PROBIOTIC EFFECTS OF SACCHAROMYCES CEREVISIA ON LAYING CHICKEN FED PALM KERNEL CAKE-BASED DIETS.

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PROBIOTIC EFFECTS OF SACCHAROMYCES CEREVISIA ON LAYING CHICKEN FED PALM KERNEL CAKE-BASED DIETS.

ABSTRACT

The study investigated the probiotic effects of Saccharomyces cerevisiae on pullets development and hen-day egg performance of the layers. A total of 120 chicks were brooded for 4 weeks, after which 100 pullets were randomly selected and placed in 4 groups (A-D) of 25 birds each. Groups A, B and C had their feed supplemented with S. cerevisiae at graded levels of 0.6, 0.8 and 1.0 g/kg of feed respectively. Group D diet did not contain S. cerevisiae (control). The diets for all the groups contained 25% PKC and they were isocaloric and isonitrogenous. The pullets were weighed weekly. At 9th, 15th and 40th week of age, 5 birds were randomly selected from each pen and 2 ml. of blood collected into an EDTA bottle for haematology. The blood sample was used to determine packed cell volume (PCV), haemoglobin concentration (HbC), total leukocyte count (TLC) and differential leukocyte count following standard procedures. At 10th and 40th week of age, 3 pullets were randomly selected from each group and placed in four different cages. The quantity of feed consumed and faeces voided were determined, proximately analyzed and used to determine the apparent digestibility of the diets. Eggs were collected three times daily, weighed and their external qualities were assessed. At the 20th week in lay, another set of 5 layers were randomly selected from each group and 5 ml of blood were collected from each bird and allowed to clot. The serum harvested was used to evaluate the serum biochemistry of the layers. At the 22nd week in lay, ten eggs were randomly selected from each group and were used to determine the egg cholesterol content. Five layers from each treatment group were randomly selected at the 70th week in lay, slaughtered and their gastrointestinal tract (GIT) carefully removed. The weight and length of the different sections of the gastrointestinal tract were determined. At the 24th month of age, another set of 5 layers were randomly selected from each group and used to evaluate the carcass quality. Repeat measure and one-way analysis of variance (ANOVA) were used to analyze the results. Variant means were separated using Duncan’s new multiple range test. Significance was accepted at p< 0.05. Group C birds had significantly (p<05) higher mean live weight than the group D birds (control). The PCV of group C birds was significantly higher (p < 0.05) than those of all other groups at weeks 9 and 15 of age. The group C birds also had significantly higher (p<0.05) TLC and lymphocytes counts than the control group (D). Apparent digestibility coefficient of dry matter, organic matter, crude protein and crude fibre were significantly (p<0.05) higher in the probiotic supplemented groups than the control. Group C had an overall significantly (p<0.05) higher hen-day egg performance of followed by groups B and A, while group D had the least hen-day egg performance. Birds in the supplemented groups had significantly higher (p<0.05) serum total proteins and significantly lower serum cholesterol when compared to the control. Eggs from the supplemented groups had significantly (p<0.05) lower cholesterol content when compared to the control. Group C birds had a significantly (p<0.05) longer colon than the control. There was no significant difference (p>0.05) in egg qualities (egg size, egg weight and shell thickness) between the supplemented groups and the control. The economic returns from sale of eggs was significantly higher (p<0.05) in group C birds in the first year of lay, but in the second year of lay, all supplemented groups had significantly higher (p<0.05) monthly revenue from eggs compared to the control. It was concluded that supplementation with probiotic S. cerevisiae significantly (p<0.05) enhanced pullet development, hen-day egg performance and monthly revenue from eggs, and significantly (p<0.05) lowered serum and egg cholesterol levels. The probiotic supplementation was most effective at the level of 1.0 g/kg of feed, and this is recommended.

