EFFECTIVE MICROORGANISMS AS AN ALTERNATIVE TO ANTIBIOTICS

By ESLAM SAID RAGHEB HEGAZY B. Sc. Agric. (Biochemistry), Al-Azhar University, 2006

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of MASTER OF SCIENCE

In AGRICULTURAL SCIENCES (Biochemistry)

Department of Biochemistry Faculty of Agriculture, Cairo Al-Azhar University

1434 A.H. 2013 A.D. TITLE: EFFECTIVE MICROORGANISMS AS AN ALTERNATIVE TO ANTIBIOTICS NAME: ESLAM SAID RAGHEB HEGAZY

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of MASTER OF SCIENCE

In AGRICULTURAL SCIENCES (Biochemistry)

Department of Biochemistry Faculty of Agriculture, Cairo Al-Azhar University

1434 A.H. 2013 A.D.

Supervision Committee: Prof. Dr. ASSEM FATHY EL-MAGHRABY ………………. Prof. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University Prof. Dr. AMINA ABD EL-HAKEM EL-FARAMAWY ………………. Prof. of Animal nutrition, Dept. of Food irradiation, National Center for Radiation Research and Technology, Egypt Prof. Dr. HALA AHMED HUSSIEN ..………...... Prof. of Microbiology, Dept. of microbiology, Center for Radiation Research and Technology, Egypt Cairo Dr. MOHAMED MABROUK EL-DANASOURY ……………….. Prof. Assistant of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University

APPROVAL SHEET

NAME: ESLAM SAID RAGHEB HEGAZY TITLE: EFFECTIVE MICROORGANISMS AS AN ALTERNATIVE TO ANTIBIOTICS

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of MASTER OF SCIENCE

In AGRICULTURAL SCIENCES (Biochemistry)

Department of Biochemistry Faculty of Agriculture, Cairo Al-Azhar University

1434 A.H. 2013 A.D.

Approved by:

Prof. Dr. MAHMOUD ABD EL-RAZEK DOHEM ………………. Prof. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Elzakazek University. Prof. Dr. MOHAMED GABER ABD EL-FADEL ………………. Prof. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University Prof. Dr. ASSEM FATHY EL-MAGHRABY ………………. Prof. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University Dr. MOHAMED MABROUK EL-DANASOURY ………… …… Asst. Prof. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University

ACKNOWLEDGEMENT

I bow my head before “ALLAH”, the Lord of the Worlds, The omnipotent, Gracious, The Merciful, who blessed me with good health and conducive environment and enabled me to work and complete my dissertation. He is entire source of all knowledge and wisdom endowed to the humanity. His blessings are unlimited.

My special praise to Prophet Muhammad (BPUH) from deepest core of my heart, who is forever a model of guidance and knowledge for the whole humanity. The very special entity ALLAH has brought in our lives whose saying “learn from cradle to grave “awakened the strong desire in myself to undertake this course of study and write up of this manuscript.

I am greatly thankful to respectable members of my supervisory committee, for their encouragement and fruitful guidance for this study. I take this opportunity to express my deep gratitude and sincere thanks to my supervisor Prof. Dr. Assem Fathy EL-Maghraby, Professor. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University and my deepest gratitude to Prof. Dr. Amina Abd EL-Hakem EL-Faramawy, Professor. of Animal nutrition, Dept. of Food Irradiation, National Center for Radiation Research and Technology, Egypt and Prof. Dr. Hala Ahmed

Hussien, Professor. of Microbiology, Dept. of Microbiology, National Center for Radiation Research and Technology, Egypt and Dr. Mohamed Mabrouk EL-Danasoury Assistant Professor. of Biochemistry, Dept. of Biochemistry, Faculty of Agriculture, Cairo, Al-Azhar University, for their supervision, their keen interest, skilful guidance and encouragement through the study and preparation of this manuscript. They are really devoted and ideal teachers and scientists.

I would like to express my deep thanks for all staff of Biochemistry Department, Faculty of Agriculture, AL-Azhar University and for all staff of Food Irradiation Department, National Center for Radiation Research and Technology, Egypt.

NO Contents page 1. INTRODUCTION 1 2. REVIEW OF LITRATURE 4 3. MATERIALS AND METHODS 34 4. RESULTS AND DISCUSSION 57 4.1. Traits of growth performance 57 4.1.1. Body weight 57 4.1.2. Body weight gain 61 4.1.3. Feed intake 66 4.1.4. Feed conversion ratio 71 4.1.5. Mortality rate 76 4.1.6. Performance index (%) 79 4.1.7. Meat yield% and internal organs% 83 4.2. Blood parameter: 87 4.2.1. Serum cholesterol, triglyceride, HDL, LDL, GPT and 87 GOT 4.2.2. Immune response and thyroid hormones 94 4.3. carcass chemical composition 98 4.3.1. Cholesterol and triglyceride in both breast and thigh meat 98 4.3.2. Amino acids profile in breast meat 101 4.3.3. amino acids profile in thigh meat 105 4.3.4. Fatty acids profile in breast meat 108 4.3.5. Fatty acids profile in thigh meat 111 4.4. fecal microbial 117 5. SUMMARY AND CONCLUSION 121 6. REFERENCES 126 الملخص العربي

List of Tables

NO Title Page 1 The ingredients and nutritional value of the basal starter, grower and 36 finisher diets and composition of Rovigypt premix. 2 Effect of addition of probiotic or antibiotic with different levels of RO 60 on average body weight in broiler chicks. 3 Effect of addition of probioticor antibioticwith different levels of (RO) 64 on body weight gain in broiler chicks. 4 Effect of addition of probioticor antibioticwith different levels of (RO) 69 on feed intake in broiler chicks. 5 Effect of addition of probioticor antibiotic with different levels of RO 74 on Feed conversion ratio in broiler chicks. 6 Effect of addition of probioticor antibiotic with different levels of RO 78 on mortality RATE in broiler chicks. 7 Effect of addition of probioticor antibiotic with different levels of RO 81 on performance index % in broiler chicks. 8 Effect of addition of probioticor antibiotic with different levels of RO 85 on Meat yield% and internal organs% in broiler chicks. 9 Effect of addition of probioticor antibiotic with different levels of RO 92 on Blood parameter in broiler chicks. 10 Effect of addition of probiotic or antibiotic with different levels of RO 97 on the immune response and thyroid hormones of broiler. 11 Effect of addition of probioticor antibiotic with different levels of RO 100 on cholesterol, triglyceride, in breast and thigh meat of broiler chicken at 42 days of age. 12 Amino acids composition (mg/g meat tissue) of breast chicken fed 104 probiotic or antibiotic with diet containing different levels of RO. 13 Amino acids composition (mg/g meat tissue) of thigh chicken fed 106 probiotic with diet containing different levels of RO. 14 Fatty acids (mg/g meat tissue) of breast chicken fed probiotic with diet 110 containing different levels of RO. 15 Fatty acids (mg/g meat tissue) of thigh chicken fed probiotic with diet 113 containing different levels of RO. 16 Effect of addition probiotic with different levels of RO on the total 120 count of bacteria, lactic acid bacteria, staph and E. coli in the faeces of broiler.

List of figures

NO Title Page 1 Effect of addition of probiotic or antibiotic with different levels of RO 61 on average body weight at the starter, grower and finisher periods of broiler chicks. 2 Effect of addition of probiotic or antibiotic with different levels of RO 65 on average weight gain at the starter and grower periods of broiler chicks. 3 Effect of addition of probiotic or antibiotic with different levels of RO 65 on average weight gain at the finisher and whole period of broiler chicks. 4 Effect of addition of probiotic or antibiotic with different levels of RO 70 on feed intake at the starter and grower periods in broiler chicks. 5 Effect of addition of probiotic or antibiotic with different levels of RO 70 on feed intake at the finisher and whole period in broiler chicks. 6 Effect of addition of probiotic or antibiotic with different levels of RO 75 on Feed conversion ratio (FCR) at the starter and grower periods in broiler chicks. 7 Effect of addition of probiotic or antibiotic with different levels of RO 75 on Feed conversion ratio (FCR) at the finisher and whole period in broiler chicks. 8 Effect of addition of probiotic or antibiotic with different levels of RO 79 on Mortality rate of broiler in the whole period of rearing 9 Effect of addition of probiotic or antibiotic with different levels of RO 82 on performance index % at the starter and grower periods in broiler chicks. 10 Effect of addition of probiotic or antibiotic with different levels of RO 82 on performance index % at the finisher and whole period in broiler chicks. 11 Effect of addition of probiotic or antibiotic with different levels of RO 86 on thigh and breast weight% in broiler chicks. 12 Effect of addition of probiotic or antibiotic with different levels of RO 86 on liver, gizzard, heart and spleen weight% in broiler chicks. 13 Effect of addition of probiotic or antibiotic with different levels of RO 93 on serum cholesterol and triglycerides levels in broiler chicks. 14 Effect of addition of probiotic or antibiotic with different levels of RO 93 on HDL, LDL, GOT and GPT in broiler chicks. 15 Effect of addition of probiotic or antibiotic with different levels of RO 97 on Antibody titer, T4 and T3 in broiler chicks. 16 Effect of addition of probiotic or antibiotic with different levels of RO 100 on cholesterol and triglycerides in breast of broiler chicks. 17 Effect of addition of probiotic or antibiotic with different levels of RO 101 on cholesterol and triglyceride in thigh of broiler chicks.

1. INTRODUCTION

Nowadays the production of safe and healthy food becomes a continuous and growing demand from the professional public and consumers. Therefore new additives have been increased for positively affecting meat production and quality with no adverse effect on human health.

Since decades antibiotics have been used in poultry production as therapeutic agents to treat bacterial infection that decrease performance and caused diseases. However, due to negative effects of using antibiotics on animal's health and production such as residue in the final products, development of bacterial resistance, accumulation in poultry excretion with consequent environmental pollution (Edens,2003)an increasing interest in finding alternatives to antibiotics becomes necessity.

Probiotics are live microbial feed supplement which have beneficial influence on: intestinal microflora balance (Kabir et al., 2005); immune response (Nayebpor et al., 2007 and Apata, 2008); serum total cholesterol and triglycerides (Ignatova et al., 2009) in addition to fat and cholesterol content of the chicken meat and yolk (Mansoup, 2011).On the other hand proved that probiotics improved economic efficiency and performance index (Awad et al.,2009).

Poultry required fat in the diet as a source of essential fatty

1 acids to improve the absorption of fat-soluble vitamins, increases the palatability of the rations, and increase the efficiency of the consumed energy. Furthermore, it reduces the passage rate of the digesta in the gastrointestinal tract which allows a better absorption of all nutrients present in the diet(Baiao and Lara, 2005).

The recent increment in the prices of oils as well as their wide use to raise the energy levels in poultry diet (El-Gendy, 1993) forced us to search for new cheap alternatives with high energy value not used for human feeding.

Use of oxidized oil (Anjume et al., 2004 and Karamouz et al., 2009), sun flower oil production wastes (Alizadeh et al., 2012) or semi-refined oils (Moraes et al., 2009) are new trends in poultry nutrition research works for economic efficiency with lowering feed cost and energy obtaining via cheaper sources. It was documented that oxidation of oil had no negative effect on its metabolizable energy levels for poultry (Hussein and Kratzer, 1982) but on other hand oxidized oil may be hazardous for birds and can lower feed efficiency and body weight (Anjum et al., 2004) and increase the levels of serum low density lipoprotein(LDL), triglyceride(TRIG) and cholesterol in serum and tissues of broilers (El-Faramawy, 2006 and Karamouz et al., 2009).

Various studies were carried out to lower cholesterol content

2 of chicken meat and egg either through the use of additives, dietary fiber or polysaturated fatty acids (Bakalli et al., 1995; Skrivanova et al., 2001; Ayerza et al., 2002; Skrivanova and Marounek, 2002). Lowering of cholesterol level in meat and egg has recently received more attention than before due to the increase in cardiovascular diseases in man mainly atherosclerosis, hypertension and coronary heart disease.

One of the main roles of probiotics bacteria is control of serum cholesterol and triglyceride levels. There are some evidences proposing that lactobacillus feed supplementation reduces the carcass cholesterol and the saturated fatty acid composition blood and carcass broiler chicks(Kalavathy et al., 2006; Ignatova et al., 2009 and Mayahi et al., 2010).

Since probiotics has hypolipidemic and hypocholesterolemic effect the aim of the present study is to evaluate growth performance, net profit, liver enzymes, serum lipid profile and humoral immune response in addition to carcass fatty acids (FA), amino acids (AA), cholesterol and triglycerides in broiler chicken fed different levels of RO with probiotic.

3 2. REVIEW OF LITERETURE

During the past 50 years, antibiotics have been used in poultry production as therapeutic agents to treat bacterial infections that decrease performance and cause diseases. Many of the antibiotics used in the poultry industry have been used in human medicine as well. Shortly after the initiation of widespread use of antibiotics in the animal industries, they were placed under increased scrutiny because of concern over development of bacterial resistance to the usual microbiocidal effects of the antibiotics (Edens, 2003). The DANMAP 1997 report indicated that the use of low levels of antibiotics in food animal feed leads to the development of resistance in zoonotic organisms of animal origin. Around the world, controversy has surrounded this report, but the impact of the work has been extremely influential as it has caused unprecedented changes in the way food animal production is being conducted today. In June of 1999 the European Union (EU) banned the use of some growth promoting antibiotics in poultry feeds. In the year 2006, the EU will officially ban the usage of all antibiotics for the sole purpose of growth promotion in poultry and livestock. Therapeutic use of appropriate antibiotics will be allowed via prescription only through a veterinarian. Therefore, the concept of probiotics for use in the poultry industry as an alternative to antibiotic growth promoters has been widely used.

4 Probiotics definition:

Over the years the word probiotic has been used in several different ways. It was originally used to describe substances produced by one protozoan which is stimulated by another (Lilly and Stillwell, 1965), but it was later used to describe animal feed supplements which had a beneficial effect on host animal by affecting its gut flora (Parker‚ 1974). Crawford (1979) defined probiotcs as "a culture of specific living micro- organisms which is implanted in the animal to ensure the effective establishment of intestinal populations of both beneficial and pathogenic organisms". In 1989, Fuller suggested this definition of probiotics, which has been widely used: "A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance". The US National Food Ingredient Association presented probiotic as a source of live naturally occurring microorganisms and this includes bacteria, fungi and yeast (Miles and Bootwalla, 1991). More precisely, probiotics are live microorganisms of nonpathogenic and nontoxic in nature, which when administered through the digestive route, are favorable to the host’s health (Guillot, 1998). According to the currently adopted definition by FAO/WHO (2001) probiotics are: "live microorganisms which when administered in adequate amounts confer a health benefit on the host".

5 Probiotic selection:

The perceived desirable traits for selection of functional probiotics are many. The probiotic bacteria must fulfill the following conditions: it must be a normal inhabitant of the gut, and it must be able to adhere to the intestinal epithelium to overcome potential hurdles, such as the low pH of the stomach, the presence of bile acids in the intestines, and the competition against other micro-organisms in the gastro-intestinal tract (Chateau et al., 1993). Many in vitro assays have been developed for the pre-selection of probiotic strains (Ehrmann et al., 2002 and Koenen et al., 2004). The competitiveness of the most promising strains selected by in vitro assays was evaluated in vivo for monitoring of their persistence in chickens (Garriga et al., 1998). In addition, potential probiotics must exert its beneficial effects (e.g., enhanced nutrition and increased immune response) in the host. Finally, the probiotic must be viable under normal storage conditions and technologically suitable for industrial processes (e.g., lyophilized).

Ideal probiotic (Lactic Acid Bacteria):

 General description:

The term Lactic Acid Bacteria (LAB) was gradually accepted in the beginning of the 20th century. Other terms as “milk souring” and “lactic acid producing” bacteria had previously been used for the same bacteria.LAB are generally associated

6 with habitats rich in nutrients, such as various food products (milk, milk product, meat, beverages and bakery products).They also are commonly found and known to proliferate in fermentation process. LAB have been detected in soil, water, manure and sewage and were isolated from milk. But some are also members of the normal flora of the mouth, intestine of mammals and birds. They require a nutrient rich environment in order to establish. Besides carbohydrates, they also need to be supplied with amino acids, peptides, salts and vitamins among others (Carr et al., 2002).

LAB are involved in the fermentation of a range of milk, meat, cereal and vegetable foods (McKay and Baldwin, 1990). The antimicrobial compounds produced by LAB can inhibit the growth of pathogenic bacteria of possible contaminants in the fermented products (Cintas et al., 1998).(LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as ethanol, H2O2, CO2, diacetyl, reuterin and bacteriocins (El- Ziney, 1998).

 Mode of action: Different modes of action of lactic acid bacteria beneficially affect the treated chicks include:

7 1. Maintaining normal intestinal microflora by competitive exclusion and antagonistic activity (Kizerwelter-swide and Binek, 2009).

a. Competitive exclusion (CE):

Metchnikoff (1961) specified that detrimental microbial in the intestinal tract produce harmful substances to the host could be neutralized by beneficial organisms in yoghurt.

The results on competitive exclusion by lactic acid bacteria are sometimes contradictory or confusing. Fuller (1977) reported that host-specific Lactobacillus strains 59 and 74/1 were able to decrease E. coli in crop and small intestine, but not in caeca of gnoto-biotic chickens. Muralidhara et al (1977) found that the homogenates of washed intestinal tissues dosed with L. lactis had higher numbers of attached Lactobacilli and lower E. coli counts than control birds. Jin et al (1996 c) found that only 26% of the isolates of Lactobacillus spp. from chicken intestines were able to attach moderately or strongly to the ileal epithelial cells of chickens. The ability of Lactobacillus to adhere in vitro to intestinal epithelial cells varies considerably among species and among strains of the same species.

b. Antagonistic activity:

The antagonistic activity of LAB against different pathogenic microorganisms can be related to the production of bacterial substances as bacteriocins, organic acids, and hydrogen

8 peroxides. Lactobacilli have been extensively studied for the production of antagonistic substances, which are referred to as bacteriocins. These include well-characterized bacteriocins (Joerger and Klaenhammer, 1986), bacteriocin-like substances (Vincent et al., 1959), and other antagonistic substances not necessarily related to bacteriocins (Shanhani et al., 1976).

2. Altering metabolism by increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production (Yoon et al., 2004).

a. Digestive enzyme activity:

Lactobacillus spp. have been shown to produce digestive enzymes in vitro and the enzymes may enrich the concentration of intestinal digestive enzymes. Szylit et al(1980) reported that two of five strains of Lactobacillus isolated from male chicks showed amylase activity. Lactobacillus spp., which are used in cheese products were found to have amylolytic, lipolytic, and proteolactic activities (Lee, 1990).

Jin et al (1996 b) reported that all 12 Lactobacillus spp. isolated from chicken’s intestine were found to secrete amylase, protease, and lipase, either extracellularly or intracellularly, or both extracellularly and intracellularly. They also found that amylase activity in small intestine increased when Lactobacillus cultures were fed to the broilers, but there was no effect on

9 lypolytic and proteolytic activities (Jin et al., 1996a).

b. Bacterial enzyme activity:

Goldin and Gordbach (1977) reported that the activities of nitroreductase, azoreductase and 3-glucuronidase in the gut of rates could be reduced by feeding supplements of L. acidophilus. The same results were observed in humans. A similar reduction in glucuronidase has been detected in chickens fed 40% yoghurt in the drinking water (Coloe et al., 1984)

c. Ammonia production

Suppressing ammonia production and urea’s activity can be beneficial for improving animal health and enhancing growth as ammonia can cause damage to the surface of cells. Chiang and Hsien (1995) reported that probiotics containing L. acidophilus, S. faecium, and B. subtilis reduced the concentration of ammonia in the excreta and litter of broilers.

3. Increasing feed intake and digestion(Awad et al., 2006).

The intestinal bacterial flora of domestic animals has an important role in the digestion and absorption of feed. It participates in the metabolism of dietary nutrients such as carbohydrates, proteins, lipids, and minerals and in the synthesis of vitamins.

