EFFECTS OF SEASONAL VARIATIONS ON THERMOREGULATION OF OSTRICH

By HAMDY ABD Al-RAHMAN MOHAMED BASUONY High Diploma in (Applied Poultry Technology) Fac. of Agric. Al-Azhar University, 2000 M.Sc. Agric. (Animal Production-Animal Physiology) Fac. of Agric. Al-Azhar University, 2005

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Animal Production-Animal Physiology)

Department of Animal Production Faculty of Agriculture, Cairo Al-Azhar University

1432 A.H 2011 A.D TITLE: EFFECTS OF SEASONALVARIATIONS ON THERMOREGULATION OF OSTRICH

NAME: HAMDY ABD Al-RAHMAN MOHAMED BASUONY

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Animal Production-Animal Physiology)

Department of Animal Production Faculty of Agriculture, Cairo Al-Azhar University

1432 A.H 2011 A.D

Supervision Committee: Prof. Dr. M. H. KHALIL……………………………… Prof. of Animal Physiology, Dept. of Animal Production, Faculty of Agric., Al-Azhar Univ. Prof. Dr. H. H. KHALIFA ……………………………... Prof. of Animal Physiology, Dept. of Animal Production, Faculty of Agric., Al-Azhar Univ. Prof. Dr. M. A. El-SAYED …………………………...... Associate Prof. of Poultry Physiology, Biology Department, Nuclear Research Center, Atomic Energy Authority, Cairo

APPROVAL SHEET

NAME: HAMDY ABD Al-RAHMAN MOHAMED BASUONY

TITLE: EFFECTS OF SEASONAL VARIATIONS ON THERMOREGULATION OF OSTRICH

THESIS Submitted in Partial Fulfillment of the Requirements for the Degree

Of DOCTOR OF PHILOSOPHY

In AGRICULTURAL SCIENCES (Animal Production-Animal Physiology) Department of Animal Production Faculty of Agriculture, Cairo Al-Azhar University 1432 A.H 2011 A.D

Approved by: Prof. Dr. H. H. KHALIFA ……………………………... Prof. of Animal Physiology, Dept. of Animal Production, Faculty of Agric., Al-Azhar Univ. Prof. Dr. H. M. S. SHOKRY ……………………………. Prof. of Poultry Physiology, Dept. of Animal Production, Faculty of Agric., Al-Azhar Univ. Prof. Dr. M. A. EL-KHASHAB ……………………….... Prof. of Animal Physiology, Dept. of Animal Production, Faculty of Agric., El-Fayoum Univ.

Date: 28 / 7 / 2011 I

LIST OF CONTENTS Title Page 1- INTRODUCTION 1 2- REVIEW OF LITERATURE 6 2-1-Thermoregulation and thermoneutral-zone 6 2-2-Thermoregulatory mechanisms 7 2-3- Feed and water consumption 8 2-4-Body temperature 13 2-5- Hematological studies 22 2-6- Blood biochemical studies 25 2-7- Blood hormone levels 31 2-8- Blood electrolytes 35 2-9- Total body water 39 2-10- Protein profile 41 3- MATERIAL AND METHODS 44 3-1- Management of experimental animals 44 3-2-Blood samples 46 3-3-Feed and water consumption 46 3-4- Body temperature 46 3-5-1-Hematological studies 47 3-5-2-Hemoglobin (Hb) 48 3-5-3-Packed cell volume (PCV) 48 3-5-4-Mean corpuscular volume (MCV) 49 2-5-5-Mean corpuscular hemoglobin (MCH) 49 3-5-6-Mean corpuscular hemoglobin concentration 49 (MCHC) 3-6-Biochemical studies 49 3-7-Estimation of serum concentrations of Calcium, 51 Sodium, Potassium and Phosphorus 3-8-Estimation of hormone levels 51 3-9- Determination of total body water 51 3-10- Proteins electrophoresis 52 II

3-11- Statistical analysis 56 4- RESULTS AND DISCUSSION 57 4-1-Effect of seasonal variations on feed and water 57 consumption 4-2-Effect of seasonal variations on body 62 temperature 4-3-Effect of seasonal variations on hematological 66 parameters 4-4-Effect of seasonal variations on blood 71 biochemical parameters 4-5- Effect of seasonal variations on serum 82 electrolytes 4-6- Effect of seasonal variations on total body 88 water. 4-7- Effect of seasonal variations on some hormone 89 levels 4-8-Effect of seasonal variations on blood proteins 96 profile. 5- SUMMARY AND CONCLUSION 104 6- REFERENCES 109 ARABIC SUMMARY

III

LIST OF APREVIATIONS

Appreviation Meaning

ACTH Adrenocorticotrophic hormone ALT Alanine transaminase Amo. Amount of protein ANP Antipyren AST Aspartate transaminase BWT Body weight Ca++ Calcium ions CA Cold-acclimated EB Electrolytes balance EHL Evaporation heat loss Gr Growth rate Hb Hemoglobin value

HCo 3 Carbonate Hct Hematocrit value HP Heat production HS Heat stress Hsp Heat shock protein K+ Potassium ions K.Da Kilo Dalton L.B.W Live body weight M.W Molecular weight MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volume Na Sodium ions

NaHCO 3 Sodium bicarbonate OD Optical density IV

OM Organic matter PCV Packed cell volume Pi Phosphorus ions PM Peritoneal macrophages R.f Rate of flow RBC s Red blood cells RH Relative humidity RT Rectal temperature SPE Serum proteins electrophoresis T.B.S Total body solids T.B.W Total body water

T3 Triiodothyronine

T4 Thyroxin Ta Ambient temperature Tb Body temperature TM Thermal manipulation TN Thermo-neutral zone TP Total proteins WBC s White blood cells

V

LIST OF TABLES

Table No. Title Page

Table (1) The ingredients and the chemical analysis of 45 the experimental diet. Table (2) Effect of seasonal variations on hematological 69 parameters. Table (3) Effect of diurnal variations on hematological 70 parameters in each season. Table (4) Effect of seasonal variations on serum total 74 protein, albumin, globulin concentrations and A/G ratio. Table (5) Effect of diurnal variations on serum total 75 protein, albumin, globulin concentrations and A/G ratio in each season. Table (6) Effect of seasonal variations on serum 80 allanine transaminase (ALT), aspartate transaminase (AST), total lipids, triglycerides, cholesterol, and glucose concentrations. Table (7) Effect of diurnal variations on serum allanine 81 transaminase (ALT), aspartate transaminase (AST), total lipids, triglycerides, cholesterol, and glucose concentrations in each season. Table (8) Effect of seasonal variations on serum 86 electrolytes concentration. Table (9) Effect of diurnal variations on serum 87 electrolytes concentration concentration in each season Table (10) Effect of seasonal variations on total body 89 water. VI

Table (11) Effect of seasonal variations on serum 94 hormone levels. Table (12) Effect of diurnal variations on serum 95 hormone levels in each season. Table (13) Physiological epigenetic sub level expressed 101 as native protein in different samples of ostrich blood serum.

Table (14) Similarity Index and genetic distance between 102 blood samples of ostrich under study.

VII

LIST OF FIGURES

Fig. No. Title Page Figure (1) Effect of seasonal variations on food 61 consumption. Figure (2) Effect of seasonal variations on water 61 consumption. Figure (3) Effect of seasonal variations on body 65 temperature Figure (4) Electrophoretic pattern by native-PAGE 98 of protein fraction of ostrich during seasonal variation (summer and winter).

Figure (5) Densitograms of protein pattern 99 separation of Marker (Biolab).

Figure (6) Densitograms of protein pattern 99 separation of ostrich winter morning.

Figure (7) Densitograms of protein pattern 100 separation of ostrich summer morning. Figure (8) Densitograms of protein pattern 100 separation of ostrich summer afternoon.

VIII

Prayerful thanks, at first to our Merciful God who gives me every thing I have .

My deepest gratitude and appreciation to Prof. Dr. M. H. Khalil professor of Animal Physiology, Department of Animal Production, Faculty of Agriculture, Al-Azhar University, for supervision, planning the experimental design providing, text revision, his strong support, his useful criticism, and his continuous encouragement during the course of this study. I’m greatly indebted to Prof. Dr. H. H. khkhaaaalifalifalifalifa professor of Animal Physiology, Department of Animal Production, Faculty of Agriculture, Al-Azhar University, for his kind guidance, valuable advice, planning the experimental design providing, the facilities, text revision, his continuous encouragement during the course of this study and helping to understand the statistics for my data. A very grateful acknowledgment and sincere thanks are extended to Prof. Dr. M. A. Elaroussi professor of Poultry Physiology, Biological Application Department, Nuclear Research Center Atomic Energy Authority, in Egypt, for their great helps during this work. I wish to express my sincere gratitude and deepest thanks to Associate Prof. Dr. M. A. Elsayed Associate Professor of Poultry Physiology, Biological Application Department, Nuclear Research Center Atomic Energy Authority, in Egypt, For his strong support, his useful IX criticism, and encouragement, valuable evinces, keep interest and providing guidance throughout he lift span of this work. I wish to express my sincere gratitude and deepest thanks to Prof. Dr. Ibrahim AbuelyazidAbuelyazid,,,, Professor of Molecular Biology, Biological Application Department, Nuclear Research Center, Atomic Energy Authority, Egypt, for his strong support, his useful criticism, encouragement, and continuous to finish my study. I would like to express my wish and thanks all staff member and workers of the animal unit, Prof. Dr. Habeeb,Habeeb, A. A. M. and also,, Prof. Dr. Farghaly, H. M. Laboratory in Applied Biological Department, Nuclear Research center, Atomic Energy Authority for their great help during the this work. Also, I would like to express my wish and thanks all staff member and workers of the poultry unit, Laboratory in Applied Biological Department, Nuclear Research center, Atomic Energy Authority for their great help during the this work. Finally my thanks are also extended to my family (my wife and my sons, Ahmed, Ghada and Eman) for their continuous encouragement, patience, interest and helpful co- operation which made this study possible.

HHHamdyHamdy Abd AlAl----rahmanrahman Basuony ABSTRACT

HAMDY ABD Al-RAHMAN MOHAMED BASUONY TITLE: EFFECTS OF SEASONAL VARIATIONS ON THERMOREGULATION OF OSTRICH Twelve ostrich aged 7 months old were used during summer and winter from the breeding flock of the ostrich farm, at the Nuclear Research Center in Inshas, of Atomic Energy Authority, Egypt. In the study all birds were exposed to ambient temperatures in summer and winter, and the birds were fed grower ration ad libitum (19% protein and 2450 K cal ME /Kg). The present study was carried out to evaluate the effect of temperature variation during summer and winter seasons and diurnal effect on changes in some physiological and blood chemical parameters, the daily feed consumption (g/bird/day) and water consumption (ml/bird/day) these parameters were measured during 7 days in each season. Cloacal temperatures was measured and blood samples were taken twice, one in the morning at 7 am and once in the afternoon at 3 pm during a representative 7 hot days of June (40±2ºC) (summer) and the 7 cold days of January (18±2ºC) (winter). Red blood cell (RBCs) counts and total white blood cell (WBCs) counts, hemoglobin concentration (Hb) and packed cell volume (PCV) were determined. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHC) were calculated. Serum, total protein (TP), albumen (A) and globulin (G) concentrations were measured. Furthermore, serum alanine transaminase (ALT), aspartate transaminase (AST), glucose and triglycerides concentrations were determined. Also, serum triiodothyronine (T 3), and aldosterone levels were estimated. Also, the amount of total body water was determined by the antipyren method. Finally, serum protein profile it was conducted by NativePAGE method (Native polyacrylamide gel electrophoresis) on vertical slab gel to determine protein profiles in blood proteins of ostrich. Results indicated that feed consumption unlike water consumption was significantly increased during winter than in summer season. Moreover, body temperature increased significantly during the summer season as compared with the winter season and was significantly elevated at the afternoon than at the morning. Blood picture showed that heamoglobin concentration (Hb), packed cell volume (PCV), red blood cells (RBCs) counts and total white blood cell (WBCs) counts were significantly decreased in the summer than in winter months at the two diurnal periods. While both, mean corpuscular volume (MCV) and Mean corpuscular hemoglobin (MCH) were significantly increased in summer than winter season at the morning and afternoon periods. However, mean corpuscular hemoglobin concentrations (MCHC) was significantly decreased in summer season at morning only. In addition, at summer season, results showed that (Hb) concentration was significantly increased at the morning than afternoon while there were no significant differences in all of the other parameters. In addition, at winter season it was found that Hb concentration, PCV and both (RBCs) and (WBCs) counts were significantly elevated at morning as compared with at afternoon values. In the mean time, no significant differences between the morning and afternoon in MCV, MCH and MCHC measurements were observed. Serum total protein, albumin, globulin concentrations and A/G ratio were significantly increased during the winter than the summer seasons at morning and afternoon. It was found that both, total protein and albumin concentrations were significantly increased at morning than the afternoon during both summer and winter seasons. Serum ALT, AST, total lipids, triglycerides, cholesterol, and glucose concentrations were significantly increased during summer season as compared with winter season at morning and afternoon periods. The increase in those serum parameters was highly significant during the afternoon as compared with at morning periods, both in summer and winter seasons.

Serum triiodothyronine (T 3) concentration was highly significantly (P ≤ 0.001) increased during the winter than in the summer season with the morning values higher than the afternoon in both seasons. Unlike T 3, the increase in aldosterone hormone level was significant during summer as compared to winter season with the morning higher in both seasons than in the afternoon. The heat stress of the summer season caused increased significant increased (P ≤ 0.001) in TBW by 15.04% and a significant decreased in TBS by 20.33% although LBW was not affected by the season. The average TBS value lost during summer was higher than the average increase in (TBW). In general the increase in temperature during the summer season caused a quantitative shift in serum proteins as indicated in gel electrophoresis experiment in the M.W of 65.9 K.Da type, it is high in winter than that of summer season may be used in acclimatization process. This quantitative shift in protein were indicated as protein amount ratio as 1.35, 1.39, 1.6, and 1.58 at the bands 5, 5, 8, and 12, respectively.

Key wards: Thermoregulation, Ostrich, Poultry, Heat stress, Cold stress, Biochemical studies, Hematology, T3, aldosterone, hormones.

Introduction

1- INTRODUCTION

Birds are like mammals, homoeothermic and in contrast to the poikilothermic reptiles; they have to maintain a relatively constant temperature of the vital organs. Therefore, heat must be lost or conserved in response to changes in the environment. The internal temperature of birds is higher than that of mammals. Deep body temperatures of the common domestic species (pigeons, ducks, geese, turkeys and chickens) range from 41.2°C to 42.2°C, in contrast to 36.4°C to 39°C for the common domesticated mammals (Borges et al., 2003a). There are several inherent factors which influence deep body temperature. Newly hatched chicks have body temperatures approximately 2.5°C below that of the adult bird; adult levels are not reached until about 6 day's post- hatch. This increase is related to feather cover and the increases of metabolic activity associated with growth. Body temperature also varies with size, breed and sex (Borges et al., 2003b). In general, the body temperature varies inversely with body weight, but this is only true between among breeds and may not hold entirely within a breed. White Leghorns, on the average, have higher rectal temperatures than New Hampshire at the same age, and generally, males have higher body temperatures than females in cooler environments, but this difference is not apparent at higher ambient temperatures. The ostrich (Struthio camelus), the largest living bird, is an inhabitant of semi–arid and desert areas of Africa and, until exterminated, the Near East and the Arabian Peninsula. When exposed to the heat stress of a hot desert, it must use water for evaporation in order to avoid overheating. While its size prevents it from taking advantage of microclimates to the extent that small desert birds and mammals can, its large size is an advantage in its water economy (Sehmidt- Nielsen1964).

-١- Introduction

The ostrich was featured in ancient Egypt and the pharaohs and Egyptian priests adorned ostrich feather. Some examples of ostrich use are displayed in Cairo museum as eggs and feather. A detailed account of the historical aspects of ostrich production in Egypt is reported (Manlius 2001). Prehistoric occupations in the eastern Sahara of Egypt were linked to climatic change during the past 12,000 years (Kuper and Kropelin 2006). Ostrich was bred for meat, egg, and feather production. In Egypt, ambient temperature may exceed than 35 oC, for more than 5 months yearly. While, in some region especially in Upper Egypt, ambient temperature may decrease to below 15 oC, during winter which may cause cold stress on heat adapted birds. Ostrich meat has received increasing attention and become popular throughout the world because of its favorable fatty acid profile and low intramuscular fat content (Sales, 1995 ). Almost all birds fly except a group of flightless bird called ratites. They are characterized by beautiful feather and absence of the keel of the sternum. They are not closely related, they include: the Kiwi of New Zealand, Rea of South Africa, Emus and Cassowary of Australia and ostrich of Africa (Brown et al ., 1982). The ostrich could maintain their body temperature at a stable level during prolonged exposure to a range of ambient temperatures of 15-50 oC. At the highest temperatures they become slightly hypothermic, but many could still maintain their body temperature at a stable level below 40 oC for periods as long as 8 hr at 50 oC ambient temperature. In fact, if the initial cloacal temperature were high due to struggling for instance, some ostriches were able to lower their cloacal temperature at any ambient temperature. These observations suggest that the body temperature could have been maintained for longer periods if the exposure had been continued. The variability among ostrich in body temperature at a given

-٢- Introduction ambient temperature is due to individual differences that resulted undoubtedly from the fact that the birds were restrained and showed various degrees of nervousness or excitement. There are also several environmental factors involved. Such as increased physical activity, caloric intake and environmental temperature all tend to increase body temperature. The extents of these effects are modified according to the diurnal rhythms changes. In species active in daylight, which includes most domestic species, the highest body temperatures occur during the day. By reversing the period of daylight one can also reverse the diurnal body temperature cycle, even though the environmental temperature cycle remains the same (Borges et al., 2003a). The effect of high environmental temperature on the general health and productivity of poultry was concerned long time. With the rapid development of the poultry industry worldwide, especially in developing countries, importation of temperature-zone high performance stocks to hot regions is on the rise ( Yalcin et al., 1997a). The low resistance to heat stress is a big problem for those birds reared in tropic and sub-tropic areas ( Vo and Boone, 1975 and Deaton et al . 1978). Birds subjected to high ambient temperature tends to reduce theis feed consumption and increase water consumption (Yalcin et al., 2001). These negative effects of high ambient temperature (AT) have been found to be more pronounced in birds with higher body weight (BW), and more growth rate (GR) than in those with lower BW and GR (Deep and Cahaner, 2001). Additionally, Veldkamp et al., (2000) found that feed intake and body weight gain were significantly lower for the high ambient temperature AT compared with low AT group while, feed consumption and mortality rate increased in low than high AT.

-٣- Introduction

Heat adaptability indices used for predicting the productive animals under hot conditions are based on their production level, water balance, protein balance, thermal response and heat induced changes in hormonal levels (Habeeb et al., 2007 and 2008). Studies on temperature regulation in birds indicated that they had the ability to regulate their core body temperature within a constant level of 40 °C to 41 °C within a wide range of ambient temperature (Hillmn et al ., 1985). The respiration rate is higher in birds exposed to high temperature than those raised under normal ambient temperature (Kalamah, 2001). Birds have no sweat glands, and under heat stress they rely upon increased evaporation from the respiratory system as a major avenue for heat dissipation. Knut, et al., (1969) too old interested in the role of the respiratory system in evaporation, and particularly in the sites of evaporation, but in mammals an increased ventilation causes alkalosis, in birds the presence of large air sacs connected to the respiratory system may have radically different effect on the gas exchange in the lung. The fact that the ventilation of the respiratory system can be modified by heat stress without change in the rate of oxygen consumption may provide an avenue for investigation of the poorly understood air-sac system of birds. Egypt was mentioned as one of many countries (Transvaal, Australia Algeria, Tunis, Sudan, Madagascar and the U.S.A.) involved in ostrich production in 1913. An attempt to recapitulate the distribution of ostriches in Egypt from prehistoric times was attempted (Manlius 2001). Scanty literature is available about thermoregulatory mechanisms of local Egyptian breeds against heat and cold. Also the determination of lower and upper critical temperatures of ostrich will help in avoiding the effect of cold and heat stress on their production by using managerial

-٤- Introduction mitigation of heat and cold above and below the thermoneutral-zone. Therefore, the present study was designed to evaluate the effect of seasonal and diurnal variation in summer and winter climates, on thermoregulatory mechanisms and changes in some physiological and blood biochemical parameters in ostrich.

-٥- Review of literature

2- REVIEW OF LITERATURE

2-1-Thermoregulation and thermoneutral-zone Adult birds are homeothermic and provided with physiological mechanisms by which they can maintain their deep body temperature constant within the Thermoneutral- zone. Birds are ‘heat stressed’ if they have difficulty achieving a balance between body heat production and body heat loss. Heat is produced by metabolism within the body, which includes maintenance, growth and egg production. Heat production is affected by body weight, species and breed, level of production, level of feed intake, feed quality and, to a lesser extent, by the amount of activity and exercise. While, heat can be lost in a variety of ways such as radiation, convection, conduction and evaporation, if heat production becomes greater than ‘maximum heat loss’ either in intensity (acute heat stress) or over long periods (chronic heat stress), birds may die. The body temperature of the broiler must remain very close to 41°C. If body temperature rises more than 45°C above this, the bird will die (Defra, 2005). This can occur at all ages and in all types of poultry. When conditions mean the ‘upper critical temperature’ is exceeded, birds must lose heat actively by panting. Panting is a normal response to heat and is not initially considered a welfare problem. But as temperatures increase, the rate of panting increases. A Thermoneutral-zone is defined within which the heat production of a bird is independent of body temperature. In this zone the heat produced by a bird is related to its live weight and feed intake. The zone is bounded by a lower critical temperature and an upper critical temperature. The lower critical temperature is the air temperature which must be maintained at all times to ensure that feed energy is not diverted unnecessarily for production purposes (Nikita- Martzopoulou et al ., 1985 and Wachenfelt et al ., (2001)).