TABLE OF CONTENTS

Title Page – – – – – – – – i

Certification – – – – – – – – ii

Acknowledgement – – – – – – – – iii

Abstract – – – – – – – iv

List of Tables – – – – – – – v

List of Figures – – – – – – vi

Table of Contents – – – – – – – vii

CHAPTER ONE: INTRODUCTION

1.1 Background of the Study – – – – – 1

1.2 Statement of the Problem – – – – – 8

1.3 Objectives of the Study – – – – – 8

CHAPTER TWO: REVIEW OF RELATED LITERATURE

2.1 Probiotics: An Overview – – – – – – 9

2.1.1 Historical Perspectives – – – – – 9

2.1.2 Characteristics of Good Probiotics – – – – 14

2.1.3 Benefits/Advantages of probiotics – – – – 14

2.1.4 Some of the beneficial effects of the practical use of probiotics 15

2.2 Probiotics in Human Medicine – – – – 18

2.2.1 Effects of S. boulardii on Gastrointestinal Diseases – 19

2.2.1a Antibiotic Associated Diarrhoea – – – 19

2.2.1b Recurrent Clostridium Difficile Associated Diseases – 20

2.2.1c Traveller’s Diarrhoea – – – – – 21

2.2.1d Acute Diarrhoea in Children – – – – – 22

2.2.1e Diarrhea in Patients with Total Enteral Feeding – – 23

2.2.1f AIDS – Associated Diarrhoea – – – – 24

2.2.1g Inflammatory Bowel Disease – – – – 24

2.2.1h Irritable Bowel Syndrome – – – – – 25

2.2.2 Safety of Administration – – – – 25

2.2.3 Experimental effects of S. boulardii – – – 26

2.2.3a Anti-Inflammatory Effects – – – – – 26

2.2.3b Inhibition of NF-KB and MAPK Activation – – 27

2.2.3c Decrease of Nitric Oxide – – – – 29

2.2.3d Enhancement of PPAR-g Expression – – – 29

2.2.3e Modulation of T cell Migratory Behaviour – – 30

2.2.4 Immuno-Modulatory Effects – – – – 30

2.2.4a. Saccharomyces boulardii effect on Innate Immunity – 31

2.2-4b. Saccharomyces boulardii effect on Adaptive immunity 31

2.2.5 Inhibition of Hydroelectralytic Secretions – – – 33

2.2-6 Neutralization of Bacterial Toxins – – – – 34

2.2-7 Decrease of Bacterial Adhesion to Intestinal Epithelial Cells 35

2.2-8 Maintenance of Epithelial Integrity – – – – 36

2.2-9 Trophics Effects on intestinal Mucosa – – – 38

2.2-10 Applications of Probiosis to Poultry – – – – 40

2.3a Probiotics for Chicken – – – – – – 43

2.3b Probiotic Effects on Laying Hens – – – – 45

2.3c Effects on Broiler Performance – – – – 46

2.3d Probiotic Effects on other Poultry – – – – 47

2.3e Probiotic Effects on other Livestock species – – – 48

2.5 The Poultry Industry in Nigeria – – – – 49

CHAPTER THREE: MATERIALS AND METHODS

Materials – – – – – – – – 52

Probiotics – – – – – – – – 52

Palm Kernel Cake – – – – – – 52

Experimental Diets – – – – – 53

Experimental Birds – – – – 57

3.2 General Experimental Design – – – – – – 57

3.3 Methods – – – – – – – – 58

3.3.1 Determination of Effects of Varied Levels of Probiotic – 58

Inclusion on Pullet Development – – – – 58

3.3.2 Haematological Determinations – – – – 58

3.3.2a Determination of Packed Cell Volume – – 59

3.3.2b Determination of Haemoglobin Concentration – – – 59

3.3.2c Total White Blood Cell Count – – – – 60

3.3.2d Differential White Blood Cells Count – – 62

3.3.3 Determination of Digestibility of the Experimental Diets 61

3.3.3a Proximate analyses – – – – – 61

3.3.3b Determination of Dry Matter of Feed and Faeces – – 62

3.3.3c Determination of organic Matter of Feed and Faeces – 63

3.3.3d Determination of Crude Protein of Feed and Faeces – 63

3.3.3e Determination of Crude Fibre of Feed and Faeces – 64

