PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

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1 PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Effect of relative humidity during incubation at a set eggshell temperature and brooding temperature posthatch on embryonic mortality and chick quality C. W. van der Pol,* 1 I. A. M. van Roovert-Reijrink,* C. M. Maatjens,* H. van den Brand, and R. Molenaar * * HatchTech B.V., PO Box 256, 3900 AG Veenendaal, the Netherlands; and Adaptation Physiology Group, Wageningen University, PO Box 338, 6700 AH Wageningen, the Netherlands ABSTRACT Previous studies have shown that RH during incubation of chicken eggs influences water loss from the egg and embryonic mortality. In those studies, eggshell temperatures (EST) were not monitored or controlled. Because RH influences the egg s heat loss through evaporation, EST might have been different between RH treatments, influencing embryonic mortality and development. To eliminate the effect of EST, in the current study eggs were incubated at an EST of 37.8 C from embryonic d (E) 0 until E18 and at a high (55 to 60%) or low (30 to 35%) RH from E2 until hatch. Embryonic mortality, hatch curve, and several chick quality characteristics (length, weight, navel quality, organ weights, and DM of the yolk free body mass and yolk) were determined on E18 and at hatch. Low RH increased egg weight loss between E0 and E18 (+3.0%) and third week embryonic mortality (+3.0% of fertile eggs) and decreased hatch of fertile eggs ( 2.9% of fertile eggs) compared with high RH. Hatch duration and INTRODUCTION During incubation, a chicken egg loses a certain amount of weight because of water evaporation (Rahn et al., 1977). This is essential to create an air cell sufficient to allow embryonic lung ventilation after internal pipping and a successful hatch (Ar and Rahn, 1980). According to Ar and Rahn (1980), the highest hatchability in broiler chicken eggs is reached when total diffusive water loss is 12 to 14% of the fresh egg weight at d 18 of incubation [embryonic day (E) 18]. Embryonic mortality increases when water loss is lower than 9.1% (Davis et al., 1988; Buhr, 1995) or higher than 18.5% (Tullet and Burton, 1982; Davis and Ackerman, 1987; chick quality characteristics did not differ between RH treatments. To assess the effect of RH during incubation on posthatch performance under suboptimal conditions, hatchlings were brooded at a normal (35.0 C at d 0, decreasing to 27.0 C at d 4) or cold (27.8 C at d 0, decreasing to 25.6 C at d 4) temperature until 4 d posthatch. Incubation RH and brooding temperature significantly interacted with posthatch growth but not development. Both low and high RH cold brooding temperature resulted in lower ( 6.9 and 6.0 g, respectively) BW than high RH normal brooding temperature at 4 d of age. The cold brooding temperature resulted in lower daily feed intake ( 1.3 g/chick) than the normal brooding temperature. In conclusion, incubating eggs at a low RH compared with a high RH and maintaining the EST at 37.8 C decreased hatch of fertile eggs, but had little effect on chick quality or posthatch performance. Key words: relative humidity, incubation, brooding temperature, chick quality 2013 Poultry Science 92 : Poultry Science Association Inc. Received January 2, Accepted April 14, Corresponding author: cvdpol@hatchtech.nl Davis et al., 1988; Packard and Packard, 1993; Buhr, 1995). Water evaporation from the egg can be manipulated by changing the RH inside the incubator, as water vapor deficit directs H 2 O exchange (Hoyt, 1979; Tullet and Burton, 1982; Davis et al., 1988; Buhr, 1995). A decrease in RH in the incubator leads to an increase in water evaporation from the egg (Hamdy et al., 1991). It can be speculated that RH in the incubator influences embryo temperatures because the energy required to evaporate water from the eggshell is subtracted from the egg. This means that heat loss could be increased in eggs incubated at a low RH compared with a high RH. Therefore, eggs incubated at a low RH may require a different machine temperature than eggs incubated at a high RH to maintain the same embryonic temperature. Eggshell temperature (EST) is often used as a measure of embryonic temperature because it is difficult to measure embryonic temperature without killing the embryo. It has been shown that an EST between 2145

