MAKERERE UNIVERSITY

COLLEGE OF NATURAL SCIENCES

SCHOOL OF BIOSCIENCES

DEPARTMENT OF PLANT SCIENCES, MICROBIOLOGY AND BIOTECHNOLOGY

GROWTH PERFORMANCE OF RATTUS NORVEGICUS FED ON VARYING CONCENTRATIONS OF AZOLLA MEAL

BY

OKELLO ISAAC OPIO

BACHELOR OF SCIENCE IN BIOTECHNOLOGY

16/U/1021

A RESEARCH REPORT SUBMITTED TO THE DEPARTMENT OF PLANT SCIENCES, MICROBIOLOGY AND BIOTECHNOLOGY IN PARTIAL FULFIILMENT OF THE REQUIREMNETS FOR THE AWARD OF BACHELOR OF SCIENCE DEGREE IN BIOTECHNOLOGY OF MAKERERE UNIVERSITY

AUGUST, 2019

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DEDICATION This report is dedicated to my beloved parents, Mr. James Opio and Mrs. Jasinta Opio for their moral care, financial supports, and encouragement during the process of developing and conduction of this research work. And to my siblings; Scovia, Anyes, Dorine, Jacob, David and Aron for their trusts and believe in my abilities.

I also dedicate to my friends beloved friends; Mary, Sandra, Godfrey, Isaac, Paul, Justin, Tonny, Alex, and Daniel.

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ACKNOWLEDGEMENT I thank the God Almighty father for His abundant grace, knowledge, wisdom, protection, and strengths that He granted me to start this piece of work.

I am also grateful to all the staffs of the department for their guidance, knowledge, time, and tireless assistance that resulted to a successful finish of this proposal.

Appreciations also go to my fellow students whom I study with for their discussions and technical assistance towards writing this report.

Special thanks also go to my University supervisors; Dr. Ssenku E. Jamilu for dedicating his times to guide me and give me all kinds of academic advice.

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TABLE OF CONTENTS DECLARATION ...... i APPROVAL ...... ii DEDICATION ...... iii ACKNOWLEDGEMENT ...... iv LIST OF FIGURES ...... vii LIST OF ACRONYMNS AND ABBREVIATIONS ...... viii CHAPTER ONE ...... 1 1.0 INTRODUCTION ...... 1 1.1 Background of the study ...... 1 1.2 Problem statement ...... 2 1.3 Objectives ...... 3 1.3.1 General objectives ...... 3 1.3.2 Specific objectives ...... 3 1.4 Justification ...... 3 1.5 Significance of the study ...... 3 1.6 Hypotheses ...... 3 CHAPTER TWO ...... 4 2.0 LITERATURE REVIEW ...... 4 2.1 Introduction ...... 4 2.2 Fodder production ...... 4 2.3 Taxonomy of Azolla ...... 4 2.4 Distribution of Azolla ...... 5 2.5 Morphology of Azolla ...... 5 2.6 Reproduction in Azolla ...... 5 2.7 Growth of Azolla ...... 6 2.8 Nutrient composition of Azolla ...... 6 2.9 Utilization of Azolla ...... 7 CHAPETR THREE ...... 8 3.0 MATERIALS AND METHODS ...... 8 3.1 Nature of study design ...... 8 3.2 Study area...... 8

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3.3 Sample Collection and Survey ...... 8 3.4 Study materials ...... 9 3.5 Sampling strategy ...... 9 3.6 Preparation of Azolla meal ...... 9 3.7 Treatments...... 10 3.8 Data collection ...... 11 3.9 Statistical analysis ...... 12 CHAPTER FOUR ...... 13 4.0 RESULTS AND DISCUSSION ...... 13 CHAPTER FIVE ...... 15 5.0 CONCLUSION AND RECOMMENDATION ...... 15 5.1 Conclusion ...... 15 5.2 Recommendations ...... 15 REFERENCES ...... 16 APPENDICES ...... 21 Appendix 1: Raw data of the life weights of Rattus norvegicus taken during the feeding experiment .. 21