3.3.4 Determination of Effects of Varied Levels of Probiotic

Inclusion on Hen-Day Egg Production and Egg Quality – 65

3.3.5 Determination of External Egg Quality – – – 66

3.3.6 Serum and Egg Biochemistry Determinations – – 66

3.3.6a Determination of Serum Cholesterol – – – – 67

3.3.6b Determination of Egg Cholesterol – – – – 68

3.3.3c Determination of Total Protein – – – – 69

3.3.3d Alamine Phosphatase (ALP) Determination – – – 70

3.3.3e Determination of Serum Alamine Amino Transferase (ALT) 71

3.3.3f Determination of Aspartate Amino Transferase (AST) – 72

3.3.3g Determination of Uric Acid in Serum – – – 73

3.3.7 Hormonal Assay – – – – – – – 74

3.3.8 Determination of Effects of Varied Levels of Probiotic

Inclusion on the Anatomy of the Gastrointestinal Tract – 76

Determination of Effects of Varied Levels of Probiotic

Inclusion on Water Consumption – – – – 77

3.3.10 Carcass Analysis of the Spent Layers – – – 77

3.3.11 Benefits-Cost Analysis of the Experimental Diets – 78

3.3.12 Data Analysis – – – – – – – 78

CHAPTER FOUR: RESULTS

Quality of cerevisiae used for the Study – – – 79

Effects of Varied Levels of Probiotic Inclusion

on Pullet Development – – – – – – 79

Results of Haematogical Determinations – – – 80

Effects of Varied Levels of Probiotic Inclusion

on Digestibility of the Experimental Diets- – – – 81

Results of Varied Levels Probiotic Inclusion on Hen-Day

Egg Production and Egg Quality – – – – 82

4.6 Effect of varied levels of Probiotic inclusion on Serum and Egg Biochemistry – – – – 83

4.7 Effects of Varied Levels of Probiotic Inclusion on Serum levels

of LH, FSH and progesterone – – – 83

4.8 Effects of Varied Levels of Probiotic Inclusion on the

Anatomy of the Gastrointestinal Tract – – – 84

4.9 Effects of Varied Levels of Probiotic Inclusion on Water

Consumption – – – – – – – 84

4.10 Effects of Varied Levels of Probiotic Inclusion on Carcass

Quality of the Spent Layers – – – – – 84

4.11 Results of the Benefits-Cost Analysis of the

Experimental Diets – – – – 85

CHAPTER FIVE: DISCUSSION, CONCLUSION AND RECOMMENDATIONS – – – – 108

REFERENCES – – – – – – – 115

CHAPTER ONE

INTRODUCTION

There have been several definitions for the word probiotics over the years. Lilly and Stillwell (1965) used it to describe substances produced by one protozoan which stimulates another. But Parker (1974) described it as animal feed supplements which had a beneficial effect on the host animal by affecting its gut flora. Fuller (1989) revised the definition and described probiotics as a live microbial feed supplement, which beneficially affects the host animal by improving its intestinal microbial balance. Other workers had also given their own definitions of the term probiotic. Donohue et al (1998) described probiotic bacteria as viable bacteria which when applied in a single or mixed culture, exhibit a beneficial effect on the health of the host. The most recent definition was by Schrezenmeir and De Vrese (2001). They defined probiotics as viable microbial food supplements which beneficially influence the health of the host. This new definition clearly points out the health promoting effect of probiotic agents. Probiotics were more recently defined by a group of experts convened by the Food and Agriculture Organization of the United Nations (FAO, 2005) as “live microorganisms administered in adequate amounts which confer a beneficial health effect on the host”.

