Physiological response of broiler embryos to different incubator temperature profiles and maternal flock age during incubation. 1. Embryonic metabolism and day-old chick quality

Physiological response of broiler embryos to different incubator temperature profiles and... Abstract Broiler strain, maternal age, and incubation temperature influence embryo metabolism. Hatching eggs were obtained from young (Y; 28 to 34 wk, $$\bar{\rm x}$$ = 31.2 wk), mid (M; 36 to 45 wk, $$\bar{\rm x}$$ = 40.5 wk) and old (O; 49 to 54 wk, $$\bar{\rm x}$$ = 51.4 wk) Ross 708 (n = 88; Experiment 1) and Ross 308 [(n = 45; Experiment 2: (Y; 25 to 34 wk, $$\bar{\rm x}$$ = 30.5 wk), (M; 35 to 44 wk, $$\bar{\rm x}$$ = 40.2 wk), and (O; 49 to 54 wk, $$\bar{\rm x}$$ = 51.6 wk)] breeders. Eggs were stored for 2 to 4 d (18°C, 73% RH), and incubated for 14 d at 37.5°C and 56% RH. At 15 d (E15), 8 fertile eggs per flock age were incubated in individual metabolic chambers at 36.0, 36.5, 37.0, or 37.5°C until E21.5. Each temperature was repeated one additional time. O2 consumption and CO2 production were used to calculate embryonic heat production (EHP). Embryo temperature was measured as eggshell temperature (EST). Initial egg weight was used as a covariate; significance was assessed at P < 0.05. In Ross 708, daily EHP tended to be higher in M and O than Y treatments at E16; EHP of M was higher than Y and O eggs at E18; M and O were higher than O eggs at E19. Incubation at 37.0°C resulted in the highest EHP from E15 to E21, except at E17. Embryos at 37.5°C had reduced EHP beyond E17. Daily EST from E15 to E21 was higher at 37.5 and 37.0°C than at 36.0 and 36.5°C. In Ross 308, daily EST was highest at 37.5°C except at E20. Incubation temperature and EST were highly correlated (R2 = 0.90 to 0.89; P < 0.001). Ross 708 chicks were longer and hatched earlier at 37.0°C than at 36.0 and 37.5°C. EST and EHP increased with incubation temperature in Ross 708. In Ross 308, maternal flock age and incubation temperature did not impact EHP. However, EST was highest at 37.5°C except at E20. Ross 708 was more sensitive to incubation temperature than Ross 308. INTRODUCTION Genetic selection of modern broiler chicken strains for increased growth, feed efficiency, and breast meat yield has influenced embryonic development (Tona et al., 2004; Druyan, 2010; Nangsuay et al., 2015a). Modern broiler embryos are heavier than layer embryos as a result of faster growth from 11 to 19 d of incubation (Ohta et al., 2004). Genetic selection of egg-strain chickens for reproductive traits has resulted in more efficient lipid metabolism, whereas selection of meat-type chickens for growth and efficiency has resulted in increased metabolic rate (Buzala et al., 2015). The consequences of increased metabolic rate in broilers include stunted liver and heart tissues, which could result in metabolic inefficiencies in embryos (Lindgren and Altimiras, 2011). Ross broiler embryos have showed higher embryonic O2 consumption (Hamidu et al., 2007; Druyan, 2010), higher heart beats per minute, and increased metabolic hormone levels and growth rate compared to Cobb broiler embryos, demonstrating differences in developmental patterns and metabolic characteristics (Druyan, 2010). Daily embryonic heat production (EHP) is a measure of embryonic metabolism; Ross 308 embryos had higher daily EHP than Cobb 500 embryos during early (1 to 7 d; Hamidu et al., 2007) and late (15 to 21 d) incubation (Hamidu et al., 2007; Nangsuay et al., 2015a). To optimize incubation conditions, the effects of maternal flock age on embryo metabolism must be considered. Embryos from older breeder flocks have increased O2 consumption, metabolic heat production, high eggshell surface temperature, and a metabolic shift from glycolysis to gluconeogenesis to meet energy demands compared to embryos from younger flocks (Tona et al., 2001; Hamidu et al., 2007; De Oliveira et al., 2008; Leksrisompong et al., 2007, 2009; Lourens et al., 2007; Christensen et al., 2008; Druyan, 2010; Molenaar et al., 2010). Differences in embryo metabolism exist in lines from different primary breeders, and even within strains from a single company. Therefore, to optimize incubation conditions, we need to know how differences in metabolism affect incubation requirements, and how those requirements are affected by flock age (Hamidu et al., 2007). Although the effects of flock age and breeder strain on embryo metabolism have been extensively researched (Tona et al., 2004; O’Dea et al., 2004; Hamidu et al., 2007), the interaction of incubation temperature with these factors on the physiological growth of embryos and subsequent impacts on hatchling quality are not well understood. Additionally, the confounding effect of maternal flock age on egg weight cannot be discounted. However, in studies in which eggs were selected to be the same weight among various flock ages, increasing flock age also increased embryonic metabolism (O’Dea et al., 2004; Hamidu et al., 2007). These results appear to suggest that maternal flock age, rather than egg weight, is the main factor influencing embryonic metabolism. Incubation temperature influences embryogenesis and post-hatch chick development (Yildirim and Yetisir, 2004; Feast et al., 1998; Elibol and Brake, 2006; Piestun et al., 2009; Janisch et al., 2015). It is interesting to note that moderate incubation temperatures (37.2 or 38.3°C) from 17 to 21 d of incubation reduced late incubation embryo mortality compared to lower (36.1°C) and higher (39.9°C) incubation temperatures (Yildirim and Yetisir, 2004). Although the initial impact of sub-optimal incubation temperatures is first seen in changes in embryonic metabolism, the subsequent effects on other physiological activities of embryos are not well understood. Chick length and disappearance of residual yolk have been used as quality indicators for newly hatched chicks (Lourens et al., 2005, 2007; Molenaar et al., 2011). Eggshell temperature (EST) is a well-investigated technique to assess embryonic development and shown to influence chick quality (Lourens et al., 2005, 2006, 2007; Molenaar et al., 2011; Romanini et al., 2013), but its continuous measurement requires sophisticated techniques and equipment (Tong et al., 2016). Measurement of heat production as an indicator of embryonic development is well established (Meijerhof and Van Beek, 1993; Tona et al., 2004 and Lourens et al., 2005, 2006, 2007), and EST has been used as an indicator of EHP. However, there is no clearly established relationship between EST and EHP, although experimental results imply that EHP may be reflected by EST changes. (Lourens et al., 2007; Nangsuay et al., 2017). Therefore, it makes sense to determine a relationship and even complex equations that can explain the reason behind this frequent use of EST to reflect EHP. Therefore, the relationship between incubation temperature and EST must be determined for EST to be used as a tool to assess the appropriateness of late-stage incubation temperature, as influenced by broiler strain and breeder age. This will underscore the importance of managing incubation temperature on embryo development and chick quality. Sub-optimal incubation temperatures, especially during the plateau stage of O2 consumption from embryonic age 17 to 19 (E17 to E19) may increase late incubation embryo mortality and the proportion of weak chicks (Dietz et al., 1998; De Oliveira et al., 2008; Abudabos, 2010). This effect might influence embryos and chicks from diverse hen ages differently. Therefore, the interaction of breeder flock age and incubation temperature on embryonic metabolism must be examined more closely. Previously, at 37.0°C, there were differences in embryonic metabolism between genetic strains and flock ages (Hamidu et al., 2007). Thus, we hypothesized that within each genetic strain, increased embryonic metabolism with increasing breeder flock age would result in different optimal incubation temperatures from 15 to 21.5 d of incubation. The objectives of the current research were to evaluate the impact of incubation temperature and maternal flock age on embryonic metabolism, eggshell temperature, and early chick quality within each of Ross 708 and Ross 308 broilers. MATERIALS AND METHODS Experimental Design On 8 separate occasions, fresh broiler breeder eggs were obtained from a commercial hatchery from each of 3 different parent flock ages: young (Y; 28 to 34 wk, $$\bar{\rm x}$$ = 31.2 wk), mid (M; 36 to 45 wk, $$\bar{\rm x}$$ = 40.5 wk), and old (O; 49 to 54 wk of age, $$\bar{\rm x}$$ = 51.4 wk) Ross 708 (Experiment 1) hens. These eggs were typical of hatching eggs in commercial incubation (52 to 70 g). The eggs were stored for 2 to 4 d at 18°C and 74% RH. Eggs were assigned to one of 4 incubation temperature treatments (36.0, 36.5, 37.0 (control), or 37.5°C) from E15 to E21.5. The control temperature of 37.0°C was similar to that used in commercial hatcheries during the last 3 d of incubation (Elibol and Brake, 2006). For each set of eggs, a single incubation temperature was applied within the incubator; each temperature was replicated 2 times in a random order. For each replicate in Experiment 1, a total of 11 eggs per each of the 3 flock ages was obtained, weighed, and incubated for 14 d at 37.5°C and 56% RH in a Jamesway AVN incubator. Beginning at E15, fertile eggs (n = 8) from each of the 3 parent flock ages were individually transferred into one of 24 one-liter metabolic chambers placed inside the incubator as described previously (Hamidu et al., 2007). Each one-liter metabolic chamber had one inlet for fresh air intake and an outlet from which air samples were analyzed for O2 and CO2. During each replication, 7 additional eggs per flock age were placed in a desiccator, covered in desiccant, and the eggs weighed back at the same time every d for 9 consecutive days. Eggshell conductance (G), which is the ability of gases and moisture to diffuse across the eggshell (O’Dea et al., 2004; Hamidu et al., 2007) was calculated using the formula of Ar et al., (1974). The value for G (mg/d per mmHg) was calculated from the rate of water loss per d from each egg (mg/d) divided by the change in water vapor pressure of the egg content and water vapor pressure in the environment surrounding the egg (mm Hg). Since the egg was covered in desiccant, the water vapor pressure in the environment surrounding the outside of the egg was equal to zero. In Experiment 2, 15 eggs per flock age were obtained from Y (25 to 34 wk, $$\bar{\rm x}$$ = 30.5 wk), M (35 to 44 wk, $$\bar{\rm x}$$ = 40.2 wk), and O (49 to 54 wk of age, $$\bar{\rm x}$$ = 51.6 wk) from Ross 308 breeders on 8 separate occasions as described above. The eggs were collected, weighed, and incubated according to temperature treatments as in Experiment 1. Eggshell conductance was not investigated in Experiment 2, due to its having been investigated previously in Ross 308 embryos in our lab (Hamidu et al., 2007). Incubation conditions followed standard commercial hatchery procedures, with eggs placed in the dark, with hourly turning interval. Proper ventilation was maintained with fresh air by connecting a tube from the incubator damper to the hatchery roof and dampers adjusted when necessary to maintain RH at 56%. For simplicity, the flock ages for both Ross 708 and Ross 308 have been defined as young (Y; 26 to 34 wk), mid (M; 35 to 45 wk), and old (O; 46 to 55 wk). In addition, all experimental procedures were approved by the University of Alberta Animal Care and Use Committee in accordance with the Canadian Council on Animal Care (2009) guidelines. Hatch Analysis At 18 d of incubation, the incubator turning mechanism was stopped to simulate the conditions of a commercial hatcher. Beginning at 452 h of incubation, the eggs were checked at 6-hour intervals to establish the times each embryo was required to externally pip and hatch. All chicks remained in the metabolic chambers, and after 518 h of incubation, they were removed, weighed, and euthanized by decapitation. The carcasses were dissected; the residual yolk sacs (RYS) were removed from the chicks and weighed. The dry matter weight of the yolk sac was determined at an oven temperature of 65°C for 4 days. All sample weights were expressed as percentage of initial chick weight. Gas Exchange and Eggshell Temperature The metabolic chambers used have been previously validated (Hamidu et al., 2007, 2010, 2011). In each run, embryonic O2 consumption and CO2 production in each of 24 metabolic chambers were measured by computer-software controlled O2 and CO2 analyzers. Each chamber was sampled sequentially for 5 min, 6 times per d (returning to the same chamber at 2-hour intervals). The values within each d were averaged for determination of average daily O2 consumption and CO2 production, which were used in the calculation of daily EHP, a measure of embryonic metabolism. The ambient air inside the incubator was sampled to determine O2 