ABSTRACT. preheating temperature, early incubation temperature, and different turning frequency on

ABSTRACT LIN, YUN-MEI AMY. Effect of Preheating, Early Incubation, and Different Turning Frequency, on Embryonic Development and Broiler Performance. (Under the direction of Dr. John T. Brake). A series of experiments was conducted to study the effects of the interaction between preheating temperature, early incubation temperature, and different turning frequency on embryonic development and broiler performance. Ross 344 x 708 broiler hatching eggs were used exclusively and 53% relative humidity was maintained during incubation. Two early incubation temperatures (38.1 C and 37.5 C), two preheating temperatures (29.4 and 23.9 C), and two turning frequencies (24 n/d and 96 n/d) were investigated. Four experiments, in various manners, measured the effects of incubation temperature, turning frequency, and preheating temperature interactions on embryo development. Experiment IV measured broiler live performance, carcass yield, organ development, and blood carrying membrane development. Chick length due to 96 n/d turning to E18 in combination with 38.1 C (to E3) was increased, while with 96 n/d turning to E15 in combination with 37.5 C (to E3) was decreased. These data suggested that with higher initial incubation temperature (to E3) turning 96 n/d was required for a longer period (to E18). Turning 96 n/d apparently dehydrates the egg at critical period of incubation, in the presence of current machine ventilation, which depressed embryonic development as evidenced by a smaller and shorter embryo. This depression of embryonic development had effects up to 35 d of age in terms of carcass yield. On the other hand, 38.1 C to E5 increased the metabolic rate and improved the development of the embryo, which improved breast muscle in the presence of greater chick

length. In parallel Israeli research, beneficial effects of all thermal manipulation treatments with 24 n/d turning were shown for BW, total Pectoralis weight, wattles, and testes. Some of these aspects were not evident in Experiment IV due to the absence of the same negative control that did not involve any preheating and the lower temperature to E5 as used in Experiment IV. Another mitigating factor may have been that the strain of broiler was different between the experiments, where Israeli Experiments A and B used Cobb broilers and Experiment IV was conducted with Ross birds. These strains have become well known to differ in thermosensitivity, breast meat yield, and feed conversion.

Effect of Preheating, Early Incubation, and Different Turning Frequency, on Embryonic Development and Broiler Performance by Yun-mei Amy Lin A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Poultry Science Raleigh, North Carolina 2012 APPROVED BY: J. T. Brake, Chair C.R Stark W.L. Flowers

DEDICATION This thesis is dedicated to my parents, Jeen-Kuo Lin and Rui-Hui Tu. ii

BIOGRAPHY Yun-mei Amy Lin, first daughter of Jeen-Kuo Lin and Rui-Hui Tu, was born and raised in Taipei, Taiwan. In 2006, after obtaining her diploma from National Dali Senior High School, she decided to apply to National Pingtung University of Science and Technology from where she received her Bachelor of Science degree in Animal Science in 2009. In her junior and senior years as an undergraduate student, she joined the animal nutrition and physiology lab and participated in research concerning the effects of antibacterial growth promoters and antibiotic-free alternatives on growth performance of broilers under the supervision of Dr. Chi Yu. With these experiences, she changed her mind about studying veterinary medicine after graduation, and she decided to pursue a MS in Poultry Science under the direction of Dr. John T. Brake. iii

ACKNOWLEDGEMENTS The author is deeply indebted to her supervisor Dr. John T. Brake for his guidance, understanding, patience, and most importantly encouragement, throughout the entire effort. Appreciation is also extended to Dr. Charles R. Stark, and Dr. Billy Flowers for serving on the graduate committee, and Dr. Shlomo Yahav for providing a great amount of knowledge and suggestions. Also, the author thanks all the support, help, and friendship from a great laboratory team and staff that included Susan Creech, Corina Rosiuta, Dr. Nirada Leksrisompong, Dr. Mireille Argüelles Ramos, Wilmer Pacheco, Basheer M. Nusairat, Rasha Qudsieh, Yi Frank Xu, and Bing Hu. Special thanks are also acknowledged to the staff members of the Lake Wheeler Road Field Laboratory Chicken Educational Unit who were always available to help regardless of the situation. The author wants to thanks her family, Jeen-Kuo Lin, Rui-Hui Tu, and Arena Lin for all the encouragement and emotional support despite the distance. Finally, the author wants to express her deep gratitude to Wu-yueh Hu for all the laughter, fun, inspiration, and comfort. iv

TABLE OF CONTENTS Page LIST OF TABLES....... LIST OF FIGURES..... LIST OF ABBREVIATIONS..... viii xv xvi INTRODUCTION.... 1 LITERATURE REVIEW.... 3 Effects of Preheating... 3 Effects of Incubation.. 6 Effects of Turning.... 9 Yolk Sac Membrane (YSM)..... 11 Chorioallantoic Membrane (CAM)...... 12 Factors Affecting Chick Length. 14 Factors Affecting Yolk Sac Absorption...... 14 Parallel Research Results.. 15 References... 24 v

Page MATERIALS AND METHODS...... 34 Experiment I. Replication 1 34 11 Experiment I. Replication 2.......... 36 Experiment II. Replication 1...... 36 Experiment II. Replication 2.. 38 Experiment III........ 39 Experiment IV. Incubation.. 40 Experiment IV. Sampling 41 Experiment IV. Broilers. 42 RESULTS AND DISCUSSION......... 47 Experiment I. Replication 1 and 2..... 47 Experiment II. Replication 1 and 2. 51 Experiment III...... 60 Experiment IV. Incubation. 67 Experiment IV. Broilers. 73 References... 162 vi

Page OVERALL DISCUSSION AND CONCLUSIONS. 164 Ventilation..... 164 Primordial Germ Cells (PGCs)... 165 Chick Quality Evaluation... 166 Preheating and Embryo Development... 166 Conclusions. 167 References... 170 vii

LIST OF TABLES... Literature Review Page Table LR-1A. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of female broiler chickens at 35 d of age in Experiment A.... 20 Table LR-2A. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of male broiler chickens at 35 d of age in Experiment A.... 21 Table LR-1B. The effect of Control, Preheating, Manipulation, and PreManipulation on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of female broiler chickens at 35 d of age in Experiment B..... 22 Table LR-2B. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of male broiler chickens at 35 d of age in Experiment B..... 23 Results: Experiment I Table I-1. Effect of 24 or 96 daily turning frequency to E18 at 38.1 C (100.5 F) initial incubation temperature1 on initial egg weight and embryonic development at E14 in two replications of Experiment I... 49 Table I-2. Effect of 24 or 96 daily turning frequency to E18 at 38.1 C (100.5 F) initial incubation temperature1 on chick body weight, absolute and relative yolk sac weight, yolk free body weight, and chick length at hatching in two replications of Experiment I... 50 viii