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LIST OF FIGURES Figure 1: Marking of the animals...... 9 Figure 2: Rattus norvegicus in one of the cages ...... 9 Figure 3: Azolla cake on the paper bag ...... 10 Figure 4: Weighing of Azolla cake ...... 10 Figure 5: Changing of beddings ...... 11 Figure 6: Weighing of experimental animal ...... 11 Figure 7: Changes in the body weight of the control and treatment groups during the feeding trial...... 13

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LIST OF ACRONYMNS AND ABBREVIATIONS AZM Azolla meal CLPC Crantz Leaf Protein Concentrates CP Crude protein Dr Doctor FAO Food and Agricultural Organization FBW Final body weight IBW Initial body weight LVFO Lake Victoria Fisheries Organization MWE Ministry of Water and Environment NDF Neutral Detergent Fibre

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ABSTRACT

Plant proteins have been reported to be useful in animal nutrition in this period of sincere efforts of reducing cost of production attributed to high cost of protein sources. This study was conducted to investigate how far Rastrineobola argentea, locally known as ‘Mukene’ can be replaced with Azolla meal. A total of 20 Albino rats (Rattus norvegicus) eight-to-ten weeks of age (average initial weight 100±5g) were used in a growth trial to investigate the potential of Azolla based diets on their growth performance. The feeding trial which lasted 38 days, dietary treatments were assigned to five caged groups of Rattus norvegicus balanced for ancestry, sex, and body weight. The groups were assigned to five experimental diets in a completely randomized design (CRD) consisting of five treatments and four replicates with one rat. Five dietary treatment groups with Azolla replacing Mukene; 0% (control), 25%, 50%, 75% and 100% inclusion levels as protein source. A cake of Azolla meal was developed and fed to Rattus norvegicus. Data were collected on body weight after every 4 days for 24 days and were subjected to analysis of variance statistically using Graph pad prism software, Version 5.01 at 95% confidence level. When Mukene was substituted for Azolla, the average daily feed intake, was not different across all diets, but average weight gain was highest (P<0.05) for the 50% than other levels of substitution. The final body weight of were significantly (P<0.05) affected by feeding on 0%, 25% and 50% Azolla meal formulation. The final body weight of Rattus norvegicus were insignificantly (P<0.05) affected by feeding on 75% and 100% Azolla meal formulation. This study revealed that Azolla can be used as a replacement for Mukene. However, the inclusion must not exceed 50%. This indicated that Azolla can be used to supplement protein source but not totally substituting Mukene as the sole protein source for animal feeds.

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CHAPTER ONE

1.0 INTRODUCTION

1.1 Background of the study Agriculture is playing inevitable role in enhancing a lucrative livelihoods of the people in Uganda. This is evidenced by the poverty assessment report from the World Bank (2016), where poverty reduction among households working in agriculture accounts to over 79% of national poverty reduction observed between 2006 and 2013. More or less two thirds of rural households in developing countries are partly or completely reliant on livestock for their livelihoods (Pica- Ciamarra, Baker, Morgan, Zezza, Azzarri, Nsiima, Nouala, Okello & Sserugga, 2015). This is because livestock rearing provides them with a large number of benefits, such as income, savings, food, manure, insurance, social status, and renewable energy (Kugonza, Kirembe, Tomusange, Lutalo & Drani, 2012). Whereas successful agriculture holds the answer to the poverty gap between various nations, it should be noted that availability of quality feed at a reasonable cost is a key to successful animal farming operation (Basak, Pramanik, Rahman, Tarafdar & Roy, 2002).

In many African countries, protein ingredients, specifically fishmeal remain the most increasingly expensive part of the feed, contributing to 70 to 80% of the production cost (Fiaboe & Nakimbugwe, 2017). This is in support of the claim by Parthasarathy, Kadirvel & Kathaperumal (2001) that feed is the most expensive of all inputs and about 70% of production cost is accounted for feed alone. There is a substantial decline in fodder production and modern livestock production is faced with the challenges of producing high quality animal protein at low prices owing to the decreasing area under forest and increase in the prices of conventional protein feedstuffs due to competition among ingredients used in the manufacture of animal feed (Kugonza et al., 2012). The shortages of animal feeds are however being compensated with commercial feeds, which results to an increased cost of production of animal products (Pillai, Premalatha, & Rajamony, 2002). In Uganda, the protein is usually obtained from Rastrineobola argentea, available in the water bodies whose market price is high but available quantities are

Page | 1 way below the demand (Fiaboe & Nakimbugwe, 2017). There is thus an enormous need for alternative protein sources that can supply in high demand for such ingredients (Chinsoro, 2015).