-٦- Review of literature

2-2-Thermoregulatory mechanisms It is postulated that 5-hydroxytryptamine or acetylcholine, rather than nor-epinephrine, may be an important neurotransmitters in the neural pathways for thermoregulation in chickens, even though their action on thermoregulation is no minor compared with or epinephrine (Hillman, 1980). Adult birds take about five days to acclimatize to high temperatures. Birds are more susceptible to sudden, large changes in temperature. The first very hot days after a cool spring often result in increased incidence of heat stress. Some of this will be due to poor acclimatization, but some will be due to managers being less well prepared than later in the summer (Defra, 2005). The cellular mechanism in response to heat stress is characterized by a reduction in protein synthesis (Craig, 1988). Sandercock et al. (2001 ) reported that exposure to acute heat stress significantly increased deep-body temperatures, panting induced acid/base disturbances, and plasma Creatine kinase activities, reflective of heat stress induced sympathy. Exposing birds to heat stress has been reported to reduce feed intake and increase water consumption (Krista et al ., 1979; Van Kampen 1981), to reduce the metabolic rate and hence heat load which is one of the possible mechanisms of thermo tolerance that is regulated to a large extent by triiodothyronine (T 3) (McNabb and King, 1993). In addition, changes in the blood system are part of the thermoregulatory response acquired by birds to enable them to withstand heat stress. Stressed birds established a cascade of physiological adaptive responses include elevation of glucose and electrolytes (Pech-Waffenschmidt et al ., 1995). Increased mineral excretion is one of the important

-٧- Review of literature consequences of heat distress. High temperature affects availability of minerals (Belay et al ., 1992; Belay and Teeter, 1996; Smith et al ., 1995). Also, plasma total proteins may be used as useful criterion for heat stress in birds (Eberhart and Washburn, 1993c). Filali-Zegzouti et al., (2000) and Marjoniemi (2000) stated that glucagon is a potential mediator of non shivering thermogenesis (NST) in birds. They concluded that the thermogenic action of glucagons in birds is probably indirect and involves at least the mobilization of lipids and sympathy- adrenal stimulation. The changes in peripheral noradrenergic activity during cold acclimation could be associated with adaptive changes leading to (NST). Bedu et al., (2001) found that liver contribution to glucagon-induced thermogenesis in vivo was estimated to be 22 % in Thermoneutral (25 oC) and 12 % in cold-acclimated (4oC) ducklings. Glucagon stimulated gluconeogenesis from lactate in duckling liver and the stimulation was 2.2-fold higher in cold-acclimated (CA) than in thermoneutral fasted birds. These results indicate a stimulated hepatic oxidative metabolism in (CA) ducklings but hepatic glucagon-induced thermogenesis (as measured by LVO2) was not improved. The role of the liver is suggested in duckling metabolic acclimation to cold through an enhanced hepatic gluconeogenesis under glucagon control.

2-3- Feed and water consumption Because feed is more expensive than any other input applied to the production of broilers, Hatlow and Ivey (1994); Cooper and Washburn (1998); Deeb and Cahaner (1999) and Koh and Macleod (1999) studied the effect of ambient temperature (AT) on body temperature (BT), body weight, feed consumption and feed conversation. They found

-٨- Review of literature that as AT increased, body weight, feed consumption and conversion decreased while water consumption and body temperature increased. These negative effects of high AT have been found to be more pronounced in chickens with higher body weight, and more growth rate (GR) than in those with lower BW and GR (Emmans and Kyriazkis, 2000 and Deep and Cahaner, 2001). These results are in agreement with May and Lott (2000) which studied the effects of environmental temperature on growth and feed: gain ratio. They showed that heat stress had a deleterious effect in both growth rate and feed conversion. The amount of water consumed per day is more on days immediately after the temperature rise than it is a few days later. For example, the rate of water consumption was doubled immediately after environmental temperature was increased from 21.2 to 37.8°C (Donkoh, 1989). In addition, at high ambient temperature, evaporative cooling is the most important mechanism of body temperature control. Water consumption increases when chickens are exposed to high environmental temperatures (North and Bell 1990), and survival during heat stress is dependent upon water consumption. Fasting improves resistance to heat exposure under some situations. Belay and Teeter (1996) reported that water intake and excretion increased (P<0.05) by 78 and 133% respectively, during heat distress. They suggested that broiler chicks adjust water intake and renal handling of water during acute heat distress and that such handling influences evaporative heat loss (EHL). Wiernusz and Teeter (1993) found that the increased heat load, with elevated feed consumption, was dissipated by increased sensible heat loss solely, as evaporative heat loss remained constant in order to maintain body temperature of 41-42 °C. When environmental temperature increases, this

-٩- Review of literature limit sensible heat loss, while evaporative heat loss (Latent heat) increased, and feed consumption decreased. Also, Veldkamp et al., (2000) studied the effects of ambient temperature on feed consumption, body weight gain and mortality rate. They found that temperature had a clear effect on performance during all age periods. They concluded that feed intake and body weight gain were significantly lower at the high AT compared with low AT group. While water consumption and mortality rate were more increased in high AT than in lower AT, the actual quantity of water is dependent, however, on many factors, i.e. body weight, physical activity, rate of egg production as well as environmental temperature (Zulkifli et al., 2009). However, when water consumption was measured for one week after this rise in temperature it was only 35 percent greater at 37.8 than at 21.2°C. A high initial increase in the intake of water also took place when the ambient temperature was raised from 21.2 to 26.6°C and from 26.6 to 32.2°C respectively (Siegel, 1995). Similarly, Lin et al., (2006) found that laying hens consumed 1.5 to 1.7 times as much water as feed in the environmental temperature range 6.7 to 4.5°C and about five times as much water as feed at 37.8°C. Olanrewaju et al., (2007) showed that increasing the environmental temperature caused an increase in water consumption by pullets. They reported that water consumption ranged from 150 cm /bird /24 hours at 35°C to 299 cm at – 2.5°C. The ratio of water to feed was approximately 2: 1 when air temperature was 18.3°C and 4.7: 1 at 35°C. The same results were found by Ahmad and Sarwar (2006) and (Derjant et al ., 2002) whose indicated that water consumption increased as temperature increased. The amount of water loss increased from 5 g per hour at normal temperatures to 30 g per hour when the bird was panting (Guyton and Hall 2001).

- ١٠- Review of literature

Feed consumption is influenced by, breed, age, sex, nutritional state and dietary factors as well as by diurnal variations activity, feathering state and group size. But for a given time the major factor that influence the feed intake is the environmental temperature (Meltzer, 1987). A bird in high ambient temperature has been reported to reduce feed intake and water consumption and feed conversion efficiency (Guo, et al., 1998). Smith (1994) demonstrated that acclimation to high ambient temperature was influenced by sex. Both sexes responded differently to heat stress. Male broilers consumed up to 37 % more water than similarly raised females. Since the past five decades researchers have shown that feed consumption is inversely related to high environmental temperature (Xin et al ., 1992; Mitchell et al ., 1992). Teeter, et al . (1992); Wideman et al . (1994) and Saiful et al. (2001) found that birds previously acclimated to cycling heat distress temperature (24 to 35 °C) for two 24-h cycles exhibited 24% lower feed consumption than birds previously housed at 24 °C and experiencing their first heat distress exposure. They suggested that acclimation may help broilers to cope with heat stress. During the winter, standing was an important behavior at similar levels to that observed for ostriches during the summer, presumably due to vigilance particularly during feeding (Ross and Deeming, 1998). Behaviors' involving activity, such as pacing and walking, occurred at a low frequency during the winter. Feeding and foraging were the most important behaviors for both genders during the winter. The proportion of the time budget taken up by eating during the colder weather was 5-6 times higher for males and doubled for females than that previously reported for these behaviors per formed by ostriches during summer months ( Mc Keegan and Deeming, 1997; Ross and Deeming, 1998). Moreover,

- ١١- Review of literature gender differences described for adult ostriches in the summer were not present during winter months. During the summer, few consistent patterns of time of day on activity have been reported, although delivery of the concentrate feed was related to a reduction in foraging activity in both genders (Mc Keegen and Deeming, 1997) during the winter, feeding activity was highest during the morning, and foraging activity was highest during the afternoon. This pattern appeared to be related to the presence of concentrate feed, which was delivered during the morning before the observation periods started. Casual observation made during the latter part of the study suggested that foraging in the afternoon was higher in enclosure were the concentrate had been consumed. This observation needs confirmation. (Deeming, 1997) suggested that the winter conditions would influence the behavior of ostrich. Maintenance of body temperature is probably the main reason for such high rates of food consumption in both genders. A study of the influence of specific weather condition during winter months also found significant effects of climate on the frequency of feeding in adult ostriches ( Deeming, 1998; Para et al. 2002 and Marder et al. 2003). Two important problems currently affected broiler production are: the sensitivity of rapid-growing birds to high temperature and the excess of water excreted by birds. Alleman and Leclereq (1997) and Mariam et al. (2001) were reported that water consumption for heat-stressed chickens raised under 32 °C (2.845 and 3.066 liter/bird) compared with those of control reared under 20 °C (1.687 and 1.925 liter/bird). Water \ gain ratios were 3.052 and 3.396 compared with 6.977 and 6.669 ml water\g body gain for control and heat-stressed birds, respectively.

- ١٢- Review of literature

2-4- Body temperature Birds, like all homoeothermic animals, maintain a constant body temperature (BT) over a wide range of ambient temperatures (AT). However, when the physiological and behavioral response to high AT is inadequate, an elevation in BT occurd, causing a decrease in appetite, growth rate, and productivity (Ain Baziz et al., 1996; Geraert et al., 1996). The ability of an animal to maintain its BT with the normal range depends on a balance between the internally produced heat and the rate of heat dissipation. The amount of internally produced heat depends on BW and feed intake. The rate of heat dissipation depends on environmental factors, mainly AT, and feather coverage. Midtgard (1989) reported that low temperature caused increase in heat production of commercial turkey hens. Donkoh (1989) indicated that the body temperature of chickens started to rise when they suddenly exposed to an ambient temperature above (30 °C). If the ambient temperature was higher than that of the bird’s body, but birds were exposed at a slower rate, they maintained a “normal” body temperature until the ambient temperature reached 91 °F (32.8 °C). There have been many reports on the influence of ambient temperature on the rectal temperature of birds (Ahmed et al ., 1987; May et al ., 1987 and Geraert et al ., 1996) which reported that increased body temperature is one of the responses associated with heat stress and declared that rectal temperature in domestic fowl is good indicator of heat stress. In a consecutive succeeding, Deep and Cahaner (2001) determined the livability of White Leghorn Chickens that were exposed to heat stress 40.6 – 43.3 °C for 6 or 24 hr with those birds exposed to 37.5 °C (control). The body temperature was measured after 20 minutes post treatment.

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They reported that significant relationship (r =-0.454) between body temperature and survival time were negatively correlated. Mac Leod et al., (1993b) showed that body temperature was significantly different between strains of chickens according to their body weights when exposed to heat stress 50 °C up to 4 hours. The response to heat in their experiment was assessed by heat prostration. Muiruri and Harrison (1991) reported that rectal temperature of heat stressed fasted and fed hens with a 34±1oC roost were increased 1.20 and 1.32 °C, respectively, whereas those of hens with the water-cooled roosts increased only 0.22 and 0.28 oC during the heat stress period, respectively. The results obtained by Abdel-Mutaal (2003) suggested that rectal temperature was higher in acclimated birds than for those reared under normal temperature. Recently, El-Badry (2004) worked on Muscovy and Domyati ducks, reported that the rectal temperature after exposure to 42 °C at 8 and 12 weeks of age heat stress was significantly higher than in the unstressed control. Rectal temperature decreased significantly in early-heated birds than heat stressed birds at 8 and 12 weeks of age. He noted that early heat exposure caused decrease in rectal temperature in Muscovy compared with Domyati ducks at different ages. A significant interaction between breed and heat stress for rectal temperature was reported. Hoffmann (1991b) stated that when ambient temperature was altered hourly in 5oC steps from 40 to 45oC there was a parabolic relation between heat production of broilers and environmental temperature at medium and high temperature. At low ambient temperature increases from 5 to about 20 oC heat production decrease. Thermoneutral temperature was dependent on age (live weight) and environmental temperature. Thermoregulatory heat production

- ١٤- Review of literature decreased with increasing age and ranged from (24 KJ/kg 0.75 at the beginning to 8kJ/kg 0.75 day-1oC) at the end of the experiments. Birds being warm blooded, are non-sweating “homeothermic” which means that their body temperature is maintained at a relatively narrow range. The upper limit of their circadian rhythm is about 43.0 °C and the lower limit about 40.6 °C. They are also “endothermic” their body temperature might raise by 1 or 2°C which they exposed to a hot ambient temperature (Etches et al ., 1995) or performing physical activity by the later is particularly true under conditions of mild or moderate heat stress (Sturkie, 1986). The thermoneutral-zone is the range of ambient temperature where metabolic rate is not changed or the homeostasis is efficient. Thermoregulation is achieved by both physiological and behavioral means. The limits to this range are referred to as the upper and lower critical temperature. Some variation may be observed according to the humidity and perhaps other physiological factors (Van Kampen, 1981). The hens' ability to dissipate heat is influenced by the skin temperature rather than by the deep body temperature. As the temperature of the air surrounding the bird increases, the blood vessels dilate, increasing the flow of blood thereby increasing the amount of heat lost (North and Bell, 1990). In chicken, more extensive efforts to decrease heat load involve panting by the vaporization of moisture from the damp lining of the respiratory tract. This is a major method of heat elimination from the body of the bird when the ambient temperature is high (Hales, 1983 and Yalcin et al., 2001). When the temperature at the bird surface is greater than adjacent air, heat is lost from the body by radiation. Therefore, the rate of heat loss is influenced by ambient temperature.

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Heat stress conditions occur in chickens when temperature is greater than 30 °C and relative humidity is greater than 50%. High environmental temperature and metabolism of food are major suppliers of body heat. This suggestion was confirmed later by El-Gendy and Washburn (1989) who indicated that in response to heat stress, as the body temperature increased at a relatively slower rate, the bird appeared to be more resistant to heat than mammals. Studies on temperature regulation in birds indicated that they had the ability to regulate their core body temperature within a constant level of 40.5 to 41.0 °C within a wide range of ambient temperature. However some birds could not withstand high air temperature. They were not able to pant fast enough to remove the heat from their body. When their body temperature rose above the physiological maximum, prostration and death occurred (North and Bell, 1990). In broilers, May et al . (1986) suggested that increased body temperature was one of responses associated with heat stress. Kutlu and Forbes (1993) confirmed the results of May et al . (1986). Marsden et al . (1987) stated “the estimated heat output of the birds increased during the course of the experiment at the lower air temperature (15, 18, and 21 °C) but decreased with time at high air temperature (30 °C). Ait-Boulahasen et al . (1993) found that under heat stress (41 °C) body temperature of broiler chickens increased compared with control (24 °C). Sturkie (1986) reported that the body temperature of chicken depends on bird size, environmental temperature, age and sex. Also, deep-body or rectal temperature is a good indicator for both heat stress and acclimation (El-Gendy and Washburn, 1995).

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Hatlow and lvey (1994) and Teeter et al . (1987) found that feed restriction of 6-wk old Arbor Acre chickens before the onset of heat stress at 35 °C had lower body temperature than those that under feed restriction after the onset of heat stress. The body temperature of laying hens increased to 42.8 °C after 4 h. of exposure to 35 °C, and declined progressively at the 2nd and the 3rd day of exposure. The mean body temperature was 41.9 °C on the 3rd day, which is the average body temperature of the adult fowl (Fujita and Yamamoto (1988). Khalifa (1991) found that under heat stress in summer (31.6-38.1 oC) the mean cloacal temperature of the normal 71- week-old white Lohman selected Loghorn (L.S.L) laying hens was (40.94±0.17 oC). Lott (1991) reported that acclimated and unacclimated broilers given access to feed had similar body temperature, but the fasted acclimated broilers had a significantly lower rectal temperature than the unacclimated group. Teeter et al . (1992) found a significant (P < 0.01) acclimation history by ambient temperature interaction, with acclimated birds having a higher rectal temperature (42.3 versus 41.2 °C) when housed at 24 °C and a lower rectal temperature (44.2 versus 44.6 °C) when exposed to 35 °C than did the unacclimated controls. Rectal temperature in the 24 °C and 35 °C environments increased linearly (P < 0.01) as feeding level increased for both acclimated and unacclimated birds. Eberhart and Washburn (1993c) compared the changes in BT of chronic heat stressed naked neck birds at 32 °C with 21 °C (control) after being exposed to acute heat stress conditions (40.5 °C). Birds grown at 32 °C had significantly lower basal BT at 8-wk BW, and significantly

- ١٧- Review of literature smaller change in BT when exposed to 40.5 °C than birds grown in 21 °C. El-Gendy and Washburn (1995) studied the genetic variation in body temperature of broilers and its response to short term acute heat stress 43.2 °C for 45 minutes. They reported an increase in mean body temperature by 1.47 °C when birds were exposed to abrupt elevation in ambient temperature. Zhou et al., (1996b) and Lin et al (2006) reported that heat production abdominal temperature shank skin temperature and respiration rate increased at (36 oC). Hai et al, (2000) found that rectal temperature of neonatal chicks was increased by high ambient temperature (35 oC) or high humidity (85%). There was significant interaction between temperature and humidity the harmful effect of high temperature on rectal temperature was aggravated by high humidity. Under low temperature (27 oC) humidity had significant effect on the rectal temperature. During the first day exposure to (27 oC) the rectal temperature of chicks had a tendency to decrease, but in subsequent days it was not affected by low temperature. They concluded that the thermoregulation mechanism in neonatal chicks is not well developed; they are more sensitive to high temperature than low temperature. Olanrewaju et al. (2007b) found that during the first day of exposure to 27 oC the cloacal temperature of neonatal chicks had a tendency to decrease, but in subsequent days it was not affected by low temperature. They concluded that the thermoregulation mechanism in neonatal chicks is not well developed; they are more sensitive to high temperature than low temperature Zhou et al., (1996b and . 1997) reported that heat production rose with increase in food intake and environmental temperature. Abdominal temperature shank

- ١٨- Review of literature skin temperatures and respiration rate also increased but, at (36 oC). El-Nabarawy (1997) found that the body temperature of Japanese quail was significantly lower in heat stressed chicks exposed either 38 °C or 42 °C compared with the control 32 °C at 3 wk of age. Additionally , Turnpenny et al., (2000) found that the prediction of body temperature for chickens was most sensitive to ambient humidity at high air temperatures, and to body resistance. Tolba (2000) and Yahav (2000) reported that body temperature of chicks was significantly increased (41.5 °C) after the exposure of heat stress compared to unexposed acclimated birds (40.4 °C). However, body temperature after the exposure were significantly less in acclimated groups (40.6 °C) compared with non acclimated group (41.5 °C) at 38 days of age. While, Malheiros et al. (2000) showed that chicks raised at low environmental temperature (20 oC) had significantly lower cloacal and surface temperatures than did other birds. The radiation heat loss was nine times higher than for the birds kept at 35 oC at 7 day of age. Also Sosnowka-Czajka and herbut (2001) observed that the increase or decrease in temperature (2 h increase or decrease of air temperature by 10 oC on day 14 from the Thermoneutral temperature did not affect rectal temperature of the broilers. Wachenfelt et al. (2001 ) found that sensible heat produced by the hens decreased as the ambient temperature increased and was lower during the day than at night. Meanwhile, latent heat production increased with increasing ambient temperature and was higher during the day than at night. Total heat production decreased with increasing

- ١٩- Review of literature temperature because the hens by thermoregulation, decreases their metabolism in order to maintain a constant body temperature. Yahav and Hurwitz (1996); Yahav and Plavnik (1999a) and De Basilio et al . (2001) reported that body temperature increased significantly as a result of a 24 h heat exposure at the age of 5 d. At the age of 42 d, a challenge of acute heat resulted in an increase in cloacal temperature, but the increase was significantly more moderate in the conditioned group than in the controls. Saiful et al. (2002) found that heat production (HP) of single Comb White Leghorn laying hens decreased with the increase of ambient temperatures (Ta) from 25 to 29 and 33 oC, and with the decrease of food intake (F1). They calculated an estimation of the increase of HP for 1 g of F1 and it was approximately 3.9 kJ at each (Ta) in the ad libitum fed hens. They stated that almost 28% of the daily HP was related to the activity at each (Ta). Brown-Bradl et al. (2003) reported that core body temperature is an important physiological measure of animal thermoregulatory responses to environmental stimuli. In another study, Yahav and Hurwitz (1996) showed that in male broiler the cloacal temperature of the surviving birds increased significantly by the end of 24 h heat exposure to 36 ± 1 °C during the 1st week of life. But the effect was decreased as the birds became older. They noted that skin temperature was not changed after the birds being exposed to heat stress at early ages; however, at 42 days of age it was significantly lower. Chalibog (1990) found that diurnal temperature from 30 °C for 4 d in 5-wk old White Plymouth Rock chicks reduced oxygen consumption, carbon dioxide production, respiratory coefficient and heat production 2 h after reaching the maximum temperature.