Results: Experiment II Table II-1. Effect of 24 or 96 daily turning frequency to E15 of incubation at 37.5 C (99.5 F) initial incubation temperature1 on initial egg weight, egg weight loss, and embryonic development in two replications of Experiment II 54 Table II-2. Effect of 24 or 96 daily turning frequency to E15 of incubation, at 37.5 C (99.5 F) initial incubation temperature1 on chick body and yolk sac weight, yolk free body weight, and chick length at hatching in two replications of Experiment II... 55 Table II-3. Effect of 24 or 96 daily turning frequency to E3, E9, and E15 of incubation, followed by 24 daily turning frequency to E18 of incubation at 37.5 C (99.5 F) initial incubation temperature1 on initial egg weight and embryonic development in second replication2 of Experiment II.. 56 Table II-4. Effect of 24 or 96 daily turning frequency to E3, E9, and E15 of incubation, followed by 24 daily turning frequency to E18 of incubation at 37.5 C (99.5 F) initial incubation temperature1 on chick body and yolk sac weight, yolk free body weight, and chick length at hatching in second replication2 of Experiment II... 58 Results: Experiment III Table III-1. Effect of incubation at 38.1 C (100.5 F) to E3 or 37.5 C (99.5 F) and 96 n/d turning frequency on initial egg weight, egg weight loss, absolute and relative embryo and yolk sac weights, and embryo length at E14 in Experiment III... 62 Table III-2. Effect of 96 daily turning frequency to E15 or E18 of incubation followed by 24 daily turning frequency to E18 with an incubation temperature of 38.1 C (100.5 F) to E3 or 37.5 C (99.5 F) on chick body and absolute and relative yolk sac weights, and chick length in Experiment III. 63 TABLE III-3. Effect of 96 daily turning frequency to E15 or E18 of incubation followed by 24 daily turning frequency to E18 of incubation temperature at 38.1 C (100.5 F) to E3 or 37.5 C (99.5 F) on chick body and absolute and relative organ weights in Experiment III.. 65 ix

Results: Experiment IV Table IV-1. Egg weight, egg weight loss, absolute and relative embryo weight, absolute and relative yolk sac weight, and embryo length from chick embryos as influenced by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature interactions at E15 in Experiment IV....... 84 Table IV-2. Effect of preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature interactions on chick body weight, and yolk free body weight (YFBW), and absolute and relative yolk sac weight, and chick length from hatched chicks at E21 in Experiment IV 88 Table IV-3. Yolk sac membrane (YSM) vasculature from E5 to E8 as influenced by preheating temperature, incubation temperature, daily turning frequency, and the interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature in Experiment IV.... 92 Table IV-4. Chorioallantoic membrane (CAM) vasculature from E7 to E10 as influenced by preheating temperature, incubation temperature, daily turning frequency, and the interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature in Experiment IV... 96 Table IV-5. Body weight of female broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV... 100 x

Table IV-6. Percentage mortality (deaths) of female broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV 104 Table IV-7. Adjusted feed conversion ratio (AdjFCR) of female broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV. 107 Table IV-8. Feed intake of female broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV 111 Table IV-9. Body weight of male broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV 114 Table IV-10. Percentage mortality (deaths) of male broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV. 117 xi

Table IV-11. Adjusted feed conversion ratio (AdjFCR) of male broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV... 120 Table IV-12. Feed intake of male broiler chickens as affected by preheating temperature, incubation temperature, daily turning frequency, and preheating temperature by incubation temperature interaction, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature interactions in Experiment IV 123 Table IV-13. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on absolute carcass yield of female broiler chickens at 35 d of age in Experiment IV.. 126 Table IV-14. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on relative carcass yield of female broiler chickens at 35 d of age in Experiment IV.. 129 Table IV-15. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on absolute carcass yield of male broiler chickens at 35 d of age in Experiment IV.. 132 xii

Table IV-16. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on relative carcass yield of male broiler chickens at 35 d of age in Experiment IV.. 135 Table IV-17. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on absolute carcass yield of female broiler chickens at 49 d of age in Experiment IV.. 138 Table IV-18. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on relative carcass yield of female broiler chickens at 49 d of age in Experiment IV.. 141 Table IV-19. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on absolute carcass yield of male broiler chickens at 49 d of age in Experiment IV... 144 Table IV-20. The effect of preheating temperature, incubation temperature, daily turning frequency, and interactions of preheating temperature by incubation temperature, incubation temperature by turning frequency, preheating temperature by turning frequency, and incubation temperature by turning frequency by preheating temperature on relative carcass yield of male broiler chickens at 49 d of age in Experiment IV.. 147 xiii

Table IV-21. Body weight and absolute and relative (g/100g BW) weights of tissues and organs from female broiler chicks as influenced by preheating temperature, incubation temperature, daily turning, and interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature at 35 d of age in Experiment IV 150 Table IV-22. Body weight and absolute and relative (g/100g BW) weights of tissues and organs from male broiler chicks as influenced by preheating temperature, incubation temperature, daily turning, and interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature at 35 d of age in Experiment IV... 153 Table IV-23. Body weight and absolute and relative (g/100g BW) weights of tissues and organs from female broiler chicks as influenced by preheating temperature, incubation temperature, daily turning, and interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature at 49 d of age in Experiment IV 156 Table IV-24. Body weight and absolute and relative (g/100g BW) weights of tissues and organs from male broiler chicks as influenced by preheating temperature, incubation temperature, daily turning, and interactions of preheating temperature by incubation temperature, incubation temperature by daily turning, preheating temperature by daily turning, and incubation temperature by daily turning by preheating temperature at 49 d of age in Experiment IV... 159 xiv

LIST OF FIGURES... Page Literature Review Figure LR-1A. Hatching progression of the four treatments in Experiment A. 18 Figure LR-1B. Hatching progression of the four treatments in Experiment B.. 19 Materials and Methods Figure MM-1. Eggs in individual pedigree bags with its tag during transfer 35 Figure MM-2. Water bath for boiling eggs... 44 Figure MM-3. Tape around the egg and cut between the tape.. 45 Figure MM-4. The yolk sac membrane (YSM) and chorioallantoic membrane (CAM) attached to the egg shell... 46 xv

LIST OF ABBREVIATIONS AdjFCR BW C CAM cm cg d E EW Em EmW F g h min RH wk YSW Adjusted feed conversion ratio, corrected for mortality Body weight Celsius Chorioallantoic membrane Centimeter Centigram Day Embryonation Egg weight Embryo Embryo weight Fahrenheit Gram Hour Minute Relative humidity Week Yolk sac weight xvi

INTRODUCTION Significant changes have taken place in the broiler industry in the past decade. Improvements in genetics, nutrition, and management have resulted in a dramatic reduction in the days to achieve broiler market weight (Havenstein et al., 2003). Hence, a chicken may now spend less than one-third of its life in an incubator. Therefore, incubation has become an even more important aspect of the poultry business. Basic principles and practices of artificial incubation have been known since ancient times by the Egyptians and Chinese, who used various forms of primitive ovens to incubate eggs and hatch chicks (Sykes, 1991). Their basic knowledge has been passed down from generation to generation. Presently, with the development of thermometers, electronics, and computers, modern incubation companies continue to try to economically incorporate what each hen does naturally into the design and mechanical efficiencies of their incubators in order to improve hatchability and chick quality (Sykes, 1991). In nature, each hen heats their eggs through direct contact with her brood patch and turns the eggs frequently at the beginning of incubation. Therefore, the two major things that a hen has control over are turning frequency and egg temperature. Olsen observed in 1930 (as reported by Landauer, 1967) that hens moved their eggs during natural incubation about 96 times daily. In his review, Wilson (1991) reported turning eggs 96 times daily to be the optimum rate. However, due to maintenance costs associated with the machines and relatively small differences in hatchability, most companies turn the eggs 24 times daily (Robertson, 1961). on the other hand, has also been considered to be one of the most influential factors on embryonic growth and development during all stages of 1

incubation. Becker and Bearse (1958) demonstrated that the hatchability of long stored eggs exhibited a greater percentage improvement when preheated prior to standard incubation than those eggs that were stored for only a short time. They also observed that eggs stored for 21 d or 28 d were more sensitive to various treatments known to affect hatchability than eggs stored for shorter periods. Hodgetts (1999) suggested that eggs should be warmed slowly to decrease temperature shock on the embryos while Wilson (1991) suggested that it was favorable to warm eggs rapidly to incubation temperature. The optimum incubation temperatures have been reported to be from 37.0 C (98.6 F) to 38.0 C (100.4 F) for chicken eggs (Insko, 1949; Romanoff, 1960; Landauer, 1967; Lundy, 1969; Wilson, 1991). Abnormal incubation temperatures have been shown to affect post-hatching growth (Romanoff, 1935, 1936; Michels et al., 1974; Decuypere, 1979) and proper organ development of avian embryos (Shafey, 2004). The objectives of the present experiments were to study the effects of preheating temperatures, daily turning frequencies, and early incubation temperature on embryo development. The effects of these factors during incubation were also evaluated in terms of chick quality at hatching and post-hatching broiler performance. 2

