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1 Corresponding author: els.willems@biw.kuleuven.be. © 2013 Poultry .... 1906. WILLEMS ET AL. ...... spen, Marcel Samain, Inge Vaesen, and Daniel Vermeu-.
PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Partial albumen removal early during embryonic development of layer-type chickens has negative consequences on laying performance in adult life E. Willems,*1 Y. Wang,* H. Willemsen,* J. Lesuisse,* L. Franssens,* X. Guo,† A. Koppenol,*‡ J. Buyse,* E. Decuypere,* and N. Everaert* *KU Leuven, Department of Biosystems, Laboratory of Livestock Physiology, Kasteelpark Arenberg 30 Box 2456, 3001 Leuven, Belgium; †College of Animal Science and Technology, Jiangxi Agricultural University, 330045, Jiangxi, China; and ‡†ILVO Animal Sciences Unit, Scheldeweg 68, 9090 Melle, Belgium ABSTRACT To examine the importance of albumen as a protein source during embryonic development on the posthatch performance of laying hens, 3 mL of the albumen was removed. At hatch, no difference in BW could be observed. Chicks from the albumen-deprived group had a lower residual yolk weight due to higher yolk utilization. During the rearing phase (hatch to 17 wk of age), the BW of the albumen-deprived pullets was lower compared with the control and sham pullets. The feed intake of the albumen-deprived pullets was also lower than the control pullets. However, during the laying phase (18 to 55 wk of age) these hens exceeded

the control and sham hens in BW, although this was not accompanied by a higher feed intake. The albumendeprived hens exhibited a lower egg production capacity as demonstrated by the reduced egg weight, laying rate, and egg mass and increased number of second grade eggs. In addition, the eggs laid by the albumendeprived hens had a higher proportional yolk and lower proportional albumen weight. In conclusion, prenatal protein deprivation by albumen removal caused a longlasting programming effect, possibly by differences in energy allocation, in favor of growth and maintenance and impairing reproductive performance.

Key words: prenatal programming, protein deprivation, posthatch performance, laying hen, egg production 2013 Poultry Science 92:1905–1915 http://dx.doi.org/10.3382/ps.2012-03003

INTRODUCTION The avian egg contains all components necessary for the development of the enclosed embryo and ensures a good preparation for the posthatch period. The egg weight and albumen/yolk ratio of the egg, however, are influenced by several factors, which may have a tremendous impact on the success and survival of the hatchling. These factors can be classified as both intrinsic factors such as season (Marion et al., 1964), age (Fletcher et al., 1981), and strain of the hens (Marion et al., 1964) and management factors such as health of the parent flock (Lensing et al., 2012), housing conditions (Varguez-Montero et al., 2012), and quantity and quality of feed given to the parent flock (Fisher, 1969; Keshavarz and Nakajima, 1995). As the hens age, the egg weight increases and the eggs contain proportionally more yolk and less albumen and shell. However, at a given age, larger eggs contain proportionally more albumen, both in laying hens (Johnston and Gous, 2007) and broiler breeders (Vieira and Moran, 1998; ©2013 Poultry Science Association Inc. Received December 28, 2012. Accepted April 3, 2013. 1 Corresponding author: [email protected]

Nangsuay et al., 2011). In addition to differences in egg size and composition, the solids content of the egg may also differ. Depending on the age and strain (Fletcher et al., 1981, 1983; Ahn et al., 1997), health (Pan et al., 2011), and the diet (Walker et al., 2012) of the breeder flock, more or less nutrients will be allocated to the egg, giving the developing embryo a more or less favorable nutritional environment to start life. Variations in the prenatal environment of an embryo during critical phases of its development cause programming effects that can have lasting consequences. For example, increasing the incubation temperature led to an improved thermotolerance at slaughter age, possibly by programming of the thyroid and adrenal axis of the broiler embryos (Piestun et al., 2008) and even led to increased mortality due to ascites, when reared under cold circumstances (Molenaar et al., 2011). Programming effects have also previously been shown by the removal of albumen from broiler (Everaert et al., 2012) and layer eggs (Muramatsu et al., 1990; Hill, 1993; Finkler et al., 1998). Everaert et al. (2012) found that removal of albumen resulted in a decreased embryonic and posthatch growth up to d 7. At hatch, the albumen-deprived chicks had a lower plasma amino acid concentration and lower expression of MuRF1 and in males also a higher expression of atrogin-1, suggesting