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In addition, it has been reported that acclimated broiler chickens had a lower body temperature when exposed to high temperature than unacclimated controls (Lott, 1991 and Teeter et al ., 1992). Yahav and Hurwitz (1996) demonstrated that 42 d-old broiler chicken exposed to acute heat stress 36± 1°C increased body temperature compared with the control. In Japanese quail, Durgan and Kestin (1998) found that increase heat exposure of the chicks from 20 °C to 42 °C (8 °C/h) for total of 150 min increased body temperature. Deeb and Cahaner, (1999) suggested that measuring body temperature can be used as indicator of the level of stress at high AT. Altan et al . (2000) reported an increase in body temperature when 44-d-pld broiler chicken exposed to high temperature 39 ± 1°C for 2 h. Marder et al. (2003) found that body temperature at a Ta of 47 oC was lower for desert birds (43.3 oC) than for temperate-zone birds (43.6 oC and rain forest birds (44.4 oC). Guerreiro et al . (2004) compared two groups of broiler chickens reared at Thermoneutral temperature up to 47 DOA then exposed to hot environment from 31 to 33 °C or exposed to gradual heat stress from 28 °C to 40 °C at a rate of 2°C/h. They showed that body temperature of birds reared at high temperature increased steeply during the first 3 h of heat stress (1.06 °C/h) and more slowly thereafter (0.59 °C/h). They added that, broilers reared at Thermoneutral temperature and exposed to heat stress later showed a small increase in the first 4 h of heat stress (0.18 °C/h) and then the body temperature increased sharply (0.72 °C/h). Nevertheless, both groups presented similar final body temperature by the end of the stress period.

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Finally, El-Sheikh et al . (2004) reported that females Japanese quail exposed during 25 days to solar radiation temperature for 12 h/d in summer (July) from 6 a.m. to 6 p.m. had significantly (P<0.01) higher body temperature and respiratory rate levels compared to unexposed groups.

2-5- Hematological studies The effect of temperature variations on hemoglobin values in turkey was studied by Yahav and Plavnik (1999). They found that hemoglobin values of 6-week old turkey exposed to 37.8 °C for 6 weeks were 11.9 compared with 12.0 g/dl for control. They postulated that the effect of high temperature on the hemoglobin value was influenced by the age of bird at exposure. However, Shlosberg et al . (1996) stated haematocrit percent (Ht %) might vary greatly, depending upon environmental factors related to ambient temperature and age of broiler chicks. Also, Yahav and Hurwitz (1996) reported that, blood haematocrit level in male broilers 42 d-old was decreased after being exposed for 6 h to heat stress (35 ± 1 °C). Also , Osman (1996) found that exposed broiler chicks to 42 °C for 2 and 4 h decreased hemoglobin concentration by 12 % in both heat treatments compared with the control birds. In general, Vo et al . (1978) reported that hemoglobin values were increased over times in male broiler than females at all temperature and there was decrease in hemoglobin values at the higher temperature that leads to hemodilution due to increase water consumption. In the same direction, Sturkie (1986) reported that heat stress increased water consumption due to heat stress exposure lead to hemodilution. This will cause a decrease in red blood cells (RBC’s) which in turn decreases hemoglobin

- ٢٢- Review of literature concentration and packed cell volume. These demonstrate the positive relationship between red blood cells and hemoglobin concentration and haematocrit. Yahav et al . (1997b) observed that the erythrocyte number of chicks exposed to high environmental temperature 6 (30 °C) from 7 to 42 days of age; was 2.78 ×10 /ml bloods 6 compared with 2.95 × 10 /ml blood in the control group. On the other hand, Yahav et al . (1997c) reported that hemoglobin concentration did not change when 8 weeks-old Cobb male broiler chicks exposed to 35 °C for 6 h when compared with control group at 22 °C. Early research has shown that haematocrit values of the adult chickens were higher in winter than in the summer. (Atta, 2002) observed that the packed cell volume (PCV) increased when chickens were exposed to lower temperature as compared with higher environmental temperatures . Krista et al . (1987) studied the effect of temperature variations (15.7, 26.8, and 37.8 °C) on haematocrit values in turkey at 6 weeks. The lowest values were observed for turkey exposed to 37.8 °C. PCV values were 36.5, 36.9, 30.8 and 34.5 % for 15.7, 26.8, 37.8 °C and control groups, respectively. Osman (1996) found that during the first day of exposure to heat stress (42 °C) the haematocrit values were decrease by 15 % and 20 % for the birds exposed to heat stress for 3 and 4 hrs, respectively, compared with those in control birds. Thereafter, the haematocrit values gradually decreased at the second and third day of exposure to heat stress. On the other hand, Yahav et al . (1997a) reported that exposed 8 week-old Cobb Male Broiler Chicks to 35 °C for 6 h did not affect heamatocrit value compared with the control group at 22 °C. But , Shlosberg et al . (1996) postulated that

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PCV might vary greatly, depending upon ambient temperature and age of the broiler chicks. With respect to the effect of early heat exposure on hematological parameters, it was observed that the mechanisms associated with the induction of thermo tolerance by early-age temperature conditioning involve homodynamic changes decrease in heamatocrit (Yahav and Hurwitz 1996 and Yahav et al ., 1997a). These results were supported by results obtained by Yahav and Plavnik (1999 ) and Uni et al . (2001) who demonstrated that, thermal conditioning reduced the increase of haematocrit with age. The same trend was noticed by Tolba (2000) who found that acclimated birds reflected in significant reduction in their haematocrit values (26.75 vs. 29.50) compared with unacclimated control birds. While, heat stressed birds showed significant decrease in their haematocrit values (23.50 vs. 29.50) compared with control birds at 38 days of age. Birds reared at high ambient temperature had lower haematocrit values than those raised at lower ambient temperature (Sturkie, 1986 and Zhou et al ., 1998). Also, El- Badry (2004) showed that heat exposure significantly reduce haematocrit values in duck 12 wks old, as compared with non- stressed ducks. Furthermore, Bhattacharyya (1990) reported that exposed Japanese quail to 42 °C for half hr led to significantly decrease in hemoglobin concentration compared with the control group that exposed to 25 °C. Also, Tolba (2000) recorded that stressed birds had significant lower hemoglobin values (9.14 g/dl) compared with control group (12.65 g/dl). While acclimated birds showed significantly higher hemoglobin concentration (10.87 g/dl) compared with heat stressed birds (9.14 g/dl) at 38 days of age.

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Finally, Mashaly et al., (2004) reported that birds exposed to low ambient temperature showed significantly reduced body weight, which coincided with a reduction in energy intake and with changes in the circulatory system to accommodate higher oxygen demand. These changes included a significant increase in haematocrit, hemoglobin concentration, and blood oxygen capacity. At the relatively high ambient temperature, changes to accommodate heat dissipation included significant increase in plasma volume and panting rate. These compensations were sufficient to control body temperature (Tb). However, the higher energy expenditure for maintenance followed by significantly higher plasma triiodothyronine (T 3) concentration, but with lower energy intake at low (Ta). Suggest a physical limitation in the ability to further increase energy intake as (Ta) declines.

2-6-Blood biochemical studies Heat stress caused a significant reduction in plasma total protein, albumin and globulins than control group (Zhou et al ., 1998; Hassan, 1999; Kalamah, 2001). In birds, plasma total protein varied between sexes and among different seasons (Lisano and Kennamer, 1977). Al- Heeti et al . (1985) found that the highest plasma total protein levels were recorded under moderate temperature and the lowest ones were recorded under high temperature. Habeeb et al., (1993) found that the overall means of total protein, albumin, and globulin significantly decreased, due to exposure of the animals to high temperature. Hattingh (1982), Trammell et al., (1988) and Morera et al., (1991) reported similar results. Pech-Waffenschmidt et al . (1995) reported that changes in the blood system are part of the thermoregulatory responses acquired by birds to enable them to with stand heat

- ٢٥- Review of literature stress, stressed birds established a cascade of physiological adaptive responses include elevation of plasma glucose. In addition , Zhou et al . (1998) reported that plasma total proteins and albumin concentration of Cobb broilers were decreased when it were exposed to 30 °C for 3 to 12 h for three day while, globulin concentration were lower in winter (2.88 gm/100 ml) than in summer (3.06 gm/100 ml). El-Nabarawy (1997) illustrated the diagrammatic representation of the serum protein fractions of Japanese quail chicks at the age of 4 wk that were exposed to chronic heat stress. The electrophoresis patterns revealed that chronic heat exposure 38 °C of birds from one-day to four weeks of age resulted in high intensity of albumin fraction compared with albumin of other birds that were kept at either 42 °C or 32 °C. El-Badry (2004) found that heat stress caused insignificant reduction in plasma total protein, A/G ratio and significant reduction in albumin than that of control ducks at different ages. He also found that early heat exposure caused an increase in plasma albumin in control birds than heat- stressed birds while plasma total protein and its fractions were almost similar to the control. There were significant differences in A/G ratio due to treatment at 8 weeks of age. Heat stress decreased A/G ratio compared with control. Also, acclimation to heat stress at early age caused an improvement for globulin value. Recent evidence suggested that plasma total protein (PTP) and the increased synthesis of a group of proteins known as the heat shock proteins (Hsp’s) might give an indication of the effect of heat stress in birds ( Al-Heeti et al ., 1985; Eberhart and Washburn, 1993c; Kutla and Forbes, 1993 and Etches et al ., 1995). On contrary, Dafaila et al ., (1987) and Essa (1992) indicated that the levels of blood total lipids and cholesterol decreased with elevation of ambient temperature.

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Morita et al. (1989) and Donkoh (1989) reported that the plasma glucose concentrations were significantly decreased at 16 hours after cold exposure (0°C for 24 hours) while plasma insulin concentration rapidly and significantly increased after 12 hrs of cold exposure. No significant difference in plasma glucagon and free fatty acids concentration when exposed to cold. They suggested that the decrease in plasma glucose concentration in hens exposed to cold might be due to the abundant release of insulin from pancreatic B-cells. In other study, broilers exposed to 40 °C and 30% relative humidity for 3 hrs and showed significant differences between heat-stressed surviving and non-surviving chickens in the blood levels of glucose, (Bogin et al ., 1996). Sands and Smith (2002) reported that under heat stress plasma glucose concentration for broilers decreased from 206 to 192 mg/dl for thermoneutral and heat stressed birds, respectively. Sahin et al . (2002) found that serum glucose concentration of broiler chicks reared under heat stress (32 °C) was 214 mg/dl. In contrast, Sands and Smith (2002) reported that under heat stress plasma glucose concentration for broilers decreased from 206 to 192 mg/dl for Thermoneutral and heat stressed birds respectively. Bogin et al . (1997) and Zulkifili et al ., (2009) reported that prolonged heat stress (32 °C) in broiler chickens for 5 wks increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities. Exposed Japanese quail female 50-d old to solar radiation temperature for 12 h/d during the summer (July) were reduced plasma total proteins compared with control. The solar radiation also reduced albumin, total lipid and

- ٢٧- Review of literature cholesterol compared with unexposed birds (El-Sheikh et al ., 2004). Malan et al. (2003) found that the fast-growing chickens had low heat production per kg metabolic body weight H/W 0.75 values compared with slow-growing lines. These fast growing breeder sires had lower plasma thyroid hormone, reduced proportional lung weights, low arterial PO 2 and high arterial pCO 2 pressures compared with the slower- growing lines. Sex of ostriches had a little influences on various plasma values, female ostriches had insignificantly lower concentrations of total protein, cholesterol, uric acid, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) than males (Levy et al ., 1989). They added that ostriches, as common with reptiles and other birds, have uric acid as the end product of nitrogen and metabolism. Okotie-Eboh et al . (1992) determined references serum biochemical values in ostrich’s blood for total protein (g/dl), albumin (g/dl), ALT (U/L), AST (U/L), cholesterol (mg/dl) and uric acid (mg/dl). The values of these components ranged between 3.0 – 5.1, 1.4 – 2.9, 6.0 – 75, 195 – 304, 36 – 148 and 5.2 – 12, respectively for males and ranged between 2.6 - 5.8, 1.2 – 2.3, 3 – 65, 22 – 270, 37 – 202 and 5.9 – 26.6, respectively for females. They added that there were no significant differences in these values according sex, except for uric acid concentration which decreased significantly (P<0.05) in male ostriches with respect to females. Another plasma biochemical values for the previous components were documented by Mushi et al . (1998), and the values ranged between 24 – 76 (g/l) for total protein, 10 – 33 (g/l) for albumin, 1.30 – 4.20 ( µmol/L) for cholesterol and 200 – 500 ( µmol/L) for uric acid. Quintavalla et al . (2001) stated the following values for some blood plasma components for males and females

- ٢٨- Review of literature ostrich respectively; total protein (4.19 and 4.88 g/dl), albumin (2.42 and 2.34 g/dl), ALT (9.31 and 9.41 U/L), AST (134.87 and 122.90 U/L) and uric acid (6.47 and 7.17 mg/dl). In addition, the values for total protein and uric acid were 39 – 56 g/L, and 351 – 649 µmol/L, respectively, in blue neck ostriches Verstappen et al . (2002). Moniello et al . (2005) reported the results of the metabolic profile obtained as a function of collection site and ostrich age. They pointed out that blood of the ostriches of two years of age have the following values of plasma chemicals elements in the African ostrich; total protein 37.36 g/L, cholesterol 1.75 mmol/L, uric acid 435.4 mmol/L, GOT 249.6 U/L and GPT 9.93 U/L. On the other side, Moniello et al . (2006) showed that total protein was higher in females (43.35 g/L) than males (38.90 g/L) and referred that to increase the level of estrogen hormones in females responsible of the high content of globulin. The same authors added that the other blood plasma parameters did not show significant differences by sex. They added that blood profiling, initially used to detect sub-clinical metabolic disorders due to incorrect management and on medium large farms, metabolic profile determination can be very useful when a decrease in production and/or reproduction is not associated to clinical sings. The same authors added that the values of blood plasma components can change according to many factors such as age, season, physiological status, sex blood collection method and stress. They also added that the variation in serum enzymes is to be attributed to the evolving of physiological conditions that change the activity of the different systems, where the higher values of plasma enzymes are found in young ostriches where tissue growth and change are higher. Borges et al ., (2003) and Tolba et al . (2005) suggested that when birds were exposed to high temperature they use compensatory mechanisms to maintain acid-base homeostasis

- ٢٩- Review of literature and concentrations of blood nutrients and physiological variable. Recently, El-Habbak et al . (2005) found that the plasma glucose level for laying hens at 46 week of age were 205 g/dl when measured at spring season. Khazraiinia et al . (2006) claimed that there were no significant differences detected between sexes of the ostrich in each of plasma blood components studied. They reported the following values for males and females, respectively; total protein (3.39 Vs 3.38 g/dl), albumin (1.49 Vs 1.50 g/dl), GPT (11.93 Vs 16.26 IU/L), GOT (359 Vs 373 IU/L), cholesterol (65 Vs 64 mg/dl) and uric acid (13.46 Vs 11.04 mg/dl). On the contrary , Niu , et al ., (2009) indicated that the levels of blood total lipids and cholesterol decreased with the elevation of ambient temperature. However, exposure of male rabbits to high temperature increased plasma cholesterol level (Arad et al., 1983; Seyrek et al., 2004). Also, Dorfman et al., (2008) who reported that chronic- intermittent cold stress in rat's hand no significant increase in serum glucose level during cold stress in winter may be due to the decrease in plasma cortisol levels in winter season. Khalil (1980; 2002; and 2005), and Eldessoki (2004) found that the increase in plasma glucocorticoid level under heat or cold stresses activates gluconeogenesis process which leads to an increase in serum glucose. It was shown that under conditions of stress, both ACTH and vasopressin secretion was increased (Aguilera, 1996), and when ACTH was secreted in large amounts, such as in conditions of stress, it could also stimulate Aldosterone secretion. Zulkifili et al., (2009) reported that prolonged heat stress (32 °C) in broiler chickens for 5 wks increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities. However, many studies reported that hyperthermia caused significant decrease in serum GOT, (El-

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Menhali 2002; and Eldessoki, 2004). Also, Ayyat et al., (2004) reported that in New Zealand White male rabbits that SGOT and SGPT were significantly lower in summer than in winter.

2-7-Blood hormone levels Hormones are produced by endocrine tissues and transported through the circulatory system to their target tissues. They provide an important link in the flow of information among cells and tissues in a bird to initiate and maintain the physiological and behavioral responses to heat stress. As a bird attempts to cope with heat stress, an intricate series of changes that is mediated by many, if not all, hormonal systems is initiated. The relative importance of each of these systems and the extent to which they are called upon depend on the severity of the heat stress. Earlier studies showed that thyroxin secretion rate in summer were half than in winter (Sturkie, 1976). The thyroid secretion rate was decreased by high temperature in chickens, but not in ducks and geese (Huston et al ., 1962). Habeeb et al., (1993) found that the overall means of thyroxin hormone (T 3) significantly decreased, due to exposure of the animals to high temperature. Hattingh (1982), Trammell et al., (1988) and Morera et al., (1991) reported similar results. Yahav and Hurwitz (1996) revealed that circulating concentration of triiodothyronine (T 3) was reduced at high temperature. Also they found that plasma (T 3) concentration decreased after a 24-h heat exposure at 5 d of age, and this ability to reduce (T 3) concentration reduced heat production and as a consequence improved thermo tolerance. Therefore, it may be anticipated that induction of thermo tolerance will be associated with modulation of plasma (T 3) concentration.

- ٣١- Review of literature

The importance of the thyroid gland in adaptation to heat stress is related to its role. Thyroid hormone secretion is depressed as ambient temperature increases and tolerance improves as thyroid function is reduced (Williams and Njoya, 1998). This finding is in agreement with the results of Buys et al., (1999) and Yahav and McMurtry, (2001).They found that plasma T 3 concentration were significantly reduced after thermal treatment, while T 4 concentrations were increased. While May et al., (1986) reported that acute exposure of growing chickens to high temperatures from 35 to 41°C did not appear consistently to affect serum (T 3) or (T 4) concentration. Bhatacharya, (1990) and Snyder et al. (1991) found that exposed birds to 10 oC an ambient temperature caused elevated serum thyroid hormone levels. The reduction in metabolic rate due to exposure to high ambient temperature may be referred to the decrease in thyroid activity. Sokolowicz and Herbut (1999) found that in both pullets and cocks reared under a high ambient temperature resulted in a reduce concentrations of thyroid hormones (triiodothyronine and thyroxine) in the blood plasma and a decreased metabolic rate, also. Czajka and Herbut (2001) observed that the increase in temperature (2 h increase of air temperature by 10 oC on days 14) resulted in a decrease of the thyroid hormone level and metabolic rate while the lowered temperature increased thyroid hormone level and air consumption. A number of studies have suggested that thyroid activity is affected by the environmental temperature. Thyroid size and secretion rate are decreased by high temperature in chickens, but not in ducks and geese (Huston et al ., 1965).

Circulating concentration of T 3 is reduced under high temperature (Yahav et al ., 1996). Therefore, it may be anticipated that induction of thermo-tolerance will be

- ٣٢- Review of literature associated with modulation of plasma T 3 concentration (Yahav and Hurwitz, 1996). High environmental temperature (35 °C) had a depressing effect on thyroid secretion (Cogburn and Harrison, 1980; 1985; Hillman et al ., 1985). The relationship between thyroid hormone and change of environmental temperature in birds has received considerable attention. The physiological importance of thyroid output is presumably through the influence of thyroid on metabolic rate as extensively evidenced in mammals and birds (Heninger et al ., 1960).