2 2146 van der Pol et al and 38.0 C from E0 until E18 is optimal for embryonic development and hatchability (Lourens et al., 2005, 2007; Leksrisompong et al., 2007; Molenaar et al., 2010, 2011). In previous studies in which the effect of RH during incubation on hatchability and chick quality was investigated, eggs were incubated at a fixed machine temperature (Hoyt, 1979; Tullet and Burton, 1982; Davis et al., 1988; Buhr, 1995). Possibly, effects found in previous studies of RH during incubation were a reflection of suboptimal EST because the machine temperature was fixed and EST was not controlled or adjusted. When eggs are incubated at the optimal EST of 37.8 C, effects on hatchability or chick quality may be attributed to the RH in the incubator and the resulting water loss. As water loss is increased, hatchling BW (Packard and Packard, 1993) and yolk free body mass (YFBM) have been found to decrease (Hoyt, 1979; Tullet and Burton, 1982; Davis and Ackerman, 1987). This has been attributed to relatively lower water content in the body, because dry body mass remained the same for incubation RH ranging between 6 to 12% (Davis et al., 1988), 12 to 19% (Tullet and Burton, 1982), and 11 to 25% (Packard and Packard, 1993). It can be speculated that egg water loss affects embryo water content, and that the effect on embryonic growth and development is limited. Posthatch performance may be influenced by incubation RH. Cold (<28 C) brooding temperatures have previously been shown to decrease broiler growth compared with normal (±30 C) or high (>32 C) brooding temperatures (Huston, 1965; Harris et al., 1975; Renwick and Washburn, 1982; Scott and Washburn, 1985; Deaton et al., 1996; Bruzual et al., 2000; Baarendse et al., 2006; Leksrisompong et al., 2009). In the current experiment, normal and cold brooding temperatures were provided to investigate if incubator RH interacts with brooding temperature to influence broiler growth. Part 1 of the experiment aimed to investigate the effect of a high (55 to 60%) or low (30 to 35%) RH during incubation, while maintaining an EST of 37.8 C, on embryonic mortality, hatch curve, and chick quality in broilers. Part 2 of the experiment aimed to investigate the effect of cold brooding temperatures in the chicks from part 1. Chicks were housed at a cold (aimed to keep rectal temperatures <40.0 C) or normal (aimed to keep rectal temperatures between 40.0 and 40.6 C) brooding temperature for 4 d posthatch and the effect of incubator RH and brooding temperature on chick quality and development posthatch was investigated. MATERIALS AND METHODS Part 1. Incubation Treatment. Part 1 of the experiment was designed to investigate the effect of low (30 to 35%) or high (55 to 60%) incubator RH applied from E2 until hatch at E21. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Wageningen University. First-grade Ross-308 broiler hatching eggs from a parent stock aged 47 wk were incubated (n = 3,600 eggs). Egg weights averaged 64.9 g and ranged from 52.7 to 79.7 g at E0 (based on n = 600 individually weighed eggs). Storage and Incubation from E0 to E18. The eggs were stored on setter trays for 5 d at 18 C. Thereafter, eggs were placed in 1 of 2 HatchTech Picoclimer setters (HatchTech B.V., Veenendaal, the Netherlands) with a capacity of 4,800 eggs each at a commercial hatchery (Lagerwey, Lunteren, the Netherlands). Eggs were equally divided among 12 trays per treatment with a capacity of 150 eggs/tray (n = 24 trays). Incubators were filled with additional eggs not used in the experiment to provide uniform airflow throughout the incubator. The EST were monitored using temperature sensors (NTC Thermistors: type DC 95, Thermometrics, Somerset, UK) placed on the equator of 4 individual eggs. Sensors were attached using heat-conducting paste (Dow Corning 340 Heat Sink Compound, Dow Corning GmbH, Wiesbaden, Germany) and tape. Humidity was regulated through a U-Vaporator (HatchTech B.V.) that created water droplets with a diameter of 1 to 10 microns. From E0 to E2, RH was maintained at 55 to 60% for both incubators. From E2 onward, incubator RH was maintained between 30 and 35% (low incubator RH) or 55 and 60% (high incubator RH). The incubator s temperature was adjusted automatically to maintain an EST of 37.8 C. The CO 2 concentration was maintained between 0.25 and 0.35%. Eggs were turned to an angle of 45 and then turned hourly by 90. Incubation from E18 to E21. At E18, eggs were candled and fertile eggs were transferred to 24 hatching baskets/treatment. Each basket had a maximum capacity of 75 eggs (n = 48 baskets). Baskets were placed in 1 of 2 HatchTech Picoclimer hatchers (HatchTech B.V.) with a capacity of 4,800 eggs each. Incubators were filled with other fertile eggs not used in the experiment to provide uniform airflow throughout the incubator. Incubator RH was again maintained at 55 to 60% for the high RH treatment or 30 to 35% for the low RH treatment using a U-Vaporator (HatchTech B.V.). The CO 2 concentration was maintained between 0.25 and 0.35%. Incubator temperature was adjusted manually to maintain an EST of 37.8 C until E19, measured for 4 eggs. At E19, the incubator temperature was fixed at the temperature that corresponded with an EST of 37.8 C at that moment (36.6 C for both incubators), and the EST was allowed to increase during the hatching process. From E20 onward, incubator temperature was maintained at 36.1 C to maintain rectal temperatures of hatched chicks between 40.0 and 40.6 C. Air temperature was measured by a sensor placed directly behind the egg mass. Measurements. All eggs from 12 trays per treatment were weighed per tray at E0 and E18 (candling and