The search for an alternative has led to a plant, Azolla, which holds the promise as a sustainable feed substitute for livestock (Pillai et al., 2002). Azolla is a free floating water fern that floats in the water and fixes atmospheric nitrogen because of its association with fixing cyanobacterium, Azollae anabaena (Raja, Rathaur, John & Ramteke, 2012). The Azollae anabaena symbiosis is outstanding due to its high productivity and rapid biomass production which is due to the high relative growth rate, thereby increasing the protein, carotene and β carotene contents (Lakshmanan, Kumar & Latha, 2017).

Azolla therefore appears to be a possible source of nutrients and has a considerably high feeding value (Hassiny, Rokni, Dehghanzadeh & Cheraghcheshm, 2008). Long before its cultivation as a green manure, Azolla was used as feed for domesticated animals such as pigs and duck, thus suggesting that Azolla can be used as unconventional feed with protein supplement for many species including ruminants, poultry, pigs and fish (Joysowal, Abdul Aziz, Mondal, Mohan, Singh, Boda & Chhaba, 2018). In recent days, Azolla is very much used as a sustainable feed substitute as fodder for goats (Toradmal et al., 2017), feeds for broiler chicken (Balaji et al., 2009), laying hens (Alalade et al., 2007), and buffalo calves (Indira et al., 2009), and its incorporation into animal feeds is profitable (Shoukat et al., 2015). However, potential of Azolla as protein supplement in non-ruminant animals remains unexploited, and growth performance of Rattus norvegicus fed on Azolla meal has not been well documented. Hence, this research was carried out to determine the growth performance of Rattus norvegicus fed on different concentrations of Azolla meal.

1.2 Problem statement In Uganda and Kenya, animal feed protein represents the most important constraint in animal production systems (Fiaboe & Nakimbugwe, 2017), contributing to over 70% of the production cost. Ingredients for protein source are usually obtained from Rastrineobola argentea (Mukene), which has been faced with increase in price from about Ugx 2,000/kg in 2005 (Gordon and Ssebisubi, 2012) to Ugx 5000, in 2017 (Katende, 2017). Even its cost is not expected to reduce because it is increasingly being used as an ingredient in processed human food products (Gordon and Ssebisubi, 2012) coupled with its decline in stock by 50% in Uganda’s fresh water sources

Page | 2 due to environmental degradation (Nakiyende et al., 2016). It will therefore be an unsustainable option to continue to rely on fishmeal as protein source in feed due to the concern about depletion of fish stocks (LVFO Secretariat, 2015). Thus, there is an urgent need to provide an alternative for protein source in animal feeds production system in Uganda and broadening knowledge base on potential use of Azolla as protein source supplement.

1.3 Objectives

1.3.1 General objectives To determine the weight gained by Rattus norvegicus fed on different Azolla meal concentrations.

1.3.2 Specific objectives To determine the live weight of Rattus norvegicus in diet treatments.

1.4 Justification Protein source is a major constraint to animal production, where commercially protein sources for animal feeds are expensive, making cost of production very high. Azolla has been of less focus in most studies of alternative protein sources. This study was to promote awareness on potential use of Azolla as an alternative protein supplement at an economically-friendly level which will facilitate conservation of Mukene, which is currently under threat of depletion from the water bodies around Uganda due to its consumption pressure for both animals and humans.

1.5 Significance of the study This study discovers the possible use of Azolla in replacing Mukene in the diets of Rattus norvegicus. It has shaded light onto the potential of Azolla as protein supplement in the formulation of animal feeds especially non-ruminant animals. The study generated data which will help researchers uncover important area of alternative feed ingredients for effective and least cost protein source for diet formulations for monogastric animals that many researchers were not able to investigate.

1.6 Hypotheses

H0: Difference in Azolla concentration has no effect on the weight gain of Rattus norvegicus.