Rudas and Pethes (1984) reported thyroxin (T 4) concentration was reduced after exposure to heat stress (35 °C) for 1 hr. They added that conversion of T 4 to T 3 played a major role in the early phase of temperature acclimation. Arjona et al . (1990) showed that exposure of chickens to high temperature at 42 d of age was associated with a reduction in plasma T 3 concentration regardless of previous high-temperature exposure. One of the possible mechanisms of thermo-tolerance is the ability to reduce heat production which is regulated to a large extent by triiodothyronine (T 3) (McNabb and King, 1993). Ait-Boulahsen et al . (1993) suggested that the tolerance of broiler chickens to acute heat stress might be modified by diet. When broilers were fed on diet deficient in calcium 0.45% before being exposed an increase of air temperature from 24 to 41 °C. They observed reduction in body temperature that is inversely related to feed intake. It was shown that under conditions of stress, both ACTH and vasopressin secretion was increased (Aguilera, 1996), and when ACTH was secreted in large amounts, such as

- ٣٣- Review of literature in conditions of stress, it could also stimulate Aldosterone secretion. In turkey exposed to diurnal cycle temperature the concentration of T 3 (15, 25 and 35 °C) tended to be lower during the part of the day when ambient temperature was higher. The control group of turkey exhibited significantly higher T 4 or T 3 than that in heat stressed group at 45 °C (Kalamah, 2001). Similar results were obtained by Uni et al . (2001) who demonstrated a significant decline in the major metabolic hormone, triiodothyronine, during exposure to thermal stress. Sahin et al . (2002) demonstrated that serum concentration of T 3 and T 4 in broiler chicks reared under heat stress (32 °C) were 2.13 and 11.75 mg/ml, respectively. Moreas et al. (2003) reported that temperature challenge (exposure to heat stress) decreased plasma (T3) of broilers but the decrease was greater in pre-conditioned broilers compared with controls. A similar trend was observed for triglycerides. These changes did not affect total heat production. Since decreased (T3) and triglycerides levels are part of the mechanisms for thermoregulation, these suggest that thermal conditioning during incubation can improve the broiler chicken capability for thermo tolerance at later post- hatch age. El-Badry (2004) stated the mechanisms associated with the induction of thermo tolerance by early-age temperature conditioning may involve the modulation of heat production through reduction in plasma triiodothyronine (T 3) concentration. Recently, Yahav et al. (2004) found that thermal manipulation (TM) ca used a significant reduction in chick's body temperature and significant decline in plasma thyroid hormones concentration, but had no effect on plasma corticosterone concentration.

- ٣٤- Review of literature

Finally, Mashaly et al., (2004) reported that birds exposed to low ambient temperature showed significantly reduced body weight, which coincided with a reduction in energy intake and with changes in the circulatory system to accommodate higher oxygen demand. These changes included a significant increase plasma triiodothyronine (T 3) concentration. At the relatively high ambient temperature, changes to accommodate heat dissipation included significant increase in plasma volume and panting rate. These compensations were sufficient to control body temperature (Tb). However, the higher energy expenditure for maintenance followed by significantly higher plasma triiodothyronine (T 3) concentration, but with lower energy intake at low (Ta). Moreover, Aldosterone hormone an important for the maintenance sodium balance and is involved in the absorbance of sodium or potassium ions, the increase in this hormone leads to the increase in the absorbance rate of sodium in the kidney according to Khalil (2002). This Author that there were a positive relationship between Aldosterone hormone levels and the increase in Na absorption from proximal and distal tubules. This relationship is related to the increase in Na & K ATPase enzyme activation in the proximal tubules.

2-8- Blood electrolytes The normal functions of tissues are dependent upon the stability of the total osmolarity of intracellular and extracellular fluids. The major ions of the plasma are sodium, potassium, calcium, phosphate, zinc and magnesium. The plasma concentration of each ion normally varies only within a remarkably small rang; a substantial shift in their concentration can cause serious disturbance to cells since these ions and the plasma albumin play a major role in establishing the osmotic balance between plasma and fluids bathing the cells. Therefore, our knowledge review of

- ٣٥- Review of literature literature on the effect of heat stress and cold stress (seasonal variation) on electrolytes is relatively scarce. Heat stress has been associated with decreases in broiler total mineral retention (Sahin and Kucuk, 2003). Early studies by Arad et al . (1983) showed that total plasma calcium levels were decreased in laying hens subjected to high environmental temperature 35 °C. Deetz and Ringrose (1976) reported that blood potassium concentration of hens decreased by high temperature (38 °C), the potassium requirement increased to 60% or greater at (37 °C) versus control group (25 °C). Also, they recorded that plasma potassium concentration were reduced during heat stress (40 °C) for 2 hrs to 4.2 mEq/L versus 4.9 mEq/L for unstressed control. In addition, Edens (1978) found that when broiler chicks 8 weeks old were exposed to high environmental temperature (43 °C) for 60 min, the plasma sodium level significantly decreased to 123 mEq/L versus 135 mEq/L for the non-stressed control. The plasma sodium level remained depressed in heat stressed chicks through the 120 min exposure to 112 mEq/L versus 140 mEq/L for the control. But high environmental temperature and time of exposure had no effect on plasma potassium concentration. Bowen and Washburn, (1995) reported that plasma sodium, chloride, potassium, calcium, phosphate, magnesium and albumin play a major role in establishing the osmotic balance between plasma and fluids bathing the cells and determining the pH of the body fluids. Elevation of body temperature to 44.5 – 45 °C by exposing chickens to 41 °C ambient temperature increased plasma sodium and chloride and decreased plasma potassium and phosphate. Pardu et al . (1985) reported that plasma sodium concentration was decreased in ducks after heat exposure from 39 to 43 °C. They also reported that breed had significant

- ٣٦- Review of literature influence on Na and K level. Plasma Na concentration was significantly higher in Muscovy than Domyati ducks at 8 and 12 weeks of age. Ait-Boulahsen et al . (1989) reported that heat stress + depressed plasma K concentration in chickens. In a series of studies, Ait et al . (1992) reported that decreased in ionized calcium and inorganic phosphate for broiler chickens exposed to gradual increase in temperature from 24 to 37 °C. They concluded that the increase in body temperature due to heat stress is inversely related to the chickens’ ability to maintain blood calcium level. Belay et al . (1992) reported that when broilers were subjected to either a thermoneutral temperature (24°C) or to a cycling temperature heat distress (24 to 35 °C) excretion of K, P, Mg and Zn was significantly increased (P < 0.05). For birds that were held at 35 °C for 36 h. heat distress increased total urinary (Na, Ca) excretion. Their studies provided evidence that heat distress adversely impacts bird mineral balance. The excretion varies with each mineral and heat distress severity. Also, Ait-Boulahsen et al . (1993) reported that blood ionized calcium (Ca +2) were decreased in chicken exposed to high ambient temperature 41 °C. They suggested that the increase in body temperature, during heat stresses inversely related to the chickens ability to maintain blood Ca +2. In Contrast, Samara et al . (1996) found that ٍtotal calcium concentration did not diminish in hen's acclimated to high cyclic temperature. Pech-Waffenschmidt et al . (1995) reported that changes in the blood system are part of the thermoregulatory responses acquired by birds to enable them to with stand heat stress, stressed birds established a cascade of physiological adaptive responses include elevation of plasma calcium, phosphorus and electrolytes.

- ٣٧- Review of literature

Abd-Ellah et al . (1995) reported that by exposure of 7- weeks old broiler chicks to (40 °C) for 30 min plasma potassium level was not affected. Belay and Teeter (1996) reported lower rates of sodium and phosphorous retention in broilers raised at high cyclic ambient temperature (24 to 35 °C) compared with birds housed at 24 °C. They postulated that loss of plasma Na concentration may be attributed to high urinary excretion occurred during heat stress. Belay and Teeter (1996) found lower rates of potassium retention in broilers raised at high cyclic ambient temperature (24 to 35 °C) compared with birds housed at 24 °C. Plasma sodium concentration was affected by acclimation. Early heat exposure decreased significantly plasma sodium compared with non-acclimated control at 8 weeks of age (Abdel-Mutaal, 2003). On the other hand, Abd- Ellah et al . (1995) reported that the plasma sodium did not affected by heat stress. Also Altan et al . (2000) suggested that heat exposure with water availability did not result in any changes in plasma Na concentration. Plasma potassium concentration was slightly increased by acclimation compared with control group at 8 weeks of age (Abdel-Mutaal, 2003). In contrast, Borges et al . (2003) subjected broilers to cyclic periods of heat stress, and found an increase in blood potassium (K) level. El-Badry (2004) reported that heat stress did not affect blood plasma potassium level in birds at 8 and 12 weeks of age. Also Arad et al . (1983), Ahmed and Maghraby (1995) and Altan et al . (2000) suggested that heat exposure with + water availability resulted in no changes in plasma K concentration. Rahimi (2005) exposed male broiler chicks to early heat stress for induction of thermo tolerance. Chicks were

- ٣٨- Review of literature exposed to 38 ± 1°C for 48 h at day 3 to 4 and for 72 h at day 4 to 6. Relative humidity ranged between 70-75 % throughout the rearing period. They found that plasma calcium levels was increased significantly at the end of rearing period when heat acclimated chicks were exposed to acute heat of 37 ± 1°C for 4 h.

2-9- Total body water Information on the body water of the live ostrich is important for research whether the research involves nutrition, physiology, genetic, disease and meat production. Body weight alone provides a poor index of the metabolically active tissue or the mass of tissue available for meat. Live body weight including total solids and total body water, water retention is known to vary considerably between birds during growth due to difference in the rate of accumulation of the less hydrated, fat, collagen and fibrous tissues in replacement of the more hydrated functioning protoplasmic mass and to the age difference in response to nutritional and climatic factors. Therefore when the animals or birds exposed to high environmental temperature during summer season of Egypt, most of the physiological and biochemical parameters are disturbed. The heat induced changes in each of thermoregulatory parameters as well as body fluids, protein turnover or balances and some allergenic hormonal levels may be used for evaluation the animal's adaptability to hot climate (Habeeb et al., 1993 and 1997). Detection of such phenomena in the animals could be achieved by different indices some indices based on body water balance in animals like total body water (TBW), total body solids (Live body weight-TBW) and body water to body solids ratio (TBW/TBS). Heat is the major constraint in animal's productivity in sub-tropical arid zone regions. Growth and reproductive

- ٣٩- Review of literature performance in male and female animals or birds are impaired as a result of the drastic changes in biological functions caused by heat stress (Habeeb et al., 1992, 1993, and 1997). The most adaptable animals or birds to such conditions are those which manifest the least deviations in their normal traits when reared in such conditions. However, there are individual differences in these changes between breeds and within each breed. The difference in the response of such animals under hot climatic conditions is due to their difference in heat adaptability measured by heat tolerance indices which is the ability of the animals to express its inherited production potential during its life-time when raised under the hot conditions (Kamal, 1982). The heat tolerance indices may be used for the selection of the heat tolerant animals within or between breeds. Heat adaptability indices used for predicting the productive animals under hot conditions are based on their production level, water balance, protein balance, thermal response and heat induced changes in hormonal levels (Habeeb et al., 2007 and 2008). Using the production level as an indicator of adaptability is a rather time-consuming, for example to evaluate the performance in sheep. Moreover, body weight gain of heat stressed animals is a misleading index of heat adaptability, since it may be due to the increase in water retention and not to the increase in body protein and fat (Kamal and Johnson, 1971). In other words, a unit of body weight gain in one animal may be due to the increase in body water at the expense of body tissue loss, while in the other animal, may be due to the increase in body solids (Kamal and Seif, 1969). Therefore, it is erroneous to consider the first animal as adapted to heat as the second animal, though both had similar apparent body weight gain. Studies concluded that using the production level

- ٤٠- Review of literature in estimating the heat adaptability in farm animals is impracticable but using heat tolerance coefficients in estimating the heat tolerance in farm animals is easily quickly and more reliable.

2-10- Serum proteins profiles A response of all organisms; animal, plant, or microbe to elevated temperature is the increased synthesis of a group of proteins known as the heat shock proteins (Hsp) (Lindquist, 1988; Etches et al ., 1995). There is evidence that the molecular mechanism in response to heat stress is characterized by a reduction in cellular protein synthesis, except for a set of polypeptides termed heat shock proteins (Hsp,s).There is also good evidence that (Hsp,s) synthesis is increased under stress condition i.e. elevation of ambient temperature (Craig, 1988). Therefore, heat shock protein 70 (HSP70) is a stress-induced protein. High levels of HSP 70 can be induced by cells in response to hyperthermia. These proteins their function is to protect heat stressed cells and to prevent proteins miss folding by stress. Heat shock protein function as a molecular chaperon by binding to other cellular proteins, assists in transportation of proteins across membranes within the stress cell, it stabilizes denaturing protein, refold, reversibly denature proteins and facilitate the degradation of irreversibly denature protein (Chirico et al., 1988 and Hart, 1996). Wang, and Edens, (1993) found that thermal stress, in vitro and in vivo, induced the synthesis of heat-shock proteins Hsp 90, Hsp 70, and Hsp 23 in turkey leukocytes. Heat shock protein (Hsp) induction was temperature and time dependent. Most of the work on molecular biology of heat-shock protein expression in vertebrates has been done using the

- ٤١- Review of literature chickens as a model system. The rapid heat shock response also, involves the expression of the heat stress genes and their encoded protein heat shock proteins (Hsp, s) (Sanchez and Lindquist, 1990; Lindquist, 1994). Members of the super family of (Hsp, s) are among the most conserved protein known in phylogeny with respect to both function and structure (Welch, 1993). It is tempting to speculate that part of the difference in heat tolerance of various breeds could be attributable to different alleles of heat shock protein-encoding genes. These results in different ability to respond rapidly to a heat stress, with different final concentrations of the relevant heat-shock protein in stressed birds or different ability of various heat shock protein to interact with their normal legends in the cell (Etches et al., 1995). The expression of heat-shock protein by peritoneal macrophages (PM) isolated from various avian species including chicken, turkey, quail and ducks exhibited a comparable induction of the three major families of stress proteins Hsp 23, Hsp 70 and Hsp 90 following a 1h-heat shock at 45 C. However, a lower molecular mass was observed for the duck peritoneal macrophages (PM) when protein Hsp 32 and Hsp 23 was expressed when compared with the Hsp profiles of the chicken, turkey and quail macrophages. This difference in the molecular mass of the Hsp32 and in ducks might indicate a species difference (Miller and Qureshi, 1992c) in heat stress tolerance. In a series of experiments (Miller and Qureshi 1992a,b) reported that the optimal temperature and time for induction of the three major 23 ,70,90 Kda, Hsp were 45 C or 46 C for 60 minutes with variable recovery period for each Hsp in another experiment. Chicken macrophage cell line produced the three classical 23, 70, 90 Kda stress proteins similar to those produced by Hs treatment after exposure to bacteria lipopolysaccaride (LPS), an antitoxin found in the

- ٤٢- Review of literature outer membrane gram-negative bacteria] which is known as non thermal stimuli. They also postulated major levels of heat shock proteins Hsp 70, Hsp 90 and 32-Kda in various avian species (chicken, turkey, quail, and duck) peritoneal macrophages that were exposed to 41 °C and/or 45 °C for 60 min. In another experiment, they compared the synthesis of heat shock proteins in chicken macrophages in response to thermal and non thermal stress in white leghorn females. They found that macrophages isolated from the 44 °C group synthesized Hsp 90, Hsp 70, Hsp 23 and Hsp 32 protein, but the non thermal stressors females (Lipopolysacaride) induced Hsp 120 protein. Heat stress in vitro has been limited to short periods, often less than 1h, and the HS response or survival rate of those cells was examined immediately or shortly after the heat treatments. Long-term heat conditioning leading to heat acclimation, of animals using Hsp as a marker for heat acclimation, has had limited examination in vertebrates (Wang, 1992). El-Nabarawy, (1997) showed that the profiles of serum protein electrophoresis (SPE) for heat shock treatments of Japanese quail chicks at two weeks of age revealed slight variations in the intensity of bands between the individuals at the same time of sampling due to heat treatments. Heat shock samples exhibited substantially lower intensity for alpha l- globulin only at 3 and 6 h compared with the control. Therefore, Hsp 70 is essential for cellular survival from heat stress and other types of physiological challenges. The expression of Hsp can be detected within several hours and last 3-5 days in duration (Kregel, 2002).

- ٤٣- Material and Methods

3 MATERIALS AND METHODS

The present study was carried out at the experimental farms ( Ostrich farm ) and the project laboratories of Nuclear Research Center in (Inshas), Atomic Energy Authority, Cairo Egypt.

31 Management of experimental animals Twelve immature ostriches aged 7 months old were used weight 50 ±5 Kg in summer and same number in winter from the breeding flock (Ostrich farm ). All birds were reared out doors and exposed to ambient temperatures daily during summer and winter, to evaluate the effect of ambient temperature variation (summer and winter) and diurnal effect (morning at 7 am and afternoon at 3 pm) on response changes of some physiological and chemical parameters. The birds were fed grower ration add libitum (19% protein and 2450 K cal ME /Kg).The components and chemical analyses of the diet is shown in Table (1). Ration was added once at morning and the residuals will be weighed in the next morning to measure daily feed consumption (gram of feed / bird / day) and water consumption (ml of water / bird / day). In addition, blood samples were taken at the hot day of summer (40 ±1ºC) (June month) and the cold day of winter (18±1ºC) (January month) to compare the measured parameters.

٤٤ Material and Methods

Table (1) the ingredients and the chemical analysis of the experimental ration.

Items ( kg)

Ingredients : Yellow corn 373 Soybean meal 270 Lucerne 300 DiCalcium phosphate 15 Limestone 10 Salt 5 Synthetic lysine 1 Synthetic methionine 1 Antitoxic 2 Min. 1 & Vit. Premix 2 12 Oil 10 Zinc pastracine 1 Total (kg) 1000 Chemical analysis (g/Kg) food: ME (K cal/ Kg) 2450 Protein% 19 Crude fiber g/Kg 11 Calcium, g/Kg 1.2 Phosphorus, g/ Kg 0.6

1- The mineral premix provides the following (per 4 kg): choline chloride 1800gm; manganese 225 gm; zinc 150gm; iron 60gm; copper 13gm; iodine 1.5 gm; selenium 0.60 gm; cobalt 0.90 gm; magnesium 250 gm ; CaCo3 add to 4 kg (inclusion rate 4 kg / ton). 2- The vitamin premix provides the following (per 2 kg): vitamin A 37500000 MIU; vitamin D3 11250000 MIU; vitamin E 300 gm; vitamin B1 3300 mg; vitamin B2 3300 mg; vitamin B6 4400 mg; vitamin B12 100 mg; pantothenic 20000 mg; nicotinic 60000 mg; folic acid 2000 mg biotin 200 mg; Caco3 add to 2 kg (inclusion rate 4 kg / ton).

٤٥ Material and Methods

32Blood samples: Blood samples were with drawn from the wing vein of each bird with a 21G needle connected a 5ml syringe. Birds had their heads covered in a woolen sock during sampling for ease of containment. Blood samples were taken at the hot day of summer (40 ±1ºC) (June month) and the cold day of winter (18 ± 1ºC) (January month) to compare our measured parameters. Blood samples were placed in two tubes, one containing lithium heparin to determine hematological parameters and the other without anticoagulant and left to clot. In the laboratory, samples were centrifuged at 1600 rpm for 15 minutes and the resulting serum was stocked at (20) until use. Serum samples were evaluated for different biochemical parameters during both seasons and a comparison was made between both seasons.

33Feed and water consumption: Feeders were supplemented once daily with feed at the morning and the residuals was weighed in the next morning to measure the daily feed consumption and expressed as (gram of feed consumption / bird / day) but water consumption (liter/bird/day) was recorded daily for a week too. While blood samples were collected in early morning and afternoon during summer and winter season.

34 Body temperature: Body temperatures (rectal temperatures) were obtained using a thermocouple thermometer (accuracy of ± 0.2°C) that was inserted approximately 5 cm into the colon.

٤٦ Material and Methods

35Hematological studies: 351 Red blood cells (RBCs ) and white blood cells (WBCs) counts were determined according to Natt and Herrick (1952) briefly the following was done. Blood diluents were made according to the following composition: 1 3.88 gm Na CL

2 2.50 gm Na 2SO4

3 2.91 gm Na 2 HPO 4.12 H2O

4 0.25 gm KH 2PO 4 5 7.50 cm Formalin (37%) 6 0.10 gm Methyl violet 2B The above chemicals were dissolved in distilled water in the order prescribed and diluted to a total volume of 1000 ml in volumetric flask. They were let to stand overnight and then filtered in (Whatman No. 2) and would be ready for immediate use after adjusting the solution pH to 7.3. The blood sample was diluted 1: 100 using designated pipet then shaked for one minute in order to obtain an equal cell distribution. The following was noticed: 1 The nucleus of (RBCs) were stained violet and also the cytoplasm to lessen extent. 2 Mononuclear leucocytes were stained deep blue violet the cytoplasm was stained light violet. 3 Polymorph nuclear leucocytes were stained light violet the blue violet (almost black) stained granules were completely fill the cell. The (RBCs) were counted in the 80 small squares and (WBCs) are counted in the 400 small squares. The respective factors for the total erythrocytes and leucocytes counts were the number of cells counted times 5000 and 1000, respectively.

٤٧ Material and Methods

Total erythrocyte and leukocyte counts were made directly from the same blood sample placed in the haemocytometer . 352 Hemoglobin (Hb): Were determined according to Dacie and Lewis (1991). Using the following reagents and procedure. Drabkin’s solution was used which consisted of:

1 200 mg potassium ferri cyanide K3 fe (CN6) 2 50 mg potassium cyanide (KCN)

3 1000 mg sodium bicarbonate (NaHCO 3) All these chemicals were diluted in one liter distilled water and stored in a dark bottle in the refrigerator until used. Sample assessment procedure: 1 0.02 ml of blood sample was diluted in 5 ml of Drabkin’s reagent in each tube of samples and mixed. 2 0.2 ml of standard (80 mg/ 100 ml) was diluted in 5ml of reagent for preparing standard and 0.2 of distilled water was added to 5ml of reagent as a blank then mixed. 3 All tubes were kept stand for 5 minutes. 4 The spectrophotometer was set to zero using the blank tubes and absorbency of the results solutions were measured at 540 nm against the standard. Calculation: Hb (g/100ml blood ) = O.D of sample X conc. of Std. O.D of STD 353Packed cell volume (PCV): The PCV was determined by micro haematocrit method of Hamer and Mac Henry, (1942) a model MB micro capillary centrifuge and a model CR micro capillary reader was used. Blood samples were centrifuged at 1500 rpm for five minutes and the percentage of packed red blood cells was recorded.