Nahanshon et al (1996) stated that addition of Lactobacillus cultures in maize/soybean or maize/barely/soybean diets

10 stimulated appetite and increased fat, nitrogen, calcium, phosphorus, copper, and manganese retention in layers.

4. Stimulating the immune system(Brisbin et al., 2008).

Immunity resulting from gut exposure to a variety of antigens, such as pathogenic bacteria and dietary protein, is important in the defense of young animals against enteric infections (Perdigon et al., 1995).

Dunhan et al (1993) reported that birds treated with L. reuteri exhibited longer ileal villi and deeper crypts, which are a response associated with enhanced T cell function and increased production of anti-Salmonella IgM antibodies.

Nahanshon et al (1994) found that Lactobacillus supple- mentation of layers diets increased cellularity of peyer’s patches in the ileum indicating a stimulation of the mucosal immune system that responded to antigenic stimuli by secreting immunoglobulin (IgA).

 Probiotics administration: There are four different methods for administering probiotic:

1. Introducing the treatment material into the crop by tube and syringe,

2. Introducing the treatment material into the beak using a hypodermic syringe fitted with a beaded needle,

3. Allowing each chick to drink from the tip of a pipette.

11 4. Dipping the beak of the bird in the treatment material.

Beneficial application of probiotic in broiler production:

 Growth performance: Numerous studies show that probiotics have positive effects on chicken growth performance and feed efficiency. Growth performance usually included: body weight, weight gain, feed conversion ratio and mortality rate. The supplementation of either mixture of lactobacilli cultures or preparations of lactobacilli and other bacteria in chicken's feed have given variable results.

Mohan et al (1995) reported that body weight gain could vary by 5 % to 9 % when chickens were supplemented with probiotic containing a mixture of L. acidophilus, L. Casei and Bifidobacterium bifidum.

Jin et al (1996a) treated two hundred 10 days-old broiler chicks with commercial Lactobacilli added to their diets under a hot and humid environment found that weight gain was significantly higher and feed gain ratio was significantly lower than that of control birds (P<0.05).

Jin et al (1997) reported that adding of LAB to the feed from 0 to 6 weeks of age either a single strain of L. acidophilus 126 or a mixture of Lactobacillus (adherent Lactobacillus cultures isolated from the intestine of the chickens) significantly

12 improved body weight and feed conversion ratios of broilers (P<0.05).

Yeo and Kim (1997) reported that feeding a diet containing probiotic (L. casei) significantly increased average daily gain during the first 3 weeks (P<0.05).

Zulkifli et al (2000) found that broiler chicks fed diets containing 1 g Lactobacillus culture / kg diet during the last three weeks of age (After 3 weeks of heat treatment exposure) had significantly higher feed intake and lower feed efficiency than control.

Kumar et al (2002) found that body weight of broiler chicks, which fed diets containing Lactobacillus acidophilus at 1g/kg diets, was significantly higher compared with control group.

Kalavathy et al (2003) stated that the mixture of 12 lactobacillus strains at 0.1% affected growth performance of broiler chicks from 1 to 42 days of age. They also found that there was significant increase in body weight, body weight gain and feed conversion ratio at 42 days of age compared with control group.

Huang et al (2004) indicated that lactobacillus acidophilus supplementation was able to enhance production performance of broiler chickens.

13 Anjum et al (2004) indicated that body weight, weight gain and feed conversion ratio were significantly improved in chicks fed on multi-strain probiotic (protexin) 110g/t in starter and 55g/t in finisher diet containing Lactobacillus culture compared with control diets.

Eglal (2006) indicated that broiler fed 1.0 kg primalac and 0.5 kg Bioaction / ton ration as a probiotic had the highest averages of body weight, weight gain and feed conversion ratio at the 4th and 6th weeks of age, when compared with other levels of different treatments and control group.

Timmerman et al (2006) indicated that the supplementation of a diet with multispecies probiotic resulted in a slight increase (by 1.84%) in broiler productivity, while the probiotic mixture of 7 lactobacillus species isolated from the digestive tract of chickens was prepared in fluid form raised productivity by 2.94 and 8.70%.

Khaksefidi and Ghoorchi (2006) reported the inclusion of bacillus subtilis in broiler diets improved body weight and body weight gain during 1-21 and 22-42 days.

Torres-Rodriguez et al (2007) reported that commercial lactobacillus-based probiotic significantly improved body weight, weight gain and feed conversion ratio of commercial turkey hen production.

Abdalla et al (2008) reported that supplementation layer

14 diets Lactobacillus or Biogen significant (P<0.01) affected on body weight and feed conversion ratio for the whole experimental period.

Fahmy (2009) showed that after 42 days of treatment, live body weight and weight gain were significantly (P<0.05) greater in broilers given 1% of effective microorganisms (EM) solution in the drinking water than the control and the other treatment groups.

Chen et al (2009) indicated that applied the Bacillus subtilis var. natto N21 (Bac; for greater proteolytic capacity) as a probiotics improved the growth performance of broilers.

Awad et al (2009) reported that a slight improvement in performance traits was observed in broilers fed the probiotic compared with control birds. So, concluded that probiotic displayed a greater efficacy as growth promoters for broilers.

Falaki et al (2010) indicated that application of probiotic (primalac 900g/ton) improved body weight gain and FCR compared with control.

Shabani et al (2012) showed that feeding broiler chicken with probiotics have positive effects on growth performance of chicken broilers.

There are many other investigations lacking positive effects of probiotics. Watkins and Kratzer (1984)found that feeding

15 chicken broiler with Lactobacilli isolated from the specific host (KTM, 74/1 59), and commercial products containing Lactobacilli did not find any improvement in growth performance of treated chicks.

Also Maiolino et al (1992) did not find any significant differences in live weights of chickens fed diets containing L. acidophilus and S. faecium from 8 to 60 days of age.

O‘dea et al (2006) indicated that there were no significant differences in broiler body weight between the probiotic treatments and the control group.

 Mortality rate: Abd-Elsamee and Abd El-Hakim (2002) observed no mortalities due to applications of Bio- Top as a probiotic based on lactobacillus culture during the experimental period. The total mortality averaged between 5 - 6 % was not due to treatments but could be attributed to natural cases.

Eglal (2006) indicated that mortality rate decreased in chicks fed Primalac, as a probiotic based on lactobacillus culture compared with control groups.

Timmerman et al (2006) reported that the supplementation of a diet with multispecies probiotic (consisted of 7 Lactobacillus species isolated from the digestive tract of chickens) reduced mortality rate compared with control.

16 On the other hand Ali (1999) observed the viability of broiler chicks from 1- 7 weeks of age was not significantly affected by addition of probiotics to the diets.

Zulkifli et al (2000) stated that broiler chicks fed on diets containing Lactobacillus culture (1g/kg diet) had insignificant affect on mortality rate at 3 and 6 weeks of age.

O’Dea et al (2006) indicated that there were no significant differences in broilers mortality rate between the probiotic treatments and the control group.

Willis et al (2007) concluded that mortality rate not affected by the broiler probiotic (Primalac) diet additive over the 3-wk period.

Also, Willis and Reid (2008) concluded that mortality rate was not significantly affected by probiotic treatment.

 Performance index: El-Husseiny et al (1995) concluded that, adding the probiotics to broiler chick diets improved performance index of broiler chicks.

El-Gendi et al (2000) indicated that feeding chicks on diets containing 0.5 g/1kg feed Biotonic (as a probiotic) improved performance index of broiler chicks.

Abdel-Azeem et al (2001) found that performance index significantly improved when microbial probiotics Lacto Sacc

17 (lg/kg feed) and Yea Sacc (lg/kg feed) was supplemented to Japanese quail chick’s diets.

Abd-Elsamee (2001) demonstrated that the average values of the total cost / kg body weight and performance index were improved when broilers fed diets supplemented with probiotic (Avi-Bac) at 0.05 % of broiler chick diets.

Abd-Elsamee and Abd El-Hakim (2002) observed that the addition of Bio-Top as a probiotic to broiler chick diets decreased the average values of feed cost / kg body weight and increased the average values of performance index.

Osman (2003) found that adding probiotic to broiler chick diets improved the performance index of low protein diets by 20% over the control diet containing the recommended level of protein.

Salim (2004) studied the effect of three kinds of probiotics (Primalac, Lacture and Biobadus) as a probotics based on lactobacillus culture on performance index of broiler chicks from 7-42 days of age. He showed that adding probiotics significantly improved performance index when compared with control.

Tollba et al (2004 a and b) obvious that, using biological additives 1 kg / ton feed Lacto Scicc (bacteria concentration) in broiler diets caused a significant improvement in their

18 performance index especially when reared at heat stress conditions.

Abd El-Gawad et al (2004) showed that adding lacture as a commercial probiotic containing lactic acid bacteria to broiler chick diets improved the utilization of low protein diets and gave the best performance index.

Eglal (2006) concluded that feeding chicks on diet supplemented with 0.5 kg / ton ration of Primalac or Bioaction respectively increased performance index when compared with other levels of different treatments applied.

Ghazalah et al (2006) showed that performance index was improved by using probiotics in broiler diets.

Awad et al (2009) reported that a slight improvement in performance traits was observed in broilers fed the probiotic compared with control birds. So, concluded that probiotic displayed a greater efficacy as growth promoters for broilers.

 Altering intestinal microbiota: Fuller (1977) found that host-specific Lactobacillus strains were able to decrease E. coli in the crop and small intestine.

Francis et al (1978) also reported that the addition of Lactobacillus product at 75 mg/kg of feed significantly decreased the coliform counts in the ceca and small intestine of turkeys.

19 Watkins et al (1982) similarly observed that competitive exclusion of pathogenic E. coli occurred in the gastrointestinal tract of gnotobiotic chicks dosed with L. acidophilus.

Watkins and Kratzer (1983) reported that chicks dosed with Lactobacillus strains had lower numbers of coliforms in cecal macerates than the control.

Kabir et al (2005) attempted to evaluate the effect of probiotics with regard to clearing bacterial infections and regulating intestinal flora by determining the total viable count (TVC) and total lactobacillus count (TLC) of the crop and cecum samples of probiotics and conventional fed groups at the 2nd, 4th and 6th week of age.

Mountzouris et al (2007) and Higgins et al (2007) demonstrated that probiotic species belonging to Lactobacillus, Streptococcus, Bacillus, Bifidobacterium, Enterococcus, Aspergillus, Candida, and Saccharomyces have a potential effect on modulation of intestinal microflora and pathogen inhibition.

Kizerwetter-Swida and Binek (2009) demonstrated that L. salivarius 3d strain reduced the number of Salmonella enteritidis and Clostridium perfringens in the group of chickens treated with Lactobacillus.

20  Immune response: Havenaar and Spanhaak (1994) have reported that probiotics stimulate the immunity of the chickens in two ways:

1. Flora from probiotic migrates throughout the gut wall and multiply to a limited extent.

2. Antigen released by the dead organisms are absorbed and thus stimulate the immune system. At present it is believed that there is some relationship between the ability of strain to translocate and the ability to be immunogenic.

The improvement of the immune system may be by three different ways:

1. Enhanced macrophage activity and enhanced ability to phagocytose microorganisms or carbon particles.

2. Increased production of antibodies usually of IgG & IgM classes and interferon (a nonspecific antiviral agent).

3. Increased local antibodies at mucosal surfaces such as the gut pathogen inhibition by the intestinal microbiota wall (usually IgA).

Kabir et al (2004) evaluated the dynamics of probiotics on immune response of broilers and they reported significantly higher antibody production (P<0.01) in experimental birds as compared to control ones. They also demonstrated that the differences in the weight of spleen and bursa of in these birds

21 could be attributed to different level of antibody production in response to sheep red blood cells (SRBC).

Haghighi et al (2005) demonstrated that administration of probiotics enhances serum and intestinal natural antibodies to several foreign antigens in chickens.

In addition, Dalloul et al (2005) examined the effects of feeding a Lactobacillus-based probiotic on the intestinal immune responses of broiler chickens over the course of an E. acervulina infection and they demonstrated that the probiotic continued to afford some measure of protection through immune modulation despite a fairly overwhelming dose of E. acervulina. They also suggested a positive impact of the probiotic in stimulating some of the early immune responses against E. acervulina, resulting in improved local immune defenses against coccidiosis.

Similarly, Khaksefidi and Ghoorchi (2006) reported that the antibody titer in the 50 mg/kg probiotic supplemented group was significantly higher at 5 and 10 days of postimmunization (PI) compared to control, when SRBC was injected at 7 and 14 days of age.

On the other hand Midilli et al (2008)showed the ineffectiveness of additive supplementation of probiotics on systemic IgG.

22  Serum total lipids, cholesterol and triglycerides: Jin et al (1998) reported that Serum cholesterol levels were significantly lower (P<0.05) in broilers fed the three diets containing Lactobacillus cultures at 30 day of age, and in the birds fed 0.05 or 0.10% Lactobacillus cultures at 20 day of age.

El-Gendi et al (2000) indicated that feeding chicks on the diet with fermacto as a probiotic based on lactobacilli had high significant effect on serum total lipids and cholesterol.

Abou-Zeid et al (2000) reported that the microbial probiotics supplementation in broilers chicks diets significantly decrease (P<0.01) total cholesterol and insignificantly decreased total lipids.

Kalavathy et al (2003)found that serum total cholesterol, low- density lipoprotein (LDL) cholesterol and triglycerides were significantly reduced (P<0.05) in broiler chicks fed on diets containing a mixture of 12 Lactobacillus strain at 0.1 %.

Abd El-Gawad et al (2004) reported that adding Lacture (three kinds of probiotics Premalac, Lacture or Biobadus) based on Lactobacillus strain decreased significantly (P<0.05) cholesterol (106 mg/dl) compared with control (124 mg/dl) and had no adverse effects on total lipids.

Salim (2004) studied the effect of three kinds of probiotics (Primalac, Lacture or Biobadus) as a probiotic based on

23 lactobacillus culture on blood constituents of broiler chicks at 42 days of age. He noticed that Primalac significantly decreased total lipids while cholesterol was not significantly affected. However, cholesterol was significantly decreased by Lecture.

Tolba et al (2004a and b) reported that adding the tested biological additives to broiler diets inoculated with 1 kg / ton feed Lacto Sacc (bacteria concentration) or Yea Sacc (yeast culture), respectively decreased (P<0.05) Plasma cholesterol and total lipids as compared with control groups under normal or heat stress conditions.

Eglal (2006) showed that feeding chick's diets with Primalac (content of lactic acid bacteria) decreased total lipids and cholesterol in serum. Chicks fed 1.0 kg Primalac or 0.5 kg Bioaction/ton ration had the lowest averages of total lipids and cholesterol at 28 and 51 day of age.

Safalaoh (2006) found that use of microbial preparations containing different type of microorganisms (lactobacillus plantarum, lactobacillus casei and streptococcus lactis) with broiler's diet may have potential to lower serum cholesterol and abdominal fat bad than the control.

Abdalla et al (2008) reported that serum total lipids and cholesterol concentration decreased significantly with increasing level of Lactobacillus supplementation layer diets. . Ignatova et al (2009) investigated that the probiotic addition

24 to broiler's diets reduced the serum cholesterol and triglyceride significantly and also reduced the fat content of the chicken meat.

Mansoub (2010) reported that diet supplementation with L. acidophilus and L. casei in combination with water and / or alone significantly decreased total cholesterol and triglyceride concentrations in the blood serum of broiler chickens.

Alkhalf et al (2010) concluded that the total lipids was not affected by probiotic supplementation while Chicken fed a diet containing various levels of probiotic showed a significant decrease in cholesterol concentration compared to control group.

Mayahi et al (2010) reported that supplementation of probiotic to broiler's diet decreased the cholesterol and triglyceride components of broiler chicks sera.

Mansoup (2011) revealed that the serum total cholesterol and LDL were significantly reduced by dietary with probiotics compared to the control group. They also found that there is significant decrease in the serum level of triglycerides between groups treated with probiotics supplemented in broiler diet in combination with water or alone.

To the contrary Abd El-Hady and Abd El-Ghany (2003) found that, there were no significant differences in blood parameters (cholesterol, triglyceride, HDL and LDL) due to

25 either dietary probiotic supplementation.

 Liver Enzymes: Abdel-Malak et al (1995) reported that glutamic-oxaloacetic transaminase (GOT) andglutamic-pyrovic transaminase (GPT) activities were decreased in serum of broilers received diet with higher levels of commercial probiotic when compared with the control group.

Abdel-Azeem et al (2001) reported that the microbial probiotics (Lacto Sacc (lg/kg feed) Supplementation in Japanese quail chicks diets significantly affected on the (GOT) and (GPT) values.

Kumar et al (2002) studied the adding probiotic (Lactobacillus acidophilus) at 1 g/kg in broiler chicks diet significantly decrease serum GPT and GOT values at 45 days of age compared with control group.

Eglal (2006) showed that chicks fed diet with Bioaction (as a probiotic) had the highest averages of GOT and GPT at 28 and 51 day of chick's age.

On the other hand, Many authors observed no changes in liver enzymes due to probiotic supplementation. Abd El-Hady and Abd El-Ghany (2003) found that there were no significant differences in broilers blood parameters due to probiotic supplementation.

26 Abd El-Gawad et al (2004) reported that adding three kinds of probiotics (Premalac, Lacture or Biobadus) had no deleterious effects on liver function (as measured by GOT and GOT).

Tolba et al (2004a and b) reported that adding the tested biological additives to broiler diets inoculated with 1 kg / ton feed Lacto Sacc (bacteria concentration) did not alter GPT and GOT enzyme activity under normal or high temperature conditions comparing to un-supplemented control group.

 Carcass chemical composition: There has been growing interest over recent years in the modulation of the cholesterol content and fatty acid composition in poultry products because occurrence of cardiovascular heart diseases are closely related to the dietary intake of cholesterol and saturated fatty acid (SFA) contents (Sacks, 2002). It is widely acknowledged that there is an urgent need to return to a balanced fatty acid diet by decreasing intake of cholesterol and saturated fats (Evans et al., 2002). Cholesterol content in chicken meat can be altered by varying the composition of diet, age, and gender (Wang et al., 2005).

In recent years, research has been focused to reduce fat, cholesterol and SFA contents of poultry meat by dietary supplementation of garlic (Konjufca et al., 1997), copper (Pesti and Bakalli, 1996), and n-3 fatty acid (Ayerza et al., 2002).

27 Several reports indicated that dietary supplementation of bacteria such as Lactobacillus cultures reduced cholesterol in broilers.

Tanaka and Santoso (2000) reported that feeding the fermented product from bacillus subtilis to broiler's diet decreased contents of triglyceride and cholesterol in carcass significantly compared with control.

Kalavathy et al(2006) investigated that Lactobacillus feed supplementation reduces the cholesterol and fatty acid composition of broiler chicks body.

Tsujii et al (2007) .showed that cholesterol and triglyceride concentrations in rats and laying hens as well as in egg-yolk were markedly reduced by dietary lactic acid bacteria.

Salma et al (2007) have shown that cholesterol concentration in thigh and breast muscle of the broilers had a positive correlation with the change of the cholesterol contents in serum. Thus, it is expected that with decreasing of serum cholesterol, the amount of meat cholesterol is tending to decrease too.

Ignatova et al (2009) investigated that the probiotic addition to broiler's diets reduced the fat content of the chicken meat.

Ashayerizadeh et al (2011) have shown that, In 21 day old birds, dietary supplementation with probiotic decrease cholesterol concentration when compared with birds fed the

28 control, prebiotic and antibiotic diets. Also, at 42 day of age, the probiotic supplemented group had a lower cholesterol and triglycerides concentrations (P<0.05) compared with those of control and antibiotic supplemented groups.