3 INCUBATION HUMIDITY AND BROODING TEMPERATURE transfer) to determine egg weight loss (n = 24 trays). All eggs from 2 trays per treatment were weighed individually at E0 and E18 to determine variation in egg weight loss among eggs (n = 600 eggs). Percentage of egg weight loss from E0 to E18 was calculated by the formula [(egg weight d 0 egg weight d 18)/egg weight d 0] 100. At E18, eggs were candled to identify the number of infertile eggs and embryonic mortality per week. At E21, nonhatched eggs were opened to classify embryonic mortality per week. Fertility was calculated as a percentage of set eggs. Hatch of fertile eggs and embryonic mortality per week were calculated as a percentage of fertile eggs. At 468, 474, 480, 486, 492, 498, 504, and 510 h of incubation, all hatched chicks in 4 hatcher baskets per treatment (n = 8 baskets) were counted to determine the percentage of hatched chicks (hatch curve) and the average incubation duration. To distribute hatchlings equally across the hatching period, hatchlings per basket were alternately allocated to be used for organ weight (n = 22 chicks) or DM analysis (n = 44 chicks) or to be brooded in part 2 of the experiment (n = 245 chicks). Hatchlings were individually tagged with wing tags (Record B.V., Velp, the Netherlands). The chicks were weighed, their length was recorded from the tip of the beak to the tip of the middle-toe excluding the nail (Hill, 2001), and navel condition was scored as 1 (a clean and closed navel), 2 (a black button or gap of <2 mm or presence of a black string), or 3 (a black button or gap of >2 mm; Molenaar et al., 2010). After BW determination, embryos from 11 randomly selected eggs per treatment at E18 (n = 22 embryos) and 11 chicks per treatment at E21 (n = 22 chicks) were killed by cervical dislocation and frozen and stored at 20 C for later determination of organ weight. After thawing the embryos and chicks in a plastic bag in a water bath at 40 C for 15 min, yolk, heart, stomach (proventriculus and ventriculus), liver, intestines, spleen, and bursa of Fabricius weight were determined. Yolk, heart, stomach, liver, and intestine weights were determined as a general measure of chick development. Spleen and bursa of Fabricius weights were determined as a measure of development of the main immunological organs. The YFBM was calculated as BW minus residual yolk weight. Embryos from 12 randomly selected eggs per treatment at E18 (n = 24 embryos) and 22 chicks per treatment at E21 that were equally distributed over the hatching period (n = 44 chicks) were killed by cervical dislocation and frozen and stored at 20 C for later determination of DM content of the yolk and YFBM (ISO 6496, 1998). Dry matter was analyzed as described by Molenaar et al. (2010). In addition, 10 randomly selected chicks per treatment at E (n = 20 chicks) were killed by decapitation and used for analysis of the blood parameters hematocrit and hemoglobin. Embryos from 10 other randomly selected eggs per treatment at E18 (n = 20 embryos) and 10 randomly selected chicks per treatment at E21 (n = 20 chicks) were used for bacterial analysis of general cfu and coliform cfu per gram in the residual yolk. The residual yolk tissue was 10-fold diluted in peptone saline (0.85%) solution and placed in a stomacher (Seward Inc., Port Saint Lucie, FL) during 2 min. Total aerobic plate counts were estimated in plate count agar (caseinpepton-dextrose-yeast agar, Merck, ) and incubated at 30 C for 72 h (NEN-EN-ISO 4833, 2003). Coliform count was determined by plating in Chromocult Coliform Agar (Merck ). Incubation conditions were 72 h at 30 C (Conform NEN , 2001). Typical colonies were subcultured on blood agar (5% sheep blood, Media-products, Groningen, the Netherlands) followed by biochemical identification using the API 20E system. The bacteriological tests were performed by Balis Laboratorium V.O.F. (Boven Leeuwen, the Netherlands). Bacterial data were classified for further analysis into logarithmic categories, with category 1 = 0 to 10 cfu/g, 2 = 11 to 100 cfu/g, 3 = 101 to 1,000 cfu/g, 4 = 1,001 to 10,000 cfu/g, 5 = 10,001 to 100,000 cfu/g, and 6 = 100,001 to 1,000,000 cfu/g. Statistics, Part 1. Tray was the experimental unit in the statistical analysis of egg weight loss. Individual eggs, embryos, and chicks were the experimental unit in the statistical analysis of all other individual measurements of egg weight, embryonic mortality, embryo and posthatch chick weight, chick length, navel quality, hatch time and curve, organ weight, DM content, blood parameters, and bacterial count. Model assumptions were verified by examination of the distributions of the means and residuals. All data were analyzed in SAS (SAS Institute Inc., 2004). The model was Y i = µ + RH i + ε i, [1] where Y i is the dependent variable, µ is