LITERATURE REVIEW Effects of Preheating. Rudnick (1944) indicated that the chicken egg at the time of laying was in the process of active hypoblast formation. Hays and Nicolaides (1934) concluded that pregastrula and early gastrula were the most common stages of development in embryo from young and old flock eggs, respectively. A well-advanced gastrula has been most commonly found in eggs from prime age flock eggs, which Taylor and Gunns (1939) confirmed. Due to the fact that eggs have been found to be in different developmental stages at the time of oviposition, preheating has become a part of hatchery management as preheating has provided a means to incrementally increase the temperature of eggs just prior to incubation. This has been found to be beneficial for eggs that need to be transformed into a state more ready for incubation (Hutt and Pilkey, 1930). However, researchers have argued that preheating can be harmful to some freshly laid eggs due to progressing eggs that were laid in an "optimal" stage to a "less-optimal" stage during and after the preheating treatment (Kosin and Pierre, 1956). Broiler eggs have been usually stored for 3 d to 5 d, with some stored beyond 7 d, prior to incubation in commercial hatcheries. It has been well documented that an increased length of the egg storage period created a lag in development (Christensen et al., 2001), altered metabolic rate (Fasenko et al., 2002; Christensen et al., 2001), increased incubation duration (Mather and Laughlin, 1976; Tona et al., 2003), decreased hatchability (Scott, 1933; Asmundson, 1947; Kosin, 1950; Becker, 1963; Merritt, 1964; Sittman et al., 1971; Whitehead et al., 1985; Fasenko et al., 2001), decreased chick quality at hatching (Byng and Nash, 1962; Tona et al., 2003, 2004), decreased subsequent growth performance 3

(Becker, 1960; Merritt, 1964; Tona et al., 2003, 2004), and increased post hatching mortality (Merritt, 1964; Yassin et al., 2009). Yassin et al. (2008) observed that each extra day of storage up to 7 d reduced hatchability by 0.2%, whereas the reduction increased to 0.5% per day after 7 d of storage. A longer storage period caused a delay in the initiation of the developmental process (Arora and Kosin, 1966) and that development proceeded at a slower rate during the initial portion of incubation (Mather and Laughlin, 1977; Meijerhof, 1992) following storage. As an example of the effects of increased storage time, under identical incubation and egg storage conditions, chicks that hatched from eggs stored at constant conditions for 10 d versus 1 d exhibited greater residual yolk sac weight as well as reduced liver and gizzard weight (Afsar et al., 2007). Further, BW at 42 d of age was decreased due to storage by 58 g or 138 g when the eggs were produced by 34 or 59-wk-old broiler breeder flocks, respectively (Ates et al., 2004). These data showed that extended egg storage somehow interfered with critical aspects of yolk sac absorption that adversely affected nutrient and/or hormone assimilation into the developing embryo and hatched chick and negatively influenced broiler progeny growth. This may be partially addressed by employing a more rapid increase in egg temperature during preheating prior to incubation to reduce early embryo mortality in larger eggs (Elibol and Brake, 2008) or younger flock eggs (Güçbilmez et al., 2009). Therefore, preheating of eggs prior to regular incubation was beneficial as demonstrated by an increased hatchability of chicken and turkey eggs (Kosin and Pierre, 1956; Becker and Bearse, 1958). Becker and Bearse (1958) suggested that the hatchability of long stored eggs exhibited a greater percentage improvement by preheating than those eggs that were stored for only a short time. These authors also observed that eggs 4

stored for 21 d to 28 d were more sensitive to treatments affecting hatchability than eggs stored for shorter periods. Another goal in preheating before incubation has been to reduce the temperature shock experienced by the embryo moving from low temperature storage to the higher temperatures of an incubator by allowing the eggs to warm up to an intermediate temperature before setting in the incubators (Renema et al., 2006). By increasing the egg temperature to an intermediate level, the eggs were then able to achieve their incubation temperature more rapidly when set in an incubator. This has been suggested to promote early embryonic growth (Güçbilmez et al., 2009). Embryos from heavily selected broiler strains, such as those found in the majority of commercial hatcheries worldwide, have been demonstrated to be intolerant of temperature variations with abnormalities and mortality of the embryo being the penalties for exceeding the narrow temperature range that has been thought to be optimum for incubation (Wilson, 1991; Decuypere and Michels, 1992). Brannan (2008) mentioned that preheating allowed embryos to more safely and adequately adjust to the dramatic increase in temperature between an egg cooler and an incubator. Eggs being preheated that experienced a high air velocity were warmed rapidly, while eggs at a low air velocity took several hours to warm (Elibol and Brake, 2008; Reijrink, 2010). In some machines, the difference in time between the first and last eggs reaching the final incubation temperature was as much as 24 h. Wilson (1991) and Lourens et al. (2005) suggested that it was favorable to warm eggs rapidly to incubation temperature. A prolonged time at temperatures below 35 C was reported to increase embryonic mortality and abnormal embryonic development (Wilson, 1991). However, Hodgetts (1999) suggested that eggs should be warmed slowly to decrease temperature shock on the embryos. Reijrink (2010) 5

compared two different lengths of preheating before incubation, 4 h or 24 h at 37.8 C, and found the hatching time and embryo development between the preheating treatments to not differ. However, preheating had a beneficial effect on the hatchability of long stored eggs (Proudfoot, 1966; Reijrink, 2010). Meijerhof (1994) collected eggs from flocks of two ages (37 wk and 59 wk) and preheated the eggs at 27 C for 16 h compared with preheating at 20 C for the same period. The author suggested that hatchability of eggs from younger hens was not significantly influenced by pre-warming while the hatchability of eggs from older birds was significantly reduced by preheating at 27 C. Mayes and Takeballi (1984) concluded that pre-warming improved viability and hatchability when low storage temperatures were used. No beneficial effects were reported when eggs were stored for a short period at 15 to 16 C. Effects of Incubation. has been commonly acknowledged to be the most influential factor concerning embryonic growth and development during all stages of incubation from storage to incubation to hatching. As mentioned above, broiler hatching eggs have usually been stored for 3 d to 5 d, some even stored beyond 7 d, prior to incubation in commercial hatcheries. It has become well known that embryonic development may be highly affected by hatching egg storage conditions. Optimal storage temperatures during artificial incubation have been found to vary with strain (Scott and Silversides, 2000), flock age (Mather and Laughlin, 1979), and duration of storage (Elibol et al., 2002). The relatively reduced rate of embryo development during the hatching egg storage period has been found to largely depend upon temperature (Brake et al., 1997; Fasenko, 2007), which can range 6