and CO2 concentration immediately prior to each of the metabolic chambers being sampled. The difference between the gas partial pressures of the metabolic chamber and that of the ambient air was used to calculate embryonic O2 and CO2 exchange rates (Hamidu et al., 2007, 2010). The measurement of ambient O2 and CO2 exchange rates using the inside of incubator or an empty metabolic chamber has been tested and published previously (Hamidu et al., 2010) and demonstrated that each metabolic chamber had a similar microclimate as the inside of the incubator (Hamidu et al., 2010). Therefore, it was not necessary to use additional metabolic chambers as a control. The EHP was calculated following the formula of Kleiber (1987): Heat production (mW) = [3.871 × O2 consumption (L/d) + 1.194 × CO2 production (L/d)] × 1 d/24 h × 1 h/3600 s × 1000 cal/1 kcal × 4.187 J/cal × 1000 mW/W. A custom-made temperature probe, held in place by foam attached to the egg tray and held in direct contact with the eggshell inside each metabolic chamber was used to monitor EST. The EST can be used to determine whether the embryos are being incubated at an optimum incubation temperature (Ar and Tazawa, 1999; Lourens et al., 2006; Hamidu et al., 2011). An additional temperature probe was hung in the inside of the incubator to monitor ambient temperature. Data were recorded 6 times per d for each chamber, but repeated measurements were rearranged into replications per d per chamber. Statistical Analysis Data from Experiment 1 (Ross 708) and Experiment 2 (Ross 308) were analyzed separately. All data were subjected to analysis of variance using the Proc. Mixed procedure of SAS (Wang and Goonewardene, 2004; SAS Institute, 2010) at P ≤ 0.05 (SAS Institute, 2010). The statistical model used to analyze all metabolic responses included the fixed effects of hen age (3 levels) and incubation temperature (4 levels), the interaction between the 2 main effects, and the covariate effect of initial egg weight. The original source of eggs (farm) was nested in the interaction of flock age and incubation temperature and used as the random variable for analysis. For G, hen age was the only fixed factor at 3 levels, while farm was used as the random factor. In the analysis, initial egg weight was used as a covariate (Jacobs, 2011), since it was confounded by maternal flock age (Hamidu et al., 2011). The statistical model used was: Yijkl = μ + αi + βj + αiβj + ϖ + λk(αiβj) + εijkl, where: Yijkl = effect measured, μ = overall mean, αi = main effect of maternal flock age, βj = main effect of incubation temperature, αiβj = interaction effect of maternal flock age and incubation temperature, ϖ = covariate effect of initial egg weight, λk = random effect due to farm or source of eggs, λk(αiβj) = random effect of maternal flock age and incubation temperature nested in farm, and εijkl = residual error term. Also, a regression analysis was performed to establish an appropriately fit relationship between incubation temperature and EST, as well as between EHP and EST. All differences between least square means were considered to be significant when P ≤ 0.05 (SAS Institute, 2010). RESULTS Maternal Flock Age Effect Experiment 1. Ross 708. Initial egg weight prior to incubation increased with increasing flock age (Table 1). Similarly, G was higher in eggs of M and O flocks compared to eggs of Y flocks (Table 1). Chick weight was not different among flock ages; however, chick length was different. The chicks of Y flock age were longer than chicks from M and O flock ages. The external pipping time of embryos prior to hatching was higher in M flock age than Y flock age. However, neither was different from O flock age; the hatching time among flock ages was not different (Table 1). The percentages of wet and dry RYS as well as yolk-free body mass (YFBM) were not different among maternal flock ages (P > 0.05; Table 2). Table 1. Initial egg weight and chick characteristics from eggs of different flock ages and incubated at different incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Eggshell conductance = daily egg moisture divided by saturated vapor pressure. 5Chick length = from tip of longest toe to the beak. 6Pipping time = from time of incubation until embryo breaking through eggshell. 7Hatching time = from time of incubation until chick leaving the eggs completely. 8Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg or chick. 9Standard error of means (n = 16 eggs per flock age). View Large Table 1. Initial egg weight and chick characteristics from eggs of different flock ages and incubated at different incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Eggshell conductance = daily egg moisture divided by saturated vapor pressure. 5Chick length = from tip of longest toe to the beak. 6Pipping time = from time of incubation until embryo breaking through eggshell. 7Hatching time = from time of incubation until chick leaving the eggs completely. 8Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg or chick. 9Standard error of means (n = 16 eggs per flock age). View Large Table 2. Chick carcass characteristics of day-old chicks from different flock ages and incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4YFBM: Yolk-free body mass. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Table 2. Chick carcass characteristics of day-old chicks from different flock ages and incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4YFBM: Yolk-free body mass. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large The daily embryonic O2 consumption was higher in M embryos compared to O embryos at E19 but not Y embryos, which was not different from M and O embryos. At E20, M and Y embryos showed higher O2 consumption compared to O embryos; there were no differences at the other embryonic ages (Figure 1a). The daily embryonic CO2 production was higher in Y and M embryos compared to O embryos at E20 (Figure 2a). Average over the 7 d investigated showed that the M embryos had higher O2 consumption from E15 to E21 compared to Y and O embryos (P = 0.011), even without a significant covariate effect of initial egg weight. The average embryonic CO2 production between E15 to E21 was not different. The daily EHP profiles among flock ages followed a similar pattern as the O2 consumption (Figure 3a). The Y and M embryos had greater EHP than O embryos at E15, E16, and E19. The M embryos had higher EHP than O embryos at E19 but neither was different from Y embryos; however, at E20, M and Y embryos had higher EHP than O embryos (Figure 3a). On average, EHP from E15 to E21 was higher in M embryos than Y and O embryos (P = 0.014; Table 3). The average (Table 3) and daily EST (data not shown) were not different between maternal flock ages (P > 0.05). Figure 1. View largeDownload slide Effect of maternal flock age on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic O2 consumption was significantly higher in embryos from M flock age compared to O flock age at E19 (*P ≤ 0.05); at E20, M and Y flock ages had higher consumption than O flock age in Ross 708 (***P ≤ 0.001). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 1. View largeDownload slide Effect of maternal flock age on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic O2 consumption was significantly higher in embryos from M flock age compared to O flock age at E19 (*P ≤ 0.05); at E20, M and Y flock ages had higher consumption than O flock age in Ross 708 (***P ≤ 0.001). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 2. View largeDownload slide Effect of maternal flock age on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production of Y and M embryos was higher than O embryos at E20 in Ross 708 (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 2. View largeDownload slide Effect of maternal flock age on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production of Y and M embryos was higher than O embryos at E20 in Ross 708 (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 3. View largeDownload slide Effect of maternal flock age on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production of M and Y flocks was higher than O embryos at E16 (†P ≤ 0.10). Also, M embryos had higher heat production than O embryos at E19, but neither was different from Y embryos (*P ≤ 0.05); however, at E20, M and Y embryos had higher heat production than O embryos (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 3. View largeDownload slide Effect of maternal flock age on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production of M and Y flocks was higher than O embryos at E16 (†P ≤ 0.10). Also, M embryos had higher heat production than O embryos at E19, but neither was different from Y embryos (*P ≤ 0.05); however, at E20, M and Y embryos had higher heat production than O embryos (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Table 3. Average gas exchange and embryonic heat output from eggs of different flock ages and incubation temperatures from 15 to 21 d of incubation for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Average heat production (mW) = average heat production from E15 to E21. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Table 3. Average gas exchange and embryonic heat output from eggs of different flock ages and incubation temperatures from 15 to 21 d of incubation for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Average heat production (mW) = average heat production from E15 to E21. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Experiment 2. Ross 308. Initial egg weight (P < 0.001) increased as flock age increased (Table 1). There was no effect of flock age on chick weight, chick length, external pipping time, or hatching time (Table 1) as well as percent YFBM, wet RYS, or dry RYS. There were no differences in daily O2 consumption (Figure 1b) nor daily CO2 production (Figure 2b) and EHP (Figure 3b) among maternal flock ages from E15 to E21. Similarly, the average O2 consumption and CO2 production were not affected by flock age (Table 3). There were no differences in daily EHP (Figure 3a) or EST (data not shown) among maternal flock ages. Similarly, average EHP and EST from E15 to E21 were not affected by maternal flock age (Table 3). Incubation Temperature Effects Experiment 1. Ross 708. Initial egg weight was not different among eggs assigned to the various incubation temperature groups. Chick weight was not affected by incubation temperature (Table 1). The eggs incubated at 37.0°C from E15 to E21 hatched longer chicks compared to eggs incubated at 37.5°C. The 37.0°C treatment also resulted in shorter external pipping and hatching times compared to all other incubation temperature treatments investigated. The chicks incubated at 36.0°C had the longest hatch time followed by 37.5°C and then 36.5°C groups. However, all chicks, irrespective of incubation temperature treatment, hatched in less than 21.5 d (36.0°C = 21.10 d; 36.5°C = 20.9 d; 37.0°C = 20.7 d, and 37.5°C = 20.9 d). The percentage of wet and dry RYS weights tended to be lower at 37.0°C (11.24 and 6.09%, respectively) than 36.0°C (13.23 and 7.31%, respectively), 36.5°C (13.31 and 7.14%, respectively), and 37.5°C (13.97; 7.74%, respectively; P = 0.079; P = 0.098 for wet and dry RYS, respectively; Table 2). In both cases, the proportion of RYS was highest at 37.5°C. Embryos from 36.5 and 37.0°C tended to have higher O2 consumption compared to 36.0°C at E15 (P < 0.10), but embryonic response at all 3 incubation temperatures was not different from 37.5°C incubation temperature. Embryos incubated at 37.0 or 37.5°C consumed more O2 from E16 to E17 as compared to those incubated at 36.0 or 36.5°C (Figure 4a). The embryos at 37.5°C consumed about 102 mL/d more O2 than those at 37.0°C at 17 d of incubation. After E17, daily O2 consumption by embryos incubated at 37.5°C dropped sharply below O2 consumption levels of embryos at 37.0 and 36.0°C from E18 to E20. At E19 and E20, embryos from the 37.0°C treatment consumed higher amounts of O2 compared to embryos in all other incubation temperature treatments; the O2 consumption was not different at E21. At 37.0°C, the average O2 consumption from E15 to E21 was higher compared to 36.5 and 36.0°C (Table 3). The O2 consumption was not different between 37.0 and 37.5°C incubation temperatures. Figure 4. View largeDownload slide Effect of incubation temperature treatments on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic O2 consumption was higher at 36.5 and 37.0°C compared to 36.0°C on E15 (†P ≤ 0.10); O2 consumption was higher at 36.0, 37.0, and 37.5°C incubation temperatures compared to 36.0°C incubation temperature at E16 (*P ≤ 0.05). At E17, embryonic O2 consumption was higher at 37.5°C compared to 36.0, 36.5, and 37.0°C incubation temperatures (**P ≤ 0.01); at E19 and E20, embryonic O2 consumption was higher at 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C incubation temperatures (***P ≤ 0.001). There was no