the overall mean, RH i is the incubator RH (i = low or high), and ε i is the residual error term. For analysis of organ weights, embryo weight, posthatch chick weight, DM content, blood parameters, and bacterial count, model [1] was extended with the age of the chick (Age k ; k = E18 or E21) and interactions of incubator RH with age. For analysis of chick length and navel quality, model [1] was extended with the hatch time of the chick (Hatch l ; l = 468, 474, 480, 486, 492, 498, 504, or 510 h of incubation). For the analysis of variation in individual egg weights and hatch time (the hatch curve), the SD was calculated and used for further analysis. Navel quality and bacterial count were analyzed using the Logistics procedure. For all other analyses, the GLM procedure was

4 2148 van der Pol et al. Figure 1. Overview of a HatchBrood unit with 1 = main entrance door, 2 = one of the 12 sections, containing 66 cradles divided over 3 dolleys, each containing 2 11 cradles, 3 = the corridor between the sections, 4 = perforated radiators that heat or cool the air and distribute it evenly over the cradles. Color version available in the online PDF. used. Least squares means were compared using Bonferroni adjustments for multiple comparisons. Data are presented as least squares means ± SEM. In all cases, differences were considered significant at P < Part 2. Brooding Period Treatment. Hatched chicks from part 1 of the experiment were used in part 2 of the experiment. Chicks incubated at a high (55 to 60%) or low (30 to 35%) RH were housed at a normal or cold brooding temperature from d 0 to 4 posthatch in a 2 2 factorial arrangement. The experimental protocol was approved by the Institutional Animal Care and Use Committee of Wageningen University. Housing from d 0 to 4 Posthatch. Chicks hatched in part 1 of the experiment were transported for 1 h to the brooding facility. Chicks were divided over 2 Hatch- Brood units (Figure 1). The HatchBrood is a system designed for the first 4 d of a chick s life (the early brooding period), after which they are transported to a broiler house for the remainder of their production cycle. A HatchBrood is divided into 12 sections. Each section can hold 3 dolleys with 22 cradles per dolley. Fiftyday-old-chicks are placed in one cradle with an inner surface area of 80 cm 2 /chick and outside dimensions of cm (width depth height, Figure 2). Each cradle is illuminated with light-emitting diode (LED) lights above the drinking gutter. Chicks are exposed to 24 h of light during the first day posthatch. From d 2 to 4, a light schedule of 2 h of light and 1 h of darkness is applied. From practical experience, it is found that this light schedule encourages chicks to eat and drink as well as rest at regular intervals when it is used in a HatchBrood unit. Feed is available ad libitum for the chicks from 2 feeding troughs on 2 sides of the cradle that are filled before placement. Water is available ad libitum from the drinking gutter on the illuminated side of the cradle. The cradles have a plastic grid floor through which manure can fall onto a cardboard manure plate. Air velocity, RH, air temperature, and CO 2 concentration are constantly monitored and adjusted to the settings. The fresh air inlet is regulated by CO 2 level, air pressure, or RH inside the HatchBrood and enters the system at the level of each cradle. The air flow is forced through perforated radiators (Figure 1) before entering the cradle with an air velocity of 0.3 m/s. In the current experiment, hatched chicks from each incubator RH treatment of part 1 were placed in 49 HatchBrood cradles with 5 chicks/cradle (n = 245 chicks). This was done to prevent chicks from huddling in a group at the cold brooding temperature, which can influence their body temperature. For the normal brooding temperature, 13 cradles with high incubator RH chicks and 11 cradles with low incubator RH chicks were used (n = 120 chicks). Air temperature was maintained at the standard temperature schedule with 35.0 C at d 0 and decreased to 27.0 C at d 4 (normal brooding temperature). This standard air temperature schedule was used to maintain rectal temperatures between 40.0 and 40.6 C. For the cold brooding temperature, 14 cradles with high incubator RH chicks and 11 cradles with low incubator RH chicks were used (n = 125 chicks). Cradle numbers were unequal because of unequal numbers of available chicks resulting from the difference in hatch of fertile eggs between incubator RH treatments. Air temperature was maintained at 27.8 C at d 0 and decreased to 25.6 C at d 4 (cold brooding temperature). This temperature schedule was provided to achieve a rectal temperature of <40.0 C. The CO 2 concentration was maintained between 0.10 and 0.15%. The RH was maintained at 45 to 55%. Water and a commercially available prestarter feed (ME = 3,100 kcal/kg, CP = 24.8%) were available ad libitum. The standard light schedule as mentioned above was applied.

