Abstract
The effect of seasons on aflatoxins loads of selected pig’s feedstuffs in the humid tropics was investigated, the feedstuffs sampled were cassava peels, bambara nut waste, palm kernel cake and brewer spent grains. The study lasted for thirty-two weeks. These feedstuffs were collected in two piggery farms in each of the six LGAs that make up Nsukka zone of Enugu State, in both dry and rainy seasons.The collected feedstuffsamples were analyzed in the laboratory for aflatoxins concentration levels using AOAC method of thin-layer chromatography (TLC)with some modifications. Data were collected from the aflatoxins laboratory analysis of these feedstuffs and with a structured questionnaire.The results of this study indicated the presence of aflatoxins in all the sampled  feedstuffs, however the lowest concentration of aflatoxins which is 0.011ppb is obtained in cassava peels and bambara nut wastes while  the highest concentration of aflatoxins obtained is 0.055ppb in brewer spent grains,these values are within the tolerance limit in Nigeria for total aflatoxins (B1+B2+G1+G2),which is 20ppb.The obtained aflatoxins levels are within range and poses less risk to pigs and pork consumers.The results also shows that there were no significant difference (P > 0.05) on the aflatoxins concentration levels of the feedstuffs at their sources, across the piggery farm locations, in the season and source of feeds interactions, season and the piggery farm locations interactions, as well as season and feedstuffs interactions. However the main effect of seasonon aflatoxins level which is 0.022ppb and 0.044ppb in the dry and rainy seasonrespectively were highly significant (P < 0.01).Also the main effect of aflatoxins of the feedstuffs were highly significant (P < 0.01) except in brewer spent grains were it is significant (P < 0.05). It follows that the aflatoxins  levels according to season in the comparison between the source of feedstuffs and the piggery farm locations for the sampled feedstuffs were highly significant (P< 0.01) especially in the rainy season, except bambara nut wastewhich is highly significant (P< 0.01) in the dry season. There were higher values in the aflatoxins levels in all the feedstuffs sampled in the rainy season than in the dry season.It was therefore concluded that season has an effects on the aflatoxins level of pig’s feedstuffs with rainy season  favouring toxigenic mould metabolism and growth in the feedstuffs which produces aflatoxins as their metabolites than in the dry season in this study, hence adequate preservative measures, proper feed handling and storage should be adopted. It is therefore recommended that piggery farmers should adopt adequate preventive measures and also apply the various preservative storage methods in order to reduce the aflatoxins levels infeedstuffs especially in the rainy season for better performance of the pigs and safety of pork consumers.
Table of Contents
Title Page … … … … … … … … … …i
Certification … … … … … … … … … …ii
Dedication … … … … … … … …. … …iii
Acknowledgement … … … … … … …. … …iv
Abstract … … … … … … … … … …v
Table of Contentss … … … … … … … … …vi
List of Tables … … … … … … … … …xi
List of Figures … … … … … … … … … ….xii
Chapter One
1.0 Introduction
1.1      Background of the study             …       …       …       …       …….1
1.2      Statement of Problem                …       …       …       …       …….4
1.3      Objectives of the study         …       …       …       …       ….      ……5
1.4      Justification of the study        …       …       …       …       ……6
Chapter Two
- Literature Review
-      Brief History of Aflatoxins ……       …       …       …       …       ……7
2.2      Natural Occurrence of Aflatoxins       …       …       …       ……8
2.3      Mycotoxins and Its Effects….   …       …       …       …       …       …..10
2.4      Chemical Reactions of Aflatoxins         …       …       …       ….12
2.4.1. Heat …. … … … … … … … … ….12
2.4.2   Alkalis …       …       …       …       …       …       …       …       ….12
2.4.3 Acids … … … … … … … … … ….14
2.4.4               Oxidizing Agents …    …       …       …       …       ….14
2.4.5 Reduction … ….. …… … … … … …14
2.5                  Chemical Structure of the Different Types of Aflatoxins       15
2.6                  Aflatoxin B1 Pathways…       …       …       …       …       …16
2.7                  Metabolism of Aflatoxins …       …       …       …       …16
2.7.1 Bio-activation … … … … … … … …16
2.7.2 Conjugation… … … … … … … … …16