The probiotic concept was introduced in the early 20th century by Elie Metschnikoff. Since then, different microorganisms have been used for their supposed ability to prevent and cure diseases leading to the coining of the term probiotics, or “pro-life” (Lilly and Stillwell, 1965) from two Greek wards ‘pro’ which means ‘for’ or in ‘support’ and ‘bios’ which means ‘life’. About 1900, Henry Tissier, a French Pediatrician, observed that children with diarrhea had in their stools a low number of bacteria characterized by a peculiar, Y shaped morphology. Those “bifid” bacteria were, on the contrary, abundant in healthy children (Tissier, 1906). Nobel laureate Elie Metchnikoff in 1907 advocated that the consumption of Lactobacilli helps in controlling endogenous intoxication (autointoxication) caused by wrong types of components in the intestinal flora. He pointed out that the long, healthy lives of Bulgarian peasants were the result of their consumption of fermented milk products which contained probiotics. The works of Metchnikoff and Tissier were the first to make scientific suggestions about the probiotic use of bacteria. The first clinical trials were done in the 1930s on the effect of probiotics on constipation (Koop-Hoolihan, 2001). Majority of the probiotic products in current use contain bacteria from the genera Lactobacillus or Bifidobacterium, although other genera, including Escherichia, Enterococcus, Bacillus and Saccharomyces have been marketed as probiotics (Oyetayo and Oyetayo, 2005).

In Nigeria, there is a very low animal protein intake as a result of poverty and overpopulation (Obi, 2004). Thus, the great need for a radical approach to livestock production. The present daily protein allowance for Nigerians according to Atsu et al., (2002) is as low as 4.5g of protein per head per day which is abyssimally low compared to the recommended animal protein intake of 14g per head per day by F.A.O.( 1997 ). Poultry production is very important in the agriculture of developing countries (Musangi, 1992). FAO (1997) recommended that the deficit in animal protein supply and consumption in developing countries should be ameliorated by increased poultry, pork and rabbit production. Smith (1990) suggested that the supply of poultry products in poorer countries can be rapidly expanded to meet their animal protein need. This is technically possible because poultry are able to adapt to most areas of the world, have low capital requirement, have rapid generation time and a high rate of productivity. Besides excellent nutritive value, egg possesses several health promoting, immunostimulating and therapeutic properties which makes it a versatile product. At every age and stage of life, eggs have a role to play. During pregnancy; eggs provide high quality proteins, vitamins and minerals, omega-3 fatty oils essential for both maternal and foetal health (Narahari, 2003). Narahari (2003) further stated the importance of nutrients contained in the egg as follows: Egg choline is needed for proper development of a child’s brain. The requirement of a pregnant woman for egg choline is about 450 mg/day, while the requirement for a nursing mother is 550 mg/day. Older people need choline to activate their memory. Egg leutin and zeaxanthin are essential to prevent macular degeneration in adults. There is more to eggs than just a good nutrition. They also contain constituents that help in the treatment of a wide range of human health problems from wounds and rashes to cancer and cardiovascular diseases (Narahari, 2003). Eggs will induce and increase satiety (i.e reduce hunger). This will improve compliance with weight loss diet and enhance the benefits of a weight loss regimen. Despite being the proteinous food with the highest biological value, eggs still cost lower than most other animal protein sources. For instance, the cost of 100 gm of eggs is N40, while 100 gm of beef costs N60 and 100 gm of chicken is N70.

Feed supply is the major limiting factor in poultry production in developing countries (Chantalakhana, 1990). The cost of feed alone accounts for 70 – 80% of the total cost of production (Yegany, 2002; Adegbola, 2004; Anyaehie and Irole, 2008). The greatest source of dilemma in the poultry industry is the unprecedented increase in the cost of poultry feedstuff especially maize and soya bean (Adene, 2004). The scarcity of cereal grains and protein concentrates for poultry feed and keen competition between man and livestock for same has been the major obstacle in poultry development in Nigeria (PAN, 1985). The future for efficient and profitable poultry production would therefore depend on finding cheaper and alternative energy and protein sources of conventional feed ingredients (Ojewola,1992).

Palm kernel cake (PKC) is one of the commonly recommended non-conventional feed ingredients used to partially replace maize or complement whole poultry ration (Onwudike, 1986; Okeudo et al., 2005). Okeudo et al., (2005) reported that inclusion of PKC at 28% and 35% for broiler starter and finisher respectively had no deleterious effect on production. Omeke et al., (2006) recommended that 30% PKC inclusion for broiler diets makes for optimum economic productivity.