5 INCUBATION HUMIDITY AND BROODING TEMPERATURE 2149 Figure 2. Two HatchBrood cradles. 1 = feed trough, 2 = feed stock in the trough, 3 = drinking gutter with integrated light-emitting diode lighting above the drinking gutter, 4 = plastic grid floor. Color version available in the online PDF. Measurements. Individual chick weight and length and mortality per cradle were determined daily. Dead chicks were removed from the cradle. Rectal temperatures of 5 randomly chosen chicks per treatment were measured daily. Total feed intake per cradle was determined on d 4 posthatch. Feed conversion was calculated using the following formula: total feed intake (g) per cradle/total weight gain (g) per cradle. Feed to length conversion was calculated using the following formula: total feed intake (g) per cradle/total length gain (cm) per cradle. After BW determination at 4 d posthatch, 40 chicks per treatment (n = 160 chicks) were killed by cervical dislocation and frozen and stored at 20 C. Residual yolk, heart, stomach (proventriculus and ventriculus), liver, intestines, spleen, bursa of Fabricius weight, and DM of the YBFM and residual yolk were determined after thawing the chicks in a plastic bag in a water bath at 40 C for 15 min. The YFBM was calculated as BW minus residual yolk weight at 4 d posthatch. Fifteen chicks per treatment (n = 60 chicks) were frozen and stored at 20 C for later DM analysis of the YFBM and residual yolk (ISO 6496). Dry matter was analyzed as described by Molenaar et al. (2010). Twenty chicks per treatment combination (n = 80 chicks) were decapitated on 4 d posthatch to obtain blood for analysis of hematocrit and hemoglobin levels. Their residual yolk was removed for bacterial analysis of general cfu and coliform cfu per gram. Bacterial analysis was performed as described under Part 1: Incubation. Statistics, Part 2. Individual chicks were the experimental unit in the statistical analysis of chick weight and length, organ weights, DM content, blood parameters, bacterial count, and rectal temperature. Cradle was the experimental unit in the statistical analysis of feed consumption and feed conversion ratios. Model assumptions were verified by examination of the distributions of the means and residuals. All data were analyzed in SAS (SAS Institute Inc., 2004). The model was Y ij = µ + RH i + Brooding j + interaction + ε ij, [2] where Y ij is the dependent variable, µ is the overall mean, RH i is the incubator RH (i = low or high), Brooding j is the brooding temperature (j = cold or normal), interaction is RH brooding temperature, and ε ij is the residual error term. Organ weights, DM content, blood parameters, rectal temperatures, feed intake, and feed conversion were analyzed using the GLM procedure. Posthatch chick weight and length were analyzed using the Mixed procedure with an auto-regressive covariance structure with the individual animal as the repeated factor. Bacterial count was analyzed using the Logistics procedure. Least squares means were compared using Bonferroni adjustments for multiple comparisons.

6 2150 van der Pol et al. Table 1. Egg weights at embryonic d (E) 0 and E18 of incubation, egg weight loss from E0 to E18, variation in egg weight loss, fertility, embryonic mortality per week, hatchability of fertile eggs, hatch time, and hatch curve of eggs incubated at a low (30 to 35%) or high (55 to 60%) RH from E2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 Hatch curve 5 (SD, h) Hatch time (h) Embryonic mortality Hatch of fertile eggs 4 wk 3 4 wk 2 4 wk 1 4 Fertility 3 Variation in egg weight loss 2 (SD, g) Egg weight loss E18 1 Egg weight E18 (g) Egg weight E0 (g) Item n 24 trays 24 trays 24 trays 600 eggs 24 trays 24 trays 24 trays 24 trays 24 trays 8 baskets 8 baskets RH Low (30 to 35%) b 12.7 a 1.79 a 93.7 b a 90.0 b High (55 to 60%) a 9.7 b 1.34 b 95.4 a b 92.9 a SEM P-value 0.82 <0.001 < a,b Least squares means lacking a common superscript within a column differ (P < 0.05). 1 Values expressed as a percentage of egg weight at E0. 2 Values expressed as the SD of percentage of individual egg weight loss (g). 3 Values expressed as a percentage of set eggs. 4 Values expressed as a percentage of fertile eggs. 5 Values expressed as the SD of hatch time (h). For analysis of organ weights, chick weight and length, DM content, and rectal temperatures, model [2] was extended with the hatch time of the chick (Hatch l ; l = 468, 474, 480, 486, 492, 498, 504, or 510 h of incubation). For the analysis of chick weight and length, model [2] was further extended with the repeated factor days posthatch (DPosthatch m ; m = 0, 1, 2, 3, or 4 d posthatch). Data are presented as least squares means ± SEM. In all cases, differences were considered significant at P < RESULTS Part 1. Incubation: Incubator RH Mean egg weights at E0 were comparable between RH treatments (Table 1). Low incubator RH eggs had higher egg weight loss from E0 to E18 (+3.0%; P < 0.001), higher third week mortality (+3.0%; P = 0.002), more variation in percentage of egg weight loss (SD of individual egg weights g; P = 0.02), and lower hatch of fertile eggs ( 2.9%; P = 0.005) compared with high incubator RH eggs (Table 1). Fertility was lower in the low incubator RH treatment ( 1.7%; P = 0.05) compared with the high incubator RH treatment. Incubator RH cannot have affected fertility so the difference in infertility was either coincidental or some nonhatched eggs appeared infertile, but in reality, contained embryos that had died during storage or in the first days of incubation. The RH did not affect 1st and 2nd week embryonic mortality, hatch time, or hatch curve (Table 1). The RH also did not affect chick length, navel score, YFBM, residual yolk weight, or relative organ weights (Table 2). At E18, RH did not affect embryo (43.2 g) weight, DM content of the YFBM (18.5%), or DM of the residual yolk (50.7%). At E21, RH did not affect chick weight (45.6 g), DM content of the YFBM (22.3%), or DM of the residual yolk (49.5%), hemoglobin level (4.3 g/dl of blood), hematocrit level (26.8%), bacterial count (656 cfu/g), or coliform bacterial count (0 cfu/g; all P > 0.17, data not shown). Residual yolk weight decreased with age. The YFBM and all relative organ weights, with the exception of the spleen, increased with age. Part 2. Brooding Period Incubator RH Brooding Temperature. A 3-way interaction was found between incubator RH brooding temperature and day posthatch for chick weight (P < 0.001, Figure 3) and chick length (P < 0.001, Figure 4). On d 0, 1, and 2, chick weight was comparable among treatments. On d 3, the normal brooding temperature treatments had higher chick weight compared with the cold brooding temperature treatments. On d 4, the normal brooding temperature treatments had higher chick weight compared with the low incubator RH cold brooding temperature treatment. The high incubator RH normal brooding temperature treat-