difference at E21. Also, in Ross 308, no difference existed in O2 consumption between incubation temperatures on all d of incubation. Figure 4. View largeDownload slide Effect of incubation temperature treatments on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic O2 consumption was higher at 36.5 and 37.0°C compared to 36.0°C on E15 (†P ≤ 0.10); O2 consumption was higher at 36.0, 37.0, and 37.5°C incubation temperatures compared to 36.0°C incubation temperature at E16 (*P ≤ 0.05). At E17, embryonic O2 consumption was higher at 37.5°C compared to 36.0, 36.5, and 37.0°C incubation temperatures (**P ≤ 0.01); at E19 and E20, embryonic O2 consumption was higher at 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C incubation temperatures (***P ≤ 0.001). There was no difference at E21. Also, in Ross 308, no difference existed in O2 consumption between incubation temperatures on all d of incubation. On E15, the embryos incubated at 36.5°C had higher CO2 production than those of 36.0°C. The embryos incubated at 37.0°C had higher CO2 production compared to those incubated at 36.5°C and than 36.0 or 37.5°C at E20 (37.0 > 36.5 > 36.0 and 37.5°C). But there were no differences at any other time point (Figure 5a). Average CO2 production from E15 to E21 was not affected by incubation temperature (Table 3). Similar to O2 consumption, the EHP of eggs incubated at 37.0°C invariably was higher than embryos at 36.0 and 36.5°C. At 37.5°C, EHP was greatest at E17, but dropped thereafter, reaching a low at E20, before rising after external pipping (Figure 6a). At 37.0 and 36.0°C, EHP rose steadily to about 155 mL/d from E15 to E17, reaching a plateau or decreasing from E17 to E20, and then rising sharply again until E21. At 36.5°C, EHP rose slightly from E15 to E16, decreased from E18 to E19, and then increased to E21. Apart from E17 when embryos incubated at 37.5°C had higher EHP, the embryos at 37.0°C consistently produced more heat from E15 to E21 compared to those of 36.0°C incubation temperature. The average EHP over the course of the study (from E15 to E21) was higher at 37.0°C compared to 36.5 and 36.0°C. (P = 0.001; Table 3). The average EST from E15 to E21 (Table 3) and the daily EST within that period (Figure 7a) were higher for embryos incubated at 37.0 and 37.5°C compared to those incubated at 36.5 and 36.0°C, except at E20 when EST of 37.5°C was higher than all other incubation temperatures (Figure 7a). The 36.5°C embryos had higher EST than 36.0°C embryos during each of the d considered. The EST and incubation temperature in Ross 708 embryos were strongly and positively related (y = −1.541x3 + 169.07x2 + 6,181.3x + 75,345; R² = 0.90; P < 0.001, Figure 7a; where y is eggshell temperature and x is incubation temperature). The EHP and EST in the same strain showed a very weak, though significant, relationship (y = 15.874x – 460.15; R² = 0.11, P < 0.001, where y = EHP and x = EST). Figure 5. View largeDownload slide Effect of incubation temperature treatments on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production at E15 was higher at 36.5°C compared to 36.0°C (†P ≤ 0.10); at E20, CO2 production was higher at 37.0°C incubation temperatures compared to 36.0, 36.5, and 37.5°C incubation temperatures in Ross 708 (***P ≤ 0.001). At E21, embryonic CO2 production was also significantly higher at 37.0°C than 36.0 and 37.5°C on E21 (*P ≤ 0.05). No differences were observed in Ross 308. Figure 5. View largeDownload slide Effect of incubation temperature treatments on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production at E15 was higher at 36.5°C compared to 36.0°C (†P ≤ 0.10); at E20, CO2 production was higher at 37.0°C incubation temperatures compared to 36.0, 36.5, and 37.5°C incubation temperatures in Ross 708 (***P ≤ 0.001). At E21, embryonic CO2 production was also significantly higher at 37.0°C than 36.0 and 37.5°C on E21 (*P ≤ 0.05). No differences were observed in Ross 308. Figure 6. View largeDownload slide Effect of incubation temperature treatments on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production (EHP) was higher in 37.0 and 37.5°C incubation temperatures compared to 36.0°C at E15 (†P ≤ 0.10) and lower at 36°C compared to 36.5, 37.0, and 37.5°C at E16 (**P ≤ 0.01); at E17, EHP was higher at 37.5°C incubation temperature compared to 36.0 and 36.5°C but not different from 37.0°C (**P ≤ 0.01). For E19 and E20, it was higher in 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C (***P ≤ 0.001) and E21 (*P ≤ 0.05). The EHP was not different at E18 (P > 0.05). No significant differences were observed in Ross 308 (P > 0.10). Figure 6. View largeDownload slide Effect of incubation temperature treatments on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production (EHP) was higher in 37.0 and 37.5°C incubation temperatures compared to 36.0°C at E15 (†P ≤ 0.10) and lower at 36°C compared to 36.5, 37.0, and 37.5°C at E16 (**P ≤ 0.01); at E17, EHP was higher at 37.5°C incubation temperature compared to 36.0 and 36.5°C but not different from 37.0°C (**P ≤ 0.01). For E19 and E20, it was higher in 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C (***P ≤ 0.001) and E21 (*P ≤ 0.05). The EHP was not different at E18 (P > 0.05). No significant differences were observed in Ross 308 (P > 0.10). Figure 7. View largeDownload slide Effect of incubation temperature treatments on eggshell temperature during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, EST at 37.5 and 37.0°C incubation temperatures was higher than 36.0 and 36.5°C incubation temperatures on all d except on E20, when EST at 37.0°C was higher than the other incubation temperatures (***P ≤ 0.001). Also, in Ross 308, EST at 37.5°C incubation temperature was higher than 36.0, 36.5, and 37.0°C incubation temperatures from E15 to E21, except at E20, when EST was not different between 37.0 and 37.5°C, but was higher in both temperatures compared to 36.0 and 36.5°C (***P ≤ 0.001). In both Ross 708 and Ross 308, the relationship between incubation temperature (X) and eggshell temperature (Y) was very high: R2 = 0.90 and R2 = 0.89, respectively. Figure 7. View largeDownload slide Effect of incubation temperature treatments on eggshell temperature during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, EST at 37.5 and 37.0°C incubation temperatures was higher than 36.0 and 36.5°C incubation temperatures on all d except on E20, when EST at 37.0°C was higher than the other incubation temperatures (***P ≤ 0.001). Also, in Ross 308, EST at 37.5°C incubation temperature was higher than 36.0, 36.5, and 37.0°C incubation temperatures from E15 to E21, except at E20, when EST was not different between 37.0 and 37.5°C, but was higher in both temperatures compared to 36.0 and 36.5°C (***P ≤ 0.001). In both Ross 708 and Ross 308, the relationship between incubation temperature (X) and eggshell temperature (Y) was very high: R2 = 0.90 and R2 = 0.89, respectively. Experiment 2. Ross 308. Initial egg weight was not different among incubation temperature treatments. Chick weight, chick length, external pipping time, and hatching time (Table 1), and percentage of YFBM and wet and dry RYS were not different among the treatments (Table 2). There was no difference in daily O2 consumption (Figure 4b), daily CO2 production (Figure 5b), or EHP (Figure 6b) among incubation temperature treatments from E15 to E21. The average O2 consumption, CO2 production, and EHP were not affected by incubation temperature (Table 3). From E15 to E21, daily EST at 37.5°C was higher than for each of the other incubation temperature treatments except at E20, when it was not different from the 37.0°C incubation temperature (Figure 7b). The daily EST at 37.0°C incubation temperature was also higher than the daily EST at 36.5 and 36.0°C, whereas 36.5°C showed higher daily EST compared to 36.0°C incubation temperature treatment, except at E20 when there was not a difference in EST. The effect of incubation temperature on EST showed a very strong polynomial relationship (y = −0.8369x3 + 92.397x2 − 3,399x + 41,701; R² = 0.89; P < 0.001, Figure 7b; where y is eggshell temperature and x is incubation temperature). The linear relationship between EST and EHP was relatively weak, although significant (y = 19.892x – 615.95; R² = 0.22, P < 0.001, where y = EHP and x = EST). Interaction of Maternal Flock Age and Incubation Temperature The interaction between maternal flock age and incubation temperature treatment did not affect any of the daily or average parameters measured in either genetic strain (P > 0.05). The covariate effect of egg weight or size greatly affected the data. In most cases, there were significant daily and average (over 7 d) effects of egg weight used as covariates for analyzing maternal flock age and incubation temperature on our measured parameters. DISCUSSION The influences of broiler breeder maternal flock age and incubation temperature on embryonic development and chick quality are of economic importance in incubation and may depend on egg weight, which increases with increasing flock age. This is because as flock age increases, hens tend to deposit more resources such as yolk into their eggs (Applegate et al., 1998; Ulmer-Franco et al., 2012), thus increasing the egg size. Generally, mid maternal flock age (34 to 45 wk) corresponds with peak egg production, as well as higher maternal and embryonic performance (Tona et al., 2001; Hamidu et al., 2007; Yassin et al., 2008). Eggs produced by young Ross 708 breeders tended to have lower G, O2, and CO2 exchange rates, embryonic metabolism, and heat production. These results also have been reported in previous studies using Ross 308 and Cobb 500 strains (Ar et al., 1974; O’Dea et al., 2004; Hamidu et al., 2007). It appears that for Ross 708 hens, maternal flock age rather than egg weight is the most important factor responsible for differences in EHP and therefore embryo metabolism (Nangsuay et al., 2013, 2015b). This is clearly seen in the non-significant effect observed in the covariate egg weight on G, O2 consumption, EHP, and EST. Nevertheless, the influence of egg size appears greater in Ross 308, as seen in the covariate egg weight and probabilities with no differences in any parameter due to maternal flock age and incubation temperature. Transfer of Ross 708 eggs to the 37°C incubation temperature at 15 d increased embryonic metabolism, and also increased chick length compared to other temperatures. An increase in embryonic metabolism may indicate higher embryonic development resulting in longer chicks, which is considered the most suitable estimator of initial chick quality (Molenaar et al., 2008). A consistent increase in EHP over time in Ross 708 embryos did not compromise embryonic development, but rather resulted in longer chicks at 37.0°C, even though the EST was higher than the recommended value of 37.8°C (Lourens et al., 2006). Higher EST occurs when embryos have access to O2 for respiration and metabolism following internal and external pipping, and possibly increased oxygen demand for utilization of yolk lipids (Menna and Mortola, 2002). Consequently, a high incidence of poor chick navel conditions is indicative of exposure to high incubation temperature as measured by the rectal temperature during brooding. The practice can be used to establish proper brooding practices to reduce chick mortality and increase survival post hatch, especially during the first wk (Chen et al., 2013). One of the main findings was the earlier hatch time for Ross 708 embryos incubated at 37°C than those incubated at 36.0 and 37.5°C. This was expected because of the reduced metabolism, which could lead to slower embryonic development. In each of the 4 temperature treatments, Ross 708 embryos incubated at 37°C had the most consistent and higher embryonic metabolism and also higher EST, which could have accelerated the hatching process. Similarly, in the same strain, the rapid increase in EST in the 37.5°C embryos to E17, and subsequent dramatic decrease at E20 may indicate a reduced metabolic rate in an attempt to avoid embryonic heat stress. A prominent sign of embryonic heat stress is a poorly absorbed yolk sac, leading to an increased incidence of unhealed navels in day-old chicks (Preez, 2007). These include a leaky navel, navel strings, navel buttons, and unclosed navels (Preez, 2007; Fasenko and O’Dea, 2008). The sudden drop in Ross 708 embryonic metabolism at E17 at the 37.5°C incubation temperature was also associated with a plateau in the EST (to ∼ 39°C) and may have been an adaptive response to overheating (Yalcin et al., 2008). In this study, we did not assign navel scores for the chicks; however, the greater RYS weights observed in the embryos incubated at 37.5°C could be evidence of heat stress (Shubber et al., 2012; Ozaydın and Celik, 2014). Consequences of high incubation temperature include decreased yolk consumption and mean embryonic weights due to lack of nutrients available for development (Ozaydın and Celik. 