H1: Difference in Azolla concentration has effect on the weight gain of Rattus norvegicus

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CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Introduction Azolla is a free floating water fern that floats in the water (Hussner, 2010). It fixes atmospheric nitrogen because of its association with fixing cyanobacterium anabaena (Raja et al., 2012), and holds the promise as a sustainable feed substitute for livestock (Pillai et al., 2002). The Azollae- anabaena symbiosis is outstanding due to its rapid biomass production which is due to the high relative growth rate, increased protein, carotene and β carotene contents (Lakshmanan et al., 2017). This technology has promising climate change adaptive capacity and is helpful in climate change adaptation of famers in these areas as climate change is a threat to livestock production because of the impact on quantity and quality of feed crops, fodder and forage (Kumar & Chander, 2017).

2.2 Fodder production Throughout the world, there is a substantial decline in fodder production and protein source throughout the world, owing to the decreasing area under forest (MWE, 2016) and partly attributed to drought and adverse weather conditions (World Bank, 2017). It must be noted that availability of quality feed at a reasonable cost is a key to successful animal farming operation (Basak et al., 2002). Modern livestock production is also faced with the challenges of increase in the prices of conventional protein feedstuffs due to competition among ingredients used in the manufacture of animal feed (Kugonza et al., 2012). These shortages of animal feeds are however being compensated with commercial protein sources, which results to an increased cost of production of animal products (Pillai et al., 2002), thus the consumers face the consequences of high prices of animal products.

2.3 Taxonomy of Azolla Benedetti et al., (2018) categorized Azolla to be belonging to the family of Salvinaceae, a family of heterosporous free-floating ferns. It also belongs in the order Salviniales (Raja et al., 2012). The genus was botanically established by Lamarck in 1783 (Kannaiyan & Kumar, 2006).

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However, recent taxonomists have assigned Azolla to a monotypic family, Azollaceae, separate from the genus Salvinia, and consisted of cyanobiont diversity in six species of Azolla (A. microphylla, A. mexicana, A. filiculoides, A. caroliniana, A. pinnata and A. rubra) (Kumar et al., 2019).

2.4 Distribution of Azolla Azolla is widely distributed throughout the world in both tropical and temperate regions (Parris, 2001). Azolla is native to the tropics, subtropics, and warm temperate regions of Africa, Asia, and America (Costa et al., 2009). Azolla floats on the surface of most ponds, ditches, in small pools, often in stagnant water, in quiet water of streams, lakes and rivers, less often in wet marshes (Tryon & Tryon, 1982). The above claim is supported by the fossil record that indicated a freshwater depositional environment and was interpreted as being preserved in situ due to their excellent preservation and the frequent attachment of microspore massulae to megaspores (Benedetti et al., 2018). Literature shows that, Azolla domestication dates back to the 11th century in Vietnam (Sadeghi et al., 2013).

2.5 Morphology of Azolla According to Hussner (2010), Azolla is a tiny plant of with a diameter of up to 2.5 cm generally. Plants are triangular or polygonal in shape and consist of floating branched rhizome bearing small alternate overlapping leaves with simple roots. The plants are dark green to reddish and float on the water surface, either individually or in mats, which can reach thickness of up to 20cm (McConnachie et al., 2004). It contains megaspore apparatus and microspore massulae. Benedetti et al., (2018) described the megaspore apparatus as composed of the megaspore body and three tiers of floats covered by a dense filosum, while its wall consisted of an exine and a two-layered perine, with a spongy endoperine and large, clavateexoperine. Furthermore, the microspore massulae are inconsistent in size and shape and bear aseptate glochidia with anchor- shaped tips.

2.6 Reproduction in Azolla The main mode of multiplication is vegetative and lateral branches detach from the main rhizome due to formation of abscission layer at their base (Konar, 1972). It also undergoes sexual reproduction that occurs during limited periods of the year. Sexual reproduction is carried out through structures known as sporocarps, which occur in pairs in either same sex or opposite

Page | 5 sex at the lateral branch axils on the ventral side of the rhizome. The pair includes microsporocarps and megasporocarps, where the spores are vertically transmitted to the next fern generation via the fern megaspores (Nagalingum et al., 2006). The microsporocarps are large and globular whereas the megasporocarps are relatively small and ovoid and are referred to as the male and female reproductive structures, respectively. The megaspore divides and forms the prothallus which produces archegonia with one egg cell in each archegonium. After fertilization, the zygote divides and sporophytes are formed (Raja et al, 2012).