Oil and Fats in broiler diets:

Lipids constitute the main energetic source for animals and they have the highest caloric value among all the nutrients. Linoleic acid is the only fatty acid whose dietetic requirement has been demonstrated. Besides supplying energy, the addition of fat to animal diets improves the absorption of fat-soluble vitamins, decreases pulverulence, increases diet palatability and the efficiency of utilization of the consumed energy. Furthermore, it reduces the rate of food passage through the gastrointestinal tract which allows a better absorption of all nutrients present in the diet. The energetic value of oils and fats depend on the following: the length of the carbonic chain, the number of double bonds, the presence or absence of ester bonds (triglycerides or free fatty acids), the specific arrangements of the saturated and unsaturated fatty acids on the glycerol backbone, the composition of the free fatty acid, the composition of the diet, the quantity and the type of the triglycerides supplemented in the diet, the intestinal flora, the sex and the age of the birds. In birds, body fat composition is similar to the composition of the fat from the diet. The apparent

29 digestibility of unsaturated fats is high in the first days of life of birds, whereas apparent digestibility of saturated fats is low. Considering diets with the same nutritive values, birds fed with rations containing oil present better performance than birds fed no oil. Moreover, the use of oil or fat in diets for broilers may change both the composition and the quality of the carcass. (Baiao and Lara, 2005)

Poultry feeding is one of the most important aspects of poultry production. Therefore profitable poultry rearing, provision of economical and balanced feed is must. Among the constituents of poultry feed, fats supply concentrated form of energy (2.25 times more energy than carbohydrates and protein). Although, plant oil have been used more often than animal fats in diets; however, it has been considered expensive fat supplemented especially in Egypt witch most of the edible oils are purchase from abroad. This is led to search about alternative cheap sources which are not used for human feeding (Ali et al., 2000).

Used restaurant oil or residual oil from fried fish and chickens are cheap fat source and characterized by changes in chemical and physical properties due to heating which may affected negatively their nutrition value. Oils are susceptible to oxidation and some changes, especially under the promoting effects of high temperatures. Lipid oxidation is a serious problem to both the food industry and consumer, because it shortens the shelf

30 life of meat and meat products, and increase the development of rancid odors and flavors. In addition free radicals formed during heated may be a potential health risk to consumers (Jakobsen, 1999).

Effect of residual oil (RO) on growth performance in broilers:

The usage of RO in diets can lead to growth depression in poultry and other animal species (Dibner et al., 1996; Jankowski et al., 2000and Keller et al., 2004). It has been suggested that growth depression might be due to a reduction in feed intake as a result of reduced palatability and off flavor of rancid feed, or a decrease in digestibility of the oxidized oil (Zadunczyk et al., 2002).

Eder (1999) reported that Body weight gains and food conversion rates were only slightly lower in the rats fed the oxidized oil compared to the rats fed the fresh oil.

Karamouz et al (2009) reported that short and long-term feeding of diet containing oxidized or heated oils decreased the final body weight of turkeys.

Bayraktar et al (2011) stated that oxidized dietary oil did not affect body weight gain and feed intake during the four to six week experimental periods; also there were no differences in FCR of the treatment groups.

31 Zhang et al (2011) reported that dietary supplementation with 5% oxidized oil to broilers did not show significant effect on growth performance and feed consumption compared to control group.

Effect of residual oil on carcass quality and blood parameters:

Authors using RO as oil supplement in the monogastric animals showed variable results. Narasimhamurthy and Raina (1999) observed higher cholesterol and lower triglyceride concentrations in the plasma of rats fed heated/fried oils compared to the group fed fresh oil.

Eder et al (2002) studied that rats fed all types of oxidized fats had higher concentration of free and total thyroxin in plasma than rats fed the equivalent diets with fresh oil, while the concentration of triiodothyroxine and thyroid-stimulating hormone did not differ between rats fed fresh and those fed oxidized fats.

Zadunczyk et al (2002) and Keller (2004) reported that oxidized dietary oils lead to a reduced antioxidant status and make them more susceptible to haemolysis. Eder et al (2003) reported that oxidized dietary oils reduced cholesterol and triglyceride concentrations.

Fawzy (2007) observed that the RO exhibited an increase in the saturated and monounsaturated fatty acids while the

32 polyunsaturated fatty acids were decreased. Also, he reported that feeding rats on RO enriched diet significantly increase in the concentration of serum T.C, LDL and TAG compared to the control. He also studied that feeding rats on RO enriched diet significantly raised the activities of GOT and GPT compared to the corresponding enzyme activities of rats fed on fresh oil.

Osman (2007) Stated that rats fed on frying oil had more activity of the LDL, GOT and GPT in serum as compared rats fed on fresh oil; he also, reported that rats receiving the frying oil had greater concentrations of T4 than rats receiving the fresh oil while The levels of T3 did not differ between the two groups.

Karamouz et al (2009) indicated that feeding chicks on diets containing 0.0, 2.5 and 5% food industrial oil increased cholesterol, LDL, HDL, triglyceride at day 42 day.

Bayraktar et al (2011) reported that neither protein nor fats digestibility was affected by mildly oxidize oil; also they reported that the supplementation of oxidized oil to grower diets did not affect the levels of total cholesterol in plasma. However, the triglyceride level was strongly influenced by the oxidized oil treatment. The birds fed oxidized oil had a significantly lower triglyceride level than the control group.

Zhang et al (2011) reported that dietary supplementation with 5% oxidized oil reduced higher levels of lipid oxidation in blood plasma than control group (P<0.05).

33 3. MATERIALS AND METHODS

Preparation of microorganisms:

Microorganism’s isolates: the probiotic was isolated in bioburden and toxins laboratory in National Center for Radiation Research and Technology (NCRT). They were identified in the same laboratory as lactobacillus casei, lactobacillus plantarum and enterococcus faecium; isolates were isolated from cheeses (L. casei) and pickles. The isolates were stored over MRS (DeMan, Rogosa, Sharpe Agar) slants at 4°c untilled use. Twenty four hours slants for each isolates were washed by sterile tap water and collected as a mixture in 1 liter elementary flask. The final concentration of the mixture contained 4x108lactobacilluscasei, 7x108lactobacillus plantarum and 5x107enterococcus faecium CFU/ml (Colony Forming Unit/ml) both antibiotic and probiotic was added to the drinking water.

Management of experimental birds:

A total number of 180 one-day old cubb broiler chicks of a nearly similar live body weight were used in this study. Chicks were weighed at hatch and kept under similar standard hygienic and environmental conditions. They were housed in electrically heated battery cages, where Feed and water were offered ad- libitum. Chicks were fed starter diet during the first two weeks

34 then replaced with grower diet until 4th week and then replaced with finisher diet until 6th week. The basal diet was formulated according to the recommended requirements of NRC, (1994). The RO was collected from kentacky fried chickens while fresh oil(FO) was purchased from the local markets and supplemented by 0.20, 2.4 and 3.6% in starter, grower and finisher diets respectively as presented in (table 1).

35 Table: (1). The ingredients and nutritional value of the basal starter, grower and finisher diets and composition of Rovigypt premix.

Item Starter Grower Finisher Basal diet* 99.8 % 97.6% 96.4% Fresh oil (FO) 0.20% 2.4% 3.6% Mixed oil (MO) (0.1% FO+0.1%RO) (1.2% FO +1.2%RO) (1.8% FO+ 1.8% RO) Residual oil (RO) 0.20% 2.4% 3.6% *Basal diet

Ingredients (%) Starter Grower Finisher

Corn 50.72 52.00 48.13 Soybean meal 41.08 35.03 30.56 Gluten 4.20 8.09 14.68 Dicalcium phosphate 2.46 2.20 2.06 DL-methionine 0.34 0.26 0.16 L-lysine 0.23 0.19 0.03 Vitamin permix b 0.25 0.25 0.25 Mineral permix c 0.25 0.25 0.25 Limestone - 0.05 - Salt 0.27 0.28 0.28 Calculated analysis ME (kcal/kg) 2820 2950 3045 Crude protein % 21.53 18.85 18.01 Crude fat % 4.04 5.05 6.57 Calcium % 0.93 0.83 0.80 Available P % 0.47 0.41 0.40 Methionine +cystine % 0.90 0.82 0.72

36 Rovigypt Premix

Vitamin Amount/ton Minirals Amount/ ton ration ration A (Retinol) 12000.000 IU Manganese 60 mg D (Calciferol) 2500000 IU Zinc 40 mg E (Tocopherol) 10 mg Copper 5 mg BI (Thiamine) 1 mg Iron 50 mg B2 (Riboflavin) 4 mg Iodine 3 mg B6 (Pyridoxine) 1.5 mg Selinium 0.1 mg B12(Cyanocobal-amin) 10 mg Cobalt 0.1 mg K (phylloquinone) 1 mg - - B9 (Folic acid) 29 mg - - B7 (Biotin) 50 mg - - B5 (Pantothenic acid) 10 mg - - Choline chloride 500 mg - -

Grouping birds and experimental design: From day one chicks were randomly divided into two equal groups ( 90 chicks each), the first group was supplemented with antibiotic in drinking water as (Virginiamycin) (Phibro, USA) (15 ppm), the second group was supplemented with (4x108 lactobacillus casei, 7x108 lactobacillus plantarum and 5x107 enterococcus faecium). Both groups were redivided into 3 dietary equal subgroups; the first group, A, B and C, the second group D, E and F, with 30 chicks having three replicates, divided in to three dietary group (A, B and C), with 30 chicks having three replicates, 10 birds in each. While the second group was divided in to three dietary groups (D, E and F), with 30 chicks having three replicates, 10 birds in each. The groups A

37 and D were fed basal diet supplemented with (FO), group B and E were fed basal diet supplemented with mixer (FO+RO) (1:1 w/w), and groups C and F were fed diet supplemented with RO. Body weight, feed intake and mortality rate were recorded after each stage; sheep red blood cell (SRBC) 3% was injected subcutaneously (0.5 cc)at day 14 and 21.

At the 42nd day age, three birds were randomly taken from each subgroup, the birds chosen were fasted for about 24 hours individually weighted alive to the nearest gram and then slaughtered according to Islamic tradition by cutting the jugular vein and the throat near the first vertebra with a sharp knife. Blood samples from each slaughtered bird were collected, centrifuged at 3000 rpm, sera were separated and kept frozen for determination of triiodothyronin(T3), teteraiodothyronin (T4) cholesterol, triglyceride, LDL, HDL, GPT, GOT and antibody titer. After complete bleeding, the birds were weighted and dressed by dry plucking. Shank and heads were separated. The birds were then eviscerated. The carcass and giblets (empty gizzard, liver, and heart) were separately weighted.

38 Parameters estimated and data collection:

Average body weight, weight gain and rat of growth:

Chicks were individually weighted to the nearest (g). Weight gain and rate of growth were individually calculated according to the following formulas suggested by broody (1949).

Weight gain (g) = w2– w1

w2 – w1 Rate of growth (%) = ------× 100 (w2 + w1) /2

Where: w1 and w2 are individual body weight at two successive periods.

Feed consumption and feed conversion:

Daily feed intake was estimated for all birds of each experimental subgroup then daily average was calculated. Data for feed consumption was then estimated and presented for the period from 0-2, 2-4, 4-6 and 0-6 weeks.

Feed conversion was similarly calculated as the amount of feed consumed (g) during each period of life to body weight gain (g) and given period as indicated in the following equation.

Feed consumed (g) Feed conversion = ------Body weight gain (g)

39 Mortality rate:

Percentage of mortality was calculated according to the following equation:

I – E Mortality % = ------× 100 I Whereas:

I = Initial number of birds

E= Number of live birds at end of the experimental period.

Performance index (PI):

Performance index was estimated for the previously mentioned periods according to the equation suggested by North (1981).

Live body weight (kg) Performance index (%) = ------×100 Feed conversion Faecal microflora analysis:

The faecal microflora was analyzed by the pour plate method (van der Wielen et al.,2000)In briefly, 5 g of faces was collected from the broilers and then poured into 95 ml sterilized saline water and kept shaking for 15 min. The mixture was

40 diluted to 10-7, 10-8 and 10-9 folds and cultured at 37°C for 48 h. These plates with colonies ranging from 30 to 300 were chose to analyze the microflora.

Serum cholesterol:

Serum cholesterol was estimated colorimetrically using Spinereact kits. Principle of the suggested method based on the following reactions:

Principle:

Chol. Esterase Cholesterol esters + H2O ------► Cholesterol + Fatty acids.

Chol.oxidase Cholesterol + O2------► 4-Cholestenone + H2O2

peroxidase 2H2O2 + phenol + 4-Aminophenazone ------► Quinonimine + 4H2O Reagents:

a. standard 200mg/dl (5.17mmol/L)

b. Buffer (R1):

- Pipes 90 mmol /L

- Phenol 26 mmol /L

c. Enzymes (R2):

41 - Cholesterol esterase 300 U/L

- Cholesterol oxidase 300 U/L

- Peroxidase 1250 U/L

- 4-Aminophenazone 0.4 mmol /L

Procedure:

1. Assay condition:

- Wave length : 505nm (500-550)

- Cuvette :1 cm light path

- Temperature 37 °C/15-25 °C

2. Adjust the instrument to Zero with distilled water.

3.Pipette into cuvette:

Blank Standard Sample WR (ml) 1.0 1.0 1.0 Standard -- 1.0 -- Sample -- -- 10

4. mix and incubate for 5 min. at 37 °C or 10 min. at room temperature.

5. Read and absorbance (A) of the samples and standard, against the Blank. The colour is stable for at least 60 minutes.

42 Calculation:

Calculation of serum cholesterol level was carried out as follows:

A Sample Cholesterol concentration (mg/dL) = ------x 200 A Standard

Conversion factor: mg/dl x 0.0258= mg/dl cholesterol in the sample.

HDL cholesterol:

Phosphotungstic acid and magnesium ions selectively precipitating all lipoprotein except the HDL fraction-cholesterol 10 ml of the supernatant was analyzed as that of total cholesterol using the same reagents.

Calculation of HDL cholesterol level was carried out as follows:

HDL cholesterol conc. (mg/dl) = ASample x 570

LDL Cholesterol:

To calculate LDL cholesterol in mg/dl was calculated using the following formula:

Triglyceride LDL cholesterol (mgl/dl)= Total cholesterol – (------+ HDL cholesterol) 5

43 Serum triglyceride:

Fully enzymatic determination of triglycerides in serum was estimated using Spinereact kits.

Assay principle:

Triglycerides Lipase Glycerol + fatty acids

Glycerol + ATP Glycerokinase Glycerol-3-phosphate +ADP

Glycerol-3-phosphat +O GPO Dihydroxyacetone phosphate

+ H2O2

2H2O2 + 4-Chlorophenol + 4-Aminoantipyrine Peroxidase

Quinoneimine + 4H2O

Reagents:

1.Standard triglyceride (ST) 200 mg /dl (2.29 mmol /L) 2.Reagent (R) :

-Pipes Buffer pH 7.0 50 mmol /L

- 4Chlorophenol 6.0 mmol /L

- Magnesium aspartate > 0.5 mmol /L

- Lipase >10 K U/L

- Peroxidase > 2.0 K U/L

- 4-Aminoantipyrin 1.0 mmol /L

44 - Glycerol kinase >750 U/L

- Glycerol-3-phosphate oxidase >3.5 K U/L

- ATP 1.0 mmol /L

- Sodium Azide 8.0 mml/l

Procedure:

Wave length : 546nm (500-550)

Zero adjustment : reagent blank.

Temperature :15-25 °C or 37°C

Incubation time : 10 min. at 15 -25 °C or 5 min. at 37 °C

Blank Standard sample Reagent 1.0 ml 1.0 ml 1.0 ml Standard -- 10 µl -- sample -- -- 10 µl

The solution was incubated for 10 min. at 37°C. The absorbance's of the sample (A sample) and the standard (A standard) were measured against blank, at 546nm (500-550) by Jan Way spectrophotometer.

Calculation:

A Sample Serum triglyceride conc. (mg/dL) = ------x 200 A Standard

45 Glutamic-oxaloacetic transaminase (GOT):

Assay principle:

The reaction involved in the assay system is as follows:

GOT L-Aspartate + 2-oxoglutarat ------► L-glutamate + oxaloacetate

GOT activity is measured by monitoring the concentration of oxaloacetate hydrazone formed with 2,4- dinitrophenylhydrazine.

Reagents:

Reagent 1 (R1):

- Phosphate buffer

- L-Aspartate

- Sodium Hydroxide

- Sodium Azide

Reagent 2 (R2):

- 2,4-dinitroohenyl-hydrazine

- HCl

46 Procedure:

1. Measurement against Reagent Blank: - Pipette into test tubes:

Reagent blank sample R1 (buffer) 0.5 ml 0.5 ml Sample --- 100 µl Distilled water 100 µl --- Mix and incubate for exactly 30 min. at 37 °C R2 0.5 ml 0.5 ml Mix and incubate for exactly 20 min. at 20-25 °C Sodium hydroxide 0.5 ml 0.5 ml Mix, measure absorbance of specimen against reagent blank at 546 nm after 5 minutes.

2. Measurement against sample plank:

Sample blank Sample

R1( buffer) 0.5 ml 0.5 ml

Sample --- 100 µl

Mix and incubate for exactly 30 min. at 37 °C

R2 0.5 ml 0.5 ml

Sample 100 µl

Mix and incubate for exactly 20 min. at 20-25 °C

Sodium hydroxide 0.5 ml 0.5 ml

Mix, measure absorbance of specimen against sample blank at 546 nm after 5 minutes.

47 Calculation:

Obtain the Got activity from the following table:

Absorbance U/L Absorbance U/L 0.020 7 0.100 36 0.030 10 0.110 41 0.040 13 0.120 47 0.050 16 0.130 52 0.060 19 0.140 59 0.070 23 0.150 67 0.080 27 0.160 76 0.090 31 0.170 89

Glutamic-pyrovic transaminase (GPT): The reaction involved in the assay system is as follows:

GPT L-alanine +2-oxoglutarat ------► L-glutamate + pyruvate

GPT activity is measured by monitoring the concentration of pyruvate hydrazone formed with 2,4-dinitrophenylhydrazine.

Reagents:

Reagent 1 (R1):

- Phosphate buffer 100 mmol/l

- DL- Alanine 200 mmol/L

- 2-oxoglutarat 6 mmol/L

- Sodium Azide 12 mmol/L

Reagent 2 (R2):

48 - 2,4-dinitroohenyl-hydrazine 2.0 mmol/L

- HCl

Procedure: 1. Measurement against Reagent Blank: Pipette into test tubes:

Reagent blank sample R1 (buffer) 0.5 ml 0.5 ml Sample --- 100 µl Distilled water 100 µl --- Mix and incubate for exactly 30 min. at 37 °C R2 0.5 ml 0.5 ml Mix and incubate for exactly 20 min. at 20-25 °C Sodium hydroxide 0.5 ml 0.5 ml

Mix, measure absorbance of specimen against reagent blank at 546 nm after 5 minutes.

1. Measurement against sample plank:

Sample blank Sample R1( buffer) 0.5 ml 0.5 ml Sample --- 100 µl Mix and incubate for exactly 30 min. at 37 °C

R2 0.5 ml 0.5 ml Sample 100 µl --- Mix and incubate for exactly 20 min. at 20-25 °C

Sodium hydroxide 0.5 ml 0.5 ml Mix, measure absorbance of specimen against sample blank at 546 nm after 5 minutes.

49 Calculation:

Obtain the Got activity from the following table:

Absorbance U/L Absorbance U/L 0.025 4 0.225 39 0.050 8 0.250 43 0.075 12 0.275 48 0.100 17 0.300 52 0.125 21 0.325 57 0.150 25 0.350 62 0.175 29 0.375 67 0.200 34 0.400 72

Determination of thyroid hormons (triiodothyronin T3 and tetriodothyronin T4): Radioimmunoassay of thyroid hormones:

Principles of assay:

The radioimmunoassay methods depends on the competition for a limited number of binding sites on a specific antibody between the hormone is serum and I125 labeled hormone. The proportion of I125 labeled hormone bound to antibody is inversely related to the concentration of the hormone present in serum.

The antibody bound hormone is then reacts with secondary antibody reagent By measuring the proportion of I125 labeled hormone bound in the presence of a reference standard solution, containing known amount of the hormone, the concentration of the hormone present in the unknown samples can be interpolated (Talwar, 1983).