2014). Other studies showed increased glucose oxidation in embryos incubated at higher (38.9°C) compared to normal (37.8°C) EST from E17.6 until E17.8 (Molenaar et al., 2013). Although this may be a sign of elevated metabolism, the authors suggested that high incubation temperature during the perinatal period of chicken embryos increases glucose oxidation and decreases hepatic glycogen reserves prior to the hatching process. This condition can reduce hepatic and cardiac glucose availability to support successful development of the embryo and the hatching process, which relies on a lot of glucose immediately prior to external pipping (O’Dea et al., 2004). In Ross 708, it is possible that the embryos incubated at 37.5°C were heat stressed, as is common during the plateau stage of O2 consumption (E16 to E19), and therefore had to decrease metabolic rate or reduce yolk consumption (Willemsen et al., 2010). The consequences could be increased late embryo mortality, dead in shell embryos, and lower chick weight at hatch (Willemsen et al., 2010). A most interesting pattern observed in this study was the very weak relationship that existed between EST and EHP and a strong polynomial relationship between incubation temperature and EST in both Ross 708 and Ross 308 embryos. This contradicts previous observations of a strong linear relationship between EHP and EST (Lourens et al., 2006). This is because heat production of the embryo is greatly influenced by heat loss of the egg. The latter is particularly determined by the ventilation rate, the egg density in the incubator, RH, and the egg size, and even sometimes, the size of eggshell pores and their number (French 1997; Harb et al., 2010; Molenaar et al., 2010; Noiva et al., 2014). In the future, the above factors need to be controlled because they may influence the relationship between EHP and EST. There is a long-standing perception that broiler breeder genetic selection over many years has led to increased embryo metabolism (O’Dea et al., 2004; Tona et al., 2004, Hamidu et al., 2007). The results of the current study appear to agree with the perception, considering that the Ross 708 is an upgrade over the Ross 308. However, Ross 708 was much more responsive to incubation temperature changes than Ross 308. Increases in EHP, an indicator of embryonic metabolism (Hamidu et al., 2007; Lourens et al., 2007), were previously suggested to be related to increases in EST (Lourens et al., 2007; Tong et al., 2013). Therefore, some researchers use EST as a predictor of the rate of heat production, and as an indication of overheating in incubating embryos (Lourens et al., 2005, 2007; Molenaar et al., 2010). While there may be some basis for this in some broiler breeder strains, e.g., Hybro G+ grandparent flocks (Lourens et al., 2007), our research showed very little relationship between EHP and EST in both Ross 308 and Ross 708 embryos. Therefore, EST may not be the best predictor of EHP or may be influenced by other factors such as air speed, incubation temperature, and gas exchange rate. However, more recently, it has been demonstrated in both Hybro and Ross 308 strains that a heat stress incubation temperature of 38.8°C from E10 compared to control temperature of 37.8°C throughout incubation significantly reduced relative yolk free embryo weight as well as decreased yolk consumption (Ozaydın and Celik, 2014). This suggests reduced metabolism in a heat stress environment but may not necessarily be related to EST changes in each strain. Eggs incubated at high or low temperatures responded by increasing or reducing EST, respectively. However, EHP of Ross 708 embryos increased in response to increased incubation temperature up to 37.0°C, but decreased after E17 when incubated at 37.0°C. In Ross 308, EHP was not affected by incubation temperature. Since incubation temperature had a significant impact on both embryonic metabolism and eggshell temperature especially in Ross 708, it must be closely managed for this particular strain in order to optimize embryonic development and subsequent chick quality. In modern genetic strains, heat generated by embryos at higher incubation temperatures, coupled with heat supplied by the incubator, can raise internal incubator temperature to about 41.1 to 41.7°C during the last wk of incubation (Hulet, 2007). This can be directly transferred to the eggshell surface, increasing its temperature. The higher eggshell temperature may lead to impaired embryonic development and poor post-hatch chick quality, including larger residual yolk sac and increased possibility of yolk sac infection (Rai et al., 2005; Hulet, 2007; Molenaar et al., 2010). There was no specific impact of flock age on the main parameters that influenced embryonic development and chick quality in Ross 308; however, G and EHP at E16, E19, and E20 were higher for the mid Ross 708 flocks, indicating a higher exchange of gases and embryonic metabolism, respectively. Incubation of eggs at 37.5°C up to E17 in Ross 708 was appropriate, but beyond E17, it was deleterious to embryo development. Embryonic sensitivity of Ross 708 to the higher incubation temperature resulted in reduced EHP, and therefore metabolism, from E17 to E20, although EST still remained high. It appears the primary factor responsible for the higher EST was incubation temperature rather than EHP. However, the same situation was not observed in Ross 308, which showed less sensitivity to the higher incubation temperature possibly as a consequence of inherently lower embryo metabolic rate. However, Ross 308 still had similar EST as Ross 708 at 37.5 and 37.0°C at E20 in the study period. This re-emphasizes that incubation temperature at 37.5°C beyond 15 d of incubation was harmful to embryonic development in both strains. It is worth noting that the extreme response of the Ross 708 strain could have arisen from its development as a large bird for roaster production, while the Ross 308 is typically used as a smaller, multipurpose bird (Persia and Saylor, 2006), and therefore Ross 708 may have higher heat production than Ross 308. The molecular and metabolic reasons for birds with high genetic potential for growth having high metabolism remain unclear, but the activation of metabolic genes in the embryos during incubation could be a factor. The activation of genes associated with the Krebs cycle and the electron transport chain in the mitochondrial membrane could lead to the production of energy, which would increase O2 demand. As a result, there would also be an increased demand for glucose metabolites, leading to high embryonic metabolism (Humphrey and Rudrappa, 2008). Therefore, further investigation is needed to characterize changes in the genes associated with the metabolic pathways during each of the last 7 d of incubation. In the meantime, incubation temperature must be optimized to account for not only genetic differences and breeder flock age but also depending on the daily response of embryos to incubation temperature. Although EST was still high from E15 to E21, incubation at 37.0°C was optimum for Ross 708 embryos as indicated by the greatest chick length in this treatment, which is a sign of chick quality (Molenaar et al., 2008). Chick quality indicators were not affected by incubation temperature in Ross 308 embryos. Across strain, the hatching times at 36.0, 36.5, 37.0, and 37.5°C were approximately 21.0, 20.9, 20.7, and 20.9 d, respectively. Thus, if the standard commercial chick pull time of 21.5 d was used, chicks would remain in the hatcher for 12.0, 14.4, 19.2, and 14.4 h after hatching, respectively. Therefore, chicks could spend almost 20 h in the hatcher before actual pull time, and thus could be subjected to overheating. Although not part of this study, the apparent sensitivity of Ross 708 embryos to increased incubation temperatures could lead to high embryonic mortality. It is recommended that no more than 25% of chicks should be hatched 23 h before the pull, and more than 75% of the total hatch should be hatched within 13 h of the pull (Cobb-Vantress, 2013). Therefore, the optimum pull time needs to reflect incubation temperature, and subsequent effects on embryo metabolism. Chick length at hatch is positively related to chick quality, thus the optimum incubation from 15 to 21.5 d of incubation was 37.0°C. The normal practice for most commercial hatcheries is to pull chicks at 7 am on the d of hatch. Therefore, egg set time would have to be adjusted depending on the strain, incubation temperature, the brand and type of machine, and experience of the hatchery staff. Such adjustments could reduce late incubation embryo mortality if the expected chick hatch time is known and avoid keeping the chicks in the hatcher for an extended time, thus reducing the risk of dehydration, excessive weight loss, and weakened chicks. In conclusion, incubation of Ross 708 embryos appears to be more challenging than Ross 308 embryos because of the more pronounced effects of elevated incubation temperature on embryo metabolism. Incubation at 37.5°C, which is used by most commercial hatcheries, could result in embryonic overheating. The use of EST to monitor metabolism during incubation may assist in adjusting incubation temperature to suit the needs of the embryo. Acknowledgements The authors would like to acknowledge the financial contributions from the Alberta Livestock and Meat Agency, Alberta Innovates Bio-Solutions, the Canadian Poultry Research Council, the Poultry Industry Council, the Alberta Hatching Egg Producers, the Alberta Chicken Producers, Maple Leaf Foods (Wetaskiwin, AB, Canada), Sofina Foods Hatchery (Edmonton, AB, Canada), the Natural Sciences and Engineering Research Council, and the Poultry Research Center (University of Alberta, Edmonton, AB). We also want to thank the following individuals for their help: Chris Ouellette, John Feddes, and staff and students of the Poultry Research Center, University of Alberta, especially Misaki Cho, Kerry Nadeau, Mark Phoa, and Victoria Owusu, and Sander Lourens (Wageningen University and Livestock Research). REFERENCES Abudabos A. 2010 . The effect of broiler breeder strain and parent flock age on hatchability and fertile hatchability . Int. J. Poult. Sci . 9 : 231 – 235 . 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Physiological response of broiler embryos to different incubator temperature profiles and maternal flock age during incubation. 1. Embryonic metabolism and day-old chick quality

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

Abstract Broiler strain, maternal age, and incubation temperature influence embryo metabolism. Hatching eggs were obtained from young (Y; 28 to 34 wk, $$\bar{\rm x}$$ = 31.2 wk), mid (M; 36 to 45 wk, $$\bar{\rm x}$$ = 40.5 wk) and old (O; 49 to 54 wk, $$\bar{\rm x}$$ = 51.4 wk) Ross 708 (n = 88; Experiment 1) and Ross 308 [(n = 45; Experiment 2: (Y; 25 to 34 wk, $$\bar{\rm x}$$ = 30.5 wk), (M; 35 to 44 wk, $$\bar{\rm x}$$ = 40.2 wk), and (O; 49 to 54 wk, $$\bar{\rm x}$$ = 51.6 wk)] breeders. Eggs were stored for 2 to 4 d (18°C, 73% RH), and incubated for 14 d at 37.5°C and 56% RH. At 15 d (E15), 8 fertile eggs per flock age were incubated in individual metabolic chambers at 36.0, 36.5, 37.0, or 37.5°C until E21.5. Each temperature was repeated one additional time. O2 consumption and CO2 production were used to calculate embryonic heat production (EHP). Embryo temperature was measured as eggshell temperature (EST). Initial egg weight was used as a covariate; significance was assessed at P < 0.05. In Ross 708, daily EHP tended to be higher in M and O than Y treatments at E16; EHP of M was higher than Y and O eggs at E18; M and O were higher than O eggs at E19. Incubation at 37.0°C resulted in the highest EHP from E15 to E21, except at E17. Embryos at 37.5°C had reduced EHP beyond E17. Daily EST from E15 to E21 was higher at 37.5 and 37.0°C than at 36.0 and 36.5°C. In Ross 308, daily EST was highest at 37.5°C except at E20. Incubation temperature and EST were highly correlated (R2 = 0.90 to 0.89; P < 0.001). Ross 708 chicks were longer and hatched earlier at 37.0°C than at 36.0 and 37.5°C. EST and EHP increased with incubation temperature in Ross 708. In Ross 308, maternal flock age and incubation temperature did not impact EHP. However, EST was highest at 37.5°C except at E20. Ross 708 was more sensitive to incubation temperature than Ross 308. INTRODUCTION Genetic selection of modern broiler chicken strains for increased growth, feed efficiency, and breast meat yield has influenced embryonic development (Tona et al., 2004; Druyan, 2010; Nangsuay et al., 2015a). Modern broiler embryos are heavier than layer embryos as a result of faster growth from 11 to 19 d of incubation (Ohta et al., 2004). Genetic selection of egg-strain chickens for reproductive traits has resulted in more efficient lipid metabolism, whereas selection of meat-type chickens for growth