2.7.3              Deconjugation …    …       …       …       …       …       …17
2.7.4               Aflatoxins Units and its Conversion …     …       …       …17
2.8                  Factors that favours Mould  Growth and Development         18
2.9                  Clinical Signs/Symptoms of Aflatoxicosis in Pigs…       …18
2.9.1               Acute  Aflatoxicosis… …       …       …       …       …19
2.9.2               Chronic Aflatoxicosis … …       …       …       …       …19
2.10                The Effects of Aflatoxins in Pigs           …       …       …20
2.11 Aflatoxins Tolerance Levels and Action Limits in Animal Feeds …23
2.12                Aflatoxins and Climate Change…         …       …       …27
2.13                Aflatoxins and its Economic Importance        …       …28
2.14                Aflatoxins Metabolism in Animals …       …       …29
2.15 Methods of Aflatoxins Determination and Quantificationin Samples…. 30
2.16                AflatoxinsPrevention and Control Strategies… ….32
 2.17.              Alternative Feedstuffs and their Uses in Pig Feeding      …41
 2.18               Cassava Peels Meal (CPM)…. … …       …       …       …42
2.19.1             Nutritive Value of Cassava Roots/Peels     …       …       …44
2.19.2             Utilization of Cassava Peels as feedstuffs …  …       ..…     …45
2.19.3Â Â Â Â Â Â Â Â Â Â Â Â Â Constraints to the Use of Cassava Meal/Peels as a feedstuff 47
2.20                Palm Kernel Cake (PKC)…    …       …       …       …48
2.20.1             Nutritive Value of Palm Kernel Cake as feedstuffs   …       …49
2.20.2             Utilization of Palm Kernel Cake …        …       …51
2.20.3             Constraints to the Use of Palm Kernel Cake     …       …52
2.21                Bambara Nut Waste (BNW)… …   …       …       …53
2.21.1             Nutritive Value of Bambara Nut/Waste as feedstuff  …       …54
2.21.2             Utilization of  Bambara Nut /Waste   …       …       …56
2.22                Brewer’s Spent Grain (BSG)…         …       …       …57
2.22.1             Nutritive Value of Brewer’s Spent Grains…    …       …58
2.22.2             Utilization of  Brewer Spent Grains as feedstuff…   …       …61
2.22.3             Constraint to the Use of Brewer Spent Grains      …       …62
2.23 Brewer Yeast … … …. … … … … …63
2.23.1 Nutritive Value and utilization of Brewer Yeast in Livestock feeds… …63
2.24                Effect of High Content of Fibre in Pigs Diets      …65
2.25                Anti-nutritional factors in Non-conventional Feedstuffs      66
2.25.1             Classification of Anti-nutritional Factors (ANFs)    …67
2.25.2 Factors Interfering with the Digestion and Utilization of Dietary Proteins and Carbohydrates   …       …       …       …       …       …68
2.25.2.1 Tannins … … … … … … … …68
2.25.2.2 Saponins … … … … … … … …69
2.25.2.3 Trypsin or Protease Inhibitors …. … … … … …69
2.25.2.4          Haemagglutinins…           …       ….      …       …       …70
2.6                  Factors that Interfers with availability of Minerals  …       …71
2.26.1 Phytates … … … … … … … …71
2.26.2 Oxalic Acid … … … … … … … …71
2.26.3 Glucosinolates… … … … … … … …71
2.26.4 Gossypol … … … … … … … …72
2.26.5 Phytoestrogens … … … … … … …72
2.26.6 Anti Vitamins … … … … … … … …72
2.26.7 Cynogens … … … … … … … …72
2.26.8 Lathyrogens … … … … … … … …73
2.26.9            Nitrates and Nitrite    …  …       …       …       …       …73
2.26.10          Flatulence Factors      …      …       …       …       …       …73
2.27                Pigs and its Characteristics    …      …       …       …       …73
2.28                Humid Tropics and its Characteristics         …       …       …74  Â
Chapter Three
3.0 Materials and Methods
3.1      Location of the Study…     …       …       …       …       …       …       …76
3.2      Experimental Materials…       …       …       …       …       …       …76
3.3      Experimental Procedure…  …       …       …       …       …       …       …77
3.4      Sample Preparation    …         …       …       …       …       …       …77
3.5 Procedure for Aflatoxins Determination and Quantification by (TLC)… …. …78
3.5.1   Apparatus…       …       …       …       …       …       …       …       …78
3.5.2   Reagents…     …      ….      …       …       …       …       …       …78  Â
3.5.3   Procedure of AOAC Aflatoxins Analysis using TLC  ….79
3.5.4 Method of Determination of Aflatoxins Concentration in Feedstuffs Samples … …81
