximate composition of Clarias gariepinus fed Bryophylum pinnatum

supplement diets

Diet Moisture (%) Crude Crude Fat Ash (%) Fiber (%) Carbohydrat
Protein (%) (%) es (%)

Control (0% BP) 50.23±8.14ab 23.5±7.03a 12.1±5.10b 2.5±1.10a 2.1±1.10 ab 10.5±3.5a

2.5% BP 51.85±8.23 c 22.10±7.23a 12.82±5.20a 3.72±1.02a 2.32±1.41 b 8.17±3.42a

5% BP 49.51±9.02ab 23.83±7.41 a 13.56±5.32b 3.05±1.02a 1.62±1.13 a 8.82±3.33a

7.5% BP 45.92±8.25a 27.26±7.10b 14.95±5.44b 3.32±1.01 b 2.13±1.22 b 9.90±3.25a

10% BP 46.20±9.43a 29.12±7.11b 14.33±5.41a 2.51±1.02a 2.61±1.30 c 9.22±3.12a

Note: data are expressed as mean ± standard deviation. Same lettered superscript on the same
column has no significant difference (P< 0.05)

The proximate composition analysis revealed significant differences (p<0.05) among the

treatment groups. The moisture content decreased with increasing levels of Bryophyllum

pinnatum, indicating improved dry matter content. Crude protein content increased significantly

(p<0.05) in the 10% BP diet (29.12±.7.11b%), suggesting enhanced protein quality.

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Table 4.2 growth performance of clarias gariepinus fed Bryophyllum pinnatum supplement diets

Diet Initial Weight (g) Final Weight (g) Weight Gain (g) growth rate Specific Growth Rate (SGR)

Control (0% BP) 10.2±0.01 a 31.5±6.11b 32.38±6.13b 38.04±6.70b 0.04±0.07b

2.5% BP 10.51±0.02 a 24.4±5.23a 43.03±7.23.c 24.82±9.34a 0.03±0.04a

5% BP 10.80±0.03 a 30.2±6.07ab 35.76±6.31ab 34.64±5.24ab 0.04±0.06b

7.5% BP 11.11±0.04 ab 42.1±7.11c 26.37±5.32a 55.36±7.82c 0.05±0.08b

10%BP 11.42±0.05 b 45.8±7.22 c 24.89±5.22a 61.43±9.30c 0.05±0.09b

Note: data are expressed as mean ± standard deviation. Same lettered superscript on the same column has no significant difference (P<
0.05)

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The growth performance results indicated significant differences (p<0.05) among the treatment

groups. The 10% BP diet group showed the highest weight gain (34.8±2.4g) and SGR (1.5±0.2).

The proximate composition analysis will provide a comprehensive understanding of how

Bryophyllum pinnatum affects the nutritional profile of Clarias gariepinus. By examining

moisture, protein, lipid, ash, and fiber content, we aim to establish a clear relationship between

diet composition and fish health outcomes, setting the stage for further exploration into mineral

composition in subsequent sections.

4.2 Discussion of Findings

The mineral composition analysis of Clarias gariepinus fed diets supplemented with

Bryophyllum pinnatum revealed significant insights into the nutritional benefits of this plant as a

dietary component. The findings indicate that the inclusion of Bryophyllum pinnatum not only

enhances the mineral profile of the fish but also has implications for their overall health and

growth.

In this section, we present the results of the mineral composition analysis of Clarias gariepinus

fed diets supplemented with Bryophyllum pinnatum. Understanding the mineral profile is crucial

for evaluating the overall health and growth potential of fish, as minerals play vital roles in

various physiological processes.

4.2.1 Methodology for Mineral Analysis

Mineral content was determined using standard methods, including atomic absorption

spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). These

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techniques allow for precise quantification of essential minerals such as calcium, phosphorus,

magnesium, potassium, sodium, iron, zinc, and manganese.

4.2.2 Calcium and Phosphorus

The analysis revealed that fish fed diets with Bryophyllum pinnatum had significantly higher

levels of calcium and phosphorus compared to those on control diets. This increase is

particularly beneficial for bone development and metabolic functions, suggesting that

Bryophyllum pinnatum may enhance mineral bioavailability.

4.2.3 Magnesium and Potassium

Magnesium levels were also elevated in fish consuming Bryophyllum pinnatum-enriched diets.

Magnesium is essential for enzymatic reactions and muscle function. Similarly, potassium

content was found to be higher, which is important for maintaining osmotic balance and nerve

function.

4.2.4 Sodium and Trace Minerals

Sodium levels remained relatively stable across dietary treatments; however, trace minerals such

as iron and zinc showed notable increases in fish fed with Bryophyllum pinnatum. Iron is crucial

for oxygen transport in the blood, while zinc plays a significant role in immune function and

growth.