2.7 Growth of Azolla During growth, Azolla has a surface area doubling time of 7 to 10 days under favorable conditions (Hussner, 2010) and maintained nitrogen content of 5 to 6% of the dry weight with atmospheric nitrogen as the only nitrogen source. Azolla can survive in water of pH ranging from 3.5 to 10, reported optimum growth occurring at pH 4.5 to 7.0. A. filiculoides for example grows at best 15oC to 20oC and high irradiance (Tung & Watanabe, 1983).

2.8 Nutrient composition of Azolla The use of Azolla as a feed ingredient in feeding livestock indicates that the nutritive value in terms of nutrient content and availability are known. According to Kumari et al., (2018), the dry matter content of Azolla is 4.7 percent while total organic matter is 82.66 percent. Among these includes 21.67 percent crude protein, 3.27 percent ether extract, 12.38 percent crude fiber, and 43.35 percent nitrogen free extract. The value for crude protein (CP) is slightly higher than 21.4 percent reported by Alalade et al., (2006) and the value for crude fibre is slightly lower than 12.7 percent reported by Alalade et al., (2006). The possible reason of variability in CP content is the response of Azolla strains to environmental conditions like temperature, light intensity, water availability and soil nutrients which affect chemical composition (Kumari et al., 2018; Lacoul & Freedman, 2006).

The total Ash and acid insoluble ash content is 19.33 and 3.64 percent, respectively while NDF, ADF and hemicellulose in Azolla (Azolla pinnata) is 41.84%, 28.56% and 13.28%, respectively (Kumari et al., 2018). Alalade et al., (2006) reported that the data on the amino acid analysis indicated that lysine, arginine, isoleucine, leucine, phenylalanine, glycine and valine are predominant. The mineral profile of Azolla (Kumari et al., 2018) indicates 1.64% Calcium and 0.34% Phosphorus and other minerals in trace levels. This is similar to earlier reports of Sujatha

Page | 6 et al., (2013). All these are attributed to the rapid biomass production as a result of high relative growth rate, increasing protein, carotene and β carotene contents (Lakshmanan et al., 2017).

2.9 Utilization of Azolla The green plants have long been recognized as the cheapest and most abundant potential source of proteins because of their ability to synthesize amino acids from a wide range of virtually unlimited, and readily available primary materials (Fasuyi & Aletor, 2005). Azolla has multidimensional uses like source of human food, animal feed, medicine, production of biogas, hydrogen fuel, water purifier, weed control, reduction of ammonia volatilization and because of the multifaceted uses it has been referred to as “green gold mine” (Wagner, 1997).

In the recent times, Azolla is gaining tremendous importance in phytoremediation of polluted waters (Arora, Saxena & Sharma, 2006), and these results showed that Azolla may be successfully employed in phytoremediation of polluted water bodies. In addition, its high protein content makes it suitable as a feed supplement for goats (Toradmal et al., 2017), feeds for broiler chicken (Balaji et al., 2009), laying hens (Alalade et al., 2007), and buffalo calves (Indira et al., 2009), and its incorporation into animal feeds is profitable (Shoukat et al., 2015). This technology has promising climate change adaptive capacity and is helpful in climate change adaptation of famers in these areas as climate change is a threat to livestock production because of the impact on quantity and quality of feed crops, fodder and forage (Kumar & Chander, 2017).

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CHAPETR THREE

3.0 MATERIALS AND METHODS

3.1 Nature of study design The study was carried out in College of Natural Sciences and College of Veterinary Medicine, Animal resources and Biosecurity, Makerere University. It followed a true experimental design, involving quantitative approach. This was characterized by presence of; independent variable, dependent variable, experimental group, control group, pre-test and post-test. Feeding trial involved five dietary treatments, and the Rattus norvegicus were distributed into five different dietary treatment groups having four replicates each. The experimental organisms were subjected to controlled conditions of moisture, light, and caged habitat within the designated animal house.