between 14 and 21 C (Fasenko et al., 1992). Optimal hatchability following long term storage (>14 d) was achieved when the storage temperature was 12 C (Funk and Forward, 1960), but 15 C was better for eggs stored for 8 d, and 18 C was best for eggs stored for 2 d (Kirk et al., 1980). Romanoff (1960) noted that cool temperatures before incubation were also evident in natural incubation, as the hen did not begin to incubate her eggs until she had laid several eggs for her clutch, which generally required several days. Embryonic developmental processes have been reported to begin from the time of fertilization while the egg was still being formed around the yolk and embryo in the oviduct of the hen prior to oviposition (Romanoff, 1960). During subsequent storage, the albumen ph has been reported to have increased and albumen quality diminished due to embryonic production of ammonia (Benton and Brake, 2001). Storage temperature has been suggested to control embryonic production of ammonia that was subsequently absorbed by the albumen (Benton and Brake, 2001). Another issue that has been raised concerning egg storage involved the initiation of incubation and response to incubation temperature. The discrepancies among studies (Kirk et al., 1980; Fasenko et al., 1992; Brake et al., 1997; Fasenko, 2007) may be related to the fact that different tissues of the embryo may have varying temperature requirements for initiating growth after storage due to effects on the metabolic rate of the embryo and the need to reduce embryo metabolism relative to length of storage. Therefore, investigations have been conducted to define the optimal incubation temperature that produced the maximum hatchability in terms of both numbers and quality of chicks. The commonly acknowledged incubation temperature has become known to be within the narrow range of 37 C (98.6 F) to 38 C (100.4 F) in chickens (Romanoff, 1960; Lundy, 1969; Wilson, 1991; Lourens et al., 7

2005). This range has commonly been applied to the incubation machine air temperature instead of the embryo temperature, with the assumption being that the two were equal (French, 1997). However, recent research has shown that the difference between these two temperatures can vary significantly and that measuring egg temperature was a much more accurate means of monitoring embryo growth and development than was measuring machine air temperature (Meijerhof and van Beek, 1993; French, 1997; Lourens, 2001; Leksrisompong, 2005). French (1997) identified the three main influences on temperature experienced by the embryo as machine temperature, heat exchange between the embryo and its environment, and the metabolic heat production of the embryo as it grew. During early incubation heat production by the embryo has been shown to be limited, so that the embryo behaved like a poikilotherm and relied mainly on machine air temperature to determine egg temperature (French, 1997). However, as the embryo aged and began to produce more heat, the machine air temperature has not always been reduced to reflect these developmental changes. As a result, research has demonstrated that chicks exposed to increased temperatures during late incubation did not perform well during the subsequent rearing period and were often unable to achieve the same BW as those incubated under optimal conditions (Hulet et al., 2007). There have been negative effects on the cardiovascular system and the gastrointestinal tract reported (Joseph et al., 2006; Leksrisompong et al., 2007; Lourens et al., 2007; Barri et al., 2011). Therefore, proper adjustment of the machine air temperature relative to initially low embryonic metabolism followed by an increasing metabolic rate and body temperature of the embryo (Yahav, 2009; Brake and Yahav, 2011) has been found to be a logical and beneficial approach. 8

Effects of Turning. Turning has been studied for many years, due to its important role in embryonic development and growth. Turning has been described as involving several parameters such as frequency, axis of setting and turning, turning angle, planes of rotation, and stage of incubation requiring turning (Wilson, 1991). Since Olsen first observed in 1930 (as reported by Landauer, 1967) that hens moved their eggs during natural incubation about 96 times daily, researchers have searched for an optimal turning rate. Wilson (1991) reported turning eggs 96 times daily to be the optimum rate during incubation while others suggested that eggs turned through 90 on an hourly basis (Lundy, 1969; Tullet and Deeming, 1987; Wilson, 1991; French, 1997) was sufficient. Turning eggs 96 times daily favors eggs from older breeder flocks that have poorer albumen quality (Elibol et al., 2004; 2006). Turning 96 times daily also appeared to work best when stopped by E14 (Elibol and Brake, 2003; Elibol et al., 2004; 2006). Continuous turning decreased early deads but reduced fertile hatchability (Ö zlü et al., 2008ab), which suggests that excessive turning during latter stages of incubation could be detrimental. However, rapid turning can help previously unturned eggs partially recover (Elibol et al., 2002) Proudfoot (1965) indicated that daily turning of eggs during storage of 14 d to 28 d resulted in higher hatchability compared to those that were stored the same length of time without turning. Subsequently, several authors (Lundy, 1969; Wilson, 1991; Elibol and Brake, 2006) confirmed that eggs from older flocks with relatively poorer quality albumen were found to benefit from an increased turning frequency during incubation. It has been found that eggs should not be rotated always in the same direction during turning as this can result in rupture of the yolk sac membrane, disruption of the chorion, allantois, 9

and shell membranes, twisting of the chalazae and rupture of blood vessels (Landauer, 1967). By comparison, turning should rotate the egg along the vertical axis. Funk and Forward (1953) turned eggs at angles of 20, 30, 40, and 45 from vertical and reported increasing fertile hatchability with increased turning angle, although the difference between 40 and 45 was small. In subsequent research, Funk and Forward (1960) investigated turning angles of 30, 45, 60, and 75 from vertical and found that 45 produced the best results. However, turning angles of less than 45 can be combined with increased turning frequency to achieve acceptable results (Elibol and Brake, 2006). The poultry industry standard for turning angle has remained 45 to the present time. The stage of incubation requiring turning has also been studied. Card (1926) observed that eggs turned during only the first 6 d of incubation (E0 to E6) hatched nearly as well as those turned throughout 18 d (E0 to E18) of incubation. Byerly and Olsen (1936) concluded that turning during the third week of incubation (E15 to E21) probably had little effect on hatchability. New (1957) turned eggs twice daily only from E4 to E7 and suggested that these eggs hatched in a manner similar to those turned throughout incubation and concluded that the critical period for turning was from E3 to E7 of incubation. Deeming et al. (1987) and Deeming (1989) agreed with New (1957) that E3 to E7 of incubation required turning. On the other hand, Kaltofen (1961) found that the second week of incubation (E8 to E14) was most sensitive to frequent turning. It has also been reported that turning after E13 of incubation, after closure of the chorioallantoic membrane had little, if any, beneficial effect (Byerly and Olsen, 1936; Proudfoot et al., 1981; Wilson and Wilmering, 1988). Wilson (1991), in his review, reported that three time periods (E1 to E3, E4 to E7, and E8 to E14) have been proposed as being the most critical for turning of chicken 10