The only problem with the use of PKC is the high crude fibre content (20.4%) which reduces feed intake, decreases both digestibility and efficiency of utilization of the feed (Dolberg et al, 1981, Zahari et al, 2005). Hence these researchers recommended that PKC should be treated to make it less fibrous and more digestible. Biotechnological options are available for enhancing the nutritive value of agro-industrial by products such as PKC (Kundu and Kuma, 1987). Biotechnological treatment to improve the digestibility of fibrous agricultural by-products includes either the direct use of microorganisms or microbial enzyme (Shaiful, 1982).

As a result of the ban on antibiotic growth promoters due to obvious problems of drug residues (Dipeolu et al., 2002; Dipeolu et al., 2004) microbial resistance (Chah et al., 2002) and high lipid (fat) in animal products (Lippstein et al., 1975) which are health hazards to both animals and man (Ogbe et al., 2005), there is increased interest in probiotics. These biotherapeutic agents (probiotics) have been shown to significantly increase feed efficiency and improve health status of livestock without any deleterious effects in both animals and humans (Baird, 1977; Fuller, 1992; Onifade and Babatunde, 1996; Chang et al., 2001; Ezema, 2007). The interest in these probiotics was further enhanced by the revealation by Shaiful (1992) that these microrganisms can be used to treat fibrous agroindustry by-products to improve the efficiency of their utilization.

Probiotic research and application originated from human medicine. For these earlier research workers, their interests were simply to use probiotics to promote good health. Hence, their choice of probiotics was mainly of bacteria origin. Weight gain and efficiency of feed utilization are not issues in human beings. In fact, human researchers are more interested in those products that could cause weight loss. But in livestock production, weight gain and feed efficiency are the crucial issues that will determine the productivity of the livestock. This is why we considered S. cerevisiae as the probiotic of choice for animal production. Live yeast (S. cerevisiae) has been shown to elaborate digestive enzymes which help to increase digestibility and efficiency of feed utilization especially in high fibre diets (Matsui et al, 1990; Numan, 2001; Ezema, 2007). On dry basis, yeast contains 54% crude protein and 3000 IU of inositol (a well known growth promoter) among other vitamins (Bery, 1982) as shown in table 1 below.

Table 1: The essential Amino Acids and Vitamins contained in Yeast

S/N Essential Amino Acid (g 100g drywt-1)

1 Lysine 8.2

2 Valine 5.5

3 Leucine 7.9

4 Isoleucine 5.5

5 Threonine 4.8

6 Methionine 2.5

7 Phenylalanine 4.5

8 Tryptophan 1.2

9 Cystine 1.6

10 Histidine 4.0

11 Tyrosine 5.0

12 Arginine 5.0

S/N Vitamins (ug g dry wt-1)

1 Thiamine Hcl 165

2 Riboflavin 100

3 Niacin 585

4 Pyridoxine Hcl 20

5 Folic Acid 13

6 Calcium pantothenate 100

7 Biotin 0.6

8 Paraminobenzoic Acid 160

9 Choline Chloride 2,710

10 Inositol 3000

Source: Berry, 1982

Statement of the Problem

Nutrition and diseases are the major problems of poultry production in the tropics (Adene, 2004). The cost of feeds alone accounts for about 80% of the total cost of poultry production (Adegbola, 2004). The future of efficient and profitable poultry production would therefore depend on finding cheaper and alternative energy and protein sources to conventional feed ingredients (Ojewola, 1992). Inclusion of palm kernel cake (PKC) has been known to reduce cost of feed but research workers have reported that due to its high crude fibre content, PKC should be treated for optimum efficiency of feed utilization (Okeudo et al., 2005). Kundu and Kumar (1987) observed that supplementing animal feed with probiotic is a biotechnological option to improve the nutritive value of agro-industrial by-product such as PKC. Shaiful (1992) showed that inclusion of live micro organisms or microbial enzyme will increase the efficiency of utilization of high fibre feed ingredient.

Objectives of the Study

The objectives of this study were:

To determine the effect of probiotic (Saccharomyces cerevisiae) on growth rate of pullets and hen-day egg performance of laying chicken.

To determine the appropriate level of inclusion of this probiotic for optimum pullet development and maximum egg production.

To assess the possible modes of action of the probiotic

To evaluate the cost benefit of this probiotic supplementation in table egg production

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