# Short periods of incubation, egg turning during storage and broiler breeder hens age for early development of embryos, hatching results, chicks quality and juvenile growth

Short periods of incubation, egg turning during storage and broiler breeder hens age for early... ABSTRACT An effect of modification of storage conditions of the eggs of broiler breeder flocks at the age of 49-, 52- and 70-, 73-wks of life on an early embryonic development, hatching time and synchronization, hatchability rates, chicks quality and broiler growth was investigated. The eggs were divided into 4 experimental groups: COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = were treated with 4 h pre-incubation at 30°C and 50–55% air humidity, delivered at 5 and 10 d over of 12 d of storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Eggs from older hens were characterized by poorer hatchability and poorer chicks quality. The use of 2 × 4 h pre-incubation in 12 d of eggs storage could have an effect on the initial acceleration of embryonic development in eggs of young hens, contributing to the alignment of embryos development in eggs from young and older hens to 72 h of incubation. Pre-incubation had no effect on the length of incubation period, hatching window, but it increased the hatchability of the set and apparently fertilized eggs and decreased the number of eggs not hatched, and also improved chicks quality. Eggs turning by 90° every 12 h during the storage positively affected the embryonic development, shortening the incubation time and the quality of chicks, but had no effect on hatchability rates and body weight in 42 d of life. Based on the obtained results, it can be concluded that the applied modifications can be effective in counteracting the negative effects of storage of hatching eggs from both young and older birds. INTRODUCTION In commercial hatcheries, obtaining a large number of simultaneously hatched chicks is often only possible after a period of eggs storage for several days. However, it has been proven that the length of eggs storage time is negatively correlated with hatchability rates (Fasenko et al., 2001a,b; Dymond et al., 2013) and the quality of the chicks (Becker, 1960; Tona et al., 2003a). Standard methods of counteracting these phenomena were eggs storing no longer than 7 d at a temperature below physiological zero, i.e., 20–21°C (Edwards, 1902). According to Fasenko et al. (2001a) under these conditions, blastodermal cells of the embryos exhibit mitotic activity but embryo development is inhibited. According to Reijrink et al. (2008), such a solution is not enough in case of longer eggs storage, since there is still an increase in the number of necrotic cellular indicators contributing to early mortality of the embryos. Therefore, it is important to examine and refine techniques aimed to counteract the reduction in hatching ability of eggs stored for more than 7 d. Several methods of eggs storage conditions modification in order to prolong the period in which they retain their high ability to hatch have been described in the literature. Pre-incubation, i.e., temporary heating of eggs, which is supposed to imitate the natural conditions that the birds provide to the eggs before they start to hatch them, turned out to be the most effective method (Meijer and Siemers, 1993). Fasenko et al. (2001a,b) demonstrated that a single pre-incubation (PI) allows the embryos to finish the formation of hypoblasts, which increases the ability of hatching even with 14 d of eggs storage. Dymond et al. (2013) modified this method by analyzing an effect of several PIs during the storage (SPIDES—short periods of incubation during egg storage). In turn, Elibol and Brake (2008) showed that the change in the position of eggs during the storage significantly reduces early mortality, which leads to improved hatching results. The aim of the study was to determine the applicability of SPIDES and turning of eggs during the storage and the age of broiler breeder hens on the improvement of hatching indices in industrial conditions and on the growth of the chicks. MATERIALS AND METHODS Experimental Design Two independent trials were conducted using eggs produced by younger (A) and older (B) broiler breeder flocks that were used in trials 1 (August 2017), and 2 (September 2017), respectively. Each of the two trials covered the production part carried out at the Poultry Hatching Plant belonging to CEDROB S.A. (Ujazdówek, PL), and the laboratory part carried out at the Poultry Breeding