3.5.5   Safety Issues…              …       …       …       …       …       …81  Â
3.6      Experimental Design…        ….      ….      ….      …       …       …82
3.7      Data Collection…    …       …       …       …       …       …       …82
3.8      Statistical Analysis      …       …       …       …       …       …       …83
Chapter Four
4.0 Results and Discussion
4.1 Results and Discussion of Aflatoxins Laboratory Analysis of Pigs feedstuffs … …84
4.1.1: The results of the effect of source of feedstuffs on the aflatoxins levels of pig feedstuffs 84
4.1.2: The results of the effect of farm location on the aflatoxin levels of pig feedstuffs … 85
4.1.3: The results of the effect of season on the aflatoxins levels of pig feedstuffs …. 86
4.1.4: The results of the effect of feedtype on aflatoxins levels of pig feedstuffs …. …. 86
4.1.5: The results of the effect of season on aflatoxins levels of pig feedstuffs …. …. 87
4.1.6: The results of the effect of source and season on aflatoxins levels of pig feedstuff … 88
4.1.7: The results of the effect of feedstuff and season on aflatoxins levels of pig feedstuffs 89
4.1.8: The results of the effect of season and farm location on aflatoxins levels of feedstuffs 90
4.1.9: The results of the effect of source and farm location on aflatoxins levels of feedstuffs 91
4.2     Pig Farmers Responses and Discussions   …       …     95
4.2.1   Marital status …   …       ….      …     98
4.2.2   Educational Qualifications           …       …       …       …       …     98
4.2.3   Gender               …       …       …       …       …       …       …     98
4.2.4   Number of pigs reared in the farm           ….      ….      …       …    99
4.2.5   Frequency of sourcing pigs’ feedstuffs      ….      ….      …       ….    99
4.2.6   Quantity of feedstuffs purchased in kg/tons ……       ….   100
4.2.7   How these feedstuffs are stored in the piggery farms   …       ….   100
4.2.8   Susceptibility of pig breeds to mould contaminated feedstuffs   100
4.2.9 Ingredients added to the feedstuffs to improve its nutrient availability … …. 101
4.2.10 Mould incidence/infestation of feedstuffs fed to pigs .      ….   101
4.2.11 Feedstuffs that develop moulds faster than others       ….   101
4.2.12 How does these symptoms affect the pigs  ….      ….   102
4.2.13Â Feedstuffs used by pig farmers to feed pigs in their farms 103
4.2.14 Main source of feedstuffs from where the pig farmers procure feedstuffs …. …. 104
4.2.15 Signs and symptoms shown by pigs when fed mould contaminated feeds …. …. 105
4.2.16 Preventive measures adopted to reduce mould infestation of the feedstuffs …. …. 106
4.2.17 The season/ period of the year mould infestation of feedstuffs are prevalent …. 107
Chapter Five
5.0 Summary,Conclusion and Recommendation
5.1 Summary … … … … … … … … … …109
5.2 Conclusion… … … … … … … … … …110
5.3      Recommendations      …       …       …       …       …       …110
References
Appendices
List of Tables
Tables                                                                                                              Pages
Table 2.1: The chemical formula and physical properties of different types of aflatoxins 10
Table 2.2: Tolerance limits and action levels for total aflatoxins in livestock feed 24
Table 2.3: Dry matter of the fresh and air-dried cassava peels at 105oC for 24hrs 45
Table 2.4: Proximate composition of palm kernel cake          50
Table 2.5: The amino acid composition of palm kernel cake (g/16gN)Â Â Â Â Â Â Â Â Â Â 50
Table 2.6: Mineral content s of palm kernel cake                           51
Table 2.7: Proximate composition of decorticated and undecorticated bambara nut 55
Table 2.8: Proximate composition of toasted and raw bambara nut (%DM) 55
Table 2.9 : The essential amino acid content of bambara nut           56
Table 2.10: The amino acid composition of brewer spent grains as a (%) of crude protein 59
Table 2.11: The digestibility of pigs (%) of brewer spent grains 59
Table 2.12:The dry matter (%) of brewer yeast    64
Table 2.13: The amino acid composition of brewer yeast as a (%) of crude protein 64
Table 4.1.1Effect of source of feedstuff on the aflatoxins level of the feedstuffs 84
Table 4.1.2: Effect of farmlocation on aflatoxins levels of pig feedstuffs 85
Table 4.1.3: Main effect of season on aflatoxins levels of pig feedstuffs 86
Table 4.1.4: Effect of feed type on aflatoxins levels of pig feedstuffs     86
Table 4.1.5: Effect of season on aflatoxins levels of pig feedstuffs  87
Table 4.1.6: Effect of source and season on aflatoxins levels pig feedstuffs    88