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a transient increase in muscle proteolysis. Muramatsu et al. (1990) discovered a reduced whole-body protein synthesis by the removal of albumen. As proposed by Hill (1993), the approach of albumen removal offers a unique avian model to investigate the direct effect of reduced protein availability during embryogenesis on growth and metabolism, in contrast with mammalian models, manipulating the maternal diet, also including indirect confounding factors, such as maternal endocrine effects. Recently, the effect of a low-protein diet of hens on metabolic programming of the offspring was investigated in the chicken (Rao et al., 2009). Both the investigation of the direct and indirect models of prenatal protein deprivation or undernutrition can contribute to unraveling the prenatal programming effects. The objective of the present study was to investigate the importance of albumen as a protein source during the development of the layer-type chicken embryo and its ulterior effects on their posthatch performance. For this purpose, 3 mL of albumen was removed from layertype eggs and replaced with saline. Because albumen is the main source of protein for the developing embryo (Freeman and Vince, 1974), the net effect of this treatment will be a decreased protein availability. Effects on the incubation parameters, posthatch performance, egg production capacity, and egg quality were examined.

MATERIALS AND METHODS Experimental Design Incubation. A total of 1,487 fertilized ISA Brown layer-type eggs (Vepymo, Poppel, Belgium) from a 48-wk-old breeder flock were individually numbered, weighed, and randomly divided over the 3 treatments. Ten additional eggs were boiled, and the albumen and yolk weight were determined. The eggs consisted of 25.5 ± 0.5% yolk, 62.5 ± 0.6% albumen, and 11.6 ± 0.3% egg shell. The albumen to egg weight was used to estimate the proportion of albumen present in all eggs. The eggs were incubated with the blunt end up in a forced-draft incubator (PAS Reform, Zeddam, the Netherlands) at a dry bulb temperature of 37.6°C and a wet bulb temperature of 29.0°C and were turned every hour over 90°. On embryonic day (ED) 18, all eggs were candled and those with living embryos were transferred from the turning trays to individual hatching baskets. Albumen Removal. Preliminary experiments were conducted to determine the optimal timing, amount, and method for albumen removal. After 1 d of incubation, albumen removal was established in 860 eggs (albumen-deprived group). Eggs were first disinfected with 70% ethanol and iso-Betadine (Meda Manufacturing, Merignac, France). After drilling a hole near the sharp end of the egg, using an electric drill (Dremel, Robert Bosch Tool Corporation, Mount Prospect, IL), 3 mL of albumen (on average 7.45% of the albumen present) was removed using a 18-ga needle (Neolus,