٤٨ Material and Methods

354Mean corpuscular volume (MCV): It is an index expresses the mean volume of individual red blood cell in cubic microns. To calculate MCV, PCV expressed in ml / liter is divided by the red cell count in million per mm 3 to be expressed in femtoliter (fl). 255Mean corpuscular hemoglobin (MCH): To calculate MCH, concentration is divided by RBCs count in millions per mm 3 and multiplied by 100 to be expressed in Picogram (pg). 356Mean corpuscular hemoglobin concentration (MCHC): This index expresses the percentage hemoglobin in each individual red cell. It is obtained by dividing the amount of hemoglobin in grams per 100 ml of blood by the percent PCV, and is expressed as a percentage.

36Biochemical Studies: 361 Serum total proteins was determined calorimetrically using method as described by Dumas, et al., (1975) and Doumas, et al., (1971) respectively. 362 Serum albumin was determined using DiamondEgypt Kits by the albumin calorimetric method described by (Doumas et al., 1971). In a buffered solution bromocresol green forms with albumin a green colored complex whose intensity is proportional to the amount of albumin present in the sample. 363 Serum globulin was calculated by subtracting serum albumin from serum total protein for each quail. 364 A/G ratio was mathematically calculated by dividing albumin on globulin value for each quail.

٤٩ Material and Methods

365 Serum total lipids were determined using biodiagnostic. Egypt kits calorimetric method according to (Zollner,N., and K. Kirsch 1962). Lipids react with sulfuric, phosphoric acids and vanillin to form pink colored complex. 366 Serum triglycerides were determined calorimetrically according to (Trinder, 1951). 367 Serum Cholestrol was measured using biodiagnostic. Egypt kits by the cholesterol colorimetric method (Richmond, 1973). The cholesterol is determined after enzymatic hydrolysis and oxidation. The quinoneimine is formed from hydrogen peroxide and 4aminoatipyrine in the presence of phenol and peroxidase.

Cholesterol – ester + H 2O ———► cholesterol + fatty acids

Cholesterol + O 2 ———► cholesterol 3 one + H 2O2

2H2O2 + phenol + 4 – Aminoantipyrine ———►

Quinoneimine + 4H2O 368 Serum Glucose was determined calorimetrically according to (Tietz, 1995). 369 Serum alanine transaminase GPT (ALT) and aspartate transaminase GOT (AST) were determined using Biodiagnostic. Egypt Kits by a calorimetric method according to Reitman, A. and Frankel, S. (1957). GPT (ALT) catalyses the following reaction: Alanine + αketoglutarate ←→ pyruvate + glutamate The keto acid pyruvate formed is measured in its derivative form 2, 4,dinitrorophenylhdrazine. GOT (AST) catalyses the following reaction: Aspartate + αKetoglutamate ←→ oxaloacetate + glutamate

٥٠ Material and Methods

The keto acid oxaloactate formed is measured in its derivative form 2, 4,dinitrorophenylhydrazine.

37Estimation of serum concentration of Calcium, Sodium, Potassium and Phosphorus: Serum total calcium, Sodium and Potassium concentrations were determined by flame atomic absorption spectrophotometer using a Perkin Elmer Model 5000 (Perkin Elmer Corporation, Norwalk, CT), (Comnerty and Briggs, 1966). But serum inorganic Phosphorus concentrations were determined calorimetrically according to (Lehman and Henery, 1984).

38Estimation of Hormone levels: Serum analyses were undertaken to determine serum Triiodothyronine (T3) by the method of (Larsen, 1972), and determined aldosterone level by Commercial Kit. All were estimated by (RIA) technique and counted at gamma counter model RIA SAR, Packard, Model 0540501.

39 Determine total body water content: The amount of total body water was determined by the method according to Habeeb, (2010) who estimating total body water (TBW/ml) in animal by dividing concentration of antipyren (ANP) injected () by concentration of ANP in plasma sample (/ml) (Dilution factor Equilibration technique). TBW=ANP injected ()/ANP in plasma sample (/ml). The dilution factor multiplied in 2 (estimation was carried out in 1/2 (2/4ml) ml plasma and also in 100/93 (percentage of water content in serum).

٥١ Material and Methods

Preparation the injection does of antipyren (ANP) 1 Standard does is 1gram ANP each 100 kg live body weight (LBW). Assuming that animal weight 50 ± 5kg need to 500mg ANP. If the experiment was carried out on 8 birds two times one under mild climate and the other under hot climate. Then we need 500mg × 8 × 2 = 8 grams ANP. 2 Dissolve 10 gram ANP in physiological saline solution and complete the final solution to 50 ml →10 g/50 ml →500 mg / 5 ml →100 mg / 1ml. 3 Injection each bird (weight 50 ±5 kg) with 5 ml (contains 500 mg ANP) in the left wing vein and blood samples intake from write wing vein after 1/2 h from injection.

310 Protein electrophoresis analysis: It was conducted by NativePAGE method (Native poly acrylamide gel electrophoresis) on vertical slab gel to determine protein profiles in blood protein of ostrich according to Laemmli, (1970).

AReagents: The following reagents were obtained from Adwic Company. 1Acrylamide /bis (30%) Acrylamide (28.2 gm) and 1.8 gm bisacrylamide were dissolved in 100 ml distilled water. The solution was filtered and stored at 4°C in dark glass bottle. 2 Sepration or resolving buffer (pH 8.8) Trishydroxymethyl aminomethane (18 gm) were dissolved in 100 ml distilled water. HCL were added until pH 8.8, then the solution was filtered and stored at 4°C.

٥٢ Material and Methods

3 Stacking buffer (pH 6.8) Trishydroxymethyl aminomethane (6 gm) were dissolved in 100 ml distilled water. The solution was filtered and stored at 4°C. 4 Tank buffer Trishydroxymethyl aminomethane (12 gm), 57.6 gm glycin and 40 ml were dissolved in 100 ml distilled water. In native protein experiment SDS was not added. 5 TEMED N, N, N,tetramethylethylenediamine kept in dark glass bottle at 4°C. 6Ammonium persulphate (10 %) (APS) Ammonium persulphate (2 mg) were dissolved in 2 ml distilled water. This solution is unstable and made up just before use. 7 Glycerol. 8 2Mercaptoethanol 10 %. 9 Coomassie brilliant blue 1 % (stock solution) Coomassie brilliant blue (2 gm) were dissolved in 200 ml distilled water. Then the stain was filtered and stored at room temperature. Stain solution: 62.5 ml stain (stock solution) 250 ml methanol and 50 ml acetic acid were completed to 500 ml with distilled water. 10 Tank buffer Methanol (500 ml) were added to 100 ml acetic then completed to 1000 ml with distilled water.

٥٣ Material and Methods

11 Marker The used marker was the wide range prestained SDSPAGE standards mixture BioRAD. The molecular weight ranged between 205 to 45 kDa. It was applied to the first well. 12 Loading sample buffer Stacking buffer pH 6.8 2.5 ml Glycerol 2 ml 2mercaptoethanol (10 %) 1 ml Distilled water. 0.5 ml Bromophenol blue 1 ml (0.l gm was dissolved in 100 ml distilled water). 13 Stacking (collecting gel preparation) Acrylamide/bis 30 % 3.4 ml Stacking buffer pH 6.8 2.5 ml Distilled water 13.6 ml 10 % ammonium per sulphate 200 l TEMMED 20 l Total volume 19.7 ml 14 Separating (tracking gel preparation) Acrylamide/bis 30 % 20 ml Separating buffer pH 8.8 15 ml Distilled water 23.8 ml 10 % ammonium per sulphate 600 l TEMMED 24 l Total volume 60 ml

٥٤ Material and Methods

B Procedure: 1 – 30 l of serum sample were mixed with 30 l loading buffer, and then heated to 100°C in boiling water bath for 10 seconds. 2The separating gel was poured between the glass plates and allowed to be polymerized for 2040 min at room temperature. 3Stacking gel was poured on the top of separating gal and the plastic comb was inserted to form sample wells. 4 The slab gel apparatus chamber was filled with tank buffer. 5 After polymerization, the comb was removed without tearing the edges of the polyacrylamide wells. 6 The prepared serum samples were loaded into wells. 7 The power supply was connected to the gel tank and electrophoresis was conducted at a constant volt (30 volt for 9 hours) till the tracking dye reaches the bottom of the gel and the current is switched off. 8The gel was removed by prying the glass plated apart gently, then the separated protein gel was stained with coomassie brilliant blue R250 overnight. 9 After gel staining, the gel was transferred to destining solution for several times, the destining solution removed the background dye form the gel leaving stained proteins as visible blue bands. 10 The slab gel of proteins was photographed. 11 Molecular weight, band density and Rf were determined using a densitometry gel proanalyzer software 3.0 (USA, 1998) according to the method of Chrambach (1985).

٥٥ Material and Methods

Data analysis: 1 Gel plate was photographed, scanned and then analyzed using a gel pro Analyzer (Version 3.1Medie Cybernetics USA) for the analysis of tested samples. This program is a comprehensive computer software application designed to determine the relative fragmentation, the molecular weights and the amounts of peptide chains as well as scanned graphical presentation of the fractionated bands of lane. 2 The similarity index (S.I) compares patterns within, as well as, between blood samples of ostrich under study using the formula: S.I. = (2 Nab/Na+Nb) Nei and Li, (1979). Na and Nb are the number of bands in individuals (a) and (b). Nab is the number of shared bands between (a) and (b). The similarity values were converted into genetic distance (D) using the formula: D = 1S.

311Statistical analysis: Data were statistically analyzed using the General Linear Models Procedure of the (SAS 1997 ). Paired Ttest (Procedure means of SAS) was run to test the effect of diurnal variation on different measurements within each season, student (Ttest of SAS) was used to test the effect of season on different measurements within each period (day and afternoon) to study the effect of seasonal variation (summer and winter season) and diurnal effect (morning and afternoon) on thermoregulation mechanisms of ostrich (changes in some physiological and blood chemical parameters).

٥٦ Results and Discussion

4 RESULTS AND DISCUSSION

41 Effect of seasonal variations on feed and water consumption Figure (1 and 2) demonstrate the effect of seasonal variation on feed and water consumption. It was found that feed consumption was significantly increased during winter than in the summer season. Unlike feed consumption, the increase in water consumption was highly significant during summer than the winter season. It is generally documented that high ambient temperature (AT) reduces feed consumption and growth rate of hens and increases water consumption (Geraert et al, 1996; May and Lott, 2000 and Yalcin et al., 2001). While, low ambient temperatures significantly increase feed consumption and decreases water consumption (Ahmad and Sarwar, 2006). These negative effects of high AT have been found to be more pronounced on birds with higher body weight (BW), and faster growth rate (GR) than in those with lower BW and GR (Emmans and Kyriazkis, 2000 and Deep and Cahaner, 2001). May et al . (1987) suggested that, feed intake was reasonably depressed in hot environments in order to reduce the metabolic rate and heat production. The present results are in agreement with May and Lott (2000); Yahav and Hurwitz (1996) who showed that heat stress had a deleterious effect in both growth rate and feed conversion. At high ambient temperature, evaporative cooling is the most important mechanism of body temperature control. Water consumption increases when chickens are exposed to high environmental temperatures and survival during heat stress is dependent upon water consumption North and Bell (1990).

٥٧ Results and Discussion

May and Lott (1992) postulated that broilers exposed to 3 days of cyclic temperatures consumed more water than controls during a subsequent exposure to temperatures up to 40.8 °C. They also showed that the increased water consumption due to high, cyclic temperatures arise from changes that occur during some times of the day and no changes occur during other times. The increase in water consumption precedes reduction in feed consumption. Teeter et al . (1992) found that heat stress lowered feed consumption. Rectal temperature increased linearly as feeding level increased. Feed intake plays a significant role in the acclimation process; that bird acclimated to heat distress has metabolic effects independent of feed consumption; and that either: (1) the heatdistressed acclimation bird has the capacity to shift exothermic processes from the heatdistressed to cooler time periods; (2) that heat dissipation mechanisms require greater than 12 h to restore normal body temperature, or (3) the birds' set point is elevated when exposed to cycling high ambient temperature distress. Wiernusz and Teeter (1993) found that the increased heat load, with elevated feed consumption, was dissipated by increased sensible heat loss solely, as evaporative heat loss remained constant. Those data indicate that heat production increases with feeding level, that broilers preferentially dissipate heat as sensible heat loss when environmental conditions permit, and that feed consumption exacerbates bird heat content rise environmental conditions limit sensible heat loss. Deeming, (1997) suggested that the winter conditions would influence the behavior of ostrich. Maintenance of body temperature is probably the main reason for such high rates of food consumption in both genders. During the winter, standing was an important behavior at similar levels to that observed for ostriches during the summer, presumably due to vigilance particularly during feeding (Ross and Deeming,

٥٨ Results and Discussion

1998). Behaviors' involving activity, such as panting and walking, occurred at a low frequency during the winter. Feeding and foraging were the most important behaviors for both genders during the winter. ( Mc Keegan and Deeming, 1997; Ross and Deeming, 1998). Moreover, gender differences described for adult ostriches in the summer were not present during winter months. Alleman and Leclercq (1997) reported that water consumption for heatstressed chickens raised under 32 °C was lower (2.9 and 3.1) liter / bird compared with those of control reared under 20 °C (1.7 and 1.9) liter /bird. Water / gain ratios were (3.052 and 3.396) compared with (6.977 and 6.669 ml water / g body gain) for control and heatstressed birds, respectively. Additionally, Veldkamp et al. (2000) studied the effects of ambient temperature on feed consumption; body weight gain and mortality rate and found that temperature had a clear negative effect on performance during all age periods. The authors concluded that feed intake and body weight gain were significantly lower at the high AT compared with low AT group while water consumption and mortality rate were more increased in high AT than in lower AT. The actual quantity of water is dependent, however, on many factors, i.e. body weight, physical activity, rate of egg production as well as environmental temperature Zulkifli et al., (2009). The amount of water consumed per day is more on days immediately after the temperature rise than it is a few days later. For example, the rate of water consumption was doubled immediately after environmental temperature was increased from 21.2 to 37.8°C (Donkoh, 1989). However, when water consumption was measured for one week after this rise in temperature it was only 35 percent greater at 37.8 than at 21.2°C. A high initial increase in the intake of water also took place when the ambient temperature was raised from 21.2 to 26.6°C and from 26.6 to 32.2°C respectively (Siegel, 1995).

٥٩ Results and Discussion

Similarly, Lin et al. (2006) and Olanrewaju et al. (2007) reported that laying hens consumed 1.5 times as much water as feed at 36.7°C and about five times as much water as feed at 37.8°C.

٦٠ Results and Discussion

2.60 2.50 2.40 2.30 2.20

KG2.10 /BIRD/DAY Series1 2.00 1.90 feed W feed S SEASONAL VARIATION

FEED CONSUMPTION DURIND SEASONAL VARIATION

Fig.(1): Effect of seasonal variations on feed consumption.

12

10

8

6

4

LITE2 R/BIRD/DAY Series1

0 WATER W WATER S SEASONAL VARIATION

WATER CONSUMPTION DURING SEASONAL VARIAION

Fig.(2): Effect of seasonal variations on water consumption.

٦١ Results and Discussion

42 Effect of seasonal variations on body temperature An increase in body temperature (BT) was recorded between two seasons, where ostrich in winter lower (BT) temperature than ostrich in summer. Exposing birds to high temperature (40 °C) increased (BT). This result could contribute to some extent to heat dissipation of the body surface (sensible heat loss) were ostrich in summer are highly tolerant to heat stress. Birds, like all homoeothermic animals, maintain a constant body temperature (BT) over a wide range of ambient temperatures (AT).However, when the physiological and behavioral response to high AT is inadequate to faced with this stress, an elevation in BT occurs, causing a decrease in appetite, growth rate, and productivity (Ain Baziz et al., 1996 and Geraert et al ., 1996). The ability of an animal to maintain its BT within the normal range depends on a balance between the internally produced heat and the rate of heat dissipation. The amount of internally produced heat depends on BW and feed intake. The rate of heat dissipation depends on environmental factors, mainly AT, and feather coverage. Birds are “endothermic” their body temperature might raise by 1 or 2°C as the ambient temperature rises (Etches et al ., 1995). These results are in agreement with Yahave and Hurwiz (1996); ElGendy and Washburn (1995); Tolba (2000) in broilers and Yassein (1998) in ducks. They demonstrated that body temperature increased in response to heat stress exposure. AitBoulahsen et al . (1993) found that under heat stress (41 °C) body temperature of broiler chickens increased compared with control (24 °C). Body temperature reached the highest value at afternoon and the lowest value was recorded in the morning. Washburn et al . (1980) showed that body temperature was significantly different among strains of chickens

٦٢ Results and Discussion according to their body weights when exposed to heat stress (50 °C) up to 4 hours. The response to heat in their experiment was assessed by heat stress. ElGendy and Washburn (1995) studied the genetic variation in body temperature of broilers and its response to short term acute heat stress 43.2 °C for 45 minutes. They reported an increase in mean body temperature by 1.47 °C when birds were exposed to abrupt elevation in ambient temperature. Zhou et al . (1997) found that whole blood viscosity (WBV) of broilers decreased significantly when they were exposed to high ambient temperature. The decrease in (WBV) may be advantageous in reducing peripheral resistance and the load on heart, and increasing tissue perfusion and circulatory distribution, including the blood supply to heat exchange surfaces. However, some birds could not withstand high air temperature. They were unable to pant fast enough to remove the heat from their body. In other world heat loss by evaporation (insensible heat loss) is much less than total heat production, when their body temperature rose above the physiological maximum, prostration and death occurred (North and Bell 1990). Deeb and Cahaner (1999) suggested that measuring body temperature can be used as indicator of the level of stress at high AT. Our results revealed that body temperature increased significantly during the summer season (high ambient temperature) as compared with the winter season. In addition, Muiruri et al., (1991) reported on the effect of roost temperature on rectal temperature (RT) of heat stressed fasted and fed hens at 34 ±1 oC RT increased 1.20 and 1.32 oC, respectively, whereas hens using the watercooled roosts, RT increased only 0.22 and 0.28 oC during the heat stress period, respectively.

٦٣ Results and Discussion

Moreover, Eberhart and Washburn (1993) reported an increase in body temperature when heat acclimation birds (32 °C) were exposed to acute heat stress (40 °C). They also small body weight of birds had a higher body temperature and smaller change in body temperature when exposed to acute heat stress than large body weight birds. Yahav and Hurwitz (1996) showed that in male broiler the rectal temperature but not the skin temperature increased significantly by the end of a 24 h exposure to heat (36 ±1°C) during the 1st week of life. However the skin temperature was significantly lower later in life at 24 days of age. ElNabarawy (1997) found that the rectal temperature of Japanese quail was significantly lower in stressed chicks exposed to heat stress, and that the birds tried to resist and cope with heat stress via the thermosensitive areas in the hypothalamus and probably via the endocrine system (metabolic thermoregulation). Tan et al. (2010) found that under heat stress (41 °C) body temperature of broiler chickens increased as compared with the control (24 °C).

٦٤ Results and Discussion

40.6

40.4

40.2

40

39.8

39.6

Bodytemperature Series1 39.4

39.2

39 BT Win. BT Win.after BT Sum. BT Sum.after morning noon morning noon Seasonal variation

Fig. (2): Effect of seasonal variation on body temperature.

Fig.(3): Effect of seasonal variations on body temperature.