Table 4.1.7: Effect of feedstuff and season on the aflatoxins levels feedstuffs                   89
Table 4.1.8: Effect of season and farm location on the aflatoxins level of the feedstuffs                                                                                                             90
Table 4.1.9: Effect of source and farm location on the aflatoxins levels of pig feedstuffs 91
Table 4.1.10: The questionnaire responses of pig farmers 96
List of Figures
Figures                                                                                                          Pages
Fig.1: Structure of Aflatoxin B1, B2, G1 and G2Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 13
Fig. 2: Structure of Aflatoxin M1, M2, B2A and G2AÂ Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â Â 14
Fig. 3: Aflatoxin B1 Metabolism Pathways                                              15
Fig. 4: Bar chart showing the various feedstuffs used by piggery farmers in feeding pigs in their farms                                                       98
Fig 5: Bar chart showing the main source of feedstuffs from where the pig farmers procure feeds                                                                                 98
Fig 6: Bar chart showing the signs/ symptoms shown by pigs when fed mould contaminated feeds                                                                            99
Fig 7: Bar chart showing the preventive measures adopted to reduce mould infestation of feedstuffs                                                            99
Fig 8: Bar chart showing the season/ period of the year mould infestation of
feedstuffs are prevalent                                                                   100
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the Study
The consumption of animal protein is very essential for meeting the protein requirement of man for normal growth and healthy development. The protein sources from animal origin are good sources of lysine and sulphur amino acids, which are lower in protein sources from plant origin (Omole, 1991). The shortage of protein, particularly animal protein is prevalent in most parts of Africa where it is estimated that on the average 10 grams of animal protein is consumed per day compared to the recommended daily intake of 35 grams (ILCA,1980; FAO, 1986). The level of animal protein consumption was estimated at 8 grams per caput per day, this is about 20 grams less than the National Research Council of United States of America’s recommendation (Obioha, 1992; FAO, 1997). Ogini (2001) stated that Nigeria, despite its vast land area of 9,237,680km2 , the greater percentage of her citizens of over 120 million are still battling with abject poverty and hunger. Atsu (2002) reported that there is inadequate intake of animal protein in Nigeria.
The high cost of feeds and feed ingredients, especially the conventional energy and protein feed sources such as maize, wheat, sorghum, soybean cake, groundnut cake and fish meal were as a result of the stiff competition between human consumers for food, for livestock feeds/feeding and industrial raw materials e.g. cereal grains for baby food formulas and biofuel production, especially maize (Agbede et al., 2002; Adegbola and Asaolu, 2008). Maize is a major source of energy in monogastric feeds, especially pigs, poultry and rabbits. In Nigeria, cereal grains are in high demand and its production has never been adequate to meet the needs of the increasing population; consequently there is little or no excess grains for livestock feeding. When available, it is always very expensive (Rhule,1999). The cost of conventional feedstuffs which are major sources of energy and protein in monogastrics diets has continued to increase (Onu and Madubuike, 2006; Defang et al., 2008) due to their short supply. In 2005 the price of maize rose from about N30 to N70 per kilogram (Rhule,1999). As the competition between man, farm animals and industrial uses for grains continues to increase, substitutes for grains are being increasingly used in poultry and livestock feeding to reduce the high cost of feed (Babatunde and Hamzat, 2005). This continuous increase in the cost of conventional feed ingredients used in formulating poultry and livestock feeds has necessitated an intensive search for cheap alternative feed ingredients that will be able to replace a given portion, if not all of the conventional feed ingredients (Babatunde, 1985; Onyimonyi, 2002; Adesehinwa et al., 2011).