eggs. Elibol and Brake (2004) reported E0 to E2 and E3 to E8 to be the most critical periods for turning of commercial broiler hatching eggs with respect to early and late embryonic deaths, respectively. This suggested that different periods of early development were ultimately responsible for different stages of longer term embryonic development. Caldwell and Cornwell (1975) believed that egg turning was involved in redistribution of the heat from the brood patch of the hen. However, turning was found to still be required in force-draught incubators where temperature gradients did not normally exist within individual eggs (Drent, 1975). The common explanation for the necessity of turning has been that it prevented the adhesion of the embryo to the inner egg shell membrane during early development (Eycleshymer, 1906; New, 1957; Drent, 1975; Freeman and Vince, 1974; Skutch, 1967; Wilson, 1991). Wilson and Wilmering (1988) showed that cessation of turning at E10 of incubation decreased hatchability but that no effect was observed when cessation of turning occurred after E16. Tona (2005) concluded that egg turning was required during incubation until E12, but should not be stopped until after E15. Likewise, Deeming (2009) mentioned that there was no evidence to suggest that the absence of turning after E15 had any negative effect on chicken embryonic development or hatchability. Yolk Sac Membrane (YSM). The yolk sac membrane (YSM) of the chick embryo was found to be composed of two germ layers, the endoderm and vascular mesoderm. The endoderm has been described as a thick layer with villus-like projections and corrugations running in a generally meridional direction, and was responsible for absorption of nutrients from the yolk. The highly vascularized outer vascular mesoderm developed from flattened cells that 11

performed a supportive role. During the first week of incubation, the YSM was described as floating on the yolk but eventually grew around and enclosed the entire yolk (Romanoff, 1960). The YSM not only played a major role in the transport of nutrients from the yolk to the chick embryo (Yadgary et al., 2011) but was also a site for the production of blood and synthesis of specific proteins. During the first few days of development, nutrients have been shown to move to the tissues from the yolk and egg shell by simple diffusion. Following development of circulatory tissues, chick embryos received nutrients and hormones from the yolk by the blood stream in a transport scheme much as would an adult bird. Therefore, blood and blood vessels in the YSM were formed before vessels developed in the embryo body (intraembryonic vessels) and were continuously changing with the stage of embryo development throughout incubation (Romanoff, 1960). The YSM has been found to function in a manner similar to the placenta in mammalians, as both tissues provided nutrients to the embryo. On the other hand, Haller (1758) observed that the endoderm of the YSM was continuous with the gut endoderm and was an extension of the intestine, which differed from the placenta. Other than nutrients, the yolk has also been reported to contain many hormones that can modulate the growth of the embryo (Hayward et al., 2004). Therefore, the development of the YSM could be described as controlling both yolk sac absorption and ultimately contributing to the quality of the hatched chicks. Chorioallantoic Membrane (CAM). The chorion has been demonstrated to grow in such a manner as to become contiguous with the inner shell membrane, and have no significant function until the allantois invaded it to form the chorioallantoic membrane (CAM), which 12

has been demonstrated to play a pivotal role in embryonic physiology (Romanoff, 1960; Freeman, 1974). The allantois was found to be the last extra-embryonic membrane to appear with development apparent around E2 of incubation as an appendage of the hind gut. The allantois has been described as a double walled membrane, with one of its walls forming the allantoic sac that accumulated kidney excretions while the other grew within the chorion to aid in respiration. The allantoic portion that accumulated kidney secretions has been reported to grow as it fills and the sac was reported to stop growing if the flow of secretions was hindered experimentally (Romanoff, 1960). The chick chorioallantoic membrane (CAM) has been described as a very simple extraembryonic membrane that served multiple functions during embryo development. The allantois was reported to initially fuse with the chorion between E5 and E6 of incubation to form the CAM. Further, about E9 the CAM folded around the albumen that remained in the small end of the egg to form the albumen sac and continued to develop until about E11 or E12 of incubation (Romanoff, 1960). The CAM has been found to be rich in blood capillaries that lined the inner surface of the egg shell membranes where it was the site of exchange of respiratory gases, calcium transport from the eggshell, acid-base homeostasis in the embryo, and ion and water reabsorption from the allantoic fluid (Baggott et al., 2002). All these functions have been shown to be accomplished by the chorionic and the allantoic epithelium through unique differentiation that created a wide range of structural and molecular peculiarities with highly specialized ion transporting epithelia. As the embryo grew, the vasculature of the yolk sac membrane (YSM) decreased in proximity to the egg shell surface, therefore carrying less oxygen to the embryo. 13

The CAM has been found to assume the role of oxygen transport as the capacity of the YSM diminished (Romanoff, 1960; Etches 1996). Factors Affecting Chick Length. Although Deeming (2005) doubted the usefulness of chick length as a meaningful indicator of chick quality, many researchers have suggested chick length to be a convenient way to determine chick quality (Wolanski et al., 2003; Decuypere and Bruggeman, 2007; Reijrink, 2010). Studies by Wolanski et al. (2003; 2007) showed that chick length at hatching was positively correlated with breeder strain. Kampula (2004) confirmed this and reported that egg size could influence chick length. Incubation processes that optimally supply the needs of the chicken embryo have also been reported to favorably influence chick length (Hill, 2001; Reijrink and Molenaar, 2008). Embryos subjected to high incubation temperature (Reijrink and Molenaar, 2008) exhibited a lower yolk free body mass (YFBM), shorter chick length, and open navels (Lourens et al., 2005, 2007; Hulet et al., 2007; Leksrisompong et al., 2007). The negative effect of prolonged egg storage on chick length was evident when storage time was increased (Reijrink, 2010). Reijrink (2010) also investigated preheating, where the eggs were preheated for 4h or 24 h and concluded that preheating affected embryonic mortality during the first 2 d of incubation of stored eggs; however chick quality including chick length was not affected. Factors Affecting Yolk Sac Absorption. Yolk sac weight has been another common parameter by which to access chick quality (Tona et al., 2003). Wolanski et al. (2004; 2006) found that the amount of residual yolk varied between 0.8 and 10.6 g and the relationship 14

between the amount of residual yolk and the YFBM were contrary. Several investigations have shown that yolk sac weight increased with the flock age (Suarez et al., 1997; Vieira and Moran, 1998; Sklan et al., 2003; Hamidu et al., 2007) while Wolanski et al. (2006) reported highly significant positive correlations between yolk sac weight and day-old chick weight for a number of breeder lines and ages. However, Nangsuay et al. (2011) later commented that in those trials, the correlation was due to egg size, not flock age. Therefore, in their subsequent study, eggs from two breeder flock ages were separated, and then divided into small and large egg categories. The results confirmed that the influence was through egg weight, irrespective of yolk availability and absorption. Finkler et al. (1998) conducted a series of experiments that manipulated egg quality by removing 20% of the albumen or yolk content from eggs prior to setting. It was interesting that although the yolk sac weight decreased in the yolk manipulation treatment at E20 of incubation, the YFBW did not differ between treatments. Parallel Research Results. The following experiments were also funded by BARD as part of the same grant as the present data, and had a similar experimental design as Experiment IV of the present thesis. Both Experiments A and B were conducted by the research team at The Volcani Center in Israel, headed by Dr. Shlomo Yahav. Experiment A. Experiment A was carried out in winter (January, 2010). Eggs were collected from a commercial Cobb 500 broiler breeder flock at the age of 33 wk. The eggs that 15

weighed between 62 g and 67 g were randomly assigned to one of 4 treatments. Eggs were stored for 1-2 d prior to setting. Experiment B. Experiment B was conducted in summer (June, 2010). The Cobb 500 broiler breeder flock was at the age of 39 wk when the eggs were collected. The eggs that weighed between 59 g and 73 g were randomly divided into the 4 treatments. Eggs were stored for 9 d prior to setting. For both experiments 180 eggs was assigned to each of the following four treatments: Control no preheating (stored at 18 C) with 37.5 C incubation temperature; Preheating - preheating at 30.2 C for 12 h with 37.5 C incubation temperature; Manipulation no preheating (stored at 18 C) with 38.1 C incubation temperature from E0 to E5, followed by 37.5 C; PreManipulation preheating at 30.2 C for 12 h with 38.1 C incubation temperature from E0 to E5, followed by 37.5 C. The eggs were incubated in 2 automatic incubators (Type 65Hs, Masalles, Barcelona, Spain). Turning frequency was 24 n/d to E18 of incubation. Hatching period, hatchability, and BW data were collected during and at hatching. At 35 d, BW, carcass parts, and organ weights were measured. Figures LR-1A and LR-1B show the percentage of chicks hatching during each hour of the hatching period for each of the four treatments in Experiments A and B. The Control group started hatching later than the other three treatments and had the least number of chicks hatched. The PreManipulation treatment started and finished hatching earlier than the groups that were treated preheating and manipulation alone. Table LR-1A presents the effects of Control, Preheating, Manipulation, and PreManipulation on BW at 0 d and 35 d, percentage carcass yield, and relative organ weights of female broiler chickens at 35 d of age in 16