and efficiency has resulted in increased metabolic rate (Buzala et al., 2015). The consequences of increased metabolic rate in broilers include stunted liver and heart tissues, which could result in metabolic inefficiencies in embryos (Lindgren and Altimiras, 2011). Ross broiler embryos have showed higher embryonic O2 consumption (Hamidu et al., 2007; Druyan, 2010), higher heart beats per minute, and increased metabolic hormone levels and growth rate compared to Cobb broiler embryos, demonstrating differences in developmental patterns and metabolic characteristics (Druyan, 2010). Daily embryonic heat production (EHP) is a measure of embryonic metabolism; Ross 308 embryos had higher daily EHP than Cobb 500 embryos during early (1 to 7 d; Hamidu et al., 2007) and late (15 to 21 d) incubation (Hamidu et al., 2007; Nangsuay et al., 2015a). To optimize incubation conditions, the effects of maternal flock age on embryo metabolism must be considered. Embryos from older breeder flocks have increased O2 consumption, metabolic heat production, high eggshell surface temperature, and a metabolic shift from glycolysis to gluconeogenesis to meet energy demands compared to embryos from younger flocks (Tona et al., 2001; Hamidu et al., 2007; De Oliveira et al., 2008; Leksrisompong et al., 2007, 2009; Lourens et al., 2007; Christensen et al., 2008; Druyan, 2010; Molenaar et al., 2010). Differences in embryo metabolism exist in lines from different primary breeders, and even within strains from a single company. Therefore, to optimize incubation conditions, we need to know how differences in metabolism affect incubation requirements, and how those requirements are affected by flock age (Hamidu et al., 2007). Although the effects of flock age and breeder strain on embryo metabolism have been extensively researched (Tona et al., 2004; O’Dea et al., 2004; Hamidu et al., 2007), the interaction of incubation temperature with these factors on the physiological growth of embryos and subsequent impacts on hatchling quality are not well understood. Additionally, the confounding effect of maternal flock age on egg weight cannot be discounted. However, in studies in which eggs were selected to be the same weight among various flock ages, increasing flock age also increased embryonic metabolism (O’Dea et al., 2004; Hamidu et al., 2007). These results appear to suggest that maternal flock age, rather than egg weight, is the main factor influencing embryonic metabolism. Incubation temperature influences embryogenesis and post-hatch chick development (Yildirim and Yetisir, 2004; Feast et al., 1998; Elibol and Brake, 2006; Piestun et al., 2009; Janisch et al., 2015). It is interesting to note that moderate incubation temperatures (37.2 or 38.3°C) from 17 to 21 d of incubation reduced late incubation embryo mortality compared to lower (36.1°C) and higher (39.9°C) incubation temperatures (Yildirim and Yetisir, 2004). Although the initial impact of sub-optimal incubation temperatures is first seen in changes in embryonic metabolism, the subsequent effects on other physiological activities of embryos are not well understood. Chick length and disappearance of residual yolk have been used as quality indicators for newly hatched chicks (Lourens et al., 2005, 2007; Molenaar et al., 2011). Eggshell temperature (EST) is a well-investigated technique to assess embryonic development and shown to influence chick quality (Lourens et al., 2005, 2006, 2007; Molenaar et al., 2011; Romanini et al., 2013), but its continuous measurement requires sophisticated techniques and equipment (Tong et al., 2016). Measurement of heat production as an indicator of embryonic development is well established (Meijerhof and Van Beek, 1993; Tona et al., 2004 and Lourens et al., 2005, 2006, 2007), and EST has been used as an indicator of EHP. However, there is no clearly established relationship between EST and EHP, although experimental results imply that EHP may be reflected by EST changes. (Lourens et al., 2007; Nangsuay et al., 2017). Therefore, it makes sense to determine a relationship and even complex equations that can explain the reason behind this frequent use of EST to reflect EHP. Therefore, the relationship between incubation temperature and EST must be determined for EST to be used as a tool to assess the appropriateness of late-stage incubation temperature, as influenced by broiler strain and breeder age. This will underscore the importance of managing incubation temperature on embryo development and chick quality. Sub-optimal incubation temperatures, especially during the plateau stage of O2 consumption from embryonic age 17 to 19 (E17 to E19) may increase late incubation embryo mortality and the proportion of weak chicks (Dietz et al., 1998; De Oliveira et al., 2008; Abudabos, 2010). This effect might influence embryos and chicks from diverse hen ages differently. Therefore, the interaction of breeder flock age and incubation temperature on embryonic metabolism must be examined more closely. Previously, at 37.0°C, there were differences in embryonic metabolism between genetic strains and flock ages (Hamidu et al., 2007). Thus, we hypothesized that within each genetic strain, increased embryonic metabolism with increasing breeder flock age would result in different optimal incubation temperatures from 15 to 21.5 d of incubation. The objectives of the current research were to evaluate the impact of incubation temperature and maternal flock age on embryonic metabolism, eggshell temperature, and early chick quality within each of Ross 708 and Ross 308 broilers. MATERIALS AND METHODS Experimental Design On 8 separate occasions, fresh broiler breeder eggs were obtained from a commercial hatchery from each of 3 different parent flock ages: young (Y; 28 to 34 wk, $$\bar{\rm x}$$ = 31.2 wk), mid (M; 36 to 45 wk, $$\bar{\rm x}$$ = 40.5 wk), and old (O; 49 to 54 wk of age, $$\bar{\rm x}$$ = 51.4 wk) Ross 708 (Experiment 1) hens. These eggs were typical of hatching eggs in commercial incubation (52 to 70 g). The eggs were stored for 2 to 4 d at 18°C and 74% RH. Eggs were assigned to one of 4 incubation temperature treatments (36.0, 36.5, 37.0 (control), or 37.5°C) from E15 to E21.5. The control temperature of 37.0°C was similar to that used in commercial hatcheries during the last 3 d of incubation (Elibol and Brake, 2006). For each set of eggs, a single incubation temperature was applied within the incubator; each temperature was replicated 2 times in a random order. For each replicate in Experiment 1, a total of 11 eggs per each of the 3 flock ages was obtained, weighed, and incubated for 14 d at 37.5°C and 56% RH in a Jamesway AVN incubator. Beginning at E15, fertile eggs (n = 8) from each of the 3 parent flock ages were individually transferred into one of 24 one-liter metabolic chambers placed inside the incubator as described previously (Hamidu et al., 2007). Each one-liter metabolic chamber had one inlet for fresh air intake and an outlet from which air samples were analyzed for O2 and CO2. During each replication, 7 additional eggs per flock age were placed in a desiccator, covered in desiccant, and the eggs weighed back at the same time every d for 9 consecutive days. Eggshell conductance (G), which is the ability of gases and moisture to diffuse across the eggshell (O’Dea et al., 2004; Hamidu et al., 2007) was calculated using the formula of Ar et al., (1974). The value for G (mg/d per mmHg) was calculated from the rate of water loss per d from each egg (mg/d) divided by the change in water vapor pressure of the egg content and water vapor pressure in the environment surrounding the egg (mm Hg). Since the egg was covered in desiccant, the water vapor pressure in the environment surrounding the outside of the egg was equal to zero. In Experiment 2, 15 eggs per flock age were obtained from Y (25 to 34 wk, $$\bar{\rm x}$$ = 30.5 wk), M (35 to 44 wk, $$\bar{\rm x}$$ = 40.2 wk), and O (49 to 54 wk of age, $$\bar{\rm x}$$ = 51.6 wk) from Ross 308 breeders on 8 separate occasions as described above. The eggs were collected, weighed, and incubated according to temperature treatments as in Experiment 1. Eggshell conductance was not investigated in Experiment 2, due to its having been investigated previously in Ross 308 embryos in our lab (Hamidu et al., 2007). Incubation conditions followed standard commercial hatchery procedures, with eggs placed in the dark, with hourly turning interval. Proper ventilation was maintained with fresh air by connecting a tube from the incubator damper to the hatchery roof and dampers adjusted when necessary to maintain RH at 56%. For simplicity, the flock ages for both Ross 708 and Ross 308 have been defined as young (Y; 26 to 34 wk), mid (M; 35 to 45 wk), and old (O; 46 to 55 wk). In addition, all experimental procedures were approved by the University of Alberta Animal Care and Use Committee in accordance with the Canadian Council on Animal Care (2009) guidelines. Hatch Analysis At 18 d of incubation, the incubator turning mechanism was stopped to simulate the conditions of a commercial hatcher. Beginning at 452 h of incubation, the eggs were checked at 6-hour intervals to establish the times each embryo was required to externally pip and hatch. All chicks remained in the metabolic chambers, and after 518 h of incubation, they were removed, weighed, and euthanized by decapitation. The carcasses were dissected; the residual yolk sacs (RYS) were removed from the chicks and weighed. The dry matter weight of the yolk sac was determined at an oven temperature of 65°C for 4 days. All sample weights were expressed as percentage of initial chick weight. Gas Exchange and Eggshell Temperature The metabolic chambers used have been previously validated (Hamidu et al., 2007, 2010, 2011). In each run, embryonic O2 consumption and CO2 production in each of 24 metabolic chambers were measured by computer-software controlled O2 and CO2 analyzers. Each chamber was sampled sequentially for 5 min, 6 times per d (returning to the same chamber at 2-hour intervals). The values within each d were averaged for determination of average daily O2 consumption and CO2 production, which were used in the calculation of daily EHP, a measure of embryonic metabolism. The ambient air inside the incubator was sampled to determine O2 and CO2 concentration immediately prior to each of the metabolic chambers being sampled. The difference between the gas partial pressures of the metabolic chamber and that of the ambient air was used to calculate embryonic O2 and CO2 exchange rates (Hamidu et al., 2007, 2010). The measurement of ambient O2 and CO2 exchange rates using the inside of incubator or an empty metabolic chamber has been tested and published previously (Hamidu et al., 2010) and demonstrated that each metabolic chamber had a similar microclimate as the inside of the incubator (Hamidu et al., 2010). Therefore, it was not necessary to use additional metabolic chambers as a control. The EHP was calculated following the formula of Kleiber (1987): Heat production (mW) = [3.871 × O2 consumption (L/d) + 1.194 × CO2 production (L/d)] × 1 d/24 h × 1 h/3600 s × 1000 cal/1 kcal × 4.187 J/cal × 1000 mW/W. A custom-made temperature probe, held in place by foam attached to the egg tray and held in direct contact with the eggshell inside each metabolic chamber was used to monitor EST. The EST can be used to determine whether the embryos are being incubated at an optimum incubation temperature (Ar and Tazawa, 1999; Lourens et al., 2006; Hamidu et al., 2011). An additional temperature probe was hung in the inside of the incubator to monitor ambient temperature. Data were recorded 6 times per d for each chamber, but repeated measurements were rearranged into replications per d per chamber. Statistical Analysis Data from Experiment 1 (Ross 708) and Experiment 2 (Ross 308) were analyzed separately. All data were subjected to analysis of variance using the Proc. Mixed procedure of SAS (Wang and Goonewardene, 2004; SAS Institute, 2010) at P ≤ 0.05 (SAS Institute, 2010). The statistical model used to analyze all metabolic responses included the fixed effects of hen age (3 levels) and incubation temperature (4 levels), the interaction between the 2 main effects, and the covariate effect of initial egg weight. The original source of eggs (farm) was nested in the interaction of flock age and incubation temperature and used as the random variable for analysis. For G, hen age was the only fixed factor at 3 levels, while farm was used as the random factor. In the analysis, initial egg weight was used as a covariate (Jacobs, 2011), since it was confounded by maternal flock age (Hamidu et al., 2011). The statistical model used was: Yijkl = μ + αi + βj + αiβj + ϖ + λk(αiβj) + εijkl, where: Yijkl = effect measured, μ = overall mean, αi = main effect of maternal flock age, βj = main effect of incubation temperature, αiβj = interaction effect of maternal flock age and incubation temperature, ϖ = covariate effect of initial egg weight, λk = random effect due to farm or source of eggs, λk(αiβj) = random effect of maternal flock age and incubation temperature nested in farm, and εijkl = residual error term. Also, a regression analysis was performed to establish an appropriately fit relationship between incubation temperature and EST, as well as between EHP and EST. All differences between least square means were considered to be significant when P ≤ 0.05 (SAS Institute, 2010). RESULTS Maternal Flock Age Effect Experiment 1. Ross 708. Initial egg weight prior to incubation increased with increasing flock age (Table 1). Similarly, G was higher in eggs of M and O flocks compared to eggs of Y flocks (Table 1). Chick weight was not different among flock ages; however, chick length was different. The chicks of Y flock age were longer than chicks from M and O flock ages. The external pipping time of embryos prior to hatching was higher in M flock age than Y flock age. However, neither was different from O flock age; the hatching time among flock ages was not different (Table 1). The percentages of wet and dry RYS as well as yolk-free body mass (YFBM) were not different among maternal flock ages (P > 0.05; Table 2). Table 1. Initial egg weight and chick characteristics from eggs of different flock ages and incubated at different incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Eggshell conductance = daily egg moisture divided by saturated vapor pressure. 