According to Ugwu et al. (2008), the main factors militating against the rapid expansion of animal production industry in Nigeria is the problem of inadequate supplies of feedstuffs at economic prices. The scarcity and high cost of conventional feedstuffs is largely responsible for the present high price of finished animal products such as eggs, meat and milk (Rhule,1999; Adesehinwa et al., 2011). Ijaiya et al. (2004) pointed out that feed cost is perhaps the most expensive input in intensively reared stocks and constitutes about 70 – 80% of the real cost of animal production. Onyimonyi and Onukwufor (2003) had earlier reported that a major solution to the problem of rising costs, scarcity of energy and protein sources for monogastrics is to seek for new and non-conventional feed resources which are able to replace a certain percentage proportion of the known conventional feedstuffs without any deleterious effect on the animal performance. Esonu et al. (2001) indicated that such substitute which have been successfully used to replace the conventional feedstuffs are brewer spent grains, stem/tuber residues and cereal by-products such as wheat offal, rice bran, maize bran as well as bambara nut waste and cassava or yam peels.
Fetuga et al. (1977), Iyayi et al. (2005) and Onyimonyi and Ugwu (2007) had studied the feeding value of such non-conventional feed resources as palm kernel cake, bambara nut offal, rice husk and cassava peels, and that they can be utilized in supplementing a given portion of the conventional feedstuffs. Onyimonyi and Ugwu (2007) further noted that these non-conventional feeding stuffs are commonly found in the South Eastern Nigeria. According to Babatunde and Hamzat (2005), these alternative feedstuffs have proved valuable in supporting the performance of livestocks and poultry and that by utilizing them in ration formulation, it will go a long way in reducing the demand and cost of conventional feedstuffs, which greatly increases the total cost of pork production. Adegbola and Oduozo (1992) reported that some of the limiting factors associated with agro-industrial by-products and crop residues utilization as animal feeds include: procurement, storage, poor feed intake, high fibre content, toxic and anti-nutrients substances as well as being low in their digestibility which subsequently lowers the animal performance.
According to Serres (1992) pigs are known to be highly prolific and very efficient in converting feed nutrients into high quality animal protein. Adesehinwa (2008) stated that pigs are used to convert a variety of feeds into meat for human consumption. The supply of animal protein by pigs is limited by many factors among which are inadequate nutrition and feeding; poor breeding stocks, poor management practices and housing; disease prevention and control in pig production (Holness,1999).
Pitt and Miscamble (1995) defined mycotoxins as toxic secondary metabolites produced by micro-organisms of the fungus kingdom commonly known as moulds. Awan (2001) reported the ability of moulds to produce toxins potentially dangerous to man and animals are common and widespread. Odoemela and Osu, (2008) reported that toxigenic moulds have been found during growth, harvest and storage of diferent foods and feeds and other agricultural produce. Abarca et al.(2001) stated that there are five major agriculturally important fungal toxins of economic importance namely; aflatoxins, deoxynivalenol, ochratoxin, zearalenone and fumunism, which are produced by fungi invasion of agricultural produces and feed ingredients under favourable conditions of temperature (25 – 30oc) and relative humidity of above 80% (Gilbert and Vargas, 2003). According to Farombi (2006), these moulds contaminate a large number of dietary staples and agricultural produce such as rice, corn, cassava, peanuts and spices, while noting that humans and animals are exposed to aflatoxins by consuming contaminated foods and feeds. These fungal metabolites which when ingested, inhaled or absorbed through the skin causes lowered performance, sickness or eventually death in both animals and humans (Kuilman et al.,1998; CAST, 2003).
Atanda et al.(2008) stated that several moulds and yeast species were isolated from grains with Fusarium verticilloides and Aspergillus flavus having the highest frequency of occurrence of 39.1% and 22.3%, respectively. Moulds have the potentials of forming aflatoxins and trichothecenes in grains, peanuts, cottonseed and other agricultural products (Atanda et al., 2008).
Adam (2000) reported that Aspergillus spp produces aflatoxins. According to Sashidhare et al.(1992), the high incidence of mycotoxins contamination could be due to physical status of grains, the moisture content, temperature, oxygen and the amount of carbon (iv) oxide in the atmosphere. These factors are reported to influence the rate of infestation and proliferation of moulds in agricultural produce and grains, especially under storage condition.
1.2 Statement of the Problem