Experiment A, where the 35 d BW in the Preheating, Manipulation, and PreManipulation treatments was significantly greater than the Control treatment. A similar effect was observed for the breast meat yield that Preheating, Manipulation, and PreManipulation treatments were significantly greater than the Control. Also, the wattle weight was greater in the Manipulation treatment than the Control and Preheating treatments. Results for the male broilers are shown in Table LR-2A. The Manipulation treatment had a significantly greater BW at 35 d than did the Control and Preheating treatments. Similarly, the wattle and testis weight were significantly greater in the Preheating, Manipulation, and PreManipulation treatments than the Control. However, the breast muscle weight was similar for all treatments. The effects of control, preheating, manipulation, and PreManipulation on BW at 0 d and 35 d, percentage carcass yield, and relative organ weights of female broiler chickens at 35 d of age in Experiment B are shown in Table LR-1B. The 35 d BW in the Preheating, Manipulation, and PreManipulation treatments was significantly greater than the Control treatment. A similar effect was seen for the breast meat yield that Preheating and Manipulation treatments were significantly greater than the Control. Also, the wattle weight was greater in PreManipulation treatment than the Control. Results for the male broilers are shown in Table LR-2B. The Preheating and PreManipulation treatments exhibited a significant greater BW at 35 d than did the Control and Manipulation treatments. Similarly, the breast muscle and testis weights were significantly greater in the Preheating, Manipulation, and PreManipulation than the Control. 17

Percentage of chicks hatched 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% Control Preheating Manipulation PreManipulation 0% 470 480 490 500 510 520 Time of incubation (h) Figure LR-1A. Hatching progression of the four treatments in Experiment A. 18

Percentage of chicks hatched 100% 90% 80% 70% Control Preheating Manipulation PreManipulation 60% 50% 40% 30% 20% 10% 0% 478 485 492 499 506 513 520 527 Time of incubation (h) Figure LR-1B. Hatching progression of the four treatments in Experiment B. 19

Table LR-1A. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of female broiler chickens at 35 d of age in Experiment A. Body Weight Carcass Parts and Organs Treatment Preheating Incubation Fat Pectoralis 0 d 35 d pad Major + Minor Heart Wattles Ovary (g) (g/100 g BW) Control 1 18.0 C 37.5 C 47.0 1781.0 b 2.2 18.9 b 0.46 0.024 b 0.020 Preheating 2 30.2 C 37.5 C 47.6 1830.0 a 2.3 19.6 a 0.46 0.025 b 0.020 Manipulation 3 18.0 C 38.1 C 47.7 1827.0 a 2.3 19.5 a 0.45 0.032 a 0.020 PreManipulation 4 30.2 C 38.1 C 47.2 1852.0 a 2.3 19.4 a 0.45 0.028 ab 0.020 a,b Means in a column that possess different superscripts differ significantly (P 0.05). 1 Eggs were not preheated, and incubated at 37.5 C. 2 Eggs were preheating at 30.2 C for 12 h prior to setting, and incubated at 37.5 C from E0 to E5. 3 Eggs were not preheated, and incubated at 38.1 C from E0 to E5. 4 Eggs were preheating at 30.2 C for 12 h prior to setting and incubated at 38.1 C from E0 to E5. 20

Table LR-2A. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of male broiler chickens at 35 d of age in Experiment A. Body Weight Carcass Parts and Organs Treatment Preheating Incubation Fat Pectoralis 0 d 35 d pad Major + Minor Heart Wattles Testes (g) (g/100 g BW) Control 1 18.0 C 37.5 C 47.2 2071.4 c 1.9 19.1 0.51 0.037 b 0.015 b Preheating 2 30.2 C 37.5 C 47.1 2103.0 bc 1.9 19.1 0.50 0.044 a 0.018 a Manipulation 3 18.0 C 38.1 C 47.6 2170.0 a 1.9 19.3 0.50 0.044 a 0.018 a PreManipulation 4 30.2 C 38.1 C 47.6 2166.0 ab 1.8 19.4 0.50 0.054 a 0.020 a a-c Means in a column that possess different superscripts differ significantly (P 0.05). 1 Eggs were not preheated, and incubated at 37.5 C. 2 Eggs were preheating at 30.2 C for 12 h prior to setting, and incubated at 37.5 C from E0 to E5. 3 Eggs were not preheated, and incubated at 38.1 C from E0 to E5. 4 Eggs were preheating at 30.2 C for 12 h prior to setting and incubated at 38.1 C from E0 to E5. 21

Table LR-1B. The effect of Control, Preheating, Manipulation, and PreManipulation on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of female broiler chickens at 35 d of age in Experiment B. Body Weight Carcass Parts and Organs Treatment Preheating Incubation Fat Pectoralis 0 d 35 d pad Major + Minor Heart Wattles Ovary (g) (g/100 g BW) Control 1 18.0 C 37.5 C 46.8 1764.9 b 2.2 19.2 b 0.39 0.026 b 0.023 Preheating 2 30.2 C 37.5 C 47.2 1789.0 a 2.2 20.0 a 0.40 0.031 ab 0.022 Manipulation 3 18.0 C 38.1 C 47.3 1833.5 a 2.2 20.0 a 0.40 0.030 ab 0.022 PreManipulation 4 30.2 C 38.1 C 47.0 1788.7 a 2.2 19.6 ab 0.39 0.036 a 0.022 a,b Means in a column that possess different superscripts differ significantly (P 0.05). 1 Eggs were not preheated, and incubated at 37.5 C. 2 Eggs were preheating at 30.2 C for 12 h prior to setting, and incubated at 37.5 C from E0 to E5. 3 Eggs were not preheated, and incubated at 38.1 C from E0 to E5. 4 Eggs were preheating at 30.2 C for 12 h prior to setting and incubated at 38.1 C from E0 to E5. 22

Table LR-2B. The effect of Control, Preheating, Manipulation, and PreManipulation treatments on body weight at 0 d and 35 d, relative carcass yield, and relative organ weights of male broiler chickens at 35 d of age in Experiment B. Body Weight Carcass Parts and Organs Treatment Preheating Incubation Fat Pectoralis 0 d 35 d pad Major + Minor Heart Wattles Testes (g) (g/100 g BW) Control 1 18.0 C 37.5 C 47.1 2080.2 c 1.8 19.2 b 0.40 0.051 0.017 b Preheating 2 30.2 C 37.5 C 46.8 2132.3 a 1.8 19.4 a 0.41 0.054 0.020 a Manipulation 3 18.0 C 38.1 C 47.4 2099.5 b 1.8 19.6 a 0.41 0.053 0.019 a PreManipulation 4 30.2 C 38.1 C 47.4 2165.4 a 1.8 19.8 a 0.41 0.059 0.019 a a-c Means in a column that possess different superscripts differ significantly (P 0.05). 1 Eggs were not preheated, and incubated at 37.5 C. 2 Eggs were preheating at 30.2 C for 12 h prior to setting, and incubated at 37.5 C from E0 to E5. 3 Eggs were not preheated, and incubated at 38.1 C from E0 to E5. 4 Eggs were preheating at 30.2 C for 12 h prior to setting and incubated at 38.1 C from E0 to E5. 23