50 Procedure of the assay:

- Pipette 50 ml of the standard (0.6, 12, 24, mg/dl ) and unknown samples into the appropriate tubes.

- Pipette 100 ml of I125 labeled hormone (tracer) in each tube.

- Pipette 200 ml of anti T3 or T4 solution in each tube.

- Vortex and incubate at 37°C for 2 hours.

- Dispense 1 ml of Polyethylene glycol (B.E.G) into each tube and vortex.

- Centrifuge all tubes for 20 min. at 3000 r.p.m.

- Decant the supernatant and keep the tubes inverted on absorbent tissue.

- Count all the tubes in gamma counter.

- Calculation of the results:

- Calculate the mean count rate for the zero standard (B0).

- Divide the count for each standard (B) by the count of zero standard and multiply by 100 B/B0 x100 = % B/B0

- Plot the % B/B0 values for each standard against T3 or T4and draw the best straight line through the duplicate points.

- Divide the count for each unknown (B) by the zero standard count (B0) and multiply % (%B/B0).

- Read of the T3 or T4 concentration from the standard curve.

51 Meat cholesterol and triglyceride:

Meat Sample Preparation:

Total lipid in meat samples was extracted following the method described by Elkin and Rogler (1990). In briefly, about 1 g of meat samples was homogenized with 12 ml of chloroform-methanol 2:1 (by volume) and filtered directly into a 50-mL volumetric flask using a glass microfiber filter. Following rehomogenization and refiltration, the meat filtrates were diluted to a final volume of 50 ml with chloroform- methanol 2:1 (by volume). In addition, to increase the concentration of lipid extract of the meat samples, the chloroform-methanol was removed by rotary evaporator (Virtis, Gardiner, NY) following centrifugation (1,000×g for 10 min) and filtration, and finally the dried extract was dissolved in 5 ml of chloroform-methanol 2:1 (by volume). The lipid extract samples were stored at−80°C until analyzed by the same kit used in serum cholesterol and triglycerides.

Antibody estimation:

Preparation of antigen:

Sheep red blood cells were used as antigen. This was obtained from sheep at Enshas farm. 2cc of blood was taken, added to anticoagulant (sodium-citrate). The sheep RBCs were washed carefully with saline solution, centrifuged at 600 rpm for 10 min. The supernatant was thrown and the process was repeated

52 three times until the supernatant become very clear. The precipitated blood was taken (0.3 cc blood) completed with saline to the concentration of 3%.

Route of administration:

Each chick was injected subcutaneously 0.5 cc, 3% sheep RBCs at day 14 of the experiment. After 21 days, the process of injection was repeated again with 0.5 cc, 3% sheep RBCs.

Principle of Antigen-antibody reaction by agglutination method:

The agglutination reaction is a method to evaluate antigen- antibody interaction where there is a specific antibody to an antigen present on surface of particles. The reaction takes place as a result of particles combination together and producing a visible clumping. The particle should have a size greater than 200-250nm to produce a visible reaction.

The reaction is sensitive enough for demonstration of as little as 10-5 to 10-8 gram of antibody (Bark et al., 1986).

Procedure: (Prowse and Jenkin, 1978).

1- For each specimen these tubes were prepared:

a- Control tube containing 0.1 ml sheep RBCs +0.1 ml saline.

b- Eight widal test tubes; a serial dilution of 0.1 ml serum, was prepared with concentration of 1/40, 1/80, 1/160, 1/320, 1/460, 1/1280, 1/1560, 1/5120.

53 2- To each tube, 0.1 ml of 3% sheep RBCs was added.

3- The tubes were mixed well and left at room temperature for 24 hr.

4- The agglutination was searched out; it appeared as a flufly shadow in the tubes.

5- The next tube without agglutination was taken as the antibody titer with special attention to the zone phenomena.

Antibody response to sheep RBCs was assayed by tube agglutination method described by Dacie and Lewis (1984).

For each chick, the humoral response was recorded as Log2 of the highest dilution that caused positive agglutination.

Lipid extraction and determination of fatty acids methyl esters by GLC:

The lipid content of dried ground chicken samples was extracted with a mixture of chloroform: methanol (2:1v/v) according to Kates (1972). The free fatty acids were methylating by methylating agent (Farag et al., 1992). The fatty acid methyl esters were extracted by ether and dried over anhydrous sodium sulphate. Fatty acids methyl esters were dissolved in chloroform (HPLC grade) and chromatographic separation was performed using hp 6890 gas chromatograph instrument equipped with a flame ionization detector using inowax-cross linked polyethylene glycol fused silica column (30

54 m long, 0.32 mm i.d. 0.5µm film thickness). Oven temperature was programmed from 150ºC for 1 min then elevated to 235ºC with a rate of 17ºC/ min then was raised again to 245ºC with a rate of 1ºC /min and hold at 245ºC for 5 min. gasses flow rates for N2 as a carrier gas, H2 and air were 1.3, 40 and 400 ml /min, respectively. Flam ionization detector and injection temperatures were 275ºC and 260ºC, respectively.

Amino acids determination:

Hydrolysis procedure:

A known amount of the defatted sample (50 mg) was hydrolyzed with HCL (5 ml, 6N) and heated in sealed test tube at 110ºC for 24h according to Suzanna (1998). The contents of each tube were filtered, evaporated until dryness and a suitable volume of sodium citrate buffer (pH 2.2) was added to dissolve the hydrolyzed sample followed by ultra filtration using a 0.2 µm membrane filter (Nellet, 1996).

Apparatus and analytical conditions:

All analyses were performed using high performance amino acids analyzer (Biochrom 20, Auto sampler version; Pharmacia Biotech) constructed at NCRRT. The amino acid analyzer was equipped with a stainless steel column (200 x 4.6 mm) packed with altropac (8µm) cation exchange resin. Elution with the sodium citrate buffers (pH 3.20, 4.25 and 6.45) resolved 17 amino acids. The flow rate was 25 ml/h for ninhydrin reagent

55 and 35 ml/h for the buffers. The reaction between the amino acids and ninhydrin was occurred at 135ºC in a 10 m PTFE reaction coil (0.3mm I. D).

Detection was performed at two wave lengths (570 and 440 nm). The data of each chromatogram was analyzed by EZ chromeTM chromatography data system tutorial and user guide- version 6.7.

Statistical analysis:

Data were statistically analyzed using general linear models procedure of SAS (1996). The statistical model used was:

Xjjk = µ + άj + βj + (άβ) ij + eIjk th Xjjk = the N observation µ = overall mean th άj = effect of the I treatments th βj = effect of the J levels (άβ)ij= the interaction between treatment and levels

eIjk = the experimental error Duncan's new multiple range tests was used for comparison between means Duncan(1955).

56 4. RESULTS AND DISCUSSION

4.1. Traits of growth performance:

4.1.1. Body weight:

The results of addition of probiotic or antibiotic with fresh oil (FO), mixed oil(MO) or residual oil (RO) on body weight of broilers presented in (Table 2 and figure 1).

The obtained results showed that significant (P<0.05) increase in body weights in the chicks treated with probiotic (LAB) at the grower and finisher periods when compared to the chicks treated with antibiotic at the same periods, while no significant (P>0.05) difference was detected in average body weights at the starter period. The Obtained results could be considered to be scientifically logic. Since the beast effect observed due to supplementation probiotic to broiler may be attributed to increase the number of lactic acid bacteria, which have several beneficial effects on growth performance of the chicks, especially in terms of improving digestion and mineral absorption . In addition, the primary role of a diet is to provide enough nutrients to fulfill metabolic requirements of the body and also to modulate different functions of the body. Probiotics are beneficial microorganisms that can be suitably used to improve growth performance and health of broiler chickens. The effect of addition LAB on broiler body weight, which was observed in the grower and finisher periods, may be attributed to

57 the fact that the biological action of probiotic needs sufficient time for improving metabolic performance and establishment of intestinal populations of both beneficial and pathogenic organisms that takes relatively long time to realize best anabolic effect.

These results agreed with those reported by many investigators (Khaksefidi and Ghoorchi, 2006; Chen et al., 2009; Falaki et al., 2010 and Shabani et al., 2012).To the contrary several authors disagree with before mentioned results (Namra et al., 2005; O'dea et al., 2006 and Willis and Reid, 2008).

Also, significant variation (P<0.05) was found in average body weight due to addition of different levels of RO to broiler's diets at the grower (2-4 wk) and finisher (4-6 wk) periods of chicks age, but no significant difference was observed at the starter period (0-2 wk) of age. There were significant (P<0.05) decreases in average body weights in the chicks fed diet containing of MO or RO compared to the chicks fed diet containing of FO. The higher mean values of average body weights in the grower and finisher periods were detected for chicks fed diet containing of FO (1484.33 gm) and (2372.50 gm) respectively, however the minimum mean values of average body weights were detected for chicks fed diet containing RO (1445.83 gm) and (2173.33) respectively. RO caused significant

58 (P<0.05) reduction in body weight at grower and finisher periods(table 2). These results were in agreement with those were noticed by (Anjum et al., 2004; Szarek et al., 2006; Dibner et al., 1996; Jankowski et al., 2000 and Keller et al., 2004).

Also Karamouz et al (2009) reported that short and long- term feeding of diet containing oxidized or heated oils decreased the final body weight of turkeys.

The interactions effect between treatments of probiotic or antibiotic and addition of different levels of RO showed significant (P<0.05) differences in average body weights as showed in (table 2 and figure 1). In the starter period no significant differences were observed in average body weights between all groups except the chicks fed probiotic with MO and chicks fed antibiotic with RO.

In the grower period there was significant increase in average body weight in the chicks fed probiotic with FO or MO compared to the other groups and chicks fed antibiotic with FO. The highest mean value was originated in the chicks fed probiotic with FO (1487.33 gm) then the chicks fed probiotic with MO (1481.33 gm) and chicks fed antibiotic with FO (1481.33). However the minimum mean value was resulted in the chicks fed antibiotic with RO (1421 gm).

In the finisher period; the chicks fed probiotic with different

59 levels of RO had a significant (P<0.05) increase in average body weights when compared to the chicks fed antibiotic with the same levels of RO, while no significant difference was found between the chicks fed probiotic with FO and the chicks fed probiotic with MO, but there was significant decrease (P<0.05) in body weight in the chicks fed probiotic with RO compared to the chicks fed probiotic with FO or MO.

Table (2): Effect of addition of probiotic or antibiotic with different levels of RO on average body weight in broiler chicks.

Independent variable and Body weight interactions Hatching starter grower Finisher Treatments Antibiotic(T1) 42.67 b 478.89 a 1449.89 b 2131.33 b (T) Probiotic(T2) 46 a 486.67 a 1479.77 a 2379.00 a

Levels of oils L1=FO 46 a 482.67 a 1484.33 a 2372.50 a (L) L2=MO 46 a 486.50 a 1464.33 b 2201.67 b L3=RO 41 b 479.17 a 1445.83 c 2173.33 c

T1xL1 46 a 482.33 ab 1481.33 a 2178.00 c T1xL2 46 a 483.67 ab 1447.33 c 2111.33 d Interactions T1xL3 36 b 470.67 b 1421.00 d 2104.67 d (T x L) T2xL1 46 a 483.00 ab 1487.33 a 2303.00 a T2xL2 46 a 489.33 a 1481.33 a 2292.00 a T2xL3 46 a 487.67 ab 1470.67 b 2242.00 b

A, b, c means in columns with different superscripts were significantly different (P<0.05).

60 Body weight in starter, grower and finisher periods

2500

2000

Antibiotic in starter 1500 Probitic in starter Antibiotic in grower Probiotic in grower 1000 Antibiotic in finisher

Body weight weight Body Probiotic in finisher

500

0 FO MO RO Levels of oil

Figure(1): Effect of addition of probiotic or antibiotic with different levels of RO on average body weight at the starter, grower and finisher periods of broiler chicks.

4.1.2. Body weight gain:

The results of addition of probiotic or antibiotic with FO, MO or RO on body weight gain of broilers presented in (table3, figure 2 and 3).

The obtained results showed significant variation (P<0.05) in average body weight gain between groups treated with probiotic and groups treated with antibiotic at the grower, finisher and the whole period (0-6 wk) of chicks age, but no significant(P>0.05) difference was observed at the starter period of chicks age.

There was significant (P<0.05) increase in average body weight gain in the chicks fed probiotic in the grower, finisher

61 and whole period of chicks age (993.11 g), (798.33 g) and (2232.11 g) compared to the chicks fed antibiotic (967.67 g), (681.44 g) and (2086.33 g) respectively.

These results are in harmony with those reported by many investigators. (Lan et al., 2003; Khaksefidi and Ghoorchi, 2006; Torres et al., 2007; Fahmy, 2009; Falaki et al., 2010 and Shabani et al., 2012). But Namra et al., 2005; O'deaet al., 2006 and Willis and reid, 2008) disagreed with before mentioned results as all reported that there was no significant differences in body weight gain of chickens fed diets containing L. acidophilus and S. faecium from 8 to 60 days of age.

Results table. 3 and figure 2 and 3 showed significant variation (P<0.05) in average body weight gain due to addition of different levels of RO to broiler's diets at the grower, finisher and whole period of chicks age, but no significant (P>0.05) difference at the starter period of chicks age.

Also there was significant (P<0.05) increase in average body weight gain in the chicks fed diet containing FO in the grower, finisher and whole period of chicks age (1001.67 g), (754.83 g) and (2193.17 g) compared to the chicks fed diet containing MO (977.83 g), (737.33 g) and (2155.67 g) or the chicks fed diet containing RO (961.67 g), (727.50 g) and (2128.83 g) respectively. Most authors observed reduced weight gain with RO, Bartov and Bornstein (1972) observed reduced weight

62 gain when oxidized oil or fat were added in the rations for broiler and guinea pigs. These results agreed with (Eder, 1999 and Anjum et al., 2004)

On contrary Bayraktar et al (2011) showed that oxidised dietary oil did not affect body weight gain of broilers during the four to six week experimental periods.

The interaction between probiotic or antibiotic and addition of different levels of RO on body weight gain followed the same trend as body weight in the starter period. Regarding the grower period the highest significant (P<0.05) increase was obtained in FO with probiotic and antibiotic, the probiotic in the grower period overcome the effect of MO and RO while the lowest body weight gain was obtained with antibiotic associated with RO then MO ones. In the finisher and whole period the beneficial effect of probiotic was evident in the three groups; all groups experienced significant (P<0.05) increase to their corresponding antibiotic ones. From the result concerning body weight and body weight gain it is evident that probiotic succeeded to a great extent in ameliorating the harmful effect of using RO at different concentrations. Therefore we can stated that addition of LAB in the water of broiler chicks improve quantitively the adverse effect of RO, but still lesser than FO and MO.

63 Table (3): Effect of addition of probiotic or antibiotic with different levels of RO on body weight gain in broiler chicks.

Independent variable and Body Weight gain interactions starter Grower finisher Whole period Treatments Antibiotic (T1) 436.22 a 967.67 b 681.44 b 2086.33 b (T) Probiotic (T2) 440.67 a 993.11 a 798.33 a 2232.11 a

L1=FO 436.67 a 1001.67 a 754.83 a 2193.17 a Levels of oil L2=MO 440.50 a 977.83 b 737.33 b 2155.67 b (L) L3=RO 438.17 a 961.67 c 727.50 b 2128.83 c

T1xL1 436.33 ab 999.00 ab 696.67 c 2132.00 c T1xL2 437.67 ab 963.67 d 664.00 d 2065.33 d

T1xL3 434.67 b 940.33 e 683.67 c 2061.67 d Interactions ( T x L) T2xL1 437.00 ab 1004.33 a 813.00 a 2245.33 a T2xL2 443.33 a 992.00 bc 810.67 a 2246.00 a T2xL3 441.67 ab 983.00 c 771.33 b 2196.00 b

A, b, c means in columns with different superscripts were significantly different (P<0.05).

64 Weight gain at starter and grower periods

1200

1000

800

Antibiotic in starter Probiotic in starter 600 Antibiotic in grower Probiotic in grower weight gain weight 400

200

0 FO MO RO Levels of oil

Figure(2): Effect of addition of probiotic or antibiotic with different levels of RO on average weight gain at the starter and grower periods of broiler chicks.

Weight gain at the finisher and whole periods

2500

2000

1500 Antibiotic in Finisher Probiotic in Finisher Antibiotic in whole Period 1000 Probiotic in whole period weight gain weight

500

0 FO MO RO Levels of oil

Figure(3): Effect of addition of probiotic or antibiotic with different levels of RO on average weight gain at the finisher and whole period of broiler chicks.

65 4.1.3. Feed intake:

Results dealing with the amount of feed consumed during the starter (0-2 wk), grower (2-4wk), finisher (4-6 wk) and whole period (0-6 wk) of age are listed in (Table 4, figure 4 and 5).

Results obtained showed a significant increase (P<0.05) in Feed intake in the chicks treated with probiotic as compared with chicks fed antibiotic at the grower, finisher and whole period, and insignificant (P>0.05) difference at the starter period. The results of current study are in line with that of (Zulkifli et al., 2000 and O‘dea et al., 2006).

On the other hand, there was significant (P<0.05) increase in feed intake in the chicks fed FO compared to the chicks fed MO and RO in grower period but no significant (P>0.05) effect was observed at starter and finisher periods. Also, no significant differences were observed on feed intake between the chicks fed MO and chicks fed RO in all periods of experiment.

These results were in harmony with those obtained by Hussien and Kratzer (1982); Award et al (1983); Anjum et al (2004) and Zadunczyk et al (2002).

On contrary Chae et al (2002) reported that chicks fed diet containing fresh oil or oxidized oil had no difference in feed intake. Similarly Bayraktar et al (2011) stated that oxidized dietary oil did not affect on feed intake during the four to six weeks experimental periods. Casado et al (2011) observed that

66 no effect in the feed intake through the growing period when peroxide or oxidized oil were included in the diet of growing rabbits.

In lucidity way, from (Table 4 and figure 4 and 5) showed no significant variation in Feed intake due to interaction at the starter period, but significant differences were observed in the grower, finisher and whole period of experiment.

In the grower period, there was significant(P<0.05) increase in feed consumed in the chicks fed probiotic with FO compared to the others groups, but no significant (P>0.05) differences were observed between the chicks fed probiotic with MO and chicks fed probiotic with RO also, no significant (P>0.05) differences were observed in the chicks fed antibiotic with different levels of RO.

In the finisher period , there was significant(P<0.05) increase in feed intake in the chicks fed probiotic with different levels of RO compared to the chicks fed antibiotic with the same levels of oils. But, no significant (P>0.05) differences were observed neither between probiotic treated groups nor between antibiotic treated groups.

In the whole period there was significant (P<0.05) increase in feed consumed in the chicks fed probiotic with FO compared to the other groups, while no significant (P>0.05) differences were observed between the chicks fed probiotic with MO and RO,

67 also no significant (P>0.05) effect was obtained between antibiotic treated group with different levels of oils. Maintaining normal intestinal microflora by competitive exclusion and antagonisms (Kabir et al., 2005 and Kizeweter-Swide and Binek, 2009), increasing digestive enzyme activity and decreasing bacterial enzyme activity and ammonia production and improving digestion (Awad et al., 2006) are part of the mode of action of LAB; this beneficial effect was reflected on improving feed intake in the interaction groups compared to the antibiotic ones.

68 Table (4):Effect of addition of probiotic or antibiotic with

different levels of RO on feed intake in broiler chicks.

Feed intake (g /bird) Independent variable and Starter Grower finisher Whole interactions period Treatments Antibiotic (T1) 463.23 a 989.20 b 2314.08 b 3766.51 b (T) Probiotic (T2) 464.94 a 1034.24 a 2395.93 a 3895.11 a

L1=FO 467.36 a 1032.16 a 2353.60 a 3853.12 a Levels of oil L2=MO 465.40 a 1002.71 b 2361.20 a 3829.31 ab (L) L3=RO 459.49 a 1000.29 b 2350.21 a 3809.99 b

T1xL1 465.60 a 1000.58 bc 2308.50 b 3774.68 c T1xL2 465.42 a 981.78 c 2325.22 b 3772.42 c

T1xL3 458.67 a 985.23 c 2308.52 b 3752.42 c Interactions (T x L) T2xL1 469.12 a 1063.73 a 2398.70 a 3931.55 a T2xL2 465.38 a 1023.63 b 2397.13 a 3886.20 b T2xL3 460.32 a 1015.35 b 2391.90 a 3867.57 b

A, b, c means in columns with different superscripts were significantly different (P<0.05).