Terumo, Tokyo, Japan) and 5-mL syringe (Terumo). Caution was taken to insert the needle as shallow as possible to minimize disturbance in the egg. The removed albumen was replaced by about the same volume of sterile saline using a 21-ga needle (Neolus, Terumo) and 5-mL syringe (Terumo), although this was not successful in every egg due to internal egg pressure. Finally, the hole was sealed using a drop of paraffin and eggs were weighed. A sham group of 306 eggs was mock-treated, similar to the albumen-deprived group, except for the actual albumen removal and saline injection. A third group of 321 eggs received no treatment (control group). The difference in the number of eggs for each treatment was based on the higher expected mortality due to albumen removal. At setting (ED 0), no differences in egg weight between the 3 groups were found, although the albumen-deprived group exhibited a slightly lower egg weight (60.9 ± 0.1 g), compared with the control (61.1 ± 0.2 g) and the sham group (61.4 ± 0.3 g). After albumen removal (ED 1), the eggs of the albumen-deprived group weighed less (59.7 ± 0.1 g) than the eggs of the control (60.6 ± 0.2 g; P = 0.007) and sham group (60.9 ± 0.3 g; P < 0.001), which were similar. Rearing and Laying Period. Per treatment, 45 female 1-d-old chicks were randomly assigned to 3 floor pens (15 pullets/pen) with wood shavings as litter. Another pen was used to house some additional pullets, used for sampling at 10 wk (n = 12, except for the sham group n = 8). All floor pens were located in one environmentally controlled room. On d 10 posthatch, the beaks were trimmed. The vaccination schedule was made in agreement with a licensed veterinarian. At first, the room temperature was set at 34°C, and this was gradually decreased until 20°C at 5 wk of age. This temperature was maintained until the end of the rearing phase (17 wk of age). At first, a 23-h light cycle was provided and this gradually decreased to 10 h at 6 wk. At 14 wk, the light cycle was prolonged to stimulate sexual maturation. A weekly 1 h increase was established to reach a 14-h light cycle at 17 wk of age. At 18 wk of age, in total 72 hens (24 hens/group) were transferred to a double sided 3-tier laying battery with 36 cages (laying phase: 18 to 55 wk of age). Twelve cages were randomly assigned to each treatment, resulting in 2 hens per cage. The different treatments were equally distributed between the upper, middle, and lower tiers to minimize cage effects. The laying battery was located in an environmentally controlled room where the temperature was set at 20°C. The light cycle was increased by half an hour at 18 and 19 wk to reach a final light cycle of 15 h. Starting from 40 wk of age, 1 h of light was given in the middle of the night to allow some extra feeding time. This schedule was maintained until the end of the experiment (55 wk of age). The hens received soy-wheat-corn based diets formulated according to the developmental requirements based on the ISA Brown Management Guide (Table 1, Research Diet Services, Wijk bij Duurstede, the Neth-

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PRENATAL UNDERNUTRITION IN CHICKEN Table 1. Overview of the consecutive diets that were fed to the hens from hatch to 55 wk of age and their ME, CP, calcium, and total phosphorus content Diet

Age (wk)

ME (kcal/kg)

CP (g/kg)

Calcium (g/kg)

Phosphorus (g/kg)

Starter Grower Pullet Prelay Layer 1 Layer 2 Layer 3

0–5 6–10 11–15 16–17 18–27 28–43 44–55

3,023 2,906 2,797 2,816 2,805 2,802 2,802

205.3 189.9 163.2 169.7 176.0 169.7 170.6

10.5 9.3 9.6 20.6 37.8 36.9 38.9

6.9 6.5 6.4 6.7 6.1 6.0 5.6

erlands). At 43 wk of age, due to problems with the feed dispensing systems, all cages remained without feed for approximately 24 h.

Data Collection Incubation Parameters. From 456 h until 518 h of incubation, the progress of hatch was monitored by checking eggs individually every 2 h for signs of internal pipping (IP), external pipping (EP), and hatch. At hatch, chicks were weighed and sexed based on the colors of their down. Relative chick weight was calculated relative to the egg weight after manipulation. The female chicks were tagged with a leg number (same as egg number) and kept for rearing, and the male chicks were culled. Unhatched eggs were opened and checked for fertility or time of death. Eggs were classified as fertile if there was liquefaction of the yolk (due to water transfer from the albumen to the yolk), subembryonic fluid, or an embryo present. Eggs were only classified as infertile if their appearance was not different from consumption eggs. Infertility was calculated as the number of infertile eggs relative to the total number of set eggs. Survival percentage was calculated as the number of hatched chicks relative to the number of fertile eggs. Early mortality was defined as dead embryos before ED 10 and mid mortality between ED 10 to 18. Embryos that died after ED 18 or were ready to hatch and alive in the shell, but had not hatched after 518 h of incubation, were classified as late embryonic mortality. Mortality rates were calculated relative to the number of fertile eggs. BW and Feed Intake. During the rearing phase (hatch to 17 wk of age), individual BW was monitored weekly for all 45 pullets per treatment and feed intake was measured in 3 pens per treatment. Feed conversion during the rearing phase was calculated as feed intake (g) divided by BW gain (g) per pen per week. During the laying phase (18 to 55 wk of age), the individual BW was measured weekly for 24 hens per treatment and the feed intake was measured in 12 cages per treatment. Feed conversion during the laying phase was calculated as feed intake (g) divided by egg mass (g) per pen per week as measured from 21 to 55 wk. Body Composition. The body composition was determined immediately after hatch, at 10 wk of age (during the rearing phase) and at the end of the experiment