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MATERIALS AND METHODS Experiment I. Replication 1. Broiler hatching eggs were obtained from Ross 344 male x Ross 708 female broiler breeders housed at the North Carolina State University Chicken Educational Unit in Raleigh, NC. The eggs were collected for 2 d at 34 wk of age. All freshly laid eggs were weighed and eggs between 53 g and 61 g were numbered individually and randomly assigned to the two turning treatments. The eggs were stored at 16 C (60.8 F) and 60% RH for 1-2 d before preheating at 23.9 C (75 F) for 12 h prior to setting. A total of 400 eggs were set in two Natureform model NOM-45 setter/hatchers (Natureform International, Jacksonville, FL) that had been modified to hold 5 trays of 180 chicken eggs each. There were 200 eggs used in each turning treatment. However, a total of 1800 eggs were set to fill the machines to insure uniform air flow and temperature. Incubators were operated at an air temperature of 38.1 C (100.5 F) and 53% RH until E3 (72 h) of incubation. From E4 to E15, the incubation air temperature was 37.5 C (99.5 F), and was thereafter lowered to 37.4 C (99.3 F) on E16, 37.3 C (99.1 F) on E18, and 36.9 C (98.5 F) to hatching. The incubators were set to turn either once hourly (24 n/d) or four times hourly (96 n/d) at 45 angle on both sides to E18. Each of the 200 individually weighed eggs in each machine constituted a replicate. At E14 of incubation, 30 eggs with live embryos from each turning treatment were randomly selected and necropsied to determine embryo length, and weights of the egg, embryo, yolk sac, and fluids. The remaining eggs were turned to E18 before transfer to hatching baskets in individual pedigree bags (Figure MM-1). At E21, BW was measured on 34

Figure MM-1. Eggs in individual pedigree bags with its tag during transfer. 35

154 chicks from each turning treatment and length was measured on 30 chicks from each turning treatment. Experiment I. Replication 2. Hatching eggs were obtained from Ross 344 male x Ross 708 female broiler breeders housed as in Replication 1. The eggs were collected for 2 d at 59 wk of age. All freshly laid eggs were weighed and numbered individually and 250 pairs of weight-matched eggs were identified. Other eggs were used to fill the trays and to obtain hatchability data. One egg of each pair with the same weight (±0.1g) was assigned to each of the two turning treatments. The eggs were stored for 2 d and preheated and incubated as in Replication 1. At E14 of incubation, 29 pairs of eggs with live embryos from each turning treatment were randomly selected and necropsied to determine embryo length and weights of the egg, embryo, yolk sac, and fluids as in Replicate 1. The remaining pairs of eggs were turned to E18 before transfer to hatching baskets in individual pedigree bags. At E21, chick BW, yolk sac weight, and chick length were measured on 209 pairs of chicks from each turning treatment. For both replicates in Experiment 1 were single factorial designs: 2 turning frequencies (24 n/d or 96 n/d). Each egg was an experimental unit. TTEST procedure of SAS Institute (2008) was used to compare variable means between the two turning treatments. Means were considered statistically different at P<0.05. Experiment II. Replication 1. Experiment II replication 1was a single factorial designs: 2 turning frequencies (24 n/d or 96 n/d). Each egg was an experimental unit. Broiler hatching eggs were obtained from Ross 344 male x Ross 708 female broiler breeders housed as in 36

Experiment I. The eggs were collected for 2 d at 38 wk of age. All freshly laid eggs were weighed and eggs that weighed between 61g and 65 g were numbered individually. There were 90 pairs of weight-matched eggs identified. All eggs were weighed and selected to provide 180 pairs of weight-matched eggs. One egg of each pair with of the same weight was assigned to each turning treatment. Non-paired eggs were used to randomly fill trays. A total of 1800 eggs were set to fill the machine to insure uniform air flow and temperature. The eggs were stored at 16 C (60.8 F) and 60% RH for 1 d before preheating at 23.9 C (75 F) for 12h prior to setting. Incubators were operated at an air temperature of 37.5 C (99.5 F) and 53% RH until E12 of incubation. From E13 to E18, the incubation air temperature was 37.3 C (99.1 F), and 36.9 C (98.5 F) thereafter upon transfer to a Natureform NMC-2000 hatcher in individual pedigree bags. The turners of the incubators were set to turn either 24 times daily (24 n/d) or 96 times daily (96 n/d) at 45 on both sides to E15, followed by 48 n/d daily turning from E16 to E18 at 40 in NMC-2000. Each of the 180 individually weighed eggs in each machine constituted a replicate. At E15 of incubation, 27 weight-matched eggs from each turning treatment were randomly selected and necropsied to determine embryo length, and weights of the egg, embryo, yolk sac, and embryonic fluids. The remaining paired eggs were transferred at E18. At E20.5, the chicks were removed from the hatcher and BW and length were measured on 115 paired chicks from each turning treatment. TTEST procedure of SAS Institute (2008) was used to compare variable means between the two turning treatments. Means were considered statistically different at P<0.05. 37

Experiment II. Replication 2. Experiment 2 replication 2 was a 2 X 3 factorial designs: 2 turning frequencies (24 n/d or 96 n/d), and 3 turning lengths (3 d, 9 d, 15 d). Each egg was an experimental unit. Hatching eggs were obtained from Ross 344 male x Ross 708 female broiler breeders housed as in Experiment I. The eggs were collected for 5 d at 51 wk of age. All freshly laid eggs were weighed and eggs that weighed between 61 g and 65 g were numbered individually. There were 90 pairs of weight-matched eggs identified. One egg of each pair of the same weight (±0.1g) was assigned to each of six turning treatments. Other eggs that fell outside the designed egg weight range were randomly selected to fill the trays and to obtain hatchability data. Two Natureform model NOM-45 setter/hatchers and one Natureform NMC-2000 setter/hatcher were used in this experiment. The eggs were stored for 2-5 d as described above and preheated in two Natureform model NOM-45 setter/hatchers at 26.7 C (80 F) for 12 h prior to setting. Incubators were operated as in Replication 1. Each NOM-45 setter/hatcher contained three turning treatment trays of eggs at setting plus two extra trays to fill each machine. One tray from each machine was moved to a NMC-1000 setter/ hatcher after incubating for 3, 9, or 15 d. The turner of each NOM-45 setter/hatcher was set to turn either 24 times daily (24 n/d) or 96 times daily (96 n/d) at 45 on both sides to E15, and the NMC-1000 setter/hatcher turned once hourly (48 n/d) at 40 on both sides. Each of the 90 individually weighed eggs in each tray constituted a replicate. At E15 of incubation, 39 weight-matched pairs of eggs with live embryos from each turning treatment were randomly selected and necropsied to determine embryo length and weights of the egg, embryo, yolk sac, and fluids as in Replicate 1. The remaining pairs of eggs were turned to E18 before transfer to hatching baskets in individual pedigree bags. From E19 to E21, the 38