69 Feed intake at the starter and grower periods

1200

1000

800

Antibiotic in starter Probiotic in starter 600 Antibiotic in grower Probiotic in grower Feed intake Feed 400

200

0 FO MO RO Levels of oil

Figure (4): Effect of addition of probiotic or antibiotic with different levels of RO on feed intake at the starter and grower periods in broiler chicks.

Feed intake at the finisher and whole period

4500

4000

3500

3000

Antibiotic in finisher 2500 Probiotic in finisher Antibiotic in whole period 2000 Probiotic in whole period Feed intake Feed 1500

1000

500

0 FO MO RO Levels of oil

Figure (5): Effect of addition of probiotic or antibiotic with different levels of RO on feed intake at the finisher and whole period in broiler chicks.

70 4.1.4. Feed conversion ratio (FCR):

Results of FCR listed in (table 5, figure 6 and 7). Results showed no significant difference (P<0.05) in FCR between the chicks treated with probiotic and chicks fed antibiotic at the starter period, while significant (P<0.05) differences was observed at the grower, finisher, and whole period of the experiment.

In each of the finisher and the whole period the mean value of FCR of chicks fed probiotic was (3.002) and (1.744) while the mean value of FCR of chicks fed antibiotic was (3.399) and (1.807), respectively. Statistical analysis showed significant (P<0.05) decrease of FCR in the chicks fed probiotic compared with the chicks fed antibiotic. It is obvious from these results that the amount of feed consumed was low whereas, the average body weight gain was increased and consequently the feed utilization was improved.

These results are in harmony with those reported by Torres et al (2007)showing that commercial lactobacillus-based probiotic significantly improved body weight, weight gain and feed conversion ratio of commercial turkey hen production. Abdalla et al (2008) reported that supplementation layer diets with Lactobacillus or Biogen significantly (P<0.01) affected body weight and FCR for the whole experimental period. Falaki et al (2010) observed that chicks fed diets supplemented with

71 probiotc have significantly higher FCR compared with control. Shabani et al (2012) showed that feeding chicken broiler with probiotics have positive effects of FCR.

The significant (P<0.05) affect on FCR was observed due to addition of different levels of RO to broiler's diets. In the starter period significant (P<0.05) decrease was only observed in the chicks fed RO compared to the chick fed FO.

In the finisher and whole period, significant(P<0.05) difference was observed on FCR between chicks fed diet containing FO and chicks fed MO or fed RO. The best value (the lowest value) of FCR was obtained in the chicks fed FO followed the chicks fed MO with significant (P<0.05) difference between them in the whole period. In the grower period the significant (P<0.05) change was observed between MO and RO.

These results are in harmony with those reported by Eschenbach and Hartfiel (1985); Cabel and Waldroup (1988) and Anjum et al (2004) noted that oxidized oil caused poorer FCR than fresh oil.

Eder (1999)concluded that body weight gains and FCR were only slightly lower in the rats fed the oxidized oil compared to the rats fed the fresh oil. Karamouz et al (2009) reported that short and long-term feeding of diet containing oxidized or heated oils decreased the FCR of turkeys. To the contrary Bayraktar et al (2011) reported that oxidized dietary oil did

72 not affect on body weight gain and feed intake during the four to six weeks experimental periods; Also there were no differences in FCR of the treatment groups.

Significant effect (P<0.05) was found due to interaction between treatment of probiotic or antibiotic and different levels of RO at all periods of experiment. In the finisher period, the best mean value of FCR was found in the chicks fed probiotic with diet containing FO (2.950) followed the chicks fed probiotic with diet containing MO (2.957) without significant difference between them.

In the whole period the best mean value of FCR was found in the chicks fed probiotic with diet containing MO (1.730) followed the chicks fed probiotic with diet containing FO (1.743) without significant (P>0.05) difference between them.

73 Table (5): Effect of addition of probiotic or antibiotic with different levels of RO on Feed conversion ratio in broiler chicks.

Independent variable and Feed conversion ratio interactions Starter Grower Finisher Hole priod Treatments Antibiotic (T1) 1.063 a 1.022 b 3.399 a 1.807 a (T) Probiotic (T2) 1.054 a 1.042 a 3.002 b 1.744 b

L1=FO 1.070 a 1.032 ab 3.133 b 1.757 c Levels of oil L2=MO 1.058 ab 1.024 b 3.230 a 1.778 b (L) L3=RO 1.048 b 1.040 a 3.238 a 1.792 a

T1xL1 1.067 ab 1.001 d 3.317 c 1.770 b T1xL2 1.067 ab 1.018 cd 3.377 a 1.823 a Interactions T1xL3 1.057 abc 1.047 ab 3.503 b 1.827 a ( T x L) T2xL1 1.073 a 1.063 a 2.950 e 1.743 cd T2xL2 1.050 bc 1.030 bc 2.957 e 1.730 d T2xL3 1.040 c 1.033 bc 3.100 d 1.760 bc

A, b, c means in columns with different superscripts were significantly different (P<0.05).

74 FCR at starter and grower periods

1.18

1.13

1.08

Antibiotic in starter Probotic in starter 1.03 Antibiotic in grower FCR Probiotic in grower

0.98

0.93

0.88 FO MO RO Levels of oil

Figure (6): Effect of addition of probiotic or antibiotic with different levels of RO on Feed conversion ratio (FCR) at the starter and grower periods in broiler chicks.

FCR at the finisher and whole period

4

3.5

3

2.5 Antibiotic in Finisher Probiotic in finisher 2 Antibiotic in whole period FCR Probiotic in whole period 1.5

1

0.5

0 FO MO RO Levels of oil

Figure (7): Effect of addition of probiotic or antibiotic with different levels of RO on Feed conversion ratio ( FCR) at the finisher and whole period in broiler chicks.

75 4.1.5. Mortality rate:

Mortality rate was estimated during the whole period as well as during the starter, grower and finisher periods. Obtained results are listed in (Table 6 and figure 8).

The obtained results showed no significant variation (P<0.05) in rate of mortality due to treatments of probiotic and antibiotic or due to addition of different levels of RO to broilers diet or interaction between them at the starter, grower and finisher, while significant variation in mortality rate was observed in the whole period. The differences of morality rate between treatments of probiotic and antibiotic was significant (P<0.05), the rate of mortality was (11.11 %) in the group treated with antibiotic while this rate significantly decrease in the group treated with probiotic (4.44 %) in the whole period.

LAB influence the immune system, it has been shown that probiotic especially lactobacilli, could modulate the systemic antibody response to antigens in the chicken (Kabir et al., 2004; Mathivanan and Kalaiarasi, 2007 and Apata, 2008) this enhancing effect of immune system most probably cause low mortality rate in probiotic group and in the interaction probotic groups compared to antibiotic especially with MO and RO.

These results are in harmony with those reported by Eglal (2006) where indicate that the mortality rate decreased in chicks fed Primalac, as a probiotic based on lactobacillus culture

76 compared with control groups. Also Timmerman et al (2006) reported that the supplementation of a diet with multispecies probiotic (consisted of 7 Lactobacillus species isolated from the digestive tract of chickens) reduced mortality rate compared with control. While disagreed with Zulkifli et al (2000) who stated that broiler chicks fed on diets containing Lactobacillus culture (1g /kg diet) had insignificant affect on mortality rate at 3 and 6 weeks of age. O’Dea et al (2006) indicated that there were no significant differences in broilers mortality rate between the probiotic treatments and the control group.

Also, significant (P<0.05) variation was observed in mortality rate due to addition of different levels of RO to broilers diet. The lowest ratio of mortality rate was observed in the group fed diet containing FO (3.33 % ) followed the group fed diet containing MO (8.33 %) then the group fed diet containing RO (11.67 %) in the whole period.

The interaction effect between treatment of probiotic or antibiotic and addition of different levels of RO showed significant differences in mortality rate in the whole period. The lowest ratio of mortality rate was observed in the group fed pribiotic with diet containing FO, the group fed probiotic with diet containing MO and the group fed antibiotic with diet containing FO (3.33 %) without significant difference between them.

77 Table: (6). Effect of addition of probiotic or antibiotic with different levels of RO on rate of mortality in broiler chicks.

Independent variable and Mortality Rate (%) interactions Starter Grower Finisher Whole period Treatments Antibiotic (T1) 6.76 a 2.22 a 2.11 a 11.11 a (T) Probiotic (T2) 3.33 a 0 a 1.11 a 4.44 b

L1=FO 3.33 a 0 a 0 a 3.33 b Levels of oil L2=MO 5.00 a 1.76 a 1.76 a 8.33 ab (L) L3=RO 6.76 a 1.76 a 3.33 a 11.76 a

A T1xL1 3.44 a 0 a 0 a 3.33 b Interactions T1xL2 6.67 a 3.33 a 3.33 a 13.33 ab ( T x L) T1xL3 10.00 a 3.33 a 3.33 a 16.76 a

T2xL1 3.33 a 0 a 0 a 3.33 b T2xL2 3.33 a 0 a 0 a 3.33 b T2xL3 3.33 a 0 a 3.33 a 6.76 ab

A, b, c means in columns with different superscripts weresignificantly different (P<0.05).

78 Mortality rate

18

16

14

12

10 Antibiotic Probiotic 8

Mortality rate 6

4

2

0 FO MO RO Levels of oil

Figure (8): Effect of addition of probiotic or antibiotic with different levels of RO on Mortality rate of broiler in the whole period of rearing.

4.1.6. Performance index (%):

Data concerning the performance index for birds of different experimental groups calculated during the starter, grower, finisher and the whole period are tabulated in (Table 7, figure 9 and 10).

Obtained results showed that treatments of antibiotic and probiotic had no significant (P<0.05) effect on the value of performance index in the starter and grower periods, But significant (P<0.05) differences were observed at the finisher and whole period. The highest value of performance index was observed on groups of birds treated with probiotic during the finisher period of age (75.73%) and during the whole rearing

79 period (130.65%).These results are in agreement with those obtained by Awad et al(2009).

On the other hand, levels of RO which added to diet showed significant (P<0.05) effect on performance index in the grower, finisher and whole rearing period. The higher value of the performance index was found during the grower period in birds fed diet containing of FO (144.18 %) followed birds fed diet containing of MO (142.85 %) without significant difference between them. Also, The higher value of the performance index was found during the finisher and whole period in birds fed diet containing of FO (71.85% %) and (127.46) followed birds fed diet containing of MO (68.90 %) and (124.03) respectively with significant difference between them.

Significant (P<0.05) variation was also detected due to the interaction effect between treatments of probiotic or antibiotic and addition of different levels of burned oil during the grower, finisher and whole rearing period. In the grower period The highest value of performance index was observed on group of birds fed antibiotic with diet containing of FO (147.93 %) followed group of birds fed probiotic with diet containing of MO (143.57 %) without significant difference between them. In the finisher period, the highest values of performance index was observed on group of birds fed probiotic with diet containing FO (77.97 %) followed group of birds fed probiotic with diet

80 containing of MO (77.51 %) without significant difference between them, while the lowest values of performance index was observed on group of birds fed antibiotic with diet containing of RO and MO. In the whole period, the highest values of performance index was observed on group of birds fed probiotic with diet containing MO (132.46 %) followed group of birds fed probiotic with diet containing FO (131.90 %) without significant difference between them.

Table: (7). Effect of addition of probiotic or antibiotic with different levels of RO on performance index % in broiler chicks.

Independent variable and Performance index (%) interactions Starter Grower Finisher Whole period Treatments Antibiotic (T1) 45.42 a 141.89 a 62.78 b 118.03 b (T) Probiotic (T2) 46.24 a 142.13 a 75.93 a 130.65 a

L1=FO 45.11 a 144.18 a 71.85 a 127.46 a Levels of oil L2=MO 46.04 a 142.85 a 68.90 b 124.03 b (L) L3=RO 46.18 a 139.00 b 67.32 c 121.39 c

T1xL1 45.21 a 147.93 a 65.73 c 123.02 c T1xL2 45.47 a 142.13 b 60.29 e 115.60 d

T1xL3 45.47 a 135.60 c 62.33 d 115.47 d Interactions A ( T x L) T2xL1 45.01 a 140.43 bc 77.97 a 131.90 a T2xL2 46.62 a 143.57 ab 77.51 a 132.46 a T2xL3 46.80 a 142.40 b 72.30 b 127.31 b

A, b, c means in columns with different superscripts were significantly different (P<0.05).

81 Performance index % at the starter and grower periods

160

140

120

100 Antibiotic in starter Probiotic in starter 80 Antibiotic in grower Probiotic in grower 60

40 Performance index % Performance

20

0 FO MO RO Levels of oil

Figure (9): Effect of addition of probiotic or antibiotic with different levels of RO on performance index % at the starter and grower periods in broiler chicks.

Performance index % at the finisher and whole period

140

120

100

80 Antibiotic in Finisher Probiotic in finisher Antibiotic in whole period 60 Probiotic in whole period

40 Performance index % Performance

20

0 FO MO RO Levels of oil

Figure (10): Effect of addition of probiotic or antibiotic with different levels of RO on performance index % at the finisher and whole period in broiler chicks.

82 4.1.7. Meat yield% and internal organs%:

The meat yield characteristics and organ traits of different groups are presented in (Table 8, figure 11 and 12). it was evident that the meat yield characteristics corresponding to the different treatments did not differ significantly (P>0.05) due to treatment of probiotic & antibiotic, addition of different levels of RO to broiler's diet or interaction between. The results found in the study are in agreement with the earlier reports of Baidya et al (1994) and Mandal et al (1994) who observed that probiotics feeding did not have any influence on the carcass yield.

The present findings also revealed that there were significant (P<0.05) differences in the weight of liver, gizzard and heart among the different groups due to treatment of probiotic and antibiotic but no significant (P>0.05) difference in spleen weight was found. The highest weights of liver, gizzard and heart obtained in the chicks fed probiotic (2.37), (2.65) and (0.30) compared with the chicks fed antibiotic (2.17), (2.42) and (0.28) respectively.

Levels of RO which added to broiler's diet showed significant decreased in liver weight and heart in the chicks fed MO and RO compared to the chicks fed FO, but no significant effect in gizzard and spleen weights.

83 The liver and gizzard are the two organs showing significant (P<0.05) effect In the interaction. The highest significant value in the liver was obtained in FO and RO probiotic group and the least significant level was experienced in the RO antibiotic group. As regard gizzard no significant (P>0.05) changes between the three groups of probiotic and FO and MO antibiotic while the RO antibiotic group was statistically decrease compared to all groups.

The present work agreed to some extent with that of (Hussein and Kartzer, 1982 and Anjum et al., 2004) as they found that oxidized oil had no significant effect on meat yield, weigh of heart ad gizzard. However the present study disagreed with Anjum et al., 2004 who observed higher weight of live when chicks fed on oxidized oil compare to control.

The increase weight of some internal organs using probiotics levels of oil or/and interaction did not reflect any harmful effect from these treatments; the increases weight in these organs especial liver must probably is due to the positive effect of probiotics on live body weight.

84 Table: (8). Effect of addition of probiotic or antibiotic with different levels of RO on Meat yield% and internal organs% in broiler chicks.

Independent variable and Meat yield% Internal organs% interactions Thigh Breast Liver Gizzard Heart Spleen Treatments Antibiotic (T1) 31.93 a 38.54 a 2.17 b 2.42 b 0.28 b 0.123 a (T) Probiotic (T2) 31.34 a 39.01a 2.37 a 2.65 a 0.30 a 0.081 a

Levels of oil L1= FO 31.00 a 38.98 a 2.37 a 2.57 a 0.29 ab 0.066 a (L) L2= MO 31.70 a 38.83 a 2.23 b 2.58 a 0.28 b 0.168 a L3=RO 32.21 a 38.51 a 2.22 b 2.46 a 0.29 a 0.073 a

T1xL1 30.67 a 39.77 a 2.28 bc 2.44 ab 0.29 a 0.052 a T1xL2 32.33 a 38.50 a 2.20 c 2.60 a 0.28 a 0.252 a Interactions T1xL3 32.80 a 37.35 a 2.04 d 2.23 b 0.28 a 0.065 a ( T x L) T2xL1 31.33 a 38.20 a 2.46 a 2.70 a 0.29 a 0.079 a T2xL2 31.07 a 39.17 a 2.26 bc 2.56 a 0.28 a 0.084 a T2xL3 31.63 a 39.67 a 2.40 ab 2.70 a 0.30 a 0.081 a

A, b, c means in columns with different superscripts weresignificantly different (P<0.05).

85 Thigh and breast weight %

45

40

35

30

Thigh weight% with antibiotic 25 Tight weight% with probiotic Breast weight with antibiotic 20 Breast weight with probiotic

15

10 Theigh and breast weight% and Theigh

5

0 FO MO RO Levels of oil

Figure (11): Effect of addition of probiotic or antibiotic with different levels of RO on thigh and breast weight% in broiler chicks.

Liver, gizzard, heart and spleen weights %

3

2.5

2 Liver weight% with antibiotic Liver weight% with probiotic Gizzard weight with antibiotic Gizzard weight with probiotic 1.5 Heart weight% with antibiotic Heart weight with probiotic

Weights % Weights Spleen weight% with antibiotic 1 Spleen weight% with probiotic

0.5

0 FO MO RO Levels of oil

Figure (12): Effect of addition of probiotic or antibiotic with different levels of RO on liver, gizzard, heart and spleen weight% in broiler chicks.

86 4.2. Blood parameter:

4.2.1. Serum cholesterol, triglyceride, HDL, LDL, GPT and GOT:

Serum cholesterol, triglyceride, HDL, LDL, GPT and GOT were estimated at 42nd days of age. Obtained results are listed in (table 9, figure 13 and 14). It was found that the level of serum cholesterol and triglycerides had decreased significantly (P<0.05) in the chicks which fed probiotic (121.67) and (132.86) compared to the chicks which fed antibiotic (164.69) and (160.81) respectively.

the significant variation (P<0.05) was also observed in serum cholesterol and triglycerides due to addition of different levels of RO. the lowest value of serum cholesterol and triglycerides was observed in the chicks fed diet containing FO( 127.05) and (137.98) followed by the group fed diet containing MO (143.51) and (148.16) then the chicks fed diet containing RO (158.99) and (154.36) respectively.

The interaction effect between treatment of probiotic or antibiotic with addition of different levels of RO showed significant (P<0.05) difference in serum cholesterol and triglycerides between all groups; The lowest value of serum cholesterol and triglycerides was observed in chicks fed probiotic with FO (109.94) and (127.01), However the higher value of serum cholesterol and triglycerides was found in the

87 chicks fed antibiotic with RO (185.11) and (169.96) respectively.

Factors affecting effectiveness of probiotic depends on stress condition of broilers (Jin et al., 1998 and lyons, 1987), capability of microorganisms to attach to the intestinal wall, their antagonism towards pathogenic bacteria and their ability to competitively exclude some pathogenic bacteria (Jin et al., 1996b). Santoso et al (1995) reported reduction of blood triglyceride levels after probiotic supplementation similar to the observation made in this study. It seems that probiotics lead to a reduction in acetyl Co-A carboxylase (limited enzyme in fatty acids synthesis) in liver and tissue. Lipogenesis reduction in liver can lead to reduction in HDL and triglycerides (Santoso et al., 1995 and Khovidhunkit et al.,2004).

The effect of probiotic in improving lipid profile is in agreement with many authors; Mohan et al (1995) reported that probiotic supplementation resulted in lowering of serum cholesterol level in white leghorn layers, also Mohan et al (1996) mentioned that chickens received probiotic had lower serum cholesterol content. Similar results were reported by Arun et al(2006) who found that serum total cholesterol and triglycerides were reduced significantly by dietary supplementation with probiotic containing L. sporogena.