5Chick length = from tip of longest toe to the beak. 6Pipping time = from time of incubation until embryo breaking through eggshell. 7Hatching time = from time of incubation until chick leaving the eggs completely. 8Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg or chick. 9Standard error of means (n = 16 eggs per flock age). View Large Table 1. Initial egg weight and chick characteristics from eggs of different flock ages and incubated at different incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 Initial egg weight Conductance4 Chick weight Chick length5 External pipping time6 Hatching time7 (g) (mg/d/mm Hg) (g) (cm) (h) Flock age Ross 708 Ross 308 Ross708 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk )1 56.53c 56.21c 9.88b 40.45 41.91 18.39a 18.16 483.8b 488.2 500.7 504.2 (64)8 (64) (56) (48) (50) (48) (50) (53) (55) (48) (50) Mid (35 to 45 wk)2 60.92b 63.61b 11.81a 41.25 41.97 17.92b 18.30 490.3a 487.0 501.4 500.8 (64) (64) (56) (51) (41) (34) (41) (61) (58) (52) (41) Old (46 to 55 wk)3 64.68a 66.93a 11.20a 40.95 42.42 17.56b 18.12 488.4a,b 485.9 501.5 499.3 (64) (64) (56) (36) (28) (28) (28) (44) (48) (36) (28) SEM9 0.62 0.62 0.44 0.53 0.46 0.13 0.16 1.8 3.16 1.58 1.84 Incubation temperature 36.0°C 61.90 65.04 – 41.73 43.24 18.03a,b 18.09 491.6a 492.19 506.5a 506.1 (48) (48) – (32) (29) (32) (29) (43) (44) (32) (29) 36.5°C 60.06 61.11 – 40.59 41.81 18.06a,b 18.21 488.4a 492.12 501.2a,b 501.4 (48) (48) – (34) (26) (34) (26) (38) (40) (34) (26) 37.0°C 59.25 61.43 – 40.64 41.51 18.46a 18.52 481.5b 482.16 496.0b 497.3 (48) (48) – (35) (30) (35) (30) (38) (40) (35) (30) 37.5°C 60.71 61.16 – 40.58 41.84 17.72b 17.95 488.5a 481.54 502.4a 500.9 (48) (48) – (34) (34) (34) (34) (39) (37) (34) (34) SEM 0.70 1.14 – 0.55 0.48 0.15 0.18 1.70 3.57 1.78 2.12 Source of variation P-values Flock age <0.001 0.001 0.005 0.520 0.746 0.039 0.699 0.033 0.878 0.885 0.303 Incubation temperature 0.099 0.105 – 0.365 0.159 0.001 0.177 0.006 0.101 0.012 0.072 Flock age*Incubation Temperature 0.948 0.472 – 0.825 0.396 0.033 0.218 0.649 0.948 0.911 0.607 Covariate egg weight – – 0.365 <0.001 <0.001 0.001 0.008 0.008 0.329 0.630 0.208 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Eggshell conductance = daily egg moisture divided by saturated vapor pressure. 5Chick length = from tip of longest toe to the beak. 6Pipping time = from time of incubation until embryo breaking through eggshell. 7Hatching time = from time of incubation until chick leaving the eggs completely. 8Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg or chick. 9Standard error of means (n = 16 eggs per flock age). View Large Table 2. Chick carcass characteristics of day-old chicks from different flock ages and incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4YFBM: Yolk-free body mass. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Table 2. Chick carcass characteristics of day-old chicks from different flock ages and incubation temperatures for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 Wet YFBM4 (%) Wet yolk sac (%) Dry yolk sac (%) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 87.23 (48)5 88.52 (50) 12.77 (48) 11.48 (50) 6.72 (48) 5.71 (50) Mid (35 to 45 wk)2 86.90 (52) 87.43 (41) 13.10 (52) 12.57 (41) 7.19 (52) 6.61 (41) Old (46 to 55 wk)3 87.05 (36) 85.54 (28) 12.95 (36) 14.46 (28) 7.30 (36) 7.99 (28) SEM6 0.77 0.94 0.77 0.94 0.47 0.62 Incubation temperature 36.0oC 86.77 (32) 86.32 (29) 13.23 (32) 13.68 (29) 7.31 (32) 7.37 (29) 36.5oC 86.69 (34) 87.38 (26) 13.31 (34) 12.62 (26) 7.14 (34) 6.68 (26) 37.0oC 88.76 (35) 88.37 (30) 11.24 (35) 11.63 (30) 6.09 (35) 6.05 (30) 37.5oC 86.03 (34) 86.57 (34) 13.97 (34) 13.43 (34) 7.74 (34) 6.98 (34) SEM 0.71 1.02 0.71 1.02 0.43 0.68 Source of variation P-values Flock age 0.938 0.180 0.938 0.180 0.752 0.106 Incubation temperature 0.079 0.485 0.079 0.485 0.098 0.563 Flock age * Incubation temperature 0.889 0.815 0.889 0.815 0.932 0.898 Covariate egg weight 0.826 0.329 0.826 0.329 1.0 0.410 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4YFBM: Yolk-free body mass. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large The daily embryonic O2 consumption was higher in M embryos compared to O embryos at E19 but not Y embryos, which was not different from M and O embryos. At E20, M and Y embryos showed higher O2 consumption compared to O embryos; there were no differences at the other embryonic ages (Figure 1a). The daily embryonic CO2 production was higher in Y and M embryos compared to O embryos at E20 (Figure 2a). Average over the 7 d investigated showed that the M embryos had higher O2 consumption from E15 to E21 compared to Y and O embryos (P = 0.011), even without a significant covariate effect of initial egg weight. The average embryonic CO2 production between E15 to E21 was not different. The daily EHP profiles among flock ages followed a similar pattern as the O2 consumption (Figure 3a). The Y and M embryos had greater EHP than O embryos at E15, E16, and E19. The M embryos had higher EHP than O embryos at E19 but neither was different from Y embryos; however, at E20, M and Y embryos had higher EHP than O embryos (Figure 3a). On average, EHP from E15 to E21 was higher in M embryos than Y and O embryos (P = 0.014; Table 3). The average (Table 3) and daily EST (data not shown) were not different between maternal flock ages (P > 0.05). Figure 1. View largeDownload slide Effect of maternal flock age on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic O2 consumption was significantly higher in embryos from M flock age compared to O flock age at E19 (*P ≤ 0.05); at E20, M and Y flock ages had higher consumption than O flock age in Ross 708 (***P ≤ 0.001). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 1. View largeDownload slide Effect of maternal flock age on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic O2 consumption was significantly higher in embryos from M flock age compared to O flock age at E19 (*P ≤ 0.05); at E20, M and Y flock ages had higher consumption than O flock age in Ross 708 (***P ≤ 0.001). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 2. View largeDownload slide Effect of maternal flock age on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production of Y and M embryos was higher than O embryos at E20 in Ross 708 (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 2. View largeDownload slide Effect of maternal flock age on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production of Y and M embryos was higher than O embryos at E20 in Ross 708 (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 3. View largeDownload slide Effect of maternal flock age on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production of M and Y flocks was higher than O embryos at E16 (†P ≤ 0.10). Also, M embryos had higher heat production than O embryos at E19, but neither was different from Y embryos (*P ≤ 0.05); however, at E20, M and Y embryos had higher heat production than O embryos (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Figure 3. View largeDownload slide Effect of maternal flock age on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production of M and Y flocks was higher than O embryos at E16 (†P ≤ 0.10). Also, M embryos had higher heat production than O embryos at E19, but neither was different from Y embryos (*P ≤ 0.05); however, at E20, M and Y embryos had higher heat production than O embryos (**P ≤ 0.01). No differences were observed in Ross 308. 1Average young flock age for Ross 708 breeders = 31.2; 2average mid flock age for Ross 708 breeders = 40.5; 3average old flock age for Ross 708 breeders = 51.4; 4average young flock age for Ross 308 breeders = 30.5; 5average mid flock age for Ross 308 breeders = 40.2; and 6average old flock age for Ross 308 breeders = 51.6. Table 3. Average gas exchange and embryonic heat output from eggs of different flock ages and incubation temperatures from 15 to 21 d of incubation for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Average heat production (mW) = average heat production from E15 to E21. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Table 3. Average gas exchange and embryonic heat output from eggs of different flock ages and incubation temperatures from 15 to 21 d of incubation for Ross 708 (Experiment 1) and Ross 308 (Experiment 2). O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 O2 consumption CO2 production Heat production4 Eggshell temperature (mL/d) (mW) (°C) Flock age Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Ross 708 Ross 308 Young (26 to 34 wk)1 631.39b (64)5 625.54 (64) 448.96 (64) 418.26 (64) 144.10b (64) 141.55 (64) 38.37 (64) 38.05 (64) Mid (35 to 45 wk)2 676.54a (64) 635.28 (64) 464.59 (64) 424.33 (64) 154.46a (64) 143.72 (64) 38.43 (64) 38.18 (64) Old (46 to 55 wk)3 638.09b (64) 575.34 (64) 433.58 (64) 382.27 (64) 144.74b (64) 130.05 (64) 38.41 (64) 38.19 (64) SEM6 11.96 20.02 9.54 16.55 2.78 4.58 0.09 0.06 Incubation temperature 36.0°C 630.35b,c (48) 599.06 (48) 435.68(48) 393.34 (48) 144.09b,c (48) 134.99 (48) 37.56c (48) 37.45d (48) 36.5°C 599.71c (48) 592.36 (48) 450.93 (48) 393.04 (48) 138.27c (48) 134.12 (48) 38.19b (48) 37.83c (48) 37.0°C 699.61a (48) 636.95 (48) 469.31 (48) 428.28 (48) 158.20a (48) 144.33 (48) 39.01a (48) 38.4b (48) 37.5°C 665.01a,b (48) 619.84 (48) 440.25 (48) 413.82 (48) 151.84a,b (48) 140.32 (48) 38.87a (48) 38.84a (48) SEM 12.26 22.95 10.28 14.41 2.91 5.26 0.10 0.06 Source of variation P-value Flock age 0.011 0.119 0.083 0.125 0.014 0.119 0.831 0.277 Incubation temperature 0.001 0.477 0.194 0.359 0.001 0.458 <0.001 <0.001 Flock age*Incubation temperature 0.299 0.687 0.674 0.790 0.419 0.705 0.337 0.703 Covariate egg weight 0.130 <0.001 0.001 <0.001 0.061 <0.001 0.832 0.001 a–cDifferent superscripts within the same column indicate significant differences between means (P ≤ 0.05). 1Average flock age for Ross 708 breeders = 31.2; average flock age for Ross 308 breeders = 30.5. 2Average flock age for Ross 708 breeders = 40.5; average flock age for Ross 308 breeders = 40.2. 3Average flock age for Ross 708 breeders = 51.4; average flock age for Ross 308 breeders = 51.6. 4Average heat production (mW) = average heat production from E15 to E21. 