3.2 Study area The laboratories and animal house within the University (000 21'00' N, 320 34'03' E), is at convenient locations for the research and safety of experimental animals. It contained the required facilities such as water sources, rat cages, qualified scientists, humidity measures, electricity, and large space among others.

3.3 Sample Collection and Survey A total of 20 Albino rats (Rattus norvegicus) eight-to-ten weeks of age (average initial weight 100±5g) were purchased from College of Veterinary Medicine, Animal Resources and Biosecurity and used in a growth trial to investigate the effect of potential Azolla based diets on growth performance. The choices of the experimental groups were at completely randomly design and each animal marked with a permanent marker from the tail. This was to ensure easy tracking and avoiding mixing. The Azolla plant were harvested from a selected swamp in Namulonge (000 31'30' N, 320 36' 54' E), following a modified method used by Pillai et al., (2002) where; plastic buckets with holes of about 1 sq cm was used to scoop the growing Azolla plants. These holes were to allow water to drip out, reducing the risk of decomposition due to dampness and under drying.

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Figure 1: Marking of the animals Figure 2: Rattus norvegicus in one of the cages

3.4 Study materials During the period of research, the following materials were used; Azolla plants, Maize bran, Rastrineobola argentea locally known as ‘Mukene’, Rattus norvegicus (Albino rats), Wooden- steel cages, Electric weighing balance, Measuring cylinder, Bucket, Mortar and pestle, Paper bags, hot air oven, Feeding and water trough among other materials.

3.5 Sampling strategy The commercial feed samples were collected randomly by buying selected Mukene and maize bran from a selected animal feed store in Kalerwe market. During choice of the animals for the experimental groups and taking measurements, availability sampling technique was used (Kothari, 2004), whereby every experimental rat was measured to obtain unbiased data.

3.6 Preparation of Azolla meal The collected Azolla plants were washed with clean water to remove contaminants, like soil particles and snails, and shade dried on cleaned open benches of Ethno botany laboratory for 5 days. These dried plants were ground using mortar and pestle, sieved to a fine powder, and then properly stored in a paper bag until the subsequent incorporation of this Azolla powder into the rat meal was done. Two commonly used commercial feed ingredients in Uganda; Maize bran and Mukene, which are carbohydrate and protein sources respectively, was used in making feed formulations.

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The formulations varied in the amount of Mukene replaced by Azolla plants, while the carbohydrate source (maize bran) was maintained at a relatively constant amount throughout the formulations. The percentage formulations were converted to their respective weight, in terms of grams, and then weighed accordingly prior feed making. Using a modified method used by Naegel (1997), Azolla meal cake were made from the powdered formulation by adding water and making dough spread into a thin layer on a tray. This was oven dried at 300 C for two days to form a cake-like meal. During supply to the animals, the cake was manually broken to smaller particles.

Figure 4: Weighing of Azolla Figure 3: Azolla cake on the cake paper bag

3.7 Treatments In the experimental feed, Azolla was used to replace Mukene on weight basis at the substitution levels of at 0% (control), 25%, 50%, 75% and 100% inclusion levels as protein source, constituting treatments 1-5 respectively. This was fed to the experimental animals, and so also water while other management practices such as weekly changing of beddings, daily cleaning of water and feeding troughs, medications, disinfection of instruments and parasitic control was carried out. Daily feed weight of 50g and water of 250 mls was given to each experimental group. No special fat source was added to the prepared feeds. The experimental animals were properly managed for housing environment, floor space, feeder and water space, litter management, lighting management, and medication as was done by Alalade et al., (2006).

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Figure 5: Changing of beddings

3.8 Data collection The experiment lasted 38 days, involving eight-to-ten week old Rattus norvegicus. Data was collected in respect of body weight after every four days. The live weights of the animals were taken using an electric weighing balance, which was always calibrated with standard weights prior to measurements. The constant reading, when the animal was at resting position was the recorded values. Initial body weights of these animals were also noted before the start of the experiment. Other observations were also made such as; palatability of the eed based on the daily feed intake, apparent digestibility of the meal basing on the texture of the fecal matter and appearance of ‘protein sores’, scabs on the neck and the back of the animal which is attributed to too much protein in the diet.