(55 wk). At hatch, 8 female chicks per treatment were killed. At 10 wk, 12 pullets per treatment were killed, except for the sham group where only 8 pullets were used. At 55 wk, 23 control and albumen-deprived and 24 sham hens were killed. At 10 wk of age, the sampling was started before the lights were turned on, so it was assumed that the pullets had fasted overnight. At 55 wk of age, the hens were fasted by removing the feeders overnight before sampling. Water was provided until decapitation. At all ages, body, liver, digestive tract, and heart weights were recorded and proportional weights relative to BW were calculated. The digestive tract was defined as starting from the end of the esophagus, containing the proventriculus, the gizzard, and the small and large intestine until the beginning of the cloaca. At hatch, the weight of the residual yolk and dry weights of the digestive tract, heart, and residual yolk were also determined, and yolk-free BW (YFBW) was calculated. Dry weights were established by drying at 65°C for 48 h and then at 105°C until a constant weight was reached. Relative residual yolk to egg weight after manipulation (ED 1) was calculated. The weight of the left side of the breast muscle (pectoralis major) was determined at 10 and 55 wk, but not at hatch. Finally, at 55 wk, the weight of the brain, the leg muscle, the ovaria, the oviduct, and the abdominal fat was measured and proportional weights relative to BW were determined. Also, the number of large yellow follicles (weight > 1 g) present at the ovaria was counted (Decuypere et al., 1993; Bruggeman et al., 2005). Egg Production. During the laying period, from 18 to 55 wk of age, eggs were daily collected and individually weighed (n = 16,600). The eggs were classified into first and second grade eggs. Second grade eggs were defined as double-yolked, shell-less, or broken eggs and were not used for calculation of laying rate and egg mass. During the laying period, the number of second grade to the total number of laid eggs (%) was calculated for the different treatments. Laying rate (LR) and egg mass (EM) were calculated per cage per week using the following formulas: LR (%) = NE/ (NH × 7) × 100, where NE = number of eggs laid per cage per week, and NH = number of hens in one cage (= 2); and

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EM (g/day per hen) = LR × MEW, where MEW = mean egg weight per cage per week (g). Egg Composition and Quality. Every 5 wk starting from wk 25, eggs were collected on several consecutive days for measurement of the egg composition and the egg quality on the day after collection. A total of 2,854 eggs were analyzed during the whole laying period. Albumen pH, water content of yolk, and albumen and yolk color were only measured at 25, 40, and 55 wk. Yolk color was also measured at 30 wk. The yolk, albumen, and egg shell of the eggs were separated using an egg separator and weighed. Proportional weights were calculated relative to egg weight. Water content of the albumen was determined by drying at a temperature of 105°C until a constant weight was reached. Water content of the yolk was obtained by drying at a temperature of 60°C for 48 h followed by a temperature of 105°C until a constant weight was reached. The albumen height measured in Haugh units (HU; Haugh, 1937) was determined at the center of the thick albumen using a standard tripod micrometer. Haugh units were calculated using the following formula (HU = 100 × log {albumen height − 5.67 × [(30 × egg weight0.37 − 100)/100] + 1.9}). The albumen was individually collected in a container and thoroughly mixed to obtain a homogenous mass. The pH of the albumen was measured using a calibrated pH electrode (pH 330, WTW, Weilheim in Oberbayern, Germany). The color of the yolk was determined by using a Roche Yolk Color Fan scale, which ranges from 1 (very light yellow) to 15 (dark orange). The shell color was determined using the transmission color value, of which the experimental setup was previously described by Kemps et al. (2010) and Mertens et al. (2010). Measurements were performed using a spectrophotometer (MCS, Zeiss) with a range between λ = 305 to 1,145 nm with a resolution of 3 nm. The integration time used was 250 ms. The rough measured spectra were corrected by the following equation (Kemps et al., 2010):