5Numbers in parentheses indicate the number of experimental units; the experimental unit was each egg. 6SEM = Standard error of means (n = 16 eggs per flock age). View Large Experiment 2. Ross 308. Initial egg weight (P < 0.001) increased as flock age increased (Table 1). There was no effect of flock age on chick weight, chick length, external pipping time, or hatching time (Table 1) as well as percent YFBM, wet RYS, or dry RYS. There were no differences in daily O2 consumption (Figure 1b) nor daily CO2 production (Figure 2b) and EHP (Figure 3b) among maternal flock ages from E15 to E21. Similarly, the average O2 consumption and CO2 production were not affected by flock age (Table 3). There were no differences in daily EHP (Figure 3a) or EST (data not shown) among maternal flock ages. Similarly, average EHP and EST from E15 to E21 were not affected by maternal flock age (Table 3). Incubation Temperature Effects Experiment 1. Ross 708. Initial egg weight was not different among eggs assigned to the various incubation temperature groups. Chick weight was not affected by incubation temperature (Table 1). The eggs incubated at 37.0°C from E15 to E21 hatched longer chicks compared to eggs incubated at 37.5°C. The 37.0°C treatment also resulted in shorter external pipping and hatching times compared to all other incubation temperature treatments investigated. The chicks incubated at 36.0°C had the longest hatch time followed by 37.5°C and then 36.5°C groups. However, all chicks, irrespective of incubation temperature treatment, hatched in less than 21.5 d (36.0°C = 21.10 d; 36.5°C = 20.9 d; 37.0°C = 20.7 d, and 37.5°C = 20.9 d). The percentage of wet and dry RYS weights tended to be lower at 37.0°C (11.24 and 6.09%, respectively) than 36.0°C (13.23 and 7.31%, respectively), 36.5°C (13.31 and 7.14%, respectively), and 37.5°C (13.97; 7.74%, respectively; P = 0.079; P = 0.098 for wet and dry RYS, respectively; Table 2). In both cases, the proportion of RYS was highest at 37.5°C. Embryos from 36.5 and 37.0°C tended to have higher O2 consumption compared to 36.0°C at E15 (P < 0.10), but embryonic response at all 3 incubation temperatures was not different from 37.5°C incubation temperature. Embryos incubated at 37.0 or 37.5°C consumed more O2 from E16 to E17 as compared to those incubated at 36.0 or 36.5°C (Figure 4a). The embryos at 37.5°C consumed about 102 mL/d more O2 than those at 37.0°C at 17 d of incubation. After E17, daily O2 consumption by embryos incubated at 37.5°C dropped sharply below O2 consumption levels of embryos at 37.0 and 36.0°C from E18 to E20. At E19 and E20, embryos from the 37.0°C treatment consumed higher amounts of O2 compared to embryos in all other incubation temperature treatments; the O2 consumption was not different at E21. At 37.0°C, the average O2 consumption from E15 to E21 was higher compared to 36.5 and 36.0°C (Table 3). The O2 consumption was not different between 37.0 and 37.5°C incubation temperatures. Figure 4. View largeDownload slide Effect of incubation temperature treatments on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic O2 consumption was higher at 36.5 and 37.0°C compared to 36.0°C on E15 (†P ≤ 0.10); O2 consumption was higher at 36.0, 37.0, and 37.5°C incubation temperatures compared to 36.0°C incubation temperature at E16 (*P ≤ 0.05). At E17, embryonic O2 consumption was higher at 37.5°C compared to 36.0, 36.5, and 37.0°C incubation temperatures (**P ≤ 0.01); at E19 and E20, embryonic O2 consumption was higher at 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C incubation temperatures (***P ≤ 0.001). There was no difference at E21. Also, in Ross 308, no difference existed in O2 consumption between incubation temperatures on all d of incubation. Figure 4. View largeDownload slide Effect of incubation temperature treatments on embryonic O2 consumption during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic O2 consumption was higher at 36.5 and 37.0°C compared to 36.0°C on E15 (†P ≤ 0.10); O2 consumption was higher at 36.0, 37.0, and 37.5°C incubation temperatures compared to 36.0°C incubation temperature at E16 (*P ≤ 0.05). At E17, embryonic O2 consumption was higher at 37.5°C compared to 36.0, 36.5, and 37.0°C incubation temperatures (**P ≤ 0.01); at E19 and E20, embryonic O2 consumption was higher at 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C incubation temperatures (***P ≤ 0.001). There was no difference at E21. Also, in Ross 308, no difference existed in O2 consumption between incubation temperatures on all d of incubation. On E15, the embryos incubated at 36.5°C had higher CO2 production than those of 36.0°C. The embryos incubated at 37.0°C had higher CO2 production compared to those incubated at 36.5°C and than 36.0 or 37.5°C at E20 (37.0 > 36.5 > 36.0 and 37.5°C). But there were no differences at any other time point (Figure 5a). Average CO2 production from E15 to E21 was not affected by incubation temperature (Table 3). Similar to O2 consumption, the EHP of eggs incubated at 37.0°C invariably was higher than embryos at 36.0 and 36.5°C. At 37.5°C, EHP was greatest at E17, but dropped thereafter, reaching a low at E20, before rising after external pipping (Figure 6a). At 37.0 and 36.0°C, EHP rose steadily to about 155 mL/d from E15 to E17, reaching a plateau or decreasing from E17 to E20, and then rising sharply again until E21. At 36.5°C, EHP rose slightly from E15 to E16, decreased from E18 to E19, and then increased to E21. Apart from E17 when embryos incubated at 37.5°C had higher EHP, the embryos at 37.0°C consistently produced more heat from E15 to E21 compared to those of 36.0°C incubation temperature. The average EHP over the course of the study (from E15 to E21) was higher at 37.0°C compared to 36.5 and 36.0°C. (P = 0.001; Table 3). The average EST from E15 to E21 (Table 3) and the daily EST within that period (Figure 7a) were higher for embryos incubated at 37.0 and 37.5°C compared to those incubated at 36.5 and 36.0°C, except at E20 when EST of 37.5°C was higher than all other incubation temperatures (Figure 7a). The 36.5°C embryos had higher EST than 36.0°C embryos during each of the d considered. The EST and incubation temperature in Ross 708 embryos were strongly and positively related (y = −1.541x3 + 169.07x2 + 6,181.3x + 75,345; R² = 0.90; P < 0.001, Figure 7a; where y is eggshell temperature and x is incubation temperature). The EHP and EST in the same strain showed a very weak, though significant, relationship (y = 15.874x – 460.15; R² = 0.11, P < 0.001, where y = EHP and x = EST). Figure 5. View largeDownload slide Effect of incubation temperature treatments on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production at E15 was higher at 36.5°C compared to 36.0°C (†P ≤ 0.10); at E20, CO2 production was higher at 37.0°C incubation temperatures compared to 36.0, 36.5, and 37.5°C incubation temperatures in Ross 708 (***P ≤ 0.001). At E21, embryonic CO2 production was also significantly higher at 37.0°C than 36.0 and 37.5°C on E21 (*P ≤ 0.05). No differences were observed in Ross 308. Figure 5. View largeDownload slide Effect of incubation temperature treatments on embryonic CO2 production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. Embryonic CO2 production at E15 was higher at 36.5°C compared to 36.0°C (†P ≤ 0.10); at E20, CO2 production was higher at 37.0°C incubation temperatures compared to 36.0, 36.5, and 37.5°C incubation temperatures in Ross 708 (***P ≤ 0.001). At E21, embryonic CO2 production was also significantly higher at 37.0°C than 36.0 and 37.5°C on E21 (*P ≤ 0.05). No differences were observed in Ross 308. Figure 6. View largeDownload slide Effect of incubation temperature treatments on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production (EHP) was higher in 37.0 and 37.5°C incubation temperatures compared to 36.0°C at E15 (†P ≤ 0.10) and lower at 36°C compared to 36.5, 37.0, and 37.5°C at E16 (**P ≤ 0.01); at E17, EHP was higher at 37.5°C incubation temperature compared to 36.0 and 36.5°C but not different from 37.0°C (**P ≤ 0.01). For E19 and E20, it was higher in 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C (***P ≤ 0.001) and E21 (*P ≤ 0.05). The EHP was not different at E18 (P > 0.05). No significant differences were observed in Ross 308 (P > 0.10). Figure 6. View largeDownload slide Effect of incubation temperature treatments on embryonic heat production during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, embryonic heat production (EHP) was higher in 37.0 and 37.5°C incubation temperatures compared to 36.0°C at E15 (†P ≤ 0.10) and lower at 36°C compared to 36.5, 37.0, and 37.5°C at E16 (**P ≤ 0.01); at E17, EHP was higher at 37.5°C incubation temperature compared to 36.0 and 36.5°C but not different from 37.0°C (**P ≤ 0.01). For E19 and E20, it was higher in 37.0°C incubation temperature compared to 36.0, 36.5, and 37.5°C (***P ≤ 0.001) and E21 (*P ≤ 0.05). The EHP was not different at E18 (P > 0.05). No significant differences were observed in Ross 308 (P > 0.10). Figure 7. View largeDownload slide Effect of incubation temperature treatments on eggshell temperature during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, EST at 37.5 and 37.0°C incubation temperatures was higher than 36.0 and 36.5°C incubation temperatures on all d except on E20, when EST at 37.0°C was higher than the other incubation temperatures (***P ≤ 0.001). Also, in Ross 308, EST at 37.5°C incubation temperature was higher than 36.0, 36.5, and 37.0°C incubation temperatures from E15 to E21, except at E20, when EST was not different between 37.0 and 37.5°C, but was higher in both temperatures compared to 36.0 and 36.5°C (***P ≤ 0.001). In both Ross 708 and Ross 308, the relationship between incubation temperature (X) and eggshell temperature (Y) was very high: R2 = 0.90 and R2 = 0.89, respectively. Figure 7. View largeDownload slide Effect of incubation temperature treatments on eggshell temperature during incubation d 15 to 21: (a) Ross 708, (b) Ross 308. In Ross 708, EST at 37.5 and 37.0°C incubation temperatures was higher than 36.0 and 36.5°C incubation temperatures on all d except on E20, when EST at 37.0°C was higher than the other incubation temperatures (***P ≤ 0.001). Also, in Ross 308, EST at 37.5°C incubation temperature was higher than 36.0, 36.5, and 37.0°C incubation temperatures from E15 to E21, except at E20, when EST was not different between 37.0 and 37.5°C, but was higher in both temperatures compared to 36.0 and 36.5°C (***P ≤ 0.001). In both Ross 708 and Ross 308, the relationship between incubation temperature (X) and eggshell temperature (Y) was very high: R2 = 0.90 and R2 = 0.89, respectively. Experiment 2. Ross 308. Initial egg weight was not different among incubation temperature treatments. Chick weight, chick length, external pipping time, and hatching time (Table 1), and percentage of YFBM and wet and dry RYS were not different among the treatments (Table 2). There was no difference in daily O2 consumption (Figure 4b), daily CO2 production (Figure 5b), or EHP (Figure 6b) among incubation temperature treatments from E15 to E21. The average O2 consumption, CO2 production, and EHP were not affected by incubation temperature (Table 3). From E15 to E21, daily EST at 37.5°C was higher than for each of the other incubation temperature treatments except at E20, when it was not different from the 37.0°C incubation temperature (Figure 7b). The daily EST at 37.0°C incubation temperature was also higher than the daily EST at 36.5 and 36.0°C, whereas 36.5°C showed higher daily EST compared to 36.0°C incubation temperature treatment, except at E20 when there was not a difference in EST. The effect of incubation temperature on EST showed a very strong polynomial relationship (y = −0.8369x3 + 92.397x2 − 3,399x + 41,701; R² = 0.89; P < 0.001, Figure 7b; where y is eggshell temperature and x is incubation temperature). The linear relationship between EST and EHP was relatively weak, although significant (y = 19.892x – 615.95; R² = 0.22, P < 0.001, where y = EHP and x = EST). Interaction of Maternal Flock Age and Incubation Temperature The interaction between maternal flock age and incubation temperature treatment did not affect any of the daily or average parameters measured in either genetic strain (P > 0.05). The covariate effect of egg weight or size greatly affected the data. In most cases, there were significant daily and average (over 7 d) effects of egg weight used as covariates for analyzing maternal flock age and incubation temperature on our measured parameters. DISCUSSION The influences of broiler breeder maternal flock age and incubation temperature on embryonic development and chick quality are of economic importance in incubation and may depend on egg weight, which increases with increasing flock age. This is because as flock age increases, hens tend to deposit more resources such as yolk into their eggs (Applegate et al., 1998; Ulmer-Franco et al., 2012), thus increasing the egg size. Generally, mid maternal flock age (34 to 45 wk) corresponds with peak egg production, as well as higher maternal and embryonic performance (Tona et al., 2001; Hamidu et al., 2007; Yassin et al., 2008). Eggs produced by young Ross 708 breeders tended to have lower G, O2, and CO2 exchange rates, embryonic metabolism, and heat production. These results also have been reported in previous studies using Ross 308 and Cobb 500 strains (Ar et al., 1974; O’Dea et al., 2004; Hamidu et al., 2007). It appears that for Ross 708 hens, maternal flock age rather than egg weight is the most important factor responsible for differences in EHP and therefore embryo metabolism (Nangsuay et al., 2013, 2015b). This is clearly seen in the non-significant effect observed in the covariate egg weight on G, O2 consumption, EHP, and EST. Nevertheless, the influence of egg size appears greater in Ross 308, as seen in the covariate egg weight and probabilities with no differences in any parameter due to maternal flock age and incubation temperature. Transfer of Ross 708 eggs to the 37°C incubation temperature at 15 d increased embryonic metabolism, and also increased chick length compared to other temperatures. An increase in