88 The obtained results showed no significant effect (P>0.05) in HDL either due to treatments of probiotic or due to addition of different levels of RO. similarly the interaction between treatment of probiotic or antibiotic and addition of different levels of RO showed insignificant difference (P<0.05)in HDL.

The value of LDL showed significant difference due to treatment of probiotic and different levels of RO. The lowest value of LDL was observed in the chicks fed probiotic (31.97). The low another value of LDL was observed in the chicks fed diet containing FO (37.71) followed by the chicks fed diet containing of MO (54.21) then the chicks fed diet containing of RO (63.72).

The interaction effect between treatments showed significant difference (P<0.05) in LDL. The lowest value of LDL was observed in the chicks fed diet containing FO with probiotic, however the highest value of LDL was observed in the chicks fed diet containing RO with antibiotic.

The obtained result showed no significant (P>0.05) effect in GPT and GOT neither due to treatments of probiotic nor due to addition of different levels of RO or due to interaction between them.

The results of the present study revealed that RO increased contents of cholesterol, triglyceride and LDL. This increase is most probably due to the high levels of trans fatty acid in RO.

89 Trans fatty acids which are harmful to human health are produced by many reactions occurring due to elevated temperature such as oxidation, isomerization, polymerization, hydrolysis and cyclization (Farag et al., 1992). Surprisingly RO had no effect on HDL and contrary to many authors (Abbey and Nestle, 1994 and Karamouz et al., 2009) as they recorded significant decrease in HDL in addition to the significant increase of other lipid profile. Abbey and Nestle (1994) explained that intake of trans fatty acid caused increase in cholesteryl ester transfer protein activity, this protein enhance transfer of cholesteryl ester from HDL or LDL which is reflected on the increase of serum LDL and decrease of HDL. It seems that the excess cholesterol synthesis in RO groups influenced the formation of LDL and the relation between total cholesterol level and LDL is more pronounced than that of LDL and HDL. The results in the present study correspond with other reports concerned with hyperlipidemia associated with RO.

The significant reduction in serum cholesterol of broiler chickens fed probiotic supplemented diet could be attributed to reduced absorption and / or synthesis of cholesterol in the gastro-intestinal tract by probiotic supplementation (Mohan et al., 1995). Also, it was speculated that lactobacillus acidophilus reduces the cholesterol in the blood by deconjugating bile salts in the intestine, thereby preventing them from acting as

90 precursors in cholesterol synthesis (Abdulrahim et al., 1996).Lactobacillus has found to have a high bile salt hydrolytic activity, which is responsible for deconjugation of bile salts (Surono, 2003). Another explanation of the mechanism by which probiotic can lower the serum cholesterol has been declared by Fukushima and Nakona (1995). The authors demonstrated that probiotic microorganisms inhibit hydroxyl- methyl-glutary-coenzyme A; an enzyme involved in the cholesterol synthesis pathway thereby decrease cholesterol synthesis.

The two liver enzymes analyzed in this experiment showed slight variation, although sGPT showed insignificant (P>0.05) variation throughout the experiment, sGOT showed only significant (P<0.05) effect in the interaction between probiotic with FO and antibiotic with RO where the significant (P<0.05) decrease in the former group was evident compared to the later group. SGPT and SGOT were chosen in the present study as a guide for the liver function tests and hence the liver status after probiotic and RO intake. Generally, both enzymes are intracellular enzymes and they are increased in serum due to their leakage from the cell after liver injury. In the present study the insignificant (P>0.05) changes of SGPT and the minor changes in the interaction groups of SGOT point to the negligible effect of probiotic or/and oil on the liver function.

91 Since total protein, albumin, SGPT and SGOT were widely used as liver function tests. The present work agreed to a great extent with the work of Alkhalf et al (2010)who reported that chicken fed on a diet containing various levels of probiotic showed no change in serum total protein and albumin tested.

Table: (9). Effect of addition of probiotic or antibiotic with different levels of RO on Blood parameter in broiler chicks.

Blood parameters Independent variable and interactions Cholesterol Triglycerides HDL LDL sGPT sGOT mg/dl mg/dl mg/dl mg/dl mmol/L mmol/L Treatments Antibiotic(T1) 164.69 a 160.81 a 58.73 b 71.79 a 8.42 a 8.41 a (T) Probiotic (T2) 121.67 b 132.86 b 63.13 a 31.97 b 8.58 a 8.12 a

Levels of oils L1=FO 127.05 c 137.98 c 61.73 a 37.71 c 8.38 a 8.27 a (L) L2=MO 143.51 b 148.16 b 59.67 a 54.21 b 8.59 a 8.39 a L3=RO 158.99 a 154.36 a 61.40 a 63.72 a 8.52 a 8.13 a

T1xL1 144.13 c 148.95 c 57.70 b 56.63 c 8.26 a 8.62 a

T1xL2 164.82 b 163.52 b 58.03 b 74.09 b 8.48 a 8.40 ab Interactions (T x L) T1xL3 185.11 a 169.97 a 60.47ab 84.65 a 8.50 a 8.20 ab T2xL1 109.94 f 127.01 f 65.77 a 18.78 e 8.49 a 7.92 b T2xL2 122.19 e 132.80 e 61.30 ab 34.33 d 8.70 a 8.37 ab T2xL3 132.88 d 138.76 d 62.33 ab 42.80 d 8.54 a 8.06 ab

A, b, c means in columns with different superscripts were significantly different (P<0.05).

92 Cholesterol and Triglycerides in serum

200

180

160

140

120 Cholesterol with antibiotic Cholesterol with probiotic 100 Triglyceride with Antibiotic Triglyceride with Probiotic 80

60

40

20 level of cholesterol and triglycerides level of cholesterol

0 FO MO RO Levels of oil

Figure (13):Effect of addition of probiotic or antibiotic with different levels of RO on serum cholesterol and triglycerides levels in broiler chicks.

HDL, LDL, GOT and GPT in serum

90

80

70

60 HDL with antibiotic HDL with antibiotic 50 LDL with antibiotic LDL with probiotic GPT with antibioitic 40 GPT with probiotic GOT with antibiotic 30 GOT with probiotic

20

10 Levels of HDL, LDL, GOT and GPT GPT and GOT HDL, LDL, Levels of

0 FO MO RO Levels of oil

Figure (14):Effect of addition of probiotic or antibiotic with different levels of RO on HDL, LDL, GOT and GPT in broiler chicks.

93 4.2.2. Immune response and thyroid hormones:

Results of immune response and thyroid hormones estimated at 42nd days of age listed in (table 10, figure 15).

Significant variation (P<0.05) in antibody total titer was detected due to treatments of probiotic, the titer had increased significantly (P<0.05) in the chicks fed probiotic (4.72) compared with the chicks fed antibiotic (4.05),while no significant variation (P>0.05) was observed in thyroid hormones in the same group.

No significant variation (P>0.05) was observed either in total titer or thyroid hormones due to addition of different levels of RO.

The interaction effect between treatment of probiotic and/or antibiotic and addition of different levels of RO showed significant difference in the total titer; the highest value was observed in the chicks fed diet containing FO with probiotic (4.66) followed the chicks fed diet containing MO with probiotic (4.55) with insignificant (P>0.05) difference between them; the lowest value of total titer was observed in the chicks fed diet containing RO with antibiotic (4.02). However, no significant different was observed between the groups fed probiotic with RO, antibiotic with FO, antibiotic with MO and antibiotic with RO. The significant difference was detected between the two groups which fed probiotic with FO or MO and

94 other groups. It is evident that probiotics especially lactic acid bacteria enhance humoral immune response (Nayebpor et al., 2007; Apata, 2008; Brisbin et al., 2008 and Fahmy, 2009). In the present study the stimulatory effect of probiotic overcome the effect of RO and all probiotic groups showed increase antibody titer.

Probiotic bacteria are normal inhabitants of microflora and confer health benefits to the host. The activation of the immune response by lactic acid bacteria requires many complex interactions; one of immune modulations by probiotics is antibody production (Perdigon et al., 1995).L. acidophilus was able to stimulate macrophage and neutrophil populations and to enhance natural killer cell activity (Matsuzaki and Chin, 2000) and increase the size of T helper lymphocyte population (Perdigon et al., 1995). Intestinal bacteria such as lactobacillus may be migrating from lumen and inhabits the omental lumphatic nodes (Fuller, 1989). This migration may be increased with the addition of probiotic to diets. Produced lymphokine in response to antigenic stimulation (migrating bacteria) by lymphocytes activate non specific immune mechanisms and consequently activate the cell mediated and humoral immunity.

Thyroid hormones play crucial roles in energy metabolism and the metabolism of thyroid hormones is influenced by

95 several nutritive factors (Eder et al., 2002). In the present study neither the use of probiotic nor the RO caused changes in both hormones (T3&T4). The present study agreed with the work of (El-Faramawy, 2006 and El-Faramawy and Fahmy, 2005) who stated that addition of RO up to 4% with or without garlic had no effect on thyroid hormones of Japanese quail.

The present work disagreed with that of Eder et al(2002) who observed increase in plasma thyroxin in rats fed thermally oxidized fats with no changes in the concentration of triiodothyronine (T3) and thyroid stimulating hormones (T.S.H).this discrepancy may be due to difference in species with consequent change in age and metabolism.

96 Table: (10) Effect of addition of probiotic or antibiotic with different levels of RO on the immune response and thyroid hormones of broiler.

Immune Thyroid hormones Independent variable and response interactions Antibody titer T4 (ng/dl) T3 (ng/dl) Treatments Antibiotic(T1) 4.05 b 112.35 a 4.05 a (T) Probiotic(T2) 4.72 a 115.14 a 4.12 a

L1=FO 4.20 a 108.25 a 4.02 a Levels of oils L2=MO 4.17 a 110.12 a 3.92 a (L) L3=RO 3.98 a 106.22 a 3.98 a

T1xL1 4.18 b 110.44 a 4.04 a T1xL2 4.11 b 109.35 a 3.97 a Interactions T1xL3 4.02 b 111.16 a 4.01 a (T x L) T2xL1 4.66 a 113.23 a 4.07 a T2xL2 4.55 a 112.51 a 3.99 a T2xL3 4.25 b 110.56 a 4.05 a

A, b, c means in columns with different superscripts were significantly different (P<0.05).

Antibody titer , T4 and T3 in serum

120

100

80 Antibody titer with antibiotic Antibody titer with probiotic T4 and T3 and , T4 T4 with antibiotic 60 T4 with probiotic T3 with antibiotic T3 with probiotic 40 Antibody titer Antibody

20

0 FO MO RO Levels of oil

Figure (15):Effect of addition of probiotic or antibiotic with different levels of RO on antibody titer, T4 and T3 in broiler chicks.

97 4.3. Carcass chemical composition:

Cholesterol, triglyceride, amino acids and fatty acids in both breast and thigh meat, were estimated at 42nd days of age.

4.3.1. Cholesterol and triglyceride in breast and thigh meat:

Cholesterol and triglyceride in both breast and thigh meat estimated at 42 days of age were illustrated in (Table 11, figure 16 and17).

The results showed that significant variation (P<0.05) in cholesterol and triglycerides in breast and thigh were detected due to treatment of probiotic. It was found that the levels of the cholesterol in breast and thigh and the levels of triglycerides in breast and thigh had decreased significantly (P<0.05) in the chicks which fed probiotic compared with the chicks fed antibiotic.

The significant variation (P<0.05) was also observed due to addition of different levels of RO. the lowest values of cholesterol and triglyceride in both kind of meat were observed in the chicks fed diet containing FO followed the chicks fed diet containing MO then the chicks fed diet containing RO.

The interaction effect between treatments of probiotic or antibiotic and addition of different levels of RO showed significant (P<0.05) differences in levels of cholesterol and triglyceride in breast and thigh; the lowest levels of cholesterol

98 and triglyceride were detected in the chicks fed probiotic with FO followed by the chicks fed antibiotic with FO with significant different between them. However the highest levels of cholesterol and triglycerides in both breast and thigh were found in the chicks fed antibiotic with RO. It should be noted that the ratio of cholesterol and triglyceride in the meat linked to the levels of cholesterol and triglyceride in the blood. The use of lactic acid bacteria as feed additives is clearly improved serum lipid profile and meat cholesterol and triglyceride. However the effect of lactic acid bacteria on serum is more profound than meat e.g the decrease in cholesterol, triglyceride and LDL reach up to 26, 17 and 55 % respectively, while the decrease of cholesterol and triglyceride in breast and thigh meat was 8, 7, 6 and 5 % of breast and thigh respectively. This may be due to either that the effect on blood is earlier or/ and probiotic affect more serum component.

The effect of probiotic in improving lipid profile is in agreement with (Salma et al., 2007; Ignatova, et al., 2009 and Ashayerzadeh et al., 2011) who reported that the supplementation of 0.04 Rhodobacter Capsulatus in diet of broiler chicken reduced (P<0.05) cholesterol and triglyceride in breast and thigh.

99 Table (11): Effect of addition of probiotic or antibiotic with different levels of RO on cholesterol, triglyceride in breast and thigh meat of broiler chicken. Cholesterol and triglycerides in meat Independent variable and Cholesterol Cholesterol Triglycerides Triglycerides interactions In breast In thigh In breast In thigh mg/100g mg/100g mg/100g mg/100g Treatments Antibiotic(T1) 110.70 a 206.34 a 172.74 a 264.64 a (T) Probiotic(T2) 100.80 b 189.98 b 161.00 b 250.68 b

Levels of oils L1=FO 89.47 c 175.07 c 157.12 c 245.80 c (L) L2=MO 107.27 b 193.35 b 166.78 b 258.98 b L3=RO 120.52 a 226.07 a 176.72 a 268.20 a

T1xL1 95.03 d 182.77 d 161.33 c 250.97 d T1xL2 112.30 b 203.47 c 172.57 b 266.00 b T1xL3 124.77 a 232.80 a 184.33 a 276.97 a Interactions T2xL1 83.90 e 167.37 e 152.90 d 240.63 e (T x L) T2xL2 102.23 c 183.23 d 161.00 c 251.97 d T2xL3 116.27 b 219.33 b 169.10 b 259.43 c

A, b, c means in columns with different superscripts were significantly different (P<0.05).

Cholesterol and triglycerides in breast

200

180

160

140

120 Cholesterol with antibiotic Cholesterol with probiotic 100 Triglycerides with antibiotic Triglyceride with probiotic 80

60

40

Levels of cholesterol and triglycerides and Levels cholesterol of 20

0 FO MO RO Levels of oil

Figure (16):Effect of addition of probiotic or antibiotic with different levels of RO on cholesterol and triglycerides in breastof broiler chicks.

100 Cholesterol and triglycerides in thigh

300

250

200

Cholesterol with antibiotic Cholesterol with probiotic 150 Triglycerides with antibiotic Triglycerides with probiotic

100

50 Levels of cholesterol and triglycerides and Levels cholesterol of

0 FO MO RO Levels of oil

Figure (17): Effect of addition of probiotic or antibiotic with different levels of RO on cholesterol and triglyceride in thigh of broiler chicks.

4.3.2. Amino acids profile in breast meat:

The results of addition of probiotic with different levels of RO on amino acids profile in breast meat of broiler were presented in (Table12 ).

There was a significant (P<0.05) increase in serine, glutamic, proline, alanine and cysteine in the chicks fed probiotic compared to chicks fed antibiotic; the other amino acids did not show any significant differences. Also, there was a significant (P<0.05) increase in aspartic, therionine, serine, glutamic, cystine and leucine in the chicks fed MO compared to chicks fed FO and/or RO.

101 In the interaction amino acid (aspartic) showed significant (P<0.5) increase in the chicks fed antibiotic with MO compared to the other groups, the amino acid therionine showed significant (P<0.05) increase in the chicks fed antibiotic with MO compared to the other groups expect the group fed antibiotic with FO, the serine gave significant (P<0.05) increase in the chicks fed probiotic with MO compared to the other groups, the glutamic showed significant (P<0.05) decrease in the chicks fed antibiotic with FO compared to the other groups, proline showed significant (P<0.05) decrease in the chicks fed antibiotic with RO compared to the other groups, glycine showed significant (P<0.05) decrease in the chicks fed antibiotic with FO compared to the other groups, alanine showed significant (P<0.05) increase in the chicks fed probiotic with MO compared to the antibiotic groups and did not show any significant differences between the probiotic groups. Cysteine experienced significant (P<0.05) increase in the chicks fed probiotic with FO compared to the chicks fed probiotic with RO. Leucine showed significant (P<0.05) increase in the chicks fed antibiotic with RO compared to the other groups except the chicks fed antibiotic with MO; the probiotic groups did not show any significant changes between them. Tryrosine gave significant (P<0.05) increase in the chicks fed antibiotic with FO compared to the chicks fed antibiotic with RO and the chicks fed probiotic with MO. The amino acids valine, methionine,

102 isoleucine, phenylalanine, histidine, lysine and arginine did not show any significant difference between the all groups.

103 (Table:12) Amino acids composition (mg/g meat tissue) of breast chicken fed probiotic or antibiotic with diet containing different levels of RO.

Independent variable Amino acids in breast and interaction

Phenyla Aspartic Therionine serine Glutamic Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tryrosine Histidine Lysine Arginine lanine

T1= A 38.2a 37.1 a 30.1 39.3b 10.3b 20.8a 23.5b 20.5b 29.0a 15.3a 31.5a 129.8 40.4a 39.6 40.4a 55. 47.8a b a a a Treatment 3 (T) T2=P 38.8 a 36.0 a 34.1 42.5a 13.1 a 21.8a 27.8a 22.8a 30.4a 15.78a 33.2a 129.1 40.4a 36.7 40.6a 56. 51.8a a a a 0a

L1=FO 35.8 b 36.1 b 29.5 39.0b 11.67 20.1a 24.5a 23.6a 30.0a 15.0a 30.8a 125.8 41.8a 39.6 40.6a 55. 50.0a b a b a a Levels of 5 oils L2=MO 40.1 a 39.1 a 35.5 42.1a 12.83 22.0a 26.6a 23.8a 28.5a 15.1a 32.5a 130.6 40.0a 34.6 39.5a 56. 49.6a (L) a a a a 5a

L3=RO 39.6 a 34.3 b 31.3 41.6a 10.67 22.0a 26.0a 17.6b 30.6a 16.5a 33.8a 132.0 39.5a 40.3 41.5a 55. 49.8a b a a a 0a

T1xL1 36.0 b 38.0ab 27.3 34.3b 11.33 18.6b 22.3c 22.6ab 29.6a 15.6a 30.6 123.3 44.0a 38.6 40.6a 55. 48.0a c a c a a a 6 T1xL2 43.3 a 41.0 a 32.6 42.3a 12.00 23.6a 23.6b 24.0ab 26.3a 15.6a 31.6a 131.6 40.6ab 41.0 40.0a 56. 49.0a b a c ab c a a Interaction 0 (T x L) T1xL3 35.3 32.3 d 30.3 41.3a 7.67 20.3a 24.6 15.0c 31.0a 16.6a 32.3a 134.6 36.6c 39.3 40.6a 54. 46.3a b bc b b bc a a 3a

T2xL1 35.6 b 34.3cd 31.6 43.6a 12.00 21.6a 26.6a 24.6a 30.3a 14.3a 31.0a 128.3 39.6ab 40.6 40.6a 55. 52.0a bc a b b b c a 3a

T2xL2 37.0 b 37.3bc 38.3 42.0a 13.67 20.3a 29.6a 23.6ab 30.6a 16.6a 33.3a 129.6 39.3bc 28.3 39.0a 57. 50.3a a a b b a 0a

T2xL3 44.0 b 36.3bc 32.3 42.0a 13.6a 23.6a 27.3a 20.3b 30.3a 16.3a 35.3 129.3 42.3ab 41.3 42.3a 55. 53.3a b b a b a 6a

A, b, c means in columns with different superscripts were significantly different (P<0.05).