7 Table 2. Navel quality and chick length after hatch [embryonic day (E) 21], yolk free body mass (YFBM), residual yolk, heart, stomach (proventriculus and ventriculus), liver, intestines, spleen, and bursa of Fabricius weights at E18 and at E21 of chicks incubated at a low (30 to 35%) or high (55 to 60%) RH from E2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 Navel quality 2 Chick Bursa of Fabricius 3 Spleen 3 Intestines 3 Liver 3 Stomach 3 Heart 3 Residual yolk (g) YFBM (g) length (cm) Item n 1 INCUBATION HUMIDITY AND BROODING TEMPERATURE RH Low (30 to 35%) High (55 to 60%) SEM Age E b 13.3 a 0.56 b 4.4 b 2.0 b 1.7 b b E a 4.6 b 0.94 a 7.0 a 3.2 a 5.6 a a SEM Source of variation RH Age <0.001 <0.001 <0.001 <0.001 <0.001 < <0.001 a,b Least squares means lacking a common superscript within a column differ (P < 0.05). 1 Chick is the experimental unit. 2 Navel quality was scored only on E21. It was scored as 1 (a clean and closed navel), 2 (a black button or gap of <2 mm or presence of a black string), or 3 (a black button or gap of >2 mm; Molenaar et al., 2010). 3 Least squares means expressed as a percentage of YFBM. ment had higher chick weight compared with the high incubator RH cold brooding temperature treatment. Chick weight increased with age in all the incubator RH and brooding temperature combinations. Within brooding temperature treatments, chick length did not differ between low and high incubator RH for each posthatch day. On d 1, 2, 3, and 4 posthatch, both normal brooding temperature treatments had longer chick length compared with the high incubator RH cold brooding temperature treatment. On d 2, 3, and 4 posthatch, both normal brooding temperature treatments had longer chick length compared with the low incubator RH cold brooding temperature treatment. Chick length increased with age in all the incubator RH and brooding temperature combinations. A 2-way interaction was found between incubator RH and brooding temperature for residual yolk weight on d 4 posthatch (P = 0.02; Table 3). Incubator RH. In part 2 of the experiment, none of the variables were found to be different between the incubator RH treatments (all P > 0.10; Tables 3 and 4). Brooding Temperature. The normal brooding temperature treatment decreased relative heart weight ( 0.09% of YFBM; P < 0.001) and relative stomach (proventriculus and ventriculus) weight ( 0.35% of YFBM; P < 0.001) and increased YFBM (+3.9 g; P = 0.02) at d 4 posthatch compared with the cold brooding temperature treatment (Table 3). Brooding temperature treatment did not affect residual yolk weight, relative liver, intestines, spleen, and bursa of Fabricius weight (Table 3). Brooding temperature furthermore did not affect relative (32.5%) and absolute (0.16 g) DM of the residual yolk and relative (25.1%) and absolute (27.4 g) DM of the YFBM. The average rectal temperature during d 0 to 4 posthatch was 40.6 C and was not affected by brooding temperature (P = 0.93). The normal brooding temperature treatment increased feed intake (+1.3 g/chick per d; P < 0.001) and decreased feed to length conversion ( 3.7 g of feed/cm of growth; P < 0.001) until d 4 posthatch compared with the cold brooding temperature treatment (Table 4). Feed conversion was comparable among treatments (P = 0.11). Brooding temperature did not affect hemoglobin level (3.6 g/dl of blood), hematocrit level (24.3%), bacterial count (58,979 cfu/g) or coliform bacterial count (2,560 cfu/g) at d 4 posthatch (all P > 0.20, data not shown). DISCUSSION 2151 Differences in gas and water vapor pressures between the internal egg and its environment affect gas and H 2 O exchange (Rahn and Paganelli, 1990). Water evaporation from the egg will increase when RH during incubation decreases (Hamdy et al., 1991). In the current study, a RH of 30 to 35% during incubation increased water loss by 3.0% at E18 compared with a RH of 55 to 60%. This is comparable with the study by Tullet and Burton (1982), who found higher water loss of 4.5% in

8 2152 van der Pol et al. Figure 3. Chick weight (g) from 0 to 4 d posthatch of chicks incubated at a low (30 to 35%) or high (55 to 60%) RH from embryonic d (E) 2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 and subsequently brooded at a low or normal environmental temperature. Weights lacking a common letter (a c) within a day differ (P < 0.05). eggs incubated at a RH of 0 to 33% compared with eggs incubated at a RH of 0 to 50%. Third week embryonic mortality was increased by 1.6% for the low incubator RH treatment (12.7% egg water loss) compared with the high incubator RH treatment (9.7% egg water loss). A factor that may explain the difference in embryonic mortality between the low and the high incubator RH embryos is that incubator RH was maintained at a relatively low level during the hatching process for the low incubator RH treatment (40%, peaking at 55%) compared with the high incubator RH treatment (55%, peaking at 70%). It can be speculated that the embryos struggled to hatch because it was observed that 2.3% of the fertile eggs in the low Figure 4. Chick length (cm) at 0 to 4 d posthatch of chicks incubated at a low (30 to 35%) or high (55 to 60%) RH from embryonic d (E) 2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 and subsequently brooded at a low or normal environmental temperature. Lengths lacking a common letter (a c) within a day differ (P < 0.05).