embryonic metabolism may indicate higher embryonic development resulting in longer chicks, which is considered the most suitable estimator of initial chick quality (Molenaar et al., 2008). A consistent increase in EHP over time in Ross 708 embryos did not compromise embryonic development, but rather resulted in longer chicks at 37.0°C, even though the EST was higher than the recommended value of 37.8°C (Lourens et al., 2006). Higher EST occurs when embryos have access to O2 for respiration and metabolism following internal and external pipping, and possibly increased oxygen demand for utilization of yolk lipids (Menna and Mortola, 2002). Consequently, a high incidence of poor chick navel conditions is indicative of exposure to high incubation temperature as measured by the rectal temperature during brooding. The practice can be used to establish proper brooding practices to reduce chick mortality and increase survival post hatch, especially during the first wk (Chen et al., 2013). One of the main findings was the earlier hatch time for Ross 708 embryos incubated at 37°C than those incubated at 36.0 and 37.5°C. This was expected because of the reduced metabolism, which could lead to slower embryonic development. In each of the 4 temperature treatments, Ross 708 embryos incubated at 37°C had the most consistent and higher embryonic metabolism and also higher EST, which could have accelerated the hatching process. Similarly, in the same strain, the rapid increase in EST in the 37.5°C embryos to E17, and subsequent dramatic decrease at E20 may indicate a reduced metabolic rate in an attempt to avoid embryonic heat stress. A prominent sign of embryonic heat stress is a poorly absorbed yolk sac, leading to an increased incidence of unhealed navels in day-old chicks (Preez, 2007). These include a leaky navel, navel strings, navel buttons, and unclosed navels (Preez, 2007; Fasenko and O’Dea, 2008). The sudden drop in Ross 708 embryonic metabolism at E17 at the 37.5°C incubation temperature was also associated with a plateau in the EST (to ∼ 39°C) and may have been an adaptive response to overheating (Yalcin et al., 2008). In this study, we did not assign navel scores for the chicks; however, the greater RYS weights observed in the embryos incubated at 37.5°C could be evidence of heat stress (Shubber et al., 2012; Ozaydın and Celik, 2014). Consequences of high incubation temperature include decreased yolk consumption and mean embryonic weights due to lack of nutrients available for development (Ozaydın and Celik. 2014). Other studies showed increased glucose oxidation in embryos incubated at higher (38.9°C) compared to normal (37.8°C) EST from E17.6 until E17.8 (Molenaar et al., 2013). Although this may be a sign of elevated metabolism, the authors suggested that high incubation temperature during the perinatal period of chicken embryos increases glucose oxidation and decreases hepatic glycogen reserves prior to the hatching process. This condition can reduce hepatic and cardiac glucose availability to support successful development of the embryo and the hatching process, which relies on a lot of glucose immediately prior to external pipping (O’Dea et al., 2004). In Ross 708, it is possible that the embryos incubated at 37.5°C were heat stressed, as is common during the plateau stage of O2 consumption (E16 to E19), and therefore had to decrease metabolic rate or reduce yolk consumption (Willemsen et al., 2010). The consequences could be increased late embryo mortality, dead in shell embryos, and lower chick weight at hatch (Willemsen et al., 2010). A most interesting pattern observed in this study was the very weak relationship that existed between EST and EHP and a strong polynomial relationship between incubation temperature and EST in both Ross 708 and Ross 308 embryos. This contradicts previous observations of a strong linear relationship between EHP and EST (Lourens et al., 2006). This is because heat production of the embryo is greatly influenced by heat loss of the egg. The latter is particularly determined by the ventilation rate, the egg density in the incubator, RH, and the egg size, and even sometimes, the size of eggshell pores and their number (French 1997; Harb et al., 2010; Molenaar et al., 2010; Noiva et al., 2014). In the future, the above factors need to be controlled because they may influence the relationship between EHP and EST. There is a long-standing perception that broiler breeder genetic selection over many years has led to increased embryo metabolism (O’Dea et al., 2004; Tona et al., 2004, Hamidu et al., 2007). The results of the current study appear to agree with the perception, considering that the Ross 708 is an upgrade over the Ross 308. However, Ross 708 was much more responsive to incubation temperature changes than Ross 308. Increases in EHP, an indicator of embryonic metabolism (Hamidu et al., 2007; Lourens et al., 2007), were previously suggested to be related to increases in EST (Lourens et al., 2007; Tong et al., 2013). Therefore, some researchers use EST as a predictor of the rate of heat production, and as an indication of overheating in incubating embryos (Lourens et al., 2005, 2007; Molenaar et al., 2010). While there may be some basis for this in some broiler breeder strains, e.g., Hybro G+ grandparent flocks (Lourens et al., 2007), our research showed very little relationship between EHP and EST in both Ross 308 and Ross 708 embryos. Therefore, EST may not be the best predictor of EHP or may be influenced by other factors such as air speed, incubation temperature, and gas exchange rate. However, more recently, it has been demonstrated in both Hybro and Ross 308 strains that a heat stress incubation temperature of 38.8°C from E10 compared to control temperature of 37.8°C throughout incubation significantly reduced relative yolk free embryo weight as well as decreased yolk consumption (Ozaydın and Celik, 2014). This suggests reduced metabolism in a heat stress environment but may not necessarily be related to EST changes in each strain. Eggs incubated at high or low temperatures responded by increasing or reducing EST, respectively. However, EHP of Ross 708 embryos increased in response to increased incubation temperature up to 37.0°C, but decreased after E17 when incubated at 37.0°C. In Ross 308, EHP was not affected by incubation temperature. Since incubation temperature had a significant impact on both embryonic metabolism and eggshell temperature especially in Ross 708, it must be closely managed for this particular strain in order to optimize embryonic development and subsequent chick quality. In modern genetic strains, heat generated by embryos at higher incubation temperatures, coupled with heat supplied by the incubator, can raise internal incubator temperature to about 41.1 to 41.7°C during the last wk of incubation (Hulet, 2007). This can be directly transferred to the eggshell surface, increasing its temperature. The higher eggshell temperature may lead to impaired embryonic development and poor post-hatch chick quality, including larger residual yolk sac and increased possibility of yolk sac infection (Rai et al., 2005; Hulet, 2007; Molenaar et al., 2010). There was no specific impact of flock age on the main parameters that influenced embryonic development and chick quality in Ross 308; however, G and EHP at E16, E19, and E20 were higher for the mid Ross 708 flocks, indicating a higher exchange of gases and embryonic metabolism, respectively. Incubation of eggs at 37.5°C up to E17 in Ross 708 was appropriate, but beyond E17, it was deleterious to embryo development. Embryonic sensitivity of Ross 708 to the higher incubation temperature resulted in reduced EHP, and therefore metabolism, from E17 to E20, although EST still remained high. It appears the primary factor responsible for the higher EST was incubation temperature rather than EHP. However, the same situation was not observed in Ross 308, which showed less sensitivity to the higher incubation temperature possibly as a consequence of inherently lower embryo metabolic rate. However, Ross 308 still had similar EST as Ross 708 at 37.5 and 37.0°C at E20 in the study period. This re-emphasizes that incubation temperature at 37.5°C beyond 15 d of incubation was harmful to embryonic development in both strains. It is worth noting that the extreme response of the Ross 708 strain could have arisen from its development as a large bird for roaster production, while the Ross 308 is typically used as a smaller, multipurpose bird (Persia and Saylor, 2006), and therefore Ross 708 may have higher heat production than Ross 308. The molecular and metabolic reasons for birds with high genetic potential for growth having high metabolism remain unclear, but the activation of metabolic genes in the embryos during incubation could be a factor. The activation of genes associated with the Krebs cycle and the electron transport chain in the mitochondrial membrane could lead to the production of energy, which would increase O2 demand. As a result, there would also be an increased demand for glucose metabolites, leading to high embryonic metabolism (Humphrey and Rudrappa, 2008). Therefore, further investigation is needed to characterize changes in the genes associated with the metabolic pathways during each of the last 7 d of incubation. In the meantime, incubation temperature must be optimized to account for not only genetic differences and breeder flock age but also depending on the daily response of embryos to incubation temperature. Although EST was still high from E15 to E21, incubation at 37.0°C was optimum for Ross 708 embryos as indicated by the greatest chick length in this treatment, which is a sign of chick quality (Molenaar et al., 2008). Chick quality indicators were not affected by incubation temperature in Ross 308 embryos. Across strain, the hatching times at 36.0, 36.5, 37.0, and 37.5°C were approximately 21.0, 20.9, 20.7, and 20.9 d, respectively. Thus, if the standard commercial chick pull time of 21.5 d was used, chicks would remain in the hatcher for 12.0, 14.4, 19.2, and 14.4 h after hatching, respectively. Therefore, chicks could spend almost 20 h in the hatcher before actual pull time, and thus could be subjected to overheating. Although not part of this study, the apparent sensitivity of Ross 708 embryos to increased incubation temperatures could lead to high embryonic mortality. It is recommended that no more than 25% of chicks should be hatched 23 h before the pull, and more than 75% of the total hatch should be hatched within 13 h of the pull (Cobb-Vantress, 2013). Therefore, the optimum pull time needs to reflect incubation temperature, and subsequent effects on embryo metabolism. Chick length at hatch is positively related to chick quality, thus the optimum incubation from 15 to 21.5 d of incubation was 37.0°C. The normal practice for most commercial hatcheries is to pull chicks at 7 am on the d of hatch. Therefore, egg set time would have to be adjusted depending on the strain, incubation temperature, the brand and type of machine, and experience of the hatchery staff. Such adjustments could reduce late incubation embryo mortality if the expected chick hatch time is known and avoid keeping the chicks in the hatcher for an extended time, thus reducing the risk of dehydration, excessive weight loss, and weakened chicks. In conclusion, incubation of Ross 708 embryos appears to be more challenging than Ross 308 embryos because of the more pronounced effects of elevated incubation temperature on embryo metabolism. Incubation at 37.5°C, which is used by most commercial hatcheries, could result in embryonic overheating. The use of EST to monitor metabolism during incubation may assist in adjusting incubation temperature to suit the needs of the embryo. Acknowledgements The authors would like to acknowledge the financial contributions from the Alberta Livestock and Meat Agency, Alberta Innovates Bio-Solutions, the Canadian Poultry Research Council, the Poultry Industry Council, the Alberta Hatching Egg Producers, the Alberta Chicken Producers, Maple Leaf Foods (Wetaskiwin, AB, Canada), Sofina Foods Hatchery (Edmonton, AB, Canada), the Natural Sciences and Engineering Research Council, and the Poultry Research Center (University of Alberta, Edmonton, AB). We also want to thank the following individuals for their help: Chris Ouellette, John Feddes, and staff and students of the Poultry Research Center, University of Alberta, especially Misaki Cho, Kerry Nadeau, Mark Phoa, and Victoria Owusu, and Sander Lourens (Wageningen University and Livestock Research). REFERENCES Abudabos A. 2010 . The effect of broiler breeder strain and parent flock age on hatchability and fertile hatchability . Int. J. Poult. Sci . 9 : 231 – 235 . 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Poultry ScienceOxford University Press

Published: Jul 11, 2018

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