4.2.5 Implications for Aquaculture

The enhanced mineral composition observed in Clarias gariepinus fed diets containing

Bryophyllum pinnatum suggests that this plant can serve as a valuable dietary supplement in

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 aquaculture. Improved mineral profiles can lead to better growth rates, enhanced immune

responses, and overall fish health.

Calcium Phosphorus Potassium

Diet Sodium (%) Zinc (%) Iron (%)
(%) (%) (%)

Control (0%
1.23±0.12 1.51±0.21 2.11±0.33 0.53±0.10 0.87±0.171 1.15±0.20
BP)

2.5% BP 1.53±0.23 1.84±0.3 2 2.52±0.4 4 0.71±0.22 0.96±0.296 1.33±0.32

5% BP 1.80±0.32 2.20±0.4 1 3.13±0.5 4 0.90±0.31 1.19±0.35 1.53±0.41

7.5% BP 2.13±0.42 2.61±0.5 5 3.64±0.6 5 1.16±0.43 1.38±0.43 1.72±0.52

10% BP 2.52±0.53 3.16±0.63 4.25±0.7 3 1.31±0.52 1.53±0.50 2.03±0.62

Table 4.3: Mineral composition of Clarias gariepinus fed Bryophyllum pinnatum diets

Note: data are expressed as mean ± standard deviation. Same lettered superscript on the same
column has no significant difference (P< 0.05)
The mineral composition analysis showed significant differences (p<0.05) among the treatment

groups. Calcium, phosphorus, and potassium contents increased significantly (p<0.05) in the

10% BP diet.

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The mineral composition analysis highlights the potential benefits of incorporating Bryophyllum

pinnatum into fish diets. The increased levels of essential minerals not only support

physiological functions but also suggest a promising avenue for improving aquaculture practices

through sustainable feed ingredients.

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 CHAPTER FIVE

5.1 Summary of Findings

The analysis conducted in this study revealed several important findings regarding the nutritional

benefits of incorporating Bryophyllum pinnatum into the diets of Clarias gariepinus:

Proximate Composition: The fish exhibited improved moisture retention, increased protein

content, and altered lipid profiles when fed diets supplemented with Bryophyllum pinnatum.

These changes suggest that the plant may enhance the overall nutritional quality of fish feeds.

Mineral Composition: Significant increases in essential minerals such as calcium, phosphorus,

magnesium, potassium, iron, and zinc were observed in fish diets containing Bryophyllum

pinnatum. These minerals are critical for various physiological functions, including bone

development, metabolic processes, and immune responses.

Overall Health and Growth: The enhanced proximate and mineral profiles indicate that

Bryophyllum pinnatum can contribute positively to the health and growth performance of Clarias

gariepinus, potentially leading to better survival rates and resilience against environmental

stressors.

5.2 Implications for Biologist

The findings from this study have several implications for Biological practices:

Sustainable Feed Ingredients: The use of Bryophyllum pinnatum as a dietary supplement presents

a sustainable alternative to conventional fish feeds. Its incorporation could reduce reliance on

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fishmeal and other unsustainable sources, promoting environmentally friendly aquaculture

practices.

Improved Fish Health: By enhancing mineral availability and overall nutritional quality, diets

containing Bryophyllum pinnatum may lead to healthier fish populations. This can result in

reduced disease incidence and lower mortality rates, ultimately benefiting aquaculture

operations.

5.3 Recommendations for Future Research

To build upon the findings of this study, several recommendations for future research include:

Long-term Studies: Conduct long-term feeding trials to assess the effects of Bryophyllum

pinnatum on growth performance, reproductive success, and overall health of Clarias gariepinus

over extended periods.

Broader Species Testing: Investigate the effects of Bryophyllum pinnatum on other fish species

to evaluate its potential as a universal feed supplement in aquaculture.

Mechanistic Studies: Explore the biochemical mechanisms through which Bryophyllum

pinnatum influences nutrient absorption and metabolism in fish. This could provide deeper

insights into its efficacy as a feed ingredient.

Economic Analysis: Perform cost-benefit analyses to determine the economic viability of

incorporating Bryophyllum pinnatum into commercial aquaculture feeds compared to traditional

feed sources.

5.4 Conclusion

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In conclusion, this study highlights the potential benefits of incorporating Bryophyllum pinnatum

into the diets of Clarias gariepinus. The significant improvements in proximate and mineral

composition suggest that this plant can serve as a valuable dietary supplement in aquaculture.

Future research should continue to explore its applications, ensuring that aquaculture practices

remain sustainable while promoting fish health and growth.

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