٦٥ Results and Discussion

43 Effect of seasonal variations on hematological parameters The effects of seasonal variation on blood hematological parameters are shown in (Table 2). It was found that both of hemoglobin concentration (Hb), packed cell volume (PCV), red blood cells counts (RBCs) and total white blood cell counts (WBCs) were significantly decreased in summer than winter season at the two diurnal periods (morning and afternoon). While both, of mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were significantly increased in summer than winter season at the two diurnal periods (morning and afternoon). However, mean corpuscular hemoglobin concentrations (MCHC) was significantly decreased in summer season at morning; the difference was not significant between the two seasons at the afternoon period. This decrease in Hb and PCV associated with increase in water consumption during heat stress in summer. MCH is important in determining the morphological characteristic of anemia. The effect of diurnal variations on blood hematological parameters is demonstrated in Table (3). At the summer season, results showed that Hb concentration increased significantly in the morning (relative low ambient temperature) than afternoon (relative high ambient temperature), there were no significant differences in all of the other parameters. In addition, at winter season it was found that Hb concentration, PCV % and both RBCs and WBCs counts were significantly elevated at mornings as compared with at afternoons. While there were no significant differences between the morning and afternoon in MCV, MCH and MCHC measurements. The data are in agreement with those of Osman (1996) and Yahav , et al . (1996) who found that exposure of broiler chicks to 42 °C for 2h, and 4h decreased hemoglobin concentration by 12 % in both heat treatments compared with the control birds. The level of hemoglobin in avian species is

٦٦ Results and Discussion influenced by age, sex, gonadal activity and intensity of production (Sturkie, 1986). In general, Sturkie, (1986); Bhattacharyya (1990); Tolba (2000) and ElBadry (2004) reported that heat stress increases water consumption and thus leads to hemodilution. This will cause a decrease in red blood cells (RBC’s) which in turn decreases hemoglobin concentration and packed cell volume. This relationship demonstrates the positive connection between red blood cells and hemoglobin concentration and hematocrit. However, Shlosberg et al ., (1996) stated hematocrit percent (Ht %) might vary greatly, depending upon environmental factors related to ambient temperature and age of broiler chicks. Also, Yahav and Hurwitz (1996) reported that, blood haematocrit level in male broilers 42 dold was decreased after being exposed for 6 h to heat stress (35 ±1 °C). On the other hand, Yahav et al. (1997b) reported that hemoglobin concentration did not change when 8 weeksold male broiler chicks were exposed to 35 °C for 6h compared with the control group at 22 °C. Yahav et al. (1997a) observed that the erythrocyte number of chicks exposed to high environmental temperature (30 °C) from 7 to 42 days of age; was 2.78 × 10 6/ ml blood compared with 2.95 × 10 6/ ml blood in the control group. Early research has shown that hematocrit values of the adult chickens were higher in winter than in the summer. Atta, (2002) observed that the packed cell volume (PCV) increased when chickens were exposed to lower temperature as compared with higher environmental temperatures . Osman (1996 ) found that during the first day of exposure to heat stress (42 °C), hematocrit values decreased by 15 % and 20 % for the birds exposed to heat stress for 3 and 4

٦٧ Results and Discussion hrs, respectively, compared with control birds. Thereafter, the hematocrit values gradually decreased at the second and third day of exposure to heat stress. Birds reared at high ambient temperature had lower hematocrit values than those raised at lower ambient temperature (Yahav, 2000; and Mashaly et al., 2004).

٦٨ Results and Discussion

Table (2) Effect of seasonal variations on hematological parameters.

Morning Afternoon Parameters Summer Winter Sign. Summer Winter Sign. Hb 11.67 ± 0.09 13.62 ± 0.12 *** 11.21 ± 0.12 12.48 ± 0.09 *** (g/dl) PCV 34.83 ±0.002 36.8 ± 0.003 *** 34.16 ± 0.003 35.6 ± 0.003 *** (%) RBC s 3.59 ± 0.07 4.56 ± 0.05 *** 3.57 ± 0.06 4.17 ± 0.07 *** (10 6/mm 3) WBC s 11416.7 ± 287.6 13000.0 ± 369.3 *** 11200.0 ± 226.6 12166.7 ± 290.6 *** (10 3/mm 3) MCV 97.71± 1.47 83.12 ± 1.30 *** 96.22 ± 2.23 85.92 ± 1.67 *** (fl) MCH 32.75 ± 0.61 29.91 ± 0.42 ** 31.54 ± 0.64 30.08 ± 0.64 NS (pg) MCHC 33.51 ± 0.33 36.02 ± 0.41 *** 32.84 ± 0.54 35.03 ± 0.52 ** (g/dl)

Mean ± stander error ** P ≤ 0.01 *** P ≤ 0.001 N.S. Non Significant

٦٩ Results and Discussion

Table (3) Effect of diurnal variations on hematological parameters in each season. Summer Winter Parameters Morning Afternoon Sign. Morning Afternoon Sign. Hb 11.67 ± 0.08 11.21 ± 0.12 * 13.62 ± 0.12 12.48 ± 0.09 * (g/dl) PCV 34.83 ± 0.002 34.16 ± 0.003 NS 37.8 ± 0.003 35.68 ± 0.003 * (%) RBC s 3.58 ± 0.07 3.57 ± 0.06 NS 4.56 ± 0.05 4.17 ± 0.07 * (10 6/mm 3) WBC s 11416.6 ± 287.6 11200.0 ± 266.5 NS 13000.1± 369.2 12166.7 ± 270.6 * (10 3/mm 3) MCV 97.71± 1.47 96.22 ± 2.23 NS 83.12 ± 1.30 85.92 ± 1.67 NS (fl) MCH 32.75 ± 0.61 31.54 ± 0.64 NS 29.91 ± 0.42 30.08 ± 0.64 NS (pg) MCHC 33.51 ± 0.33 32.84 ± 0.54 NS 36.02 ± 0.41 35.03 ± 0.52 NS (g/dl)

Mean ± stander error * P ≤ 0.05 N.S. Non Significant

٧٠ Results and Discussion

44 Effect of seasonal variations on blood biochemical parameters Table (4) presents results of the effect of seasonal variations on serum total proteins, albumin, globulin concentrations and A/G ratio. The reported data of the pervious parameters were all significantly increased during winter than summer seasons at morning and afternoon. Additionally, it was found that both of serum total protein and albumin concentrations were significantly increased at morning (relatively low ambient temperature) that that at afternoon (relatively high ambient temperature) during both summer and winter seasons as shown Table (5). There were no significant differences between morning and afternoon periods in globulin concentration during summer and winter seasons. However A/G ratio was significantly increased at morning than afternoon during summer season, and there was no significant difference between morning and afternoon during winter season Table (5). These results indicate that serum TP tended to be lower in summer than winter and in afternoon during two seasons. The significantly lower TP in summer was due to significantly lower globulin during summer season and afternoon in two seasons, so that serum albumin to globulin ratio exhibited a significant linear increase with the increase in ambient temperature. These results are in agreement with AlHeeti et al . (1985) Kutlu and Forbes (1993); Zhou et al . (1998); Hassan (1999); and Kalmah (2001). They demonstrated that the highest levels in total serum proteins were recorded under moderate temperature and the lowest levels were under high temperature and low humidity. Also, a significant effect was found by (Sahin et al., 2001; Eldossoki, 2004; and Ozbey et al ., 2004). Elaroussi, (1981) found that plasma total proteins were decreased significant when hens were exposed to 38 °C

٧١ Results and Discussion environments. At heat stress 40 °C period, plasma albumin decreased significantly while globulin concentration increased significantly due to heat stress condition. This may be due to changes in body temperature that causes a shift in tissue fluids and thus cause a change in the concentration of plasma proteins. This is in contrast to Renden and McDaniel (1984) who did not detect any significant difference in total proteins of broiler chicks between control (21.3 °C) and those subjected to high ambient temperature (36 °C). Habeeb et al., (1993) found that the overall means of total proteins, albumin, and globulin significantly decreased, due to exposure of the animals to high temperature. Hattingh (1982), Trammell et al., (1988) and Morera et al., (1991) reported similar results. When birds were exposed to high ambient temperature they use compensatory mechanisms to maintain acidbase homeostasis and concentrations of blood nutrients and physiological variable (Borges et al., 2003a and Tolba et al. 2005). Heat stress caused a significant reduction in plasma total protein, albumin and globulins than in control group (Zhou et al ., 1998; Hassan, 1999 and Kalamah, 2001). Serum total proteins varied between among different seasons (Lisano and Kenamer, 1977 ). AlHeeti et al. (1985) found that the highest serum total protein levels were recorded under moderate or low temperature and the lowest ones were recorded under high temperature. In addition, Zhou et al. (1998) reported that serum total proteins and albumin concentration of Cobb broilers were decreased when they were exposed to 30 °C for 3 to 12 hrs for 3 d while, globulin concentration were lower in winter (2.88gm/100ml) than in summer (3.06gm/100ml).

٧٢ Results and Discussion

ElNabarawy (1997) illustrated the diagrammatic representation of the serum protein fractions of Japanese quail chicks at the age of 4 wk that were exposed to chronic heat stress. The electrophoresis patterns of birds from oneday to four weeks of age revealed that chronic heat exposure at 38 °C resulted in high intensity of albumin fraction compared with albumin of other birds that were kept at either 42 °C or 32 °C. While, ElBadry (2004), found that heat stress caused insignificant reduction in serum total protein, A/G ratio and significant reduction in albumin than that of control ducks at different ages. He also found that early heat exposure caused an increase in bird's serum albumin than the heatstressed birds, while serum total protein and its fractions were almost similar to the control. There were significant differences in A/G ratio due to treatment at 8 weeks of age, heat stress decreased A/G ratio as compared with control. Also, acclimation of birds to heat stress at early age caused an improvement for globulin value. Exposure of female Japanese quail, 50d old to solar radiation temperature for 12 h/d during the summer (July) reduced serum total proteins when compared with the control. The solar radiation also reduced albumin, total lipid and cholesterol as compared with the unexposed birds (Nichelmann et al., 2002).

٧٣ Results and Discussion

Table (4) Effect of seasonal variations on serum total proteins, albumin, globulin concentrations and A/G ratio.

Morning Afternoon Parameters Summer Winter Sign. Summer Winter Sig. T. proteins 4.64 ± 0.07 6.13 ± 0.07 *** 4.05 ± 0.04 5.51 ± 0.09 *** (g/dl) Albumin 2.59 ± 0.04 3.69 ± 0.05 *** 2.10 ± 0.03 3.23 ± 0.04 *** (g/dl) Globulin 2.08 ± 0.08 2.44 ± 0.08 ** 1.94 ± 0.06 2.28 ± 0.10 ** (g/dl) A/G 1.26 ± 0.06 1.52 ± 0.06 ** 1.10 ± 0.05 1.45 ± 0.08 ** (Ratio)

Mean ± stander error ** P ≤ 0.01 *** P ≤ 0.001

٧٤ Results and Discussion

Table (5) Effect of diurnal variations on serum total proteins, albumin, globulin concentrations and A/G ratio in each season.

Summer Winter Parameters Morning Afternoon Sign. Morning Afternoon Sign. T. proteins 4.64 ± 0.07 4.05 ± 0.04 ** 6.13 ± 0.07 5.51 ± 0.09 ** (g/dl) Albumin 2.59 ± 0.04 2.10 ± 0.03 ** 3.69 ± 0.05 3.23 ± 0.04 ** (g/dl) Globulin 2.09 ± 0.08 1.94 ± 0.06 NS 2.44 ± 0.08 2.28 ± 0.10 NS (g/dl) A/G 1.26 ± 0.06 1.10 ± 0.05 * 1.52 ± 0.06 1.45 ± 0.08 NS (Ratio)

Mean ± stander error * P ≤ 0.05 ** P ≤ 0.01 N.S. Non Significant

٧٥ Results and Discussion

The data in Table (6) show the effects of seasonal variations on serum ALT, AST, total lipids, triglyceride, cholesterol, and glucose concentrations. It was found that all these parameters were significantly increased during the summer season as compared with winter season both at the relative low ambient temperature of morning and relative high ambient temperature of the afternoon. In addition it was found that all of these parameters were highly significantly increased at the afternoon as compared with the morning periods during both of summer and winter seasons as shown in Table (7). The data are in agreement with those of Morita et al. (1989) and Donkoh (1989) who reported that the plasma glucose concentrations of hens were significantly decreased at 16 hours after cold exposure (0°C for 24 hours), while plasma insulin concentration rapidly and significantly increased after 12 hrs of cold exposure. No significant difference in plasma glucagon and free fatty acids concentration when exposed to cold. They suggested that the decrease in plasma glucose concentration in hens exposed to cold might be due to the abundant release of insulin from pancreatic Bcells. They added that during low ambient temperature it was found that total lipids, triglycerides and cholesterol concentrations were significantly decreased. Blood glucose concentration may increase as a direct response to a transient increase in secretion of adrenaline (epinephrine), noradrenalin, and glucocorticoids (kolb, 1984). These results are in according with Samara et al., (1996) and Sahin et al., (2001) who found that heat stress increased serum glucose level. The increase in glucose under heat stress may be due to the increase in glucocorticoids under heat stress. Li et al., (2001) found that heat stress and other forms of stress increases glucocorticoids activity in cells. Vander et al., (1994) explained that corticosteroids are steroid hormones which are also known as glucocorticoids because of their important metabolic effect on the glucose and other

٧٦ Results and Discussion organic nutrients by promoting hydrolysis of muscle proteins to amino acids and in turn increase the level of glucose when these amino acids are converted to glucose in the liver. Dorfman et al., (2008) reported that chronic intermittent cold stress in rat's hand no significant increase in serum glucose level during cold stress in winter may be due to the decrease in plasma corticosterone levels in winter season. (Khalil 1980; 2002; and 2005) and Eldessoki (2004) found that the increase in plasma glucocorticoid level under heat or cold stresses activates gluconeogenesis process which leads to an increase in serum glucose. Also, in the present study showed the decrease in cholesterol level during winter season and diurnal had been previously reported by Ali, (2004) who found that the plasma cholesterol level of rats tended to be higher during cold stress in winter than in summer. Also, Siegel, (1971) who stated that while there is a depletion of cholesterol from the adrenal, levels in the blood, after an initial dip, increase during stress and evoked by the increased corticosteroid. As known cholesterol is the primary precursor for adrenal steroids, synthesized in the cortical tissue and it is thought that ACTH exert its major effect by increasing the concentration of the high energy compound (cyclic AMP), for the completion of corticosteroid synthesis. Therefore, it is expected that cholesterol increases under stress. Elaroussi (1981) found that plasma cholesterol levels increase under environmental stress, as one of the adaptive reactions, indicating that stress hampers the additive genes affecting and controlling cholesterol level in the blood. It's of interest also to notice the high maternal effects under both climatic conditions, especially under cold stress. However, Isoki (1985) reported that serum total cholesterol was higher in cold stressed rats than in control ones but the effect was insignificant. The reduction in serum

٧٧ Results and Discussion cholesterol level under cold stress in the present study may be due to the high conversion of cholesterol to steroid hormones because the birds of experiment were in before adult stage directly (7 months old). PechWaffenschmidt et al . (1995) reported that changes in the blood system are part of the thermoregulatory responses acquired by birds to enable them to withstand heat stress. Stressed birds established a cascade of physiological adaptive responses include elevation of plasma glucose. This finding is in agreement with Zhou et al . (1998) who stated "Glucose intake may influence blood viscosity, because blood plays an important role in the diffusion body heat. On the contrary , Niu , et al ., (2009) indicated that the levels of blood total lipids and cholesterol decreased with the elevation of ambient temperature. However, exposure of male rabbits to high temperature decreased plasma cholesterol level (Arad et al., 1983; Seyrek et al., 2004). Additionally, Sands and Smith (2002) reported that under heat stress plasma glucose concentration for broilers decreased from 206 to 192 mg/dl for thermoneutral and heat stressed birds, respectively. Borges et al . (2003) suggested that when birds are exposed to high temperatures they respond by increasing respiratory rate, which leads to a reduction in blood CO 2 levels. This reduction results in a decrease in HCO3 concentrations due to the increase in HCO 3 excretion with a reduction of H + excretion by the kidneys to maintain the acid base balance in the bird. Under heat stress challenges, broiler chicken use compensatory mechanisms when attempting to maintain acidbase homeostasis and concentrations of blood nutrients and physiological variables. They also reported that the average of plasma glucose increased from 223 to246 (mg/dl) for thermoneutral.

٧٨ Results and Discussion

Also, Zulkifili et al., (2009) reported that prolonged heat stress (32 °C) in broiler chickens for 5 wks increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities. However, many studies reported that hyperthermia caused significant decrease in serum GOT, (ElMenhali 2002; and Eldessoki, 2004). Also Ayyat et al., (2004) reported that in New Zealand White male rabbits that SGOT and SGPT were significantly lower in summer than in winter.

٧٩ Results and Discussion

Table (6) Effect of seasonal variations on serum allanine transaminase (ALT),aspartate transaminase (AST), total lipids, triglycerides, cholesterol, and glucose concentrations. Morning Afternoon Parameters Summer Winter Sign. Summer Winter Sign. ALT 18.68 ± 0.16 16.90 ± 0.17 ** 18.92 ± 0.14 17.14 ± 0.16 ** (IU/L) AST 173.58 ± 0.95 155.58 ± 0.41 ** 178.83 ± 0.63 159.33 ± 0.73 ** (IU/L) T. Lipids 3.17 ± 0.08 1.43 ± 0.06 *** 3.59 ± 0.10 2.34 ± 0.08 *** (mg/dl) Triglycerides 159.08 ± 1.31 136.67 ± 1.05 *** 164.00 ± 1.89 140.50 ± 0.76 *** (mg/dl) Cholesterol 180.3 ± 12.66 112.33 ± 2.72 *** 214.50 ± 12.93 131.08 ± 2.56 *** (mg/dl) Glucose 227.83 ± 1.58 208.17 ± 2.47 *** 240.42 ± 2.16 213.50 ± 1.71 *** (mg/dl)

Mean ± stander error ** P ≤ 0.01 *** P ≤ 0.001

٨٠ Results and Discussion

Table (7) Effect of diurnal variations on serum allanine transaminase (ALT), aspartate transaminase (AST), total lipids, triglycerides, cholesterol, and glucose concentrations in each season. Summer Winter Parameters Morning Afternoon Sign. Morning Afternoon Sign. ALT 18.68 ± 0.16 18.92 ± 0.14 * 16.90 ± 0.17 17.14 ± 0.16 * (IU/L) AST 173.58 ± 0.95 178.83 ± 0.63 ** 155.58 ± 0.91 159.33 ± 0.73 ** (IU/L) T. Lipids 3.17 ± 0.08 3.58 ± 0.16 ** 1.93 ± 0.06 2.34 ± 0.08 ** (mg/dl) Triglycerides 159.08 ± 1.31 164.00 ± 1.89 *** 136.67 ± 1.05 140.50 ± 0.76 *** (mg/dl) Cholesterol 180.33 ± 12.66 214.50 ± 2.93 *** 112.33 ± 2.72 131.08 ± 2.56 *** (mg/dl) Glucose 227.83 ± 1.58 240.42 ± 2.16 *** 208.17 ± 2.47 213.50 ± 1.71 ** (mg/dl)

Mean ± stander error * P ≤ 0.05 ** P ≤ 0.01 *** P ≤ 0.001

٨١ Results and Discussion

45 Effect of seasonal variations on serum electrolytes concentration Table (8) revealed that the decrease in ostrich serum calcium, phosphorus, sodium and potassium, during summer and winter were insignificant as the ambient temperature increased. Furthermore, it could be noticed also that all these electrolytes increased in the morning than afternoon. This decrease in plasma Ca, Pi, Na, and K levels in the summer were a companied with an increase in body temperature during heat stress which was inversely related to the ostrich ability to maintain blood ionized. Calcium, phosphorus, sodium and potassium concentrations were significantly lower when birds were exposed to heat stress conditions during the summer season. The insignificant reduction in (Na) under heat stress is in accordance with Olsson et al., (1995) who reported that increasing ambient temperature from 20 to 39.5 °C for 5 hrs decreased plasma (Na) and increased plasma aldosterone of goats. Also, Dickson and Harding (1997) showed that maternal heating decreased maternal plasma Na and K concentrations. AbdElKhalek (1997) suggested that the slightly lower K concentration in summer than in winter may be due to the increase in PCV during this season. Moreover, these results are in agreement with Odom et al . (1986); AitBoulhasen et al . (1993) who found that changes in acidbase balance during heat stress reduced the blood ionized calcium and phosphorus levels, which may in turn limit the availability of calcium for egg shell formation. Heat stress has been associated with decreases in broiler weight gain, feed intake, feed efficiency and total mineral retention (Austic, 1985; Sahin and Kucuk, 2003). Similar findings were reported by Edens (1978); Pardue et al . (1985); Belay and Teeter (1996) and ElBadry (2004). Also, Aubach et al., (1992) Findings suggestions further support the above as increased urinary Ca losses in the

٨٢ Results and Discussion animals are always being associated with simultaneous decreases in the important factors in the development of hypocalcaemia and the secondary increase in PTH secretion. Mary Jean (1986) and Khalil (2002) reported that the most important regulator of PTH secretion is the concentration of Calcium in the extra cellular fluid. Elevated Ca suppresses PTH secretion, whereas low serum Ca levels stimulate it. The mechanism of PTH secretion involves the manipulation of the enzyme that converts proOTH to PTH. Calcium inhibits the activity of this enzyme, whereas lowered Ca removes this inhibition and results in an increased conversion of the inactive proPTH to the active PTH molecule, so that the serum PTH rises rapidly. Allen and Somjen, (1983) showed that hypothermia in the chickens stimulates thermal panting and respiratory alkalosis. The elevation in blood pH would theoretically reduce the blood ionized calcium pool by increasing the concentration of calcium that is bound to protein. In addition, organic acid produced during thermal panting would increase the complexes – calcium pool, therefore, further restricting the availability of calcium for eggshell formation. While, Samara et al . (1996) reported no decrease in total calcium concentration in hens acclimated to high cyclic temperature. The interpretation of the potassium concentration was explained from the role of potassium ion electrostatic pressure around the cell. The main concentration of the potassium is presented inside the cell to keep the positive ion concentration and the electrostatic pressure in homeostasis status around the cell membrane. At the same time, the sodium ion is more capable to enter and exit from the cell membrane, which cause the electric balance and the activity of transpiration through the cell membrane in and out (Bruce et al ., 1989). Habeeb et al., (1993) found that the blood Na, K, Ca, and Pi, concentrations decreased as a result of exposure to high temperature. The more hemodilution which occurs to the

٨٣ Results and Discussion animals under heat stress conditions may be also contribute in the decline in blood (Kamal and Shebaita, 1972). Smith et al . (1995) reported that mineral excretion is one of the important consequences of heat distress. High temperature affects availability of minerals. So that, Sahin et al . (2005) found that heat stress causes an increase in excretion of minerals that results in adverse effects on poultry performance. Belay and Teeter (1996) confirmed that increased mineral excretion is one of the important consequences of heat distress when they reported lower rates of phosphorus, potassium, sodium, magnesium, manganese and zinc retention in broilers raised at high cycling ambient temperature (24 to 35 °C) compared with birds housed at 24 °C, similar results were reported in this study. Plasma sodium concentrations was reported to be affected by acclimation when Abdel Mutaal (2003) found that early heat exposure significantly decreased plasma sodium compared with control group at 8 weeks of age. Also Belay and Teeter (1996) reported lower rates of sodium retention in broilers raised at high cyclic ambient temperature (24 to 35 °C) compared with birds housed at 24 °C. While , Altan et al . (2000) and Arad et al . (1983) postulated that heat exposure with water availability did not result in any changes in plasma Na concentration. Similar to these reports, AbdEllah et al . (1995) concluded that heat stress had no effect on the plasma sodium of chickens. Huston (1978); AitBoulahsen et al. (1989); and Belay and Teeter (1996) reported that blood potassium concentration of hens decreased by high temperature. This is in agreement with the current findings that potassium was less in summer in ostrich. In contrast, Borges et al . (2003); Abdel Mutaal (2003) found that the levels of plasma potassium increased due to heat stress.