Figure 6: Weighing of experimental animal

Data quality was controlled by ensuring that measurements were always taken in the morning, before the experimental animals eating for that day. Measurements were also taken at equal time

Page | 11 intervals, animals of the similar age and weight was used. Measurements were recorded in terms of grams (g) for weights, and milliliters (mls) where liquid is involved for volumes.

3.9 Statistical analysis All generated data was entered and organized in Microsoft excel. This was then exported to GraphPad software version 5.01, and subjected to analysis of variance using 1-Way ANOVA, followed by post-hoc to compare the mean live weights gained by the experimental animals in each feeding group.

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CHAPTER FOUR

4.0 RESULTS AND DISCUSSION Appendix 1 shows the growth performance of Rattus norvegicus in response to the five dietary treatments, recorded as the live weights of the replicates during the whole period of feeding trial. The initial body weight (IBW) had no significant difference. This is important in ensuring reliable data collection. The similarity in the IBW may be attributed to the similar age of the experimental animals. However, the final body weight (FBW) in the five treatment groups were significantly (P = 0.002) different by the 24th day.

Figure 1 shows initial decrease in the body weight of all the experimental animals. This initial decrease in animal weight was due to low rate of feed intake. This is because the experimental animals were not accustomed to the Azolla meal since they were initially being fed on the standard commercial rat feeds. To increase data reliability, a 14 day period was given for the animals to get accustomed to new feeds and cages, during which period the animals were fed on the Azolla meal but no any data was being collected.

Figure 7: Changes in the body weight of the control and treatment groups during the feeding trial.

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The body weight gain were decreasing as the level of substitution of Mukene with Azolla got increased beyond 50%. However, the live weights of animals fed on 25% and 50% Azolla meal had no significant differences with the control (post-test, P<0.05) throughout the feeding period. This trend is in agreement with the earlier report of Foluke et al., (2018). The results also revealed that animals of 75% and 100% treatment groups had insignificant (P<0.05) differences in their live weights, but had significantly (P<0.05) lower life weights compared to the control group. This indicated that the rats in the control group had the best weight gain.

The above observation implies that with Azolla inclusion, the rats will ate more feeds to gain a unit of life body weight and that as the level of inclusion increased, the feed were poorly utilized due to high fibre content of plant protein diets. This result agreed with previous works carried out on high fibre diets (Nworgu et al., 2012) that revealed that the presence of high fibre level in diets can cause intestinal irritation, lower digestibility and overall decreased nutrient utilization.

Reduction in the body weight of the experimental animals fed on higher Azolla levels (75% and 100%) might be due to higher level of Neutral Detergent Fibre (NDF) (Buckingham et al., 1978) and lignin (Tamany et al., 1992) in Azolla meal which are the main limiting factors for its efficient utilization. It may be due to the lower percentage of digestible proteins that do not offer significant improvement in the digestibility of the Azolla meal (Manish et al., 2018). Furthermore, the digestibility of feed with high fibre content is very poor in non-ruminant animals due to lack of suitable digestive enzymes (Cellulase, xylanase and pectinase) in the gastrointestinal tract.

Although the daily food intake in all treatment groups was similar, the rate of intake kept decreasing with increasing concentration of Azolla in the feed. This observed trend might be associated with the poor aroma and palatability of the Azolla meal (Bested and Morento, 1985 ) when compared to that of Mukene only (0%) and increased bulkiness of Azolla (Bacerra et al., 1995), and yet the body weight gain is dependent on daily food intake.

No rat died in any treatment group during the experimental period, indicating that inclusion of Azolla in feed had no deleterious effect on Rattus norvegicus.

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CHAPTER FIVE

5.0 CONCLUSION AND RECOMMENDATION

5.1 Conclusion Azolla can be used as a replacement for Mukene, which is the conventional protein source in animal feeds. However, the inclusion must not exceed 50%. This study showed that the substitution of 50% Azolla for 50% Mukene could be the optimal substitution level in supplement for growing the rats of the type used in this study.