104 4.3.3. Amino acids profile in thigh meat:

The results of addition of probiotic or antibiotic with different levels of RO on amino acids profile in thigh meat of broilers were presented in (Table:13 ).The obtained Results showed that no significant (P>0.05) differences was found in amino acids profile in thigh meat either due to treatments of probiotic or addition of different levels of RO or interaction between them except the amino acid alanine; it was found significant (P<0.05) increase in alanine in the chicks fed probiotic compared to chicks fed antibiotic. the levels of oil did not show any significant (P>0.05) differences in amino acids profile in thigh meat; the interaction effect showed significant decrease in alanine in the chicks fed antibiotic with MO compared to the other groups except the group fed antibiotic with FO.

105 (Table: 13) Amino acids composition (mg/g meat tissue) of thigh chicken fed probiotic with diet containing different levels of RO.

Independent Amino acids in thigh variable and interactions Aspartic Therionine serine Glutamic Proline Glycine Alanine Cysteine Valine Methionine Isoleucine Leucine Tryrosine Phenyl Histidine Lysine Arginine alanine

Treatme A 40.2a 38.3a 31.3a 42.4a 10.2a 22.3a 25.0b 20.7a 32.0a 20.2a 30.5a 130.8a 39.4a 41.0a 25.7a 52.6a 49.8a nt

(T) P 40.8a 38.5a 32.7a 42.1a 12.1a 22.2a 28.4a 21.1a 31.2a 20.4a 31.2a 130.6a 41.5a 40.1a 27.3a 53.3a 50.6a

Levels of L1=FO 39.8a 37.6a 31.3a 43.3a 11.1a 22.0a 26.5a 21.8a 31.3a 20.0a 30.0a 130.3a 40.1a 39.6a 26.0a 53.6a 50.3a oils (L) L2=MO 40.3a 38.8a 32.3a 41.3a 11.0a 21.8a 25.5a 20.5a 31.5a 19.5a 31.0a 129.8a 40.3a 41.0a 27.0a 53.1a 51.1a

L3=RO 41.5a 38.8a 32.5a 42.1a 11.3a 23.0a 28.1a 20.5a 32.0a 21.5a 31.6a 131.8a 41.0a 41.0a 26.6a 52.1a 49.3a

T1xL1 40.0a 37.0a 30.3a 42.3a 10.0a 22.0a 25.0ab 21.0a 31.6a 20.3a 28.0a 130.3a 38.3a 40.0a 24.0a 53.3a 49.6a

T1xL2 40.0a 40.3a 32.0a 41.6a 10.0a 22.6a 22.3b 21.0a 32.0a 19.3a 31.6a 130.7a 39.6a 41.3a 26.3a 53.0a 51.0a

Interacti T1xL3 40.6a 37.6a 31.6a 43.3a 10.6a 22.3a 27.6a 20.3a 32.3a 21.0a 32.0a 131.3a 40.3a 41.6a 27.0a 51.6a 49.0a on

(T x L) T2xL1 39.6a 38.3a 32.3a 44.3a 12.3a 22.0a 28.0a 22.6a 31.0a 19.6a 32.0a 130.3a 42.0a 39.3a 28.0a 54.0a 51.0a

T2xL2 40.6a 37.3a 32.6a 41.0a 12.0a 21.0a 28.6a 20.0a 31.0a 19.6a 30.3a 129.0a 41.0a 40.6a 27.6a 53.3a 51.3a

T2xL3 42.3a 40.0a 33.3a 41.0a 12.0a 23.6a 28.6a 20.7a 31.6a 22.0a 31.3a 132.3a 41.6a 40.3a 26.3a 52.6a 49.6a

A, b, c means in columns with different superscripts were significantly different (P<0.05).

106 Among the 17 amino acids tested in the present study there is 10 essential AA from these essential AA probiotic caused significant (P<0.05) increase in 3 of them (therionine, proline and leucine). The only one in thigh muscle showing significant increase is nonessential AA (alanine). AA, the precursor of proteins are needed in the animal body for the synthesis and continuous regeneration of tissue proteins, formulation of hormones, enzymes and immuono globulins. These results pointed definitely to the beneficial effect of lactobacillus on protein synthesis especially in breast muscle; this is most probably due to better digestion obtained by these microflora.

Two interesting findings concerning RO have been observed; the first observation is that level of RO has no significant (P<0.05) effect on thigh muscle besides the little effect of probiotic; we may conclude that probiotic RO play minimum role of any in thigh protein formation and there is a selective carcasses role of these two treatments. The second observation is that FO showed significant (P<0.05) decrease in 5 of the 6 statistically changed AA compared to either MO or RO i.e. RO and MO intake stimulate the formation of AA, a point which need further study.

107 4.3.4. Fatty acids profile in breast meat:

The results of addition of probiotic or antibiotic with different levels of RO on fatty acids profile in breast meat of broiler was presented in (Table 14).

There was a significant (P<0.05) increase in oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3), but there were significant (P<0.05) decrease in palmatic acid (C16:0) and stearic acid, (C18:0) in the chicks fed probiotic compared to chicks fed antibiotic. there was also significant (P<0.05) increase in TUSFA in the chicks fed probiotic compared to chicks fed antibiotic.

The significant variation (P<0.05) was also found in fatty acids profile in breast meat due to addition of different levels of RO except myristic acid (C14:0). There was significant (P<0.05) increase in palmatic acid (C16:0) and stearic acid, (C18:0) in the chicks fed RO compared to chicks fed FO or MO, while There was a significant (P<0.05) decrease in palmitoleic acid (C16:1), oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3) in the chicks fed RO or MO compared with chicks fed FO. Total saturated fatty acids did not show any significant differences, but Total unsaturated fatty acids showed significant (P<0.05) decrease in chicks fed RO or MO compared to chicks fed FO.

108 The interaction groups showed significant (P<0.05) differences in fatty acids except myristic acid (C14:0). Palmatic acid (C16:0) and stearic acid, (C18:0) showed significant (P<0.5) decrease in the chicks fed probiotic with FO compared to the other groups; the highest values of palmatic acid (C16:0) and stearic acid, (C18:0) were observed in the chicks fed antibiotic with RO 24.60 and 6.31 respectively followed by chicks fed probiotic with RO 23.86 and 4.85 respectively. There was significant decrease in palmitolec acid in the chicks fed antibiotic or probiotic with MO or RO compared to the chicks fed antibiotic with FO. Oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3) showed significant (P<0.5) increase in the chicks fed probiotic with FO compared to the other groups. The lowest value in Oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3) was found in the chicks fed antibiotic with RO. The total saturated fatty acids and ratio of USFA/SFA did not show any significant differences (P>0.05); while there was significant (P<0.05) increase in total unsaturated fatty acids in the chicks fed probiotic with FO compared to other groups.

109 (Table: 14). Fatty acids composition (mg/g meat tissue) of breast chicken fed probiotic with diet containing different levels of RO.

Fatty acids in breast Independent variable Myristic Palmatic Palmitolec Stearic Oleic Linoleic Lenolenic and interactions acid acid Acid acid acid acid acid (TSFA) (TUSFAS) (TU / (C14:0) (C16:0) (C16:1) (C18:0) (C18:1) (C18:2) (C18:3) TS)

Treatment Antibiotic 0.05 a 22.72 a 8.72 a 5.12 a 39.01 b 15.49 b 0.63 b 25.23 a 63.86 b 2.98 a (T) Probiotic 0.00 a 21.43 b 8.56 a 4.16 b 42.49 a 22.24 a 1.24 a 25.59 a 74.53 a 2.96 a

Levels of L1=FO 0.00 a 19.94 c 10.82 a 3.90 c 43.33 a 22.13 a 1.17 a 23.83 a 77.45 a 3.28 a oils L2=MO 0.00 a 22.06 b 8.56 b 4.45 b 40.21 b 18.82 b 0.89 b 26.51 a 68.49 b 2.59 a (L) L3=RO 0.07 a 24.23 a 6.55 c 5.58 a 38.71 b 15.65 c 0.74 c 25.88 a 61.65 c 3.06 a

T1xL1 0.00 a 21.20 c 11.17 a 4.28 bc 42.17 b 18.80 c 0.74 d 25.48 a 72.88 b 2.86 a

T1xL2 0.00 a 22.37 bc 8.58 b 4.77 b 39.03 c 15.37 d 0.62 de 27.14 a 63.61 d 2.34 a Interaction a a c a d e e a e a (T x L) T1xL3 0.15 24.60 6.42 6.31 35.83 12.31 0.53 23.05 55.09 3.74 T2xL1 0.00 a 18.68 d 10.48 a 3.51 d 44.49 a 25.45 a 1.60 a 22.18 a 82.02 a 3.69 a

T2xL2 0.00 a 21.76 c 8.54 b 4.12 c 41.39 b 22.28 b 1.17 b 25.88 a 73.38 b 2.83 a

T2xL3 0.00 a 23.86 ab 6.67 c 4.85 b 41.60 b 19.00 c 0.94 c 28.71 a 68.21 c 2.37 a A, b, c means in columns with different superscripts were significantly different (P<0.05).

110 4.3.5. Fatty acid profile in thigh meat:

The results of addition of probiotic or antibiotic with different levels of RO on fatty acid profile in thigh meat of broilers were presented in (Table 15 ).The obtained Results showed that there was significant (P<0.05) differences in all fatty acids profile in thigh meat of broilers except palmitolec acid. There was significant (P<0.05) decrease in myristic acid (C14:0), palmatic acid (C16:0) and stearic acid, while there was significant (P<0.05) increase in oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3) in the chicks fed probiotic compared to the chicks fed antibiotic; also, there was significant (P<0.05) increase in TUSFA in the chicks fed probiotic compared to the chicks fed antibiotic, while there was significant (P<0.05) decrease in TSFA in the chicks fed probiotic compared to the chicks fed antibiotic; the ratio of USFA/SFA showed significant (P<0.05) increase in the chicks fed probiotic compared to the chicks fed antibiotic.

Significant variation (P<0.05) was found in fatty acid profile in thigh meat due to addition of different levels of RO except oleic acid (C18:1). There was significant (P<0.05) increase in myristic acid (C14:0), palmatic acid (C16:0) and stearic acid, there was significant (P<0.05) decrease in lenoleic acid (C18:2) and linolenic acid (C18:3) in the chicks fed MO or RO compared to chicks fed FO; and therefore there was a significant

111 (P<0.05) increase in total saturated fatty acids and significant (P<0.05) decrease in unsaturated fatty acids in the chicks fed MO or RO compared to the chicks fed FO; also there was a significant (P<0.05) decrease in USFA/SFA in the chicks fed MO or RO compared to the chicks fed FO.

The interaction effect between treatments of probiotic or antibiotic and addition of different levels of RO showed significant (P<0.05) differences in fatty acids; there was significant decrease in myristic acid (C14:0), palmatic acid (C16:0) and stearic acid in the chicks fed different levels of oil with probiotic compared with chicks fed different levels of oil with antibiotic, while there was significant increase in both oleic acid (C18:1), lenoleic acid (C18:2) and linolenic acid (C18:3) in the chicks fed FO or MO or RO with probiotic compared to chicks fed FO or MO or RO with antibiotic, there was also a significant (P<0.05) decrease in TAFA acids and significant (P<0.05) increase in TUSFA in the chicks fed probiotic with levels of oil compared to the chicks fed antibiotic with the same levels of oil; also there was a significant (P<0.05) increase in proportion of SFA/USFA in the chicks fed FO or MO compared to the other groups.

112 (Table:15 ).Fatty acids composition (mg/g meat tissue) of thigh chicken fed probiotic with diet containing different levels of RO

Fatty acids in thigh Independent variable and interactions Myristic Palmatic Palmitolec Stearic Oleic Linoleic Lenolenic acid acid acid acid acid acid acid (TSFA) (TUSFA) (TU / TS) (C14:0) (C16:0) (C18:0) (C18:1) (C18:2) (C18:3) (C16:1)

Treatment Antibioti 0.55 a 25.97 a 10.49 a 6.83 a 40.24 b 13.38 b 0.70 b 33.35 a 64.80 b 2.00 b (T) c Probiotic 0.00 b 22.81 b 10.26 a 5.28 b 45.36 a 19.32 a 1.44 a 28.15 b 76.38 a 2.76 a

L1=FO 0.25 c 21.75 c 12.12 a 4.20 b 44.39 a 19.72 a 1.35 a 26.20 c 77.57 a 2.99 a Levels of oils L2=MO 0.27 b 23.92 b 10.27 b 6.22 b 42.69 a 15.61 b 1.04 b 30.42 b 69.62 b 2.31 b (L) L3=RO 0.30 a 27.49 a 8.73 c 7.75 a 41.32 a 13.71 c 0.82 c 35.62 a 64.58 c 1.85 c

T1xL1 0.50 c 22.90 c 12.27 a 4.60 c 41.50 d 17.60 c 0.86 cd 28.00 c 72.22 c 2.58 c

T1xL2 0.55 b 25.41 b 10.43 b 6.63 b 40.13 e 11.70 d 0.69 de 32.59 b 62.96 e 1.93 e

Interactions T1xL3 0.60 a 29.59 a 8.76 c 9.27 a 39.09 e 10.84 d 0.55 e 39.46 a 59.23 f 1.50 f (T x L) T2xL1 0.00 d 20.60 d 11.97 a 3.81 c 47.28 a 21.84 a 1.83 a 24.41 d 82.92 a 3.39 a

T2xL2 0.00 d 22.44 c 10.11 b 5.81 b 45.25 b 19.53 b 1.40 b 28.25 c 76.28 b 2.70 b

T2xL3 0.00 d 25.39 b 8.69 c 6.23 b 43.56 c 16.59 c 1.10 c 31.78 b 69.94 d 2.20 d A, b, c means in columns with different superscripts were significantly different (P<0.05).

113 The dietetic and nutritive value of poultry meat depends on numerous factors which are formed to a high degree, in vivo. A wide range of quality attributes, both positive and negative, from the feeding manner of birds. Fats are one of the components able to add energy value to diets; they have a high caloric value and when applied in feed mixture even in small doses, they make it possible to obtain feed with desirable energy level (Lopez-Bote et al., 1997). An important issue referring to poultry feeding is the supplementation of diets with oxidized fats or waste fats of different thermal processes. However this thermal oils may result in the production of different components, including peroxides, hydro peroxides, aldehyeds, ketones and others, the majority of which may have toxic potential (Cabel et al, 1988). Negative results of lipids oxidation may also affected the meat of those birds (Line et al., 1998) hence in the present study we evaluate FA and AA of breast and thigh meat of broiler fed on probiotic with different levels of RO.

In animal carcasses, the fatty acids composition depends to a high degree on the type of diet this is evident in the present study when comparing the FA of RO with the carcass FA ( table 17) . The nutritive value of fat is determined by the concentration and composition of FA with the number of double bonds and the position of the fist double bond (Channon and

114 Trout, 2002). High content of multi unsaturated fatty acids (MUSFA) in animal products may be beneficial for human health. Several nutritional studies strongly support a relationship between SFA and the risk of cardiovascular heart diseases, and hence, there is a need to reduce consumption of SFA and increase consumption of PUSFA (Salma et al., 2007).

Linoleic (C18:2), linolineic (C18:3) and arachidonic (C20:4) are necessary constituents of mitochondria and cell wall. These FA are essential because the body cannot produce any of the FA above , unless one of them is available in the diet. Oleic , linoleic and linolenic acids each belong to different family of compounds in which unsaturation occurs at the n-9, the n-6 and n-3 carbon atoms, respectively. Recognizing that FA are absorbed from the diet and incorporated into tissue fat, producers have attempted to improve the nutritional quality of meat by incorporating various sources of n-3- PUSFA (Wood et al., 1999 and Raes et al., 2004). In the present study probiotic had a profound beneficial effect of FA profile in thigh and breast. It does not only caused significant ( P<0.05) increase in total and individual USFA but it also almost double the content of linolenic with no negative effect on the essential palmitoleic acid.

The modulation of probiotic (lactobacillus) on FA profile may be due to nature of this microflora, the efficiency of a poultry

115 digestion depends on the microorganisms which live naturally in its digestive tract. Dietary additives of lactobacillus may create favorable conditions in the animal's intestine for the digestion of feed and improve the production results of meat producing chicks.

RO had dual effect in thigh carcasses, it increase the total and individual SFA and decrease total and individual USFA especialy palmitoleic (C16:1) and linoleic (C18:2) which agreed with (El-Faramawy,2006) as she found increase in SFA and decrease in USFA in Japanese quail where RO were added at 4% instead of FO. It also agreed with (El-Faramawy and Fahmy2006) taking different concentrations of RO on the same birds.

The results of the interaction groups revealed that propiotic ameliorates the effect of oxidized oil on both SFA and USFA of thigh and breast. The variation of fatty acid composition have profound effects on meat quality, because FA composition determines firmness/oilness of adipose tissue and the oxidative stability of muscle , which in turn affects flavor and muscle colour. It is well known that high PUFA levels may produce alterations in meat flavour due to susceptibility to oxidation and the production of unpleasant volatile components during cooking (Wood et al., 1999). Therefore composing breast and thigh carcasses according to the level of TU/TS ratio we may

116 stale that breast muscle is healthier but thigh muscle is more delicious.

4.4. Fecal microbial:

The effect of addition the probiotic mixture to the water of broiler chickens on some pathogenic and beneficial gut microorganisms was presented in (Table 16). Total bacteria, lactic acid bacteria (for its beneficial effect) staph. Aureus (as gram positive Pathogenic), E. coli (as gram negative) count were estimated in the bird feces.

Obtained Results showed that significant (P<0.05) increase in count of total bacteria and lactic acid bacreia in probiotic group compared to antibiotic group while there is significant (P<0.05) decrease in staph and E. coli.

As regards the levels of RO; the results found that insignificant (P>0.05) changes were observed in lactic acid bacteria and E.coli with different levels of RO while significant decrease (P<0.05) was observed in total and staph bacteria with RO compared to other groups.

The interaction groups showed significant (P<0.05) increase in total bacteria and lactic acid bacteria in all probiotic groups compared to the antibiotic ones, while there is significant (P<0.05) decrease in staph and E. Coli in all probiotic groups compared to the antibiotic groups. In other words the use of probiotic with different levels of RO leads to significant

117 (P<0.05) increase in total bacteria and lactic acid bacteria and significant (P<0.05) decrease in staph and E. Coli bacteria.

In the present study lactic acid bacteria is the probiotic of choice, as identified from the results it has a stimulatory effect on the lactobacilli and another inhibitory effect on E. coli and staph. These results agreed with the results of Biernasiak and Slizewska (2009) who stated that there are statistical significant differences after the third week of breeding between the kind of feed additives and the number of bacteria of lacto genus in feces. It is also agreed to some extent with the work of Jin et al., 1998 who claimed that adding L-acidophilus or a mix of lactobacillus bacteria into chicken diet induced a statistically significant (P<0.05) decrease in the number of E.coli group in caecum. Kralik et al., 2004 in their turn, reported that a decrease in the number of bacteria of the Enterobacteriaceae family and colistrains after 42 days of supplementing water with a probiotic containing 5x109 CFU/g of Enterococcus faeciumM- 74. However they did not show any statistically significant differences in relation to bacteria staphylococcus SP. Similarly, Salarmoni and Fooladi, (2011) stated that addition of fermented milk containing L-acidophilus to the diets increased the number of lactobacilli in ileum and colon and also addition of either of fermented milk or commercial probiotic to the basal diet decreased the number of E. coli forms in ileum and colon.

118 Although the bacteria in the present study are enumerated from the feces some authors found that they did not reflect the number of the bacteria in the gut. Jin et al (1998) demonstrated that when the broilers received a supplement of L. acidophilus or a mix of bacteria of lactobacillus genus, it did not affected any statistical significant increase during individual weeks of breeding. Despite insignificant changes recorded by most authors there is improvement in intestinal microflora balance after bacteria analysis (Salarmoini and Fooladi, 2011).Moreover, our results denoted that supplementation of probiotic had beneficial effects over antibiotic on growth performance; immune response and hypolipidemia reflecting better digestion and absorption. In other words probiotic administration modulated the composition and, to an extent, the activities of cecal micoflora, resulting significant probiotic effect and a metabolic stimulation of the cecal microflora .