− Ti , noise T Ti , prop =  i , rough ×   100%Ti , prop , Ti , ref − Ti , noise

where Ti,noise stands for the electrical noise present at wavelength i, Ti,ref for transmission at wavelength i of the reference measurement, Ti,rough for the rough transmission through the egg at wavelength i and Ti,prop finally for the proportional transmission spectrum at wavelength i obtained after the correction. The shell thickness was determined using a micrometer gauge with spherical tips (Digimatic micrometer 0–25 mm, 0.001 mm accuracy, Mitutoyo Products, Kawasaki, Japan) and is considered to be the average of 3 equidistant measurements at the equator of the egg (Coucke et al., 1999). Their average value was used for the statistical analysis. The Acoustic Egg tester was used both for

crack detection (De Ketelaere et al., 2000) and for measurement of the dynamic strength of the egg (Coucke et al., 1999).

Statistical Analysis All data were processed with the statistical software package SAS version 9.2 (SAS Institute Inc., Cary, NC). Egg weight before incubation and after treatment, absolute, and relative residual yolk weight and YFBW were analyzed using the one-way ANOVA with treatment as variable. Survival rate, early, mid and late embryonic mortality, infertility, and percentage of second grade eggs were analyzed using the logistic regression model with treatment as the classification variable. Time of IP, EP, hatch, and their intervals and absolute and relative hatching weight were analyzed using GLM with treatment, sex, and their interaction. Body composition was analyzed using GLM with treatment, age, and their interaction. When there was a significant effect of treatment, or interaction with age, means were further compared by a post-hoc Tukey’s test. Average weekly BW, feed intake, and conversion during the rearing phase were analyzed using a repeated measurements GLM after a log-stabilizing transformation, containing age, treatment, and their interactions as variables. Average weekly BW, feed intake, and feed conversion, egg weight, arcsin-square root-transformed laying rate, and egg mass during the laying phase were analyzed using repeated measurements GLM (per pen, except BW per hen), containing age, treatment, and their interaction as variables. Leg or pen numbers were used for identification. For every egg composition and quality parameter, an average value per cage per age (every 5 wk) was calculated and used for further statistical analysis using repeated measurements GLM, containing age, treatment, and their interaction as variables. Cage numbers were used for identification. Differences between treatments were analyzed by the specification of estimates based on the significant model. For all parameters, significance was set at the 5% level. All values were expressed as mean, and when possible with their SEM.

Ethics The present research was approved by the Ethical Commission for Experimental Use of Animals of the KU Leuven.

RESULTS Incubation Parameters Survival percentage at hatch of the albumen-deprived group was lower compared with both the control (P < 0.001) and the sham group (P < 0.001), with the latter 2 also different from each other (P < 0.001, Table 2). The albumen-deprived group had a higher early mortal-

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PRENATAL UNDERNUTRITION IN CHICKEN Table 2. Survival percentage; early, mid, and late mortality; and infertility of the eggs of the control, sham, and albumen-deprived group1 Item (%) Survival Early mortality Mid mortality Late mortality Infertility

Control

Sham

Albumendeprived

P-value

87.2a 5.3c 1.2 6.2 2.1

53.9b 36.6b 1.3 8.2 2.9

38.0c 52.2a 1.7 8.0 2.2