٨٤ Results and Discussion

While, PechWaffenschmidt et al . (1995) reported that changes in the blood system are part of the thermoregulatory responses acquired by birds to enable them to with stand heat stress. Stressed birds established a cascade of physiological adaptive responses including elevation of plasma calcium, phosphorus and electrolytes.

٨٥ Results and Discussion

Table (8) Effect of seasonal variations on serum electrolytes concentrations.

Morning Afternoon Parameters Summer Winter Sign. Summer Winter Sign. Calcium 8.14 ± 0.06 8.89 ± 0.09 * 7.83 ± 0.07 8.52 ± 0.06 * (mg/dl) Phosphorus 4.83 ± 0.07 5.11 ± 0.07 ** 4.69 ± 0.06 4.92 ± 0.05 ** (mg/dl) Ca/Pi 1.69 ± 0.03 1.73 ± 0.03 * 1.67 ± 0.02 1.73 ± 0.02 * (Ratio) Sodium 186.3 ± 5.50 205.1± 5.05 ** 179.2 ± 5.89 191.69 ± 6.10 NS ( mg/dl) Potassium 17.47 ± 0.11 18.11± 0.09 * 17.19 ± 0.08 17.70 ± 0.07 * ( mg/dl)

Mean ± stander error * P ≤ 0.05 ** P ≤ 0.01 N.S. Non Significant

٨٦ Results and Discussion

Table (9) Effect of diurnal variations on serum electrolytes concentrations in each season.

Summer Winter Parameters Morning Afternoon Sign. Morning Afternoon Sign. Calcium 8.14 ± 0.06 7.83 ± 0.07 * 8.89 ± 0.09 8.52 ± 0.06 * (mg/dl) Phosphorus 4.83 ± 0.07 4.69 ± 0.06 * 5.11 ± 0.07 4.92 ± 0.05 * (mg/dl) Ca/Pi 1.69 ± 0.03 1.67 ± 0.02 NS 1.73 ± 0.03 1.73 ± 0.02 NS (Ratio) Sodium 186.3 ± 5.50 179.2 ± 5.89 * 205.08 ± 5.05 191.69 ± 6.10 * (mg/dl) Potassium 17.47 ± 0.11 17.19 ± 0.08 * 18.11± 0.09 17.70 ± 0.07 * (mg/dl)

Mean ± stander error * P ≤ 0.05 N.S. Non Significant

٨٧ Results and Discussion

46 Effect of seasonal variations on total body water. Table (10) show the results obtained on total body water of ostrich in winter and in summer and the effect of seasonal variation during summer and winter, heat stress of the summer season caused significant increase in total body water (TBW) by 15.04% and a significant decrease in total body solid (TBS) by 20.33 %, although live body weight (LBW) was not affected by the season. The average TBS value that was lost during summer was higher than the average increase in TBW. These results indicate that ostrich lost from their body solids during summer season and increased their body water (Habeeb et al., 1992). These results were agreement and confirms the with results obtained concerning the biochemical parameters, such as the decrease in total proteins, albumin, minerals, thyroid hormones (T 3), aldosterone hormone and blood picture parameters Hb, PCV, WBCs and RBCs. Therefore, the decrease in all these parameters may be due to the heamodiluation as a result to the increase in TBW in birds during heat stress conditions of the summer season (Habeeb et al., 1993 and 2001). Exposure of the birds to the stressful conditions of summer season increased significantly cortisol level, total lipids and the activities of the liver enzymes (liver function) (Habeeb et al., 1992). Heat adaptability indices used for predicting the productive animals under hot conditions are based on their production level, water balance, protein balance, thermal response and heat induced changes in hormonal levels (Habeeb et al., 2007 and 2008).

٨٨ Results and Discussion

Table (10): Effect of seasonal variations on total body water.

Parameter Winter Summer P.P LBW 50.33 ± 0.33 50.50 ± 0.42 N.S TBW 29.73 ± 0.65 34.20 ± 0.49 ***

TBS 20.46 ± 0.67 16.3 0± 0.45 ***

Mean ± stander error *** P ≤ 0.001 N.S. Non Significant

47 Effect of seasonal variations on serum hormone levels

Seasonal variation effect on serum triiodothyronin (T 3), cortisol and aldosterone hormones is demonstrated in Table (11).The results reported show that T3 hormone concentration was highly significantly increased during the winter than summer season at both of morning and afternoon periods. Unlike aldosterone hormones who was highly significantly increased during summer than winter season at both morning and afternoon periods. Moreover, results in Table (12) shown the effect of diurnal variations on serum triiodothyronine (T 3), and aldosterone hormones and is suggesting that (T3) was highly significantly increased at mornings than afternoons during the summer and winter seasons, while aldosterone hormones was highly significantly elevated at afternoon during the summer season as compared with that at the morning period. The present results in winter were similar with those of Snyder et al. (1991) who found that exposure of birds to an ambient temperature of 10 oC caused an elevated serum thyroid hormone levels. The reduction in metabolic rate due to

٨٩ Results and Discussion exposure to high ambient temperature may be referred to the decrease in thyroid activity. Furthermore, the current results are in agreement with a number of studies that have suggested that thyroid activity is affected by the environmental temperature. Thyroid secretion rate was decreased by high temperature in chickens. Calculating concentration of (T 3) is reduced at high temperature (Hillman et al ., 1985) and appears to be linearly related to feed intake (Yahav et al ., 1995; 1996; 1998). The relationship between thyroid function and change of environmental temperature in birds has received considerable attention. The physiological importance of thyroid output is presumably through the influence of thyroid on metabolic rate as extensively evidenced in mammals and birds (Heninger et al . (1960). Bowen and Washburn, (1985); Hillman et al., (1985) Sokolowicz and Herbut (1999) and ElBadry, 2004) found that rearing both pullets and cocks under a high ambient temperature resulted in a reduced concentrations of thyroid hormones (triiodothyronine and thyroxin) in blood plasma and a decreased metabolic rate. Earlier studies showed that thyroxin secretion rate in summer were half than that in winter (Sturkie, 1976). The decrease in the thyroxine hormone lead to a low protein biosynthesis ( Habeeb et al., 1989) which may explain the decrease in the live body weight, as well as, daily gain under hyperthermia conditions. From the nutritional point of view, the decline in growth performance of heat stressed ostrich may be also due to the depression in appetite and consequently feed intake. The depression in proteins biosynthesis on one hand and feed intake on the other hand, lead to pronounced decrease in each of the protein fractions and their end products, i.e., as the temperature rises.

٩٠ Results and Discussion

Habeeb et al. (1993) found that the overall means of thyroxin hormone (T 3) significantly decreased, due to exposure of the animals to high temperature. Hattingh (1982), Trammell et al., (1988) and Morera et al., (1991) reported similar results. Rudas and Pethes (1984) and Uni et al. (2001) reported that thyroxin (T 4) concentration was reduced after exposure to heat stress (35 °C) for 1 hr. They added that conversion of T 4 to T 3 played a major role in the early phase of temperature acclimation. Arjoba et al. (1990) and Sahin et al. (2002) showed that exposure of chickens to high temperature at 42 d of age was associated with a reduction in plasma T 3 concentration regardless of previous hightemperature exposure. McNabb and King (1993) postulated that reduced metabolic rate and hence body heat load is one of the possible mechanisms of thermo tolerance that is regulated to a large extent by triiodothyronine (T 3). McNabb, et al., (1993) observed also that the increase in temperature (2 hrs increase of air temperature by 10 oC on days 14) resulted in a decrease of the thyroid hormone level and metabolic rate while the lowered temperature increased thyroid hormone level and air consumption. Osman (1996); Yava and Hurwitz (1996); and El Badry (2004) found that T 3 concentration decreased significantly by the exposure to high temperature and that conversion of (T 3) into (T 4) is reduced to regulate the metabolic rate and then heat production. Yahav and Hurwitz (1996) revealed that circulating concentration of triiodothyronine (T 3) was reduced at high temperature. Also they found that plasma (T 3) concentration decreased after a 24h heat exposure at 5 day of age, and this ability to reduce (T 3) concentration reduced heat production and as a consequence improved thermo tolerance. Therefore,

٩١ Results and Discussion it may be anticipated that induction of thermo tolerance will be associated with modulation of plasma (T 3) concentration. Buys et al., (1999) showed that a higher feed intake and metabolic rate is linked to an increase in T 3 and T 4. On other hand, Williams and Njoya (1998); Uni et al., (2001) and Yahav and McMurty (2001) reported that exposure of chickens to high environmental temperature decreased feed intake by about 14% and decreased plasma T 3 but not T 4 concentration. While, May et al., (1986) reported that acute exposure of growing chickens to high temperatures from 35 to 41 °C did not appear to consistently affect serum T 3 or T 4 concentration. But chronic exposure (>2 week) reduces serum T 3 and T 4 or results in lower T 3 but higher T 4 (Williamson et al., 1985). Our results were supported by results obtained from Yahav and Plavnik (1999) who demonstrated a significant decline in the major metabolic hormone, triiodothyronine, during exposure to thermal challenge. Sahin et al . (2002) demonstrated that serum concentration of T4 and T 3 in broiler chicks reared under heat stress (32 °C) was 11.75 and 2.13 mg/ml, respectively. Yahav et al., (2004) found that thermal manipulation (TM) caused a significant reduction in chick's body temperature and significant decline in plasma thyroid hormones concentration, but had no effect on plasma corticosterone concentration. They concluded that (TM) did not affect body weight but had a positive effect on thermoregulation, most probably through reducing the metabolic rate of birds. ElBadry (2004) stated the mechanisms associated with the induction of thermotolerance by earlyage temperature conditioning may involve the modulation of heat production through reduction in plasma triiodothyronine (T 3) concentration.

٩٢ Results and Discussion

Moreover, Aldosterone hormone an important for the maintenance sodium balance and is involved in the absorbance of sodium or potassium ions, the increase in this hormone leads to the increase in the absorbance rate of sodium in the kidney according to Khalil (2002). This Author that there were a positive relationship between aldosterone hormone levels and the increase in Na absorption from proximal and distal tubules. This relationship is related to the increase in Na & K ATPase enzyme activation in the proximal tubules. It was shown that under conditions of heat stress, that both ACTH and vasopressin secretion were increased (Aguilera, 1996), and when ACTH was secreted in large amounts, such as in conditions of stress, it could also stimulate aldosterne secretion.

٩٣ Results and Discussion

Table (11) Effect of seasonal variations on serum hormone levels.

Morning Afternoon Parameters Summer Winter Sign. Summer Winter Sign. T 3 92.18 ± 1.10 106.98 ± 2.3 *** 77.97 ± 1.39 90.54 ± 1.39 *** (mg/dl) Aldosterone 3.98 ± 0.09 3.34 ± 0.18 ** 3.48 ± 0.19 2.55 ± 0.15 ** (g/ml)

Mean ± stander error ** P ≤ 0.01 *** P ≤ 0.001

٩٤ Results and Discussion

Table (12): Effect of diurnal variations on serum hormone levels in each season.

Summer Winter Parameters Morning Afternoon Sign. Morning Afternoon Sign. T 3 92.18 ± 1.10 77.97 ± 1.4 *** 106.98 ± 2.3 90.54 ± 1.39 *** (mg/dl) Aldosterone 3.98 ± 0.09 3.48 ± 0.19 ** 3.34 ± 0.18 2.55 ± 0.15 ** (g/ml)

Mean ± stander error ** P ≤ 0.01 *** P ≤ 0.001

٩٥ Results and Discussion

48 Effect of seasonal variations on serum proteins profile NativePAGE electrophoresis patterns phylogeny of blood proteins of ostrich bird during winter morning and summer at the morning and the afternoon. Subchronic heat stress of ostrich birds during seasonal variations are shown in (Figure 3), were electrophoresed to screen the physiological titer through native protein pattern. The results reported in (Table 13, 14) and (Figure 3) showed that the protein profile was revealed as (11) types of protein with molecular weight ranged from 38.3 to 219.5 K.Da and Rf ranged from 0.02 to 0.71 for the first sample at (Lane 1, winter sample), while at (Lane 2, the summer morning sample) produced 13 types of native protein with molecular weight ranged from 36.9 to 219.5 K.Da and rate of flow (Rf) ranged from 0.02 and 0.71. But, the third sample (summer at afternoon at Lane 3) had 12 types of protein, the molecular weight is ranged from 37.2 to 217.7 K.Da with (Rf) ranged from 0.02 to 0.71. In the present study, there are 8 common bands from total 15 bands (more than 50%), these bands with average molecular weight ranged from 218.9 to 37.5 K.Da, while the common bands between the summer samples recorded 12 bands from 13 with the percentage of 92.3 compared to that between the winter and summer samples (60%). Also, it was found that the characteristic bands for the samples were appeared as band 3 and 9 with molecular weight of 192.3 and 99.4 K.Da for winter sample. Another characteristic band with molecular weight of 163.2 K.Da was disappeared from the summer afternoon sample. From the other hand the epigenetic physiological activity recorded the highest level 0.96 as similarity index between the summer samples, while the lowest one, that between the summer afternoon and winter sample 90.70), that was recorded in (Table 14).

٩٦ Results and Discussion

The biological function is controlled by specific type of protein according to the central dogma (DNA gene ▬► mRNA ▬► protein ▬► characteristic), Holdane, (1917). It states that each gene produces different types of messenger (mRNA) each type of (mRNA) produces specific type of protein, this agreement with Gabriel et al . (1996) who showed that heat stress induced an increase in Hsp 70 mRNA and Hsp 70 in broiler liver. These proteins their function is to protect heat stressed cells and to prevent proteins miss folding by heat stress (Craig, 1988). The protein profile refers to pervious results, higher concentrations (total protein, Albumen and Globulin) in winter as in (Table 4 and 5) while the types of winter protein is less than the summer protein, that is may be due to the biological process in winter is less than that in summer. In general the raising in temperature during summer season caused a quantitative mutation as in the M.W of 65.9 K.Da type, it is high in winter than that of summer season, and it may be used in acclimatization process. This quantitative mutation were focused as protein amount ratio as 1.35, 1.39, 1.6, and 1.58 at the bands 5, 8, and 12, respectively.

٩٧ Results and Discussion

Fig. (4) Electrophoretic pattern by nativePAGE of protein fraction of ostrich birds during seasonal variation (summer and winter). Mar : Marker (Biolab) wide range high molecular weight unstained. W.m : Lane of winter morning . S.m : Lane of summer morning . S.a : Lane of summer afternoon.

٩٨ Results and Discussion

Lane 1

Fig. (5): Densitograms of protein pattern separation of Marker (Biolab).

Lane 2

Fig. (6): Densitograms of protein pattern separation of ostrich in the winter morning.

٩٩ Results and Discussion

Lane 3

Fig. (7): Densitograms of protein pattern separation of ostrich in the summer morning.

Lane 4

Fig. (8): Densitograms of protein pattern separation of ostrich in the summer afternoon.

١٠٠ Results and Discussion

Table (13) Physiological epigenetic sublevel expressed as native protein in different samples of ostrich blood serum. No. W.m S.m S.a Aver. Amo. M.w Ratio M.w Amo. R.f M.w Amo. R.f M.w Amo. R.f R1 219.5 8.87 0.02 219.5 8.48 0.02 217.7 7.74 0.02 219.9 1.2 2 203.2 5.77 0.05 201.4 5.11 0.05 199.6 5.18 0.05 201.4 1.1 3 192.3 7.50 0.06 192.3 4 185.0 6.23 0.07 183.2 7.18 0.07 184.1 1.2 5 163.2 9.16 0.10 163.2 6.75 0.10 163.2 1.4 6 123.3 12.17 0.16 119.6 8.76 0.16 121.5 12.2 0.16 121.5 1.4 7 110.6 6.93 0.21 110.6 8.0 0.21 100.6 1.2 8 103.2 9.42 0.27 103.2 5.88 0.27 102.8 6.67 0.27 103.1 1.6 9 99.4 8.33 0.29 99.4 10 98.1 6.55 0.31 98.8 7.31 0.31 98.5 1.1 11 87.5 8.03 0.35 88.1 8.45 0.35 88.2 1.1 12 66 8.31 0.45 66.6 5.98 0.45 65.2 5.26 0.46 65.9 1.6 13 62.5 6.87 0.40 62.5 6.30 0.48 62.5 6.18 0.48 62.5 1.1 14 58.5 9.18 0.52 58.2 11.9 0.52 57.1 10.8 0.53 57.9 1.3 15 38.3 14.4 0.71 36.9 13.2 0.71 37.2 15.1 0.71 37.5 1.1 W.m : Sample of winter morning . S.m : Sample of summer morning . S.a : Sample of summer afternoon.

١٠١ Results and Discussion

Table (14) Similarity Index and genetic distance between blood samples of ostrich under study. Sample W.m S.m S.a W.m 0.75 0.70 S.m 0.25 0.96 S.a 0.30 0.04 W.m : Sample of winter morning . S.m : Sample of summer morning . S.a : Sample of summer afternoon.

In the present study, the ostrich birds in summer exhibited higher values of protein in blood plasma than ostrich in winter. Also, heat stress caused an increase in the serum protein profiles of compared with the birds during the winter season. These results was in agreement with ElNabarawy, (1997) who showed that the profiles of serum protein electrophoresis (SPE) for heat shock treatments of Japanese quail chicks at two weeks of age revealed slight variations in the intensity of bands due to heat treatments. Heat shocked samples exhibited substantially lower intensity for alpha l globulin only at 3 and 6 h compared with the control. Also, Price and Caderwood (1991) who showed that heat stress induced activation of the Hsp 70 K.Da. Also Gabriel et al . (1996) who showed that heat stress induced an increase in Hsp 70 K.Da mRNA and Hsp 70 K.Da in broiler liver. In addition , Miller and Qureshi (1992c) they found that White Leghorn female synthesized Hsp after exposure to 44 °C. There is also good evidence that Hsp synthesis is increased under heat stress condition i.e. elevation of ambient

١٠٢ Results and Discussion temperature (Craig, 1988). Therefore, heat shock protein 70 (Hsp70) is a stressinduced protein. High levels of (Hsp70) can be induced by cells in response to hyperthermia. These proteins, function is to protect heat stressed cells and to prevent proteins miss folding by stress. Miller and Qureshi (1992a) reported that the expression of Hsp by peritoneal macrophages (PM) isolated from various avian species including chicken, turkey, quail and ducks exhibited a comparable induction of the three major families of stress proteins Hsp 23, Hsp 70 K.Da and Hsp 90 K.Da following a 1hheat shock at 45 °C. However, a lower molecular mass observed for the duck peritoneal macrophages (PM) protein Hsp 32 and Hsp 23 when compared with the Hsp profiles of the chicken, turkey and quail macrophages. These differences in the molecular masses of the Hsp 32 might indicate a species specific (Miller and Qureshi, 1992b). Therefore, Hsp 70 and others are essential for cellular survival from heat stress and other types of physiological challenges. The expression of (Hsp s) can be detected within several hours and last 35 days in duration (Kregel, 2002).