Therefore (Rada and Rychly, 1995;Lineet al., 1998andPascual et al., 1999). stated that probiotic intake should result in the creation of gut micro-ecology conditions that suppress harmful microorganisms and favor beneficial microorganisms and ultimately enhance gut health.

119 Table: (16). Effect of addition probiotic with different levels of RO on the total count of bacteria, lactic acid bacteria, staph and E. coli in the faeces of broiler.

Fecal microbe Independent variable and Total Lactic acid Staph E. coli interactions bacteria bacteria CFU/1g CFU/1g CFU/1g CFU/1g feces feces feces feces Treatments Antibiotic(T1) 5.0 x 107b 2.2 X 107 b 5.6 x 106a 4.9 x 105a (T) Probiotic (T2) 5.6 x 108a 6.9 x 108a 3.8 x 104b 2.6 x1 04b

Levels of L1=FO 3.4 x 108a 3.6 x 108a 3.0 X 106a 2.4 x 105a oils L2=MO 3.2 x 108a 3.7 x 108a 2.9 x 106a 2.6 x 105a (L) L3=RO 2.5 x 108b 3.5 x 108a 2.5 x 106b 2.6 x 105a

T1xL1 5.9 x 107d 4.8 x 107b 6.0x 106a 4.7 x 105a T1xL2 4.8 x 107d 3.4 x 107b 5.7 x 106a 4.9 x 105a Interactions T1xL3 4.0 x 107d 2.9 x 107b 4.9 x 106b 5.03 x 105a (T x L) T2xL1 6.3 x 108a 7.1 x 108a 3.8 x 104c 2.77 x 104b T2xL2 5.8 x 108b 7.0 x 108a 3.7 x 104c 2.75 x 104b T2xL3 4.6 x 108 c 6.7 x 108a 3.8 x 104c 2.43 x 104b

A, b, c means in columns with different superscripts were significantly different (P<0.05).

120 5. Summary and Conclusion

This experiment was carried out the National Center for Radiation Research and Technology, Atomic Energy Authority, Nasr City, Egypt. The study aimed to evaluate the effect of probiotic (some lactic acid bacteria) with different levels of food industrial residual oil ( RO ) in broiler commercial diets on growth performance, performance index, lipid profile, liver enzymes, thyroid hormones and carcasses cholesterol, triglycerides, amino acids and fatty acids.

One hundred and eighty one day cubb chicks about 50±3 gm each purchased from El-Wady Company were used in this study. They were equally and randomly divided into 6 groups ( 30 chicks per each group ), 3 replicates with 10 birds in each: the antibiotic with fresh oil ( FO), the antibiotic with mixed oil (MO) (FO+RO 1:1 W/W ), the antibiotic with RO, the probiotic with FO, The probiotic with MO and The probiotic with RO. Virginiamycin ( Phibro, USA ) 15 ppm, was the antibiotic, while some lactic acid bacteria is chosen as probiotic both was added to the water. During the experiment which lasted for 42 days body weight, Feed intake and mortality rate were recorded at 2th , 4th and 6th weeks of birds age then body weight gain, feed conversion ratio and economical efficiency were calculated. Sheep red blood cell 3% was injected subcutaneously (0.5 cc) at day 14 and 21 of age. At 42 day of

121 age the feces of 3 birds from each sub groups were collected for microbial determination. After slaughtered of the birds the blood was collected for cholesterol, triglycerides, HDL, LDL, GPT, GOT, antibody titer and thyroid hormones determination. Also carcass and organs were weight then cholesterol, triglycerides, amino acids and fatty acids were estimated in breast and thigh.

The results could be summarized as follow:

- Average body weight and body weight gain significantly (P<0.05) increased in groups of chicks fed probiotic with different levels of RO compared with the group of chicks fed with the antibiotic at the same levels of RO. The highest value was observed in the group of birds which fed probiotic with FO while the lowest value was showed in the group of chicks fed antibiotic with RO.

- Average of feed consumption increased significantly (P<0.05) in the groups of chicks fed probiotic with FO compared with other groups. It was observed that the rate of decreasing of average FCR differed according to broiler age. The highest decrease level was showed during the period from 4th to 6th weeks of birds age in the group which fed probiotic with FO and MO compared with other groups.

- The fecal microbial showed significant (P<0.05) variation; the total bacterial and lactic acid bacteria showed significant (P<0.05) increased in the chicks fed probiotic with FO while the

122 staph and E.coli showed significant (P<0.05) decrease in the birds fed probiotic with different levels of RO compared to the chicks fed antibiotic with the same levels of oils.

- The rate of mortality decrease significantly (P<0.05) in the groups of chicks fed probiotic with FO, MO and chicks fed antibiotic with FO without significant differences.

-The meat yield, heart and spleen weight corresponding to the different treatments did not differ significantly (P>0.05) while liver increase significantly (P<0.05) in probiotic FO and gizzard in all probiotic group and antibiotic MO.

- The highest FO and MO performance index was observed in groups of birds treated with probiotic with MO followed birds treated with probiotic FO without significant differences.

- Significant (P<0.05) differences were observed in serum cholesterol and triglycerides between all groups; the best effect showed due to supplementation of probiotic with different levels of RO. The lowest value of serum cholesterol was observed in chicks fed probiotic with different levels of oils compared to antibiotic ones.

- The HDL showed significant (P< 0.05) increase in probiotic FO compared to antibiotic FO and MO, as regards LDL the lowest significant (P<0.05) values was observed in the 3 groups of chicks fed probiotic.

123 - The obtained results of thyroid hormones showed no significant (P>0.05) change and significant (P<0.05) increase in GOT in antibiotic FO compared to probiotic FO.

-The highest value of antibody titer was observed in chicks fed diets containing FO with probiotic followed by the birds fed containing MO with insignificant (P>0.05) different between them compared to other groups .

- The levels of cholesterol and triglycerides in both breast and thigh carcasses significantly (P<0.05) decreased in the groups of chicks fed probiotic with FO compared to other groups.

- The Fatty acids of the breast and thigh meat showed significant (P<0.05) differences in some fatty acids and insignificant differences in the others. It the breast TSFA and the ratio of TUSFA/TSFA showed no significant (P>0.05) change while all probiotic and antibiotic FO group showed significant (P<0.05) increase in USFA. In thigh the probiotic FO showed the highest significant (P<0.05) in TUSFA and the ratio of USFA/TSFA.

- The amino acids in the thigh muscle showed negligible changes while in the breast muscle showed significant (P<0.05) differences in some amino acids and insignificant (P>0.05) differences in others.

124 Conclusion:

- LAB had a beneficial effect not only on the quantity of meat production but also on quality of both thigh and breast carcass. Beside it increase the resistance of birds with consequent decrease in mortality rate as evident by increase antibody titer production.

- Addition of oxidized oil (RO) in the diet of broiler chicks is at different concentration in the three stage of age had may adverse effect of the quantity and quality of birds compared to fresh oil (FO) although the net profit per birds was higher with RO

- LAB supplementation ameliorate the harmful effect of oxidized oil .

- This probiotic (LAB) can be considered an ideal supplementary diet since it exert its effect through biological mechanisms: i.e. it decrease pathogenic organism increase useful ones; increase bird resistance and improve intestinal absorption .

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153 الملخص العربي

الكائنات الحية الدقيقة الفعالة كبدائل للمضادات الحيوية

أجريت هذه الدراسة بمزرعة المركز القومي لبحوث وتكنولوجيا اإلشعاع – هيئة الطاقة الذرية – مدينة نصر – القاهرة بهدف دراسة تأثير إضافة بعض أنواع من بكتيريا لالكتيكحمض ا مع مستويات مختلفة من زيت المطاعم المستعمل كبديل للزيت الطازج في عليقه الدواجن على إنتاجية بداري التسمين وتأثيره على بعض الصفات الكيميائي ة في اللحم والدم وكذلك المحتوى البكتيري في روث الدواجن.

استخدم في هذه الدراسة 081 كتكوت كب عمر يوم متماثلة فى الوزن تم شرائها من شركة الوادي للدواجن، قسمت الكتاكيت عشوائيا إلى 6 مجموعات كل منها يحتوي على 01 كتكوت وكل مجموعة قسمت إلى ثالث مكررات كل منها يحتوى على 01 كتكوت. غذيت طيور المجموعة األولى عليقة محتوية على زيت طازج )زيت بذرة القطن( مع المضاد الحيوي، غذيت طيور المجموعة الثانية على عليقة محتوية على زيت مختلط من الزيت الطازج والزيت المستعمل بنسبة )0:0( مع المضاد الحيوي، غذيت طيور المجموعة الثالثة على عليقة محتوية على زيت مستعمل مع المضاد الحيوي، غذيت طيور المجموعة الرابعة على عليقه محتوية على زيت طازج )زيت بذرة القطن( مع بعض أنواع بكتريا حمض الالكتيك ، غذيت طيور المجموعة الخامسة على عليقة محتوية على زيت مختلط من الزيت الطازج والزيت المستعمل بنسبة )0:0 ( مع البكتريا ، غذيت طيور المجموعة السادسة على عليقة محتوية على زيت مستعمل مع .البكتريا

تم التسجيل الفردي لوزن الجسم عند الفقس ، 2، 4، 6 أسابيع من العمر. تم تقدير الزيادة المكتسبة فى وزن الجسم، معدل استهالك العليقة، معدل التحويل الغذائى، دليل كفاءة النمو: الكفاءة االقتصادية، نسبة النفوق وصفات الذبيحة واعتبرت كصفات دالة على الكفاءة اإلنتاجية كما تم تقدير الوزن لبعض األعضاء الداخلية )الكبد – القونصة – القلب - الطحال( كما تم تقدير النسبة المئوية للصدور واألوراك بالنسبة لوزن الذبيحة.

تم تقدير نسبة الكولسترول ، الجليسريدات الثالثية الليبوبروتين عالى الكثافة والليبوبروتين منخفض الكثافة واإلنزيمات الناقلة لمجموعة األمين )إنزيمات الكبد(" إنزيم اسبرتات امينو ترنسفيراز وإنزيم األنين امينو ترانسفيراز" فى الدم .كما تم تقدير أيضا هرمونات الغدة الدرقية .

تم تقدير ايضا نسبة الكولسترول والجليسريدات الثالثية فى األوراك والصدور كما تم تقدير محتوى األوراك والصدور من األحماض االمينية واأل هنية.دحماض ال

ويمكن تلخيص النتائج المتحصل عليها كما يلى:

- أظهرت الكتاكيت المغذاة على عليقة تحتوى على زيت طازج مع البروبيوتك زيادة معنوية في وزن الجسم مقارنة بالمجموعات األخرى . - ارتفع متوسط الزيادة المكتسبة فى وزن الجسم معنويا فى الكتاكيت المغذاة على عليقة تحتوى على زيت طازج مع البروبيوتك زيادة معنوية في وزن الجسم مقارنة بالمجموعات األخرى. - أظهرت الكتاكيت المغذاة على عليقة تحتوى على زيت طازج مع البروبيوتك اعلي معدل في تناول العليقة مقارنة بالمجموعات األخرى. - أظهرت الكتاكيت المغذاة على عليقة تحتوي على زيت طازج مع البروبيوتك أفضل قيمة في معدل تحويل الغذاء والذي اختلف باختالف عمر الطيور وكان أعلى قيمة انخفاض فى معدل تحويل الغذاء خالل الفترة من )4-6( أسابيع من عمر الطيور. - أظهرت الكتاكيت المغذاة على عليقة تحتوي على الزيت الطازج مع البروبيوتك أعلى متوسط دليل كفاءة النمو - انخفضت نسبة النفوق فى مجموعات الدواجن المغذاة على البروبيوتك مع الزيت العادي والزيت المختلط وكذلك الدواجن المغذاة على المضاد الحيوى مع الزيت الطازج مع عدم وجود أي اختالفات معنوية بينهم. - لم تتأثر نسبة أوزان األوراك والصدور بالنسبة لوزن الذبيحة بسبب البروبيوتك وال بسبب الزيت المستعمل حيث لم تظهر اى اختالفات معنوية بينهم. - أظهرت النتائج وجود اختالفات معنوية فى أوزان الكبد والقونصة بينما لم تظهر اختالفات معنوية فى وزن الطحال. - أظهرت النتائج أيضا وجود اختالفات معنوية فى نسبة الكوليسترول والجليسريدات الثالثية فى الدم حيث لوحظ انخفاض معنوي فى مستوى الكوليسترول والجليسريدات الثالثية بالدم فى الدواجن المغذاة على البروبيوتك مع مستويات الزيت المستعمل مقارنة بالدواجن المغذاة على البروبيوتك مع نفس المستويات من الزيت. - أظهرت النتائج أيضا وجود اختالفات معنوية فى مستوي الليبوبروتين عالى الكثافة والليبوبروتين منخض الكثافة حيث لوحظ أن أعلى قيمة كانت فى الدواجن المغذاة على العليقة المحتوية على الزيت المستعمل مع المضاد الحيوي بينما أقل قيمة كانت مع الدواجن المغذاة على العليقة المحتوية على الزيت الطازج مع البربيوتك. - لم تظهر أى اختالفات معنوية بين مستويات انزيمات الكبد فى جميع المعامالت. - أظهرت النتاتج وجود زيادة معنوية فى نسبة األجسام المضادة فى الدواجن المغذاة على عليقة محتوية على زيت طازج مقارنة بباقي المجموعات. - لم تظهر النتائج اى اختالفات معنوية فى مستويات هرمونات الغدة الدرقية بين جميع المعامالت. - أظهرت النتائج وجود انخفاض معنوي فى نسبة كل من الكولبسترول والجليسريدات الثالثية فى األوراك والصدور فى الدواجن المغذاة على عليقة محتوية على الزيت العادى مع البروبيوتك مقارنة بباقى المجموعات. - أظهرت النتائج أيضا وجود اختالفات معنوية فى نسب بعض االحماض الدهنية واألحماض األمينبة بينما لم تظهر اختالفات فى نسب بعض األحماض االخري بسبب المعامالت. اإلهــــداء أهدى هذا الجهد المتواضع إلى والدي ووالدتي اللذان ما برحا يملئان ذلك الفراغ الهائل وهما يحمالن عنا أعباء الحياة, و خي ففان عنا بحنانهما الدافئ أثقالها.....

و إلى رفيقة دربي .....إلى من سارت معي نحو الحلم.. خطوة بخطوة

بذرناه معاً.. وحصدناه معا ...ًوسنبقى معاً بإذن هللا ..زوجتي الغالية..جزاك هللا خيرا ً

الى مهج قلبى .....وأملى في الحياة...... إلى ابنائى رقية ومحمد

وإلى أخوتي وأخواتي وهم يملؤن على حياتي فأجد فيهم تلك الشموع المحترقة التي تنور ما ادلهم من الليالي وهم يشاطرونني خطواتي الواثقة .....

أهدى جهدي هذا لعله يرد الدين لمن وفر لى فرصة اإلبداع وأدعوا هللا أناء الليل والنهار أن تكون يانعة بعدما سقيت بسلسبيل الصدق ..... وهللا ولى التوفيق

إسالم سعيد حجازي الشكر والتقدير

الحمد هلل رب العالمين الذي هدانا إلى درب المعرفة والصالة والسالم على سيد المرسلين محمد وآله الطيبين الطاهرين....

بعد أن وفقني اهلل سبحانه وتعالى على إتمام رسالتي ، أتقدم بخالص شكري وامتناني وأعطر باق ات الحب والتقدير إلى األستاذ الدكتور عاصم فتحي المغربي األستاذ بقسم الكيمياء الحيوية- الزراعةكلية -جامعة األزهر وإلى األستاذة الدكتورة أمينة عبد الحكيم الفرماوي األستاذة بالمركز القومي لبحوث وتكنولوجيا اإلشعاع- هيئة الطاقة الذرية وإلى األستاذة الدكتورة هاله أحمد حسين األستاذة بالمركز القومي لبحوث وتكنولوجيا اإلشعاع- هيئة الطاقة الذرية وإلى السيد الدكتور محمد مبروك الدناصوري األستاذ المساعد بقسم الكيمياء الحيوية-كلية الزراعة-جامعة األزهر لمساعدتهم و توجيهاتهم العلمية الكبيرة طوال مدة الدراسة التي كان لها أكبر األثر في إتمام هذا البحث وإيصاله إلى المستوى المطلوب راجياً من اهلل عز وجل أن يمتعهم بوافر الصحة والعافية وأن يحفظهم من كل مكروه وان يجعل النجاح والخير رفيق دربهم.

ومن الواجب أن أتقدم بالشكر الكبير إلى جميع أعضاء قسم الكيمياء الحيوية - كلية الزراعة –جامعة األزهر. وجميع أعضاء قسم تشعيع األغذية- المركز القومي لبحوث وتكنولوجيا اإلشعاع- هيئة الطاقة الذرية

وحتما ً شكري وتقديري إلى كل من مد لي يد العون والمساعدة ودعائي له أن يجازيه اهلل خير الجزاء.

واهلل ولي التوفيق، والحمد هلل رب العالمين. الكائنات الحية الدقيقة الفعالة كبدائل للمضادات الحيوية

رسالة مقدمة من إسالم سعيد راغب حجازي العا اإلجازة لي ة )البكالوريوس( فى العلوم الزراعية )كيمياء حيوية( جامعة األزهر 6002م

استيفاء لمتطلبات الحصول على درجة التخصص )الماجستير( في العلوم الزراعية )الكيمياء الحيوية(

قسم الكيمياء الحيوية كلية الزراعة بالقاهرة - جامعة األزهر

4141هـ 6044م

أجــازهـا: أ.د/ محمود عبدالرازق جمعة دهيم ...... أستاذ الكيمياء الحيوية - كلية الزراعة – جامعة الزقازيق أ.د/ محمد جابر عبدالفضيل طه ...... أستاذ الكيمياء الحيوية – كلية الزراعة – جامعة االزهر أ.د/ عاصم فتحى المغربي ...... أستاذ الكيمياء الحيوية – كلية الزراعة – جامعة االزهر د/محمد مبروك الدناصوري ...... أستاذ الكيمياء الحيوية المساعد – كلية الزراعة – جامعة االزهر

تاريخ المناقشة: 6044/40/9 الكائنات الحية الدقيقة الفعالة كبدائل للمضادات الحيوية

رسالة مقدمة من إسالم سعيد راغب حجازي ااإلجازة الع لي ة )البكالوريوس( فى العلوم الزراعية )كيمياء حيوية( جامعة األزهر 6002م

استيفاء لمتطلبات الحصول على درجة التخصص )الماجستير( في العلوم الزراعية )الكيمياء الحيوية(

قسم الكيمياء الحيوية كلية الزراعة بالقاهرة - جامعة األزهر

4141هـ 6044م

راف:لجنة اإلش أ.د/ عاصم فتحى المغربى ...... أستاذ الكيمياء الحيوية - قسم الكيمياء الحيوية - كلية الزراعة بالقاهرة - جامعة األزهر. أ.د/ أمينة عبد الحكيم الفرماوي ...... أستاذ تغذية الحيوان– قسم تشعيع االغذية - المركز القومي لبحوث وتكنولوجيا اإلشعاع. أ.د/ هاله أحمد حسين ...... أستاذ الميكروبيولوجي - قسم الميكروبيولوجي - المركز القومي لبحوث وتكنولوجيا اإلشعاع. أ.د/ محمد مبروك الدناصوري ...... أستاذ الكيمياء الحيوية المساعد – قسم الكيمياء الحيوية - كلية الزراعة بالقاهرة – جامعة األزهر. الكائنات الحية الدقيقة الفعالة كبدائل للمضادات الحيوية

رسالة مقدمة من إسالم سعيد راغب حجازي ااإلجازة الع لي ة )البكالوريوس( فى العلوم الزراعية )كيمياء حيوية( جامعة األزهر 6002م

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