number of chicks hatched was recorded every 12 h along with the individual chick BW at that time. At E21 (pull), chick BW, YFBW, and chick length were measured on 35 chicks from each turning treatment. The general lineal model of SAS Institute (2008) was used to analyze the variables and differences among means were partitioned by LSMEANS. Means were considered statistically different at P<0.05. Experiment III. Experiment 3 was a 2 X 2 factorial design: 2 turning lengths (15 d or 18 d), and 2 early incubation temperatures (38.1 C or 37.5 C). Each egg was an experimental unit. Broiler hatching eggs were obtained from Ross 344 male x Ross 708 female broiler breeders housed as described previously. The eggs were collected for 3 d at 49 wk of age. All freshly laid eggs were weighed and eggs between 65 g and 68 g were numbered individually. There were 90 pairs of weight-matched eggs identified. One egg of each pair with the same weight (±0.1g) was assigned to each of 4 treatments. Other eggs that fell outside the desired egg weight range were randomly selected to fill the trays, and to obtain hatchability data. Each tray represented a treatment, and each egg was a replication. A total of 10 trays with 1800 eggs total were set to fill the machine to insure uniformity of air flow and temperature. The eggs were stored at 16 C (60.8 F) and 60% RH for 1-2 d before preheating at 23.9 C (75 F) for 12 h prior to setting. Incubators were operated at an air temperature of either 38.1 C (100.5 F) or 37.5 C (99.5 F) and 53% RH until E3 (72 h) of incubation. From E3 to E15, both machines were operated at the same air temperature of 37.5 C (99.5 F) from E3 to E9, 37.3 C to 37.1 C (99.2 F to 98.8 F) from E10 to E14, 37.0 C to 36.4 C (98.6 to 97.5 F) from E15 to E18, and 36.1 C (97.0 F) to hatching. The incubators were set to turn 96 times 39

daily (96X) to E18, or to E15 followed by turning once hourly (24X) to E18. The turning angle was 45 at both directions. At E14 of incubation, 15 weight-matched pairs of eggs from each treatment were randomly selected and necropsied to determine embryo length, and weights of the egg, embryo, and yolk sac. The remaining eggs were turned to E18 before transfer to hatching baskets in individual pedigree bags. At E21, chick BW, yolk sac weight, heart weight, gizzard weight, yolk free body weight, and chick length were measured on 48 pairs of chicks equally representing the four treatments. The general lineal model of SAS Institute (2008) was used to analyze the variables and differences among means were partitioned by LSMEANS. Means were considered statistically different at P<0.05. Experiment IV. Incubation. Freshly laid eggs produced by Ross 708 broiler breeders were provided by Mountaire Farms and delivered to the Piedmont Research Station, Salisbury, NC. Eggs were collected for 1d when the flock was 41 wk of age. All freshly laid eggs were weighed and eggs between 60 g and 63 g were numbered individually. There were 90 pairs of weight-matched eggs identified. One egg of each pair with the same weight (±0.1g) was assigned to each of the 8 treatment combinations in the 2x2x2 design. Other eggs that fell outside the desired egg weight range but were of similar weight were randomly selected to fill the trays and to obtain hatchability data. A total of 2880 eggs were set in 4 Natureform model I-14 setters (Natureform International, Jacksonville, FL). There were 360 eggs used in each treatment. Each setter was operated at a different setting. The eggs were stored for 1 day following weighing before preheating at 23.9 C (75 F) or 29.4 C (85 F) for 12 h prior to setting. Humidity was automatically maintained at 53% throughout the incubation period. 40

Following preheating, incubators were operated at an air temperature of either 38.1 C (100.5 F) or 37.5 C (99.5 F) until E5 (120 h) of incubation. The temperature of 37.5 C (99.5 F) employed from E5 to E9 was then gradually decreased from 37.3 C to 37.1 C (99.2 F to 98.8 F) from E10 to E14, 37.0 C to 36.4 C (98.6 to 97.5 F) from E15 to E18, and 36.1 C (97.0 F) to hatching. The turner of the setters were set to turn 45 in both directions and turn either 24 times daily (24 n/d) or 96 times daily (96 n/d) to E15 and followed by 24 n/d daily turning to E18. At E18 eggs were transfer to hatching baskets in individual pedigree bags. Thus, the experimental design was two preheating temperatures x 2 E0-E5 temperatures x 2 turning frequencies to E15. Experiment IV. Sampling. From E5 to E10, 10 eggs from each treatment per day were boiled at 80 C (176 F) in a water bath for 2 min (Figure MM-2), taped around the equator (Figure MM-3), and then cut between the tapes to open the eggs while not severely cracking the shell. The albumen and yolk were then carefully poured out in such a manner to maintain the yolk sac membrane (YSM) and chorioallantoic membrane (CAM) firmly attached to the egg shell (Figure MM-4). Photos were taken of these membranes under Wild Photomakroskop M400 with the Nikon DS-Fi1 Digital Camera and analyzed by Matlab to calculate the area of the blood vascular area within each membrane. At E14, 35 pairs of weight-matched eggs from each treatment combination were randomly selected to necropsy embryos for YFBW, egg, embryo weight, and embryo length. At E21 (pull), chick weight, YFBW, and chick length were measured on 27 chicks from each treatment. 41

Experiment IV. Broilers. The chicks that hatched at the Piedmont Research Station, Salisbury, NC were grouped into boxes according to the 8 treatment combinations. They were then transported to the NCSU-CEU in Raleigh. All chicks were then sexed and permanently identified with neck tags and placed in floor pens according to the incubation treatment combination and sex. These were 12 to 18 birds placed per pen with 4 pens of each sex per treatment combination for a total of 64 pens. There were two blocks to account for small positional differences found among house quarters. Feed and water were available for ad libitum consumption. Feed consumption was determined at 14 d, 35 d, and 49 d of age. Mortality was collected twice daily and weighed in order to calculate the AdjFCR. Group BW were determined at 1 d, 14 d, 35 d, and 49 d of age. At 35 d and 49 d of age, BW of the two sexes in the same block was calculated to derive an estimate of the mean. Subsequently, individual BW of three birds per pen that fell within ±250g of the estimated mean BW for each sex were marked with black paint. Feed was withdrawn 12 h before processing, while allowing continued access to water. At 36 d and 50 d of age, two of three marked birds per pen were chosen at random for processing. The processing sequence consisted of bleeding, scalding, plucking, sex confirmation and evisceration with care being taken not to remove that fat pad during the manual evisceration. Subsequently, the dry carcass weight without shanks, feet, and neck was recorded prior to removal of the fat pad. Carcasses were then further processed into predefined cuts consisting of ribs, wings, breast skin, Pectoralis major, Pectoralis minor, legs, and thighs. All remaining birds in the same block were euthanized, weighed, and necropsied to determine weights of the heart, gizzard, proventriculus, wattles, and testes, while the presence of an ovary was confirmed in the case of female birds. For 42

Experiment IV, a complete randomized block design was utilized for the eight incubation treatment combinations with 2 blocks in the chicken house. The general lineal model of SAS Institute (2008) was used to analyze the variables and differences among means were partitioned by LSMEANS. Means were considered statistically different at P<0.05, while differences at P<0.08 were considered to be numerical trends. 43

Figure MM-2. Water bath for boiling eggs. 44

Figure MM-3. Tape around the egg and cut between the tape. 45

Figure MM-4. The yolk sac membrane (YSM) and chorioallantoic membrane (CAM) attached to the egg shell. 46