5.2 Recommendations More works has to be done to identify the anti-nutritional factors present in Azolla meal and their pathological effects on Rattus norvegicus so as to devise ways to detoxify these factors. Also, hematological parameters such as the packed cell volume, hemoglobin concentration, red blood cell counts and serum metabolites should be carried out to assess the effect of Azolla meal on them. Also, Azolla meal can be made from fermented Azolla extract to overcome the digestion problems in monogastric animals due to fibre and lignin.

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Alalade, O.A. & Iyayi, E.A. (2006).Chemical Composition and the Feeding Value of Azolla (Azolla pinnata) Meal for Egg-Type Chicks. International Journal of Poultry Science. 5 (2): 137-141.

Arora, A., Saxena, S., & Sharma, D. K. (2006).Tolerance and phytoaccumulation of chromium by three Azolla species. World Journal of Microbiology and Biotechnology. 22(2), 97-100.

Bacerra, M., Preston, T.R., & Ogle, B. (1995). Effect of replacing whole boiled soya beans with Azolla in the diets of growing ducks. Livestock Research for Rural Development, 7: 1-11.

Balaji, K., Jalaludeen, A. C.R., Richard, P.A., Peethambaran, S., & Senthilkumar (2009). Effect of dietary inclusion of Azolla (Azolla pinnata) on production performance of broiler chicken. Indian J. Poult. Sci., 44(2):195-198. Basak, B., Pramanik, M. A. H., Rahman, M. S., Tarafdar, S. U., & Roy, B. C. (2002). Azolla (Azolla pinnata) as a feed ingredient in broiler ration. Int.J. Poult. Sci, 1(1), 29-34.

Bested, S.B. & Morento, S.E. (1985). The effect of different percentage of Azolla on fattening pigs. Journal of Mountain State Agricultural College, 17: 31-40.

Chinsoro, P. (2015). Potential use of baobab seeds in poultry diets, department of livestock and pasture science. Faculty of Science and Agriculture, University of Fort Hare. Pp 52-53

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APPENDICES

Appendix 1: Raw data of the life weights of Rattus norvegicus taken during the feeding experiment Time 0% Azolla meal Animal 1 Animal 2 Animal 3 Animal 4 (Days) 0 90.8 109.3 100.00 114.40 4 82.7 113.8 107.40 98.30 8 81.2 105.5 103.20 95.60 12 105.9 116.5 110.40 111.40 16 129.8 114.3 138.10 134.60 20 131.9 112.6 145.60 140.70 24 147.2 121.6 150.20 164.20

Time 25% Azolla meal (Days) Animal 1 Animal 2 Animal 3 Animal 4 0 100.60 109.40 118.40 112.20 4 106.40 103.50 115.80 92.30 8 102.90 107.80 100.10 107.90 12 111.90 110.60 113.50 100.80 16 101.30 122.70 119.70 125.40 20 95.60 133.20 114.60 143.50 24 96.80 137.60 121.90 153.10

Time 50% Azolla meal (Days) Animal 1 Animal 2 Animal 3 Animal 4 0 107.80 104.20 108.40 102.20 4 117.90 95.30 101.80 108.60

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8 108.90 92.30 110.30 109.20 12 100.80 102.10 113.40 103.10 16 120.20 107.30 126.30 117.00 20 144.60 105.60 130.60 123.20 24 157.20 111.70 142.90 132.10

Time 75% Azolla meal (Days) Animal 1 Animal 2 Animal 3 Animal 4 0 99.30 97.80 98.30 90.60 4 96.80 95.40 96.80 87.10 8 94.70 92.90 104.70 76.90 12 102.10 97.00 102.10 81.20 16 103.50 99.50 103.50 84.90 20 104.10 104.80 104.10 86.70 24 113.20 107.10 113.20 94.10

Time 100% Azolla meal (Days) Animal 1 Animal 2 Animal 3 Animal 4 0 107.60 95.10 104.20 97.60 4 103.30 86.70 95.30 92.60 8 93.90 83.90 92.30 89.10 12 99.40 85.10 102.10 94.30 16 100.70 85.60 107.30 95.70 20 102.10 88.10 105.60 97.20 24 102.80 89.50 111.70 98.80

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