9 INCUBATION HUMIDITY AND BROODING TEMPERATURE 2153 Table 3. Yolk free body mass (YFBM), residual yolk, heart, stomach (proventriculus and ventriculus), liver, intestines, spleen, and bursa of Fabricius weights at 4 d posthatch of chicks incubated at a low (30 to 35%) or high (55 to 60%) RH from embryonic d (E) 2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 and subsequently brooded at a low or normal environmental temperature for d 0 to 4 posthatch Item n 1 (g) YFBM Residual yolk (g) Heart 2 Stomach 2 Liver 2 Intestines 2 Spleen 2 Bursa of Fabricius 2 RH Low (30 to 35%) High (55 to 60%) SEM Brooding temperature Cold b a 5.23 a Normal a b 4.88 b SEM RH brooding temperature Low (30 to 35%) cold Low (30 to 35%) normal High (55 to 60%) cold High (55 to 60%) normal SEM Source of variation RH Brooding temperature <0.001 < RH brooding temperature a,b Least squares means lacking a common superscript within a column and treatment differ (P < 0.05). 1 Chick is the experimental unit. 2 Least squares means expressed as a percentage of YFBM. 3 Least squares means did not differ (P > 0.05) after correction for Bonferroni. incubator RH contained chicks that were alive in the eggshell at 510 h of incubation, but unable to hatch. For the high incubator RH treatment, the percentage of chicks alive in the eggshell was 0.9%. These chicks were included in the third week mortality numbers. Keeping the RH at a low level in the low incubator RH treatment during hatching was realized by increasing the inlet of fresh air. Although the air temperature measured behind the egg mass was comparable between treatments, it can be speculated that the inlet of a high amount of cold air caused a reduction of temperature at the embryo level, resulting in an increased number of late deaths and chicks that were unable to hatch. Unfortunately, this cannot be supported by EST because it is almost impossible to measure EST reliably from E19 onward because the hatching process can cause eggshell sensors to fall off the eggs. Additionally, we only measured EST at 5 eggs and during the hatching phase the variation is large among eggs. The reliability of EST measurements of only a few eggs during the hatching phase can be questioned. It can be speculated that embryonic mortality and ability to hatch are not Table 4. Feed intake (g/chick per d), feed conversion (g of feed/g of growth), and feed to length conversion (g of feed/cm of growth) of chicks incubated at a low (30 to 35%) or high (55 to 60%) RH from embryonic d (E) 2 to E21 at a fixed eggshell temperature of 37.8 C from E0 to E19 and subsequently brooded at a low or normal environmental temperature for d 0 to 4 posthatch Treatment Feed intake (g/chick per d) Feed conversion (g of feed/g of growth) Feed to length conversion (g of feed/cm of growth) RH Low (30 to 35%) High (55 to 60%) SEM Brooding temperature Cold 12.8 b a Normal 14.1 a b SEM RH brooding temperature Low (30 to 35%) cold Low (30 to 35%) normal High (55 to 60%) cold High (55 to 60%) normal SEM Source of variation RH Brooding temperature < <0.001 RH brooding temperature a,b Least squares means lacking a common superscript within a column and treatment differ (P < 0.05).