١٠٣ Summary and Conclusions

5-SUMMARY AND CONCLUSION

The present study was carried out at the ostrich project experimental farms and laboratories of the Nuclear Research Center in Inshas Atomic Energy Authority in Egypt. The effects of seasonal and diurnal variations on thermoregulation of ostrich were studied through the summer and winter seasons. In this study, 12 ostrich aged 7 months old were used in the summer and same number in the winter from the ostrich breeding flock. All birds were exposed daily to ambient temperatures during summer and winter, and the birds were fed ad libitum grower ration (19 % protein and 2450 K cal ME /Kg). 5-1 Blood samples Blood samples were with drawn from the wing vein of each bird with a 21G needle connected a 5ml syringe. Birds had their heads covered in a woolen sock during sampling for ease of containment. Blood samples were taken at the hot day of summer (40 ±1ºC) (June month) and the cold day of winter (18 ± 1ºC) (January month) to compare our measured parameters. Blood samples were placed in two tubes, one containing lithium heparin to determine hematological parameters and the other without anticoagulant and left to clot. In the laboratory, samples were centrifuged at 1600 rpm for 15 minutes and the resulting serum was stocked at (-20) until use. Serum samples were evaluated for different biochemical parameters during both seasons and a comparison was made between both seasons. The present study was carried out to evaluate the effect of temperature variation during summer and winter seasons and diurnal effect within on changes in some physiological and blood biochemical parameters. The daily feed consumption

- ١٠٤- Summary and Conclusions

(g/bird/day) and water consumption (ml/bird/day) was measured for a representative 7 days during each season. Cloacal temperatures was measured and blood samples were take twice, one in the morning at 7 am and once in the afternoon at 3 pm during a representative 7 hot days of June (40±2ºC) (summer) and the 7 cold days of January (18±2ºC) (winter). Red blood cell (RBCs) counts and total white blood cell (WBCs) counts, hemoglobin concentration (Hb) and packed cell volume (PCV) were determined. Mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHC) were calculated. In serum, total protein (TP), albumen (A) and globulin (G) concentration were measured. Furthermore, serum alanine transaminase (ALT), aspartate transaminase (AST), glucose, urea, total lipids, triglycerides and cholesterol concentrations were determined. Also, serum triiodothyronine (T 3), and aldosterone levels, all were estimated by radioimmunoassay (RIA) technique and counted at Gama counter model RIA SAR, Pacard, Model 0540501. Finally, serum protein electrophoresis was conducted by Native-PAGE method (Native polyacrylamide gel electrophoresis) on vertical slab gel to determine electrophortic pattern and possible protein profiles in blood proteins of ostrich. Results indicated that feed consumption contrary to water consumption was significantly increased during winter than in summer season. Moreover, body temperature increased significantly during the summer season as compared with the winter season and was significantly elevated at the afternoon than at the morning period. Blood picture and serum biochemical analysis could be summarized in the following:- 1-Blood picture showed that heamoglobin concentration (Hb), packed cell volume (PCV), red blood cells (RBCs) counts and total white blood cell (WBCs) counts were significantly decreased in the summer than in winter months at the two

- ١٠٥- Summary and Conclusions diurnal periods. While both, mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were significantly increased in summer than winter season at the morning and afternoon periods. However, mean corpuscular hemoglobin concentrations (MCHC) was significantly decreased in summer season at morning only. In addition, at the summer season, the present results showed that (Hb) concentration was significantly increased at the morning than in the afternoon while there were no significant differences in all of the other parameters. In addition, at winter season it was found that (Hb) concentration, PCV% and both (RBCs) and (WBCs) counts were significantly elevated at morning as compared with at afternoon values. In the mean time, no significant differences between the morning and afternoon in MCV, MCH and MCHC measurements were observed. 2- Serum total proteins, albumin, globulin concentrations and A/G ratio were significantly increased during the winter than the summer seasons at morning and afternoon. Additionally, it was found that both, total protein and albumin concentrations were significantly increased at morning than the afternoon during both summer and winter seasons. 3- Serum ALT, AST, total lipids, triglyceride, cholesterol, urea and glucose concentrations were significantly increased during summer season as compared with winter season at morning and afternoon periods. The increase in those serum parameters was highly significant during the afternoon as compared with at morning periods, both in summer and winter seasons.

4- Triiodothyronine (T 3) hormone serum level concentration was highly significantly increased during the winter than the summer season with the morning values higher than the afternoon in both seasons (summer and winter). Unlike T 3, the increase in aldosterone hormone level was significant during the summer as compared to the winter season with the

- ١٠٦- Summary and Conclusions morning higher in both seasons (summer and winter) than in the afternoon. 5- Serum electrolytes concentration, calcium, phosphorus, sodium and potassium, for ostrich during summer and winter was significantly decreased as the ambient temperature increased in summer than in winter and during the morning and afternoon periods, respectively. This decrease in serum Ca, Pi, Na, and K, levels were a companied with an increase in body temperature during heat stress which was inversely related to the ostrich ability to maintain a stable blood ionized Ca level. Calcium, phosphorus, sodium and potassium concentrations were significantly lower when birds were exposed to heat stress conditions during summer season. 6- Seasonal variations during the summer and winter affected on total body water (TBW) were significant. Heat stress of the summer season caused significant increased in TBW by 15.04% and a significant decrease in total body solids (TBS) by 20.33 % although (LBW) was not affected by the season. The average loss in TBS value during the summer was higher than the average increase in (TBW). 7- Winter and summer serum proteins were electrophoresis by Native-PAGE electrophoresis patterns phylogeny of serum proteins of winter, summer at the morning and the summer at the afternoon. Sub-chronic heat stress of ostrich birds during seasonal variations, were electrophoreses was employed to screen the physiological induced change in native protein. In the present study 8 common bands from total 15 bands (more than 50%) was obtained, these bands with average molecular weight ranging from 218.9 to 37.5 while the common bands between the summer samples recorded 12 bands from 13 with a percentage of 92.3 for the comparison between the winter and summer samples (60%). Also, we found characteristic bands for the samples of winter and the summer; it appeared that band 3 and 9 with

- ١٠٧- Summary and Conclusions molecular weight of 192.3 and 99.4 K.Da are characteristic for the winter sample. While a characteristic band with molecular weight of 163.2 Kda was disappeared from the summer afternoon sample. On the other hand the epigenetic physiology level recorded the highest level value of 0.96 for the similarity index between the summer samples while, the lowest one, was between the summer afternoon and winter sample recording 90.70, as shown in (Table 14). In general the raising in temperature during summer season causes a quantitative shift in protein bands as in the ban with M.W of 65.9 K.Da type, was higher in winter than that of summer season this may be attributed acclimatization process that took place. This quantitative shift in protein bands were focused as protein amount ratio as 1.35, 1.39, 1.6, and 1.58 at the bands 5, 5, 8, and 12, respectively.

CONCLUSION: It is concluded from the present study that heat stress has negative effect on most of the parameters studied and if the diets presented in summer is supplemented with nutrients to overcome the negative effect that will help the ostrich bird to better perform under such conditions. Problem reports have recommended nutrients like vitamins C, and E, sodium bicarbonate and yeast.

- ١٠٨- References

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St. Joseph, Mich.: American Society of Agric. Engin.; Transactions of the ASAE. 35: 315-318. Yahav, S. (2000). Relative humidity at moderate ambient temperatures: its effect on male broiler chickens and turkeys. Br. Poult. Sci. 41: 94-100. Yahav, S. and I. Plavnik (1999a). Effect of early age thermal conditioning and food restriction on performance and thermotolerance of male broiler chickens. Br. Poult. Sci. 40: 120-126. Yahav, S. and J. P. McMurtry (2001). Thermotolerance Acquisition in Broiler chickens by Temperature conditionings early in life, the effect of timing and ambient temperature. Poultry Sci., 80:1662-1666. Yahav, S. and S. Hurwitz (1996). Induction of Thermotolerance in male broiler chickens by temperature conditioning at an early age. Poultry Sci. 75: 402-406. Yahav, S.; A. Straschnow; I. Plavnik and S. Hurwitz (1996). Effecta of diurnal cyclic versus constant temperature on chickens growth and food intake. Br. Poult. Sci. 37: 43-54. Yahav, S.; A. Shamai; A. Haberfeld; G. Horev; S. Hurwitz and M.F. Einat (1997a). Induction of Thermotolerance in chickens by temperature conditioning heat shock protein expression. Pages 628- 636 in: An Update in Thermoregulation from Cellular Functions to Clinical Relevance C.M. Blatteis, ed, New York Academy of Sciences, New York, NY. Yahav, S.; A. Straschnow; I. Plavnik and S. Hurwitz (1997b). Blood system response of chickens to changes in environmental temperature. Poultry Sci. 76: 627-633.

- ١٤٠- References

Yahav, S.; Straschnow, A.; Luger, D.; Shinder, D.; Tanny, J. and Cohen, S. (2004). Ventilation, sensible heat loss, broiler energy and water balance under harsh environmental conditions. Poultry Science. 83 (2): 253- 258. Yalcin S.; S. Ozkan; L. Turkmut and B. Siegel, (2001). Responses to heat stress in commercial and local broiler stocks. 1. Performance traits. Br. Poultry Sci. 42: 149- 152. Zhou, W.T.; Fujita, M.; Ito, T. and Yamaamato, S.A.A. (1997). Effects of early heat exposure on thermoregulatory responses and blood viscosity of broilers prior to marketing. British Poultry Science. Vol. 38, No. 3, pp. 301-306, 29. Zhou, W.T.; Fujita, M.; Ito, T. and Yamaamoto, S.A.A. (1996b). Effects of intermittent short-term acclimation to 32 deg oC. On some thermoregulatory responses and standing behavior of laying hens exposed acutely to 36 deg oC. Japanese Poultry Science. Vol. 33, No. 4, pp. 221-229, 21. Zhou, W.T.; M. Fujita; S. Yamamoto; K. Iwasaki; Oyama R. Ikawa and H. Horikawa (1998). Effects of glucose in drinking water on the changes of whole blood viscosity and plasma osmolality of broiler chickens during high temperature exposure. Poultry Sci. 77: 644-647. Zollner, N., and K. Kirsch (1962 ): Z.ges. exp. Med. 135:545. Zulkifli, A. AlAqil, A. R. Omar , A. Q. Sazili and M. A. Rajion. (2009). Crating and heat stress influence blood parameters and heat shock protein 70 expression in broiler chickens showing short or long tonic immobility reactions Poult. Sci. 88:471-476

- ١٤١- ا : ى اـــ ـــ ات ا ا ااري ام ار : ا ( اآ را ا ) ام ارا اج اا ان آــــ ا راــــ ــــــ ازه اــ اـــ إاء ا ر ام آ اث او را اات ا ( اف درت اارة ا اء ) و ) آ اات ا ( اف درت اارة اح اء ) ات ا و ام ا و ا. ام ارا د ١٢ ر ام ا ٧ ا . ا ( ١٩ % و و ٢٤٥٠ آ آرى ) و اء ر ار س ى اك ا و اء ة أع آ ا و اء . س در ارة ار ا و ا ت ام ا و ا ا ( ء ) آ ا و اء . و ام س ى آ ا و ات ا إ آات ام ااء و اء و ب ا اى . آ س ى م ام آ او ا و ا و ا إ آ اآز و اات ا و اول . آ س ى

ام آ إ ALT و AST و آ ه ار T3 و و ا ون aldosterne و آ Protein electrophoresis اوت ا ا واء او ر وت ا اار (heat shock proteins (Hsp . .

١ ١ آ ا ا آ :

• ار اك ا رة اء ا ذ و ا اج ارى وال ا آ ادة اك اء اء ا اء ا ء ا ا . .

• ار در ارة ا ر رة ا اء اة ا ا ا و ا ارى رات ا وا . .

• اض ي ى آ ا و ات ا PCV و د آات ام ااء و اء ا ا و ا اء . .

• ارع ي آ MCV و MCH ا اء اة ا و ا آن اض ي ى MCHC اة ا . .

• ا آن اض ا ي اة ا ا هك اف ي اح و اء ى آ ات ا PCV و د آات ام ااء و اء و MCV و MCHC و MCH . .

• اء اح ارع ى آ ا و ا ت ا PCV و د آات ام ااء و اء اء .

٢ ٢ هك وق ى آ و MCV و MCHC و و . . MCH

• ال ارع ى آ او ا و ا و ا و ا إ ا A/G اء ا اح اء . .

• و اة ادة ا ى او ا و ا اح اء ا و اء . .

• آ ارع ى آ إت ا ALT و AST و اات ا و ا ات ا و اول و اآز ا و ءا اء و ءا . و اة ارع اه اء ر ح و ذ ا و اء . .

• و ن ار T3 ارع ى ان ءا . . آ آ ادة اة ا ا آ ا . و و . ادة ى هن ا ون aldosterne ا اء . و آ ادة اء آ ادة اء . .

• و آ ات ادم وام وام وار ات ا اض ي ت ادم وام وار ا ر اء وآ ة ا اح ا واء ا ن ادم ارع ا

٣ ٣ ة اح اء آ ات ا اض ي ن ام اء ر ا . .

• و ن ا ون ارع ى ان ا و ءا اء و ءا . و اة ار ع ى ان اح ر ء و ذ ا . .

• و ء ا ا اص وآ ا و ى ار او ا ان ام ن آ اء ا ر اء ذ اان ا و ارى و ا ء ا ا ل ا ا . .

• و Protein electrophoresis اوت ا ا واء او ر وت ا اار heat ( shock proteins (Hsp ات ا ر د ٢ اوت اردة اء ور د ٤ اوت ارة ا ا و اء اارى اء ا . .

٤ ٤ ﺘﺎﺜﻴﺭﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻝﻤﻭﺴﻤﻴﻪ ﻋﻠﻰ ﺍﻝﺘﻨﻅﻴﻡ ﺍﻝﺤﺭﺍﺭﻱ ﻓﻲ ﺍﻝﻨﻌﺎﻡ

ﺭﺴﺎﻝﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﺤﻤﺩﻯ ﻋﺒﺩ ﺍﻝﺭﺤﻤﻥ ﻤﺤﻤﺩ ﺒﺴﻴ ﻭﻨﻰ ﺩﺒﻠﻭﻡ ﺩﺭﺍﺴﺎﺕ ﻋﻠﻴﺎ ( ﺘﻜﻨﻭﻝﻭﺠﻴﺎ ﺩﻭﺍﺠﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٠ ﻡ ﻡ ﻤﺎﺠﺴﺘﻴﺭ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ - ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٥ ﻡ ﻡ

ﺍ ﺴﺘﻴﻔﺎﺀ ﻝﻤﺘﻁﻠﺒﺎﺕ ﺍﻝﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻝ ﻌﺎﻝﻤﻴﻪ ( ﺩﻜﺘﻭﺭﺍﻩ ﺍﻝﻔﻠﺴ )ﻪﻔ )ﻪﻔ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺇﻨﺘﺎﺝ ﺤﻴﻭﺍﻨﻲ - ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) )

ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ ﺒﺎﻝﻘﺎﻫﺭﺓ - ﺠﺎﻤﻌ ﺔ ﺍﻷﺯﻫﺭ

١٤٣٢ ﻫـ ٢٠١١ﻡ

ﺘﺎﺜﻴﺭﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻝﻤﻭﺴﻤﻴﻪ ﻋﻠﻰ ﺍﻝﺘﻨﻅﻴﻡ ﺍﻝﺤﺭﺍﺭﻱ ﻓﻲ ﺍﻝﻨﻌﺎﻡ

ﺭﺴﺎﻝﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﺤﻤﺩﻯ ﻋﺒﺩ ﺍﻝﺭﺤﻤﻥ ﻤﺤﻤﺩ ﺒﺴﻴﻭﻨﻰ ﺩﺒﻠﻭﻡ ﺩﺭﺍﺴﺎﺕ ﻋﻠﻴﺎ ( ﺘﻜﻨﻭﻝﻭﺠﻴﺎ ﺩﻭﺍﺠﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٠ ﻡ ﻡ ﻤﺎﺠﺴﺘﻴﺭ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ - ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٥ ﻡ ﻡ

ﺍ ﺴﺘﻴﻔﺎﺀ ﻝﻤﺘﻁﻠﺒﺎﺕ ﺍﻝﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻝ ﻌﺎﻝﻤﻴﻪ ( ﺩﻜﺘﻭﺭﺍﻩ ﺍﻝﻔﻠﺴﻔ )ﻪ )ﻪ ﻓ ﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺇﻨﺘﺎﺝ ﺤﻴﻭﺍﻨﻲ - ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) )

ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ ﺒﺎﻝﻘﺎﻫﺭﺓ - ﺠﺎﻤﻌ ﺔ ﺍﻷﺯﻫﺭ

١٤٣٢ ﻫـ ٢٠١١ﻡ

ﻝﺠﻨﻪ ﺍﻹﺸﺭﺍﻑ: ﺩ.ﺃ / ﻤﺩﺤﺕ ﺤﺴﻴﻥ ﺨﻠﻴل ﺃﺴﺘـﺎﺫ ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ – ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ - ﻜﻠﻴ ﺔ ﺍﻝﺯﺭﺍﻋﺔ- ﺠﺎﻤﻌ ﺔ ﺍﻷﺯﻫﺭ ﺩ.ﺃ / ﻫﺸﺎﻡ ﺤﺴﻴﻥ ﺨﻠﻴﻔﻪ ﺃﺴﺘـﺎﺫ ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ – ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ - ﻜﻠﻴ ﺔ ﺍﻝﺯﺭﺍﻋﺔ- ﺠﺎﻤﻌ ﺔ ﺍﻷﺯﻫﺭ ﺩ.ﺃ / ﻤﺤ ﻤﺩ ﻋﺒﺩﺍﻝﻤﺤﺴﻥ ﺍﻝﺴﻴﺩ ﻤﺒﺭﻭﻙ ﺃﺴﺘﺎﺫ ﻤﺴﺎﻋﺩ ﻓﺴﻴﻭﻝﻭﺠﻰ ﺍﻝﺩﻭﺍﺠﻥ - ﻤﺭﻜـﺯ ﺍﻝﺒﺤـﻭﺙ ﺍﻝﻨﻭﻭﻴـﺔ – ﻫﻴﺌﺔ ﺍﻝﻁﺎﻗﺔ ﺍﻝﺫﺭﻴﺔ

ﺘﺎﺜﻴﺭﺍﻻﺨﺘﻼﻓﺎﺕ ﺍﻝﻤﻭﺴﻤﻴﻪ ﻋﻠﻰ ﺍﻝﺘﻨﻅﻴﻡ ﺍﻝﺤﺭﺍﺭﻱ ﻓﻲ ﺍﻝﻨﻌﺎﻡ

ﺭﺴﺎﻝﺔ ﻤﻘﺩﻤﺔ ﻤﻥ ﺤﻤﺩﻯ ﻋﺒﺩ ﺍﻝﺭﺤﻤﻥ ﻤﺤﻤﺩ ﺒﺴﻴﻭﻨﻰ ﺩﺒﻠﻭﻡ ﺩﺭﺍﺴﺎﺕ ﻋﻠﻴﺎ ( ﺘﻜﻨﻭﻝﻭﺠﻴﺎ ﺩﻭﺍﺠﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ – ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٠ ﻡ ﻡ ﻤﺎﺠﺴﺘﻴﺭ ﻓﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ - ﺠﺎﻤﻌﺔ ﺍﻷﺯﻫﺭ ٢٠٠٥ ﻡ ﻡ

ﺍ ﺴﺘﻴﻔﺎﺀ ﻝﻤﺘﻁﻠﺒﺎﺕ ﺍﻝﺤﺼﻭل ﻋﻠﻰ ﺩﺭﺠﺔ ﺍﻝ ﻌﺎﻝﻤﻴﻪ ( ﺩﻜﺘﻭﺭﺍﻩ ﺍﻝﻔﻠﺴﻔ )ﻪ )ﻪ ﻓ ﻰ ﺍﻝﻌﻠﻭﻡ ﺍﻝﺯﺭﺍﻋﻴﺔ ( ﺇﻨﺘﺎﺝ ﺤﻴﻭﺍﻨﻲ - ﻓﺴﻴﻭﻝﻭﺠﻰ ﺤﻴﻭﺍﻥ ) )

ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ ﻜﻠﻴﺔ ﺍﻝﺯﺭﺍﻋﺔ ﺒﺎﻝﻘﺎﻫﺭﺓ - ﺠﺎﻤﻌ ﺔ ﺍﻷﺯﻫﺭ

١٤٣٢ ﻫـ ٢٠١١ﻡ

ﺠﺃ ﺯﺎـ :ﻫﺎ ﺩ.ﺃ / ﻫﺸﺎﻡ ﺤﺴﻴﻥ ﺨﻠﻴﻔﻪ أـذ ان - ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ – آـ اراـ ـ ازه /ﺩ.ﺃ ﻫﺸﺎﻡ ﻤﺤﻤﺩ ﺼﺎﻝﺢ ﺸﻜ ﺭﻱ أـذ اوا - ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ – آـ اراـ ـ ازه /ﺩ.ﺃ ﻤﻨﻲ ﻋﺒﺩﺍﻝﺘﻭﺍﺏ ﺍﻝﺨﺸﺎﺏ أـذ ان ﻗﺴﻡ ﺍﻹﻨﺘﺎﺝ ﺍﻝﺤﻴﻭﺍﻨﻲ – آـ اراـ ـ ا م

ﺘﺎﺭﻴﺦ ﺍﻝﻤﻨﺎﻗﺸﻪ ﻓﻲ : ٢٨ / ٧ / ٢٠١١ ﻡ ﻡ