10 2154 van der Pol et al. affected by high or low incubator RH as long as EST are maintained at the optimal level of 37.8 C until E19. The same possibly holds for the period between E19 and hatch, but only in the case that the RH can be maintained without severe extra ventilation, which may cause a drop in embryo temperature. When this is not possible, a high RH during the hatching process may be advisable. Although egg weight loss until E18 was 3.0% (1.9 g) lower for high incubator RH eggs than for low incubator RH eggs, embryonic and chick weight and DM content at E18 and E21 were not affected by a high or low incubator RH in the current study. It can be speculated that part of this water was lost from the extra embryonic fluids. According to literature, an embryo copes with water loss during incubation by maintaining its water content through selective reabsorption of water from the allantois (Hoyt, 1979; Davis et al., 1988; Buhr, 1995). It can be speculated that residual water in the extraembryonic fluids after hatch can be found in the eggshells and residues. However, posthatch residual water in the eggshell and residues was not measured in the current study, so this hypothesis cannot be tested by the current data. At E21, chick length, organ weights, YFBM, residual yolk weight, DM of the YFBM and residual yolk, and hatch curve were comparable between incubator RH treatments. This is in accordance with previous studies where posthatch chick weight and DM content of the YFBM and residual yolk were found to be comparable between high and low rates of water loss (Tullet and Burton, 1982; Davis and Ackerman, 1987; Davis et al., 1988; Packard and Packard, 1993). It can be hypothesized that at an EST of 37.8, the embryo could maintain its body water content at optimal levels and continue growth and development despite egg water loss of 9.7 or 12.7% from E0 until E18. The RH during incubation interacted with brooding temperature and days posthatch on chick weight and length. However, the effect of this interaction seemed to be small and only reached significance on d 4 posthatch for chick weight and on d 1 posthatch for chick length. Bruzual et al. (2000) incubated eggs at a RH of 43, 53, or 63% and consequently brooded the chicks at 26.0 to 26.8 C (cold) or 32.0 to 32.9 C (normal) for 5 d posthatch. They also did not find an effect of incubator RH on weight gain, feed consumption, or feed conversion. This result was in line with expectations because hatchling quality and development seemed comparable between incubation RH treatments (Bruzual et al., 2000). If EST are maintained at 37.8 C until E18 and egg water loss from E0 to E18 is between 9.7 and 12.7%, it appears that incubation RH does not greatly affect posthatch performance. The effect of brooding temperature on posthatch growth and development appeared much larger than the effect of incubator RH. In the current study, the cold brooding temperature treatment resulted in a lower BW from d 3 onward than the normal brooding temperature treatment. The negative effect of cold brooding temperature on BW up to 3 wk posthatch has been observed previously (Huston, 1965; Harris et al., 1975; Renwick and Washburn, 1982; Scott and Washburn, 1985; Deaton et al., 1996; Bruzual et al., 2000; Leksrisompong et al., 2009). Decreased growth for the cold brooding temperature treatment was related to decreased feed intake, because feed conversion was comparable between brooding temperature treatments. The decrease in feed intake found in the current study is likely a result of a decrease in activity of the chicks in the low brooding temperature treatment. Low brooding temperatures have previously been found to be related to a decrease in feed intake because of huddling, decreased activity, or both (Harris et al., 1975; Renwick and Washburn, 1982; Leksrisompong et al., 2009). Although feed conversion was comparable between brooding temperature treatments in the current study, feed to length conversion (g of feed/cm of chick length) was higher for the cold brooding temperature treatment compared with the normal brooding temperature treatment. In the current study, chick length was used as a measure of frame development. Chick length was lower for the cold brooding temperature treatment than for the normal brooding temperature treatment from d 1 onward. This may suggest that chicks subjected to a cold brooding temperature had a different bone development compared with those subjected to a normal brooding temperature. Moraes et al. (2002) found that bone (tibia and femur) length and weight were lower for chicks brooded at a cold temperature, which suggests a difference in frame development compared with chicks brooded at a normal temperature. It can be speculated that this difference in bone and frame development is related to decreased activity at the cold brooding temperature treatment. Decreased locomotor activity has been associated with impaired skeletal development (reviewed by Bessei, 2006) and may lead to decreased growth length in bones. Organ development was also affected by brooding temperature. The cold brooding temperature resulted in a higher relative heart weight than the normal brooding temperature treatment. It has often been observed that cold brooding temperatures resulted in increased heart weights in chickens (Deaton et al., 1969a, 1976; Moraes et al., 2002). It has been proposed that this is caused by the increased metabolic rate as a result of cold environmental temperatures, which leads to increased oxygen requirement, increased cardiac output, and therefore, higher relative heart weights. In later life, this can lead to the development of pulmonary hypertension syndrome (Deaton et al., 1969b; Julian, 1993). In the current study, EST were maintained at 37.8 C to eliminate the effect of incubator RH on EST. Results from the current study seemed to be in line with previous studies. It has been shown that an incubator RH of 30 to 35% compared with 55 to 60% throughout incubation did not lead to different hatchling weight, quality, or development and had only a minor effect on posthatch performance when the EST was maintained

11 INCUBATION HUMIDITY AND BROODING TEMPERATURE at an optimal level of 37.8 C until E18. It can be speculated that chicks can cope with low or high incubation RH as long as the EST is maintained at the right level, and RH levels are allowed to increase once the hatching process starts beyond the low incubator RH values achieved in the current study (40%, peaking at 55%). ACKNOWLEDGMENTS The authors thank hatchery Lagerwey B.V. (Lunteren, the Netherlands) for donating eggs, and hatchery Probroed & Sloot B.V. (Langenboom, the Netherlands) for the use of the HatchBrood facility. The authors thank Cargill Animal Nutrition Innovation Center Velddriel (Velddriel, the Netherlands) for the use of their laboratory facilities. REFERENCES Ar, A., and H. Rahn Water in the avian egg: Overall budget of incubation. Am. Zool. 20: Baarendse, P. J. J., B. Kemp, and H. van den Brand Earlyage housing temperature affects subsequent broiler chicken performance. Br. Poult. 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