Division, University of Life Sciences (Warsaw, PL). Due to the practical conditions in a commercial hatchery, and analysis of the development of chicken embryos below 13 d of incubation, this experiment not require application to and consent of the National Ethical Commission at the Ministry of Science and Higher Education in Poland (Directive2015/266/EC; Public Information Biulletin, 2017). Breeder Flock The study was carried out using the eggs from two broiler breeder flocks Ross 308. Flock A in 49- and 52-wk-old and flock B in 70- and 73-wk-old. Birds were fed restricted diets according to standard Ross guidelines (Aviagen, 2016). At the time the beginning of the experiment, the number of birds in flock A was 51,239, where male: female ratio was 1:7. The number of birds in flock B was 42,900, and male: female ratio was 1:10, and 20% of cocks in the 63 wk of life, were replaced with younger ones (21 wk of life). The light schedule for both flocks included 13 h of light and 11 h of darkness. The conditions for both flocks maintaining were standard: litter with a fully automated system of feeding, watering and eggs collecting. Eggs Storage Profiles and Experimental Classes Eggs after collection were laid with the large end up (LEU) on the hatching trays, which were then put on trolleys. The eggs were stored in the farm storehouse for approximately 12 h (16°C and 75% RH). The eggs were then transported using adapted trolleys with shock absorbing wheel, under controlled microclimate conditions (15–18°C and 65–75% RH). Transport to the CEDROB S. A Hatchery lasted about 2 hours, after which the eggs were placed in the hatching egg storehouse under the same conditions as on the farm. Experimental eggs (1 trolleys × 4 treatment × 2 flock × 2 trial = 86,016 eggs) were stored together with other commercial eggs in a storehouse holding once 6 million eggs. There was 5376 eggs on one trolley (32 trays x 168 eggs), which constituted one experimental group. Eggs were stored 5 and 12 d using SPIDES consisting of only two PIs, not four as described by Dymond et al. (2013), but with the concurrent introduction of turning of eggs during storage: ⇒ COI = control incubation treatment were eggs storage 5 d, at turning every 12 h ⇒ NSP = non-SPIDES control eggs were eggs storage 12 d, at turning every 12 h ⇒ SPIDES = were treated with 4 h PI at 30°C and 50–55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h ⇒ NCOI = negative control incubation were eggs storage 12 d, no turning and no PI. The turning of eggs during the storage consisted in setting the angle of trays inclination by 90° every 12 h, so the eggs were always in the 45° inclination from the vertical plane, but with the change of the side plane (up-down). PI was carried out in setter (Jamesway, Inc. P120–129,024, Cambridge, Canada), to which entire egg trolleys were introduced and then returned to the storehouse. Incubation The eggs were placed in the setter (Jamesway, Inc. P120–129,024, Cambridge, Canada). Incubations were carried out at parameters 37.5–38.5°C and 50–60% RH. Egg turning (by 90°) during the incubation was carried out at a frequency of 12 h, the CO2 level was monitored from 5 d of incubation (3.500–4.500 ppm). One incubator contained at one time 4 trolleys with eggs arranged in 2 rows. The trolleys were moved every 24 hours clockwise to eliminate the effect of different distance from the fan. Eggs candling was performed in the 6 d of incubation, all eggs rejected were considered unfertilized. In the 18 d incubation, a second candling was carried out on the basis of which the late mortality of the embryos was determined. After an candling in 18 d, the eggs were placed in breeding baskets and placed in hatcher (Jamesway, Inc. P40–43,008, Cambridge, Canada). The hatching of the chicks was carried out in the horizontal position of the eggs at 37.6°C and 65% RH. The chicks were removed exactly 504 h after the start of incubation. The hatching was carried out using a separator (Chich/Shell Separators, KL, London, Ontario, Canada) which is used for shells separation as well as counting and vaccination of the chicks. Apparent fertile eggs (%), hatchability of set eggs (%) and hatchability of fertile eggs (%) were calculated on the basis of the results obtained, where: \begin{eqnarray*} &&{Apparent\,fertile\,eggs\,}\\ && = \frac{\begin{array}{@{}l@{}} number\,of\,set\,eggs\, - \,number\,of\,rejected\,eggs\,\\ during\,candling\,at\,6\,d\,of\,incubation \end{array}}{{number\,of\,set\,eggs}}\\ \quad && \times\, 100 \end{eqnarray*} s \begin{eqnarray*} &&{Hatchability\,of\,set\,eggs}\,\\ && = \,\frac{number\,of\,hatched\,chicks}{number\,of\,set\,eggs}\times\, 100 \end{eqnarray*} \begin{eqnarray*} && Hatchability\,of\,apparent\,fertile\,eggs\,\\ && = \,\frac{number\,of\,hatched\,chicks}{number\,of\,apparent\,fertile\,eggs} \times\, 100 \end{eqnarray*} Additional 100 eggs for each treatment group (total for both trials: 1600 eggs with possibly similar weight: 55.0–62.0 g for flock A and 67.0–74.0 g for flock B), treated in the same manner in the storehouse as the eggs incubated at the hatchery, were transported to the laboratory of Poultry Breeding Division, University of Life Sciences (Warsaw, PL). The transport took about 1.5 h (119 km). The eggs were transported on trlleys adapted to transport hatching eggs with a mean of transport (Mercedes-Benz, Daimler AG, Germany). The cars are equipped with full suspension and climate control determining the microclimae conditions inside the zone with hatching eggs (15–18°C and 65–75% RH). Then, all the eggs were numbered and placed in the setter with humidity pump (OvaEasy 380 Setter, Brinsea, Weston, UK). Incubations were carried out at 37.6–38.0°C and 50–65% RH, but without the possibility of CO2 level control. The eggs were turned automatically every by 90° 12 h. At 48 and 72 h of incubation, 15 eggs (240 eggs in the entire experiment) were removed from each group to control the development of embryos. The remaining 70 eggs from each group (1120 eggs in the entire experiment) were removed from the incubators and manually candled (OvaScope Brinsea, Weston, UK) after 18 d of incubation removing the eggs unfertilized and not hatched fetuses. The remaining eggs were placed in hatcher with clear, double-glazed observation door (OvaEasy 380 Hatcher, Brinsea, Weston, UK). Determination of Embryonic Characteristics In each experimental group, 10 embryos were analyzed in 48 and 72 h of incubation. The remaining 5 out of 15 eggs for this part of the study constituted a reserve. The embryos were isolated following the methodology described by Gupta and Bakst (1993). It consisted in applying a filter ring (paper disc) on the perivitelline layer so that the entire embryo was located in the central part of the ring. Then, after moistening the ring with protein residues for better integration, one of the periphery of the ring was held with tweezers and the vitelline membrane was cut with scissors on its outer edge. The filtration ring together with the PL on which the embryo was located was transferred to the microscope slide by placing the inside up. After gentle rinsing with deionized water to remove the remaining yolk, the specimen was viewed using a stereoscopic microscope (Olympus SZX10, Shinjuku, Japan). A microscope image was taken for each embryo and its developmental stage was classified according to the scale described by Hamburger and Hamilton (1951). In order to compare the degree of embryonic development in 48 h of incubation, the number of somites, the size and shape of the heart (if it was visible), brain development, primary optic vesicles and left head flexion advancement were noted in the SEM image (Figure 1). To analyze the degree of embryos development in 72 h of incubation, the following traits were used: number of somites pairs, body length measured from the tail tip to mesencephalon tip, wing- and leg buds presence, heart development, brain development, pigmentation of the eye and head flexion advancement (Figure 2). Figure 1. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 48 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50–55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 1. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 48 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50–55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 2. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 72 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 2. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 72 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Hatching Time and Chicks quality The hatching of the chicks was carried out individually at 37.6°C and 65% RH. The equipment of the hatching apparatus with a transparent door made it possible to precisely determine the time of hatching of the first chicks in a given experimental group. From that moment, each hatching apparatus was checked every 1 h. Hatched chicks were recorded (hatching time and egg number), weighed and tagged on the right wing with an individual chick tag. Next, the chicks were transferred to the collective hatching apparatus (R COM Maru, Bandury, UK) in which they stayed until the end of hatching. Hatching time monitoring allowed to calculate the percentage of chicks hatched in particular time intervals and the total time of hatching referred to as hatch window (HW = time between first and last hatched chick). The quality of chicks hatched in the laboratory was determined in accordance with the methodology described by Tona et al. (2003a). This method involves the point evaluation of several characteristics of the hatchling according to their meaning, in which the maximum sum of points is 100. These characteristics include: activity, down and appearance, retracted yolk, eyes, legs, navel area, remaining membrane, and remaining yolk. The quality of chicks hatched in the hatchery was evaluated in a standard manner due to their large numbers. All chicks that were qualified in the hatchery for the rearing were determined as first grade chicks. The other chicks, i.e., those that were removed from the separator and considered unsuitable for rearing, were qualified as the second grade. Generally, with the practical distribution of the chicks to the first and second categories, the three characteristics are considered: down appearance: chicks with wet down, or the apparent deficiencies of down are classified in the second category; navel appearance: open, visible remains of the yolk sac and the distinct discoloration of the navel area disqualifies the chick; other visible defects such as Loco genetic defects, or non-genetic innate variations, e.g., lack or additional limbs, also disqualify the chicks (hatchery personnel, unpublished data). Chicks Growth Chicks hatched in the laboratory of Poultry Breeding Division, University of Life Sciences (Warsaw, PL) were transported to the experimental farm RZD Wilanów-Obory Goździe owned by the University of Life Sciences (Warsaw, PL). The chicks were reared in experimental groups in separate compartments without gender distribution (with an initial stock density of 11–14 chicks/m2) up to 42 d of life. From each treatment, the chickens were divided into 3 replications. In each replication depending on the numbers of hatched chicks, reared 18 to 23 chickens. The light schedule was as follows: 24L: 0D for the first 2 d, then reduced to 16L: 8D in 8 d and maintained in this schedule until the end of rearing. The temperature was consistent with the relevant guidelines for broiler chickens: 35°C for 1st wk then successively reduced for 18°C in 6th wk. Chicks were fed ad libitum a commercially produced wheat-maize-soya-based diets in a 3-stage system: 0–14 d, starter: 12.5 MJ of energy, 21.9% crude protein; 15–35 d, grower: 12.8 MJ of energy, 20.8% crude protein; 36–42 d, finisher: 13.2 MJ of energy, 19.3% crude protein. Body weight (BW) of the chicks was controlled individually every 7 d, up to 42 d of life. Only those chickens that survived until the last weighing were included in the analysis. Mortality in all groups was low and did not exceed 1.80% (data not shown). Statistical Analysis Comparison of means between various treatments of eggs and flock age was conducted based on two-way analysis of variance (ANOVA) and Duncan's multiple range test at 0.05 significance level. The first factor in ANOVA was flock age with two levels and the second factor was treatment with four levels. Effects of the factors and their interaction were evaluated on all treatments or only for selected treatments to evaluate effects of egg turning and pre-incubation. Effect of egg turning was evaluated using dataset for NSP and NCOI treatments, while effect of pre-incubation was determined using dataset for NSP and SPIDES treatments. Analysis of variance was performed according to linear model: \begin{equation*} {Y_{ijk}} = \mu + {\alpha _i} + {\boldsymbol{\beta }_j} + {\left( {\alpha \boldsymbol{\beta }} \right)_{ij}} + {\varepsilon _{ijk}} \end{equation*} where: Yijk—is observed value of the trait, —is effect of ith flock age, βj—is effect of jth treatment, (αβ)ij—is interaction of flock age and treatment, —is error term. Effects of flock age and treatment were treated as fixed and independent (not nested). Evaluation of effects of flock age and treatment and their interaction for binomial data advances in embryonic development in 48 and 72 h of incubation (according to Hamburger and Hamilton (1951)) was conducted using generalized linear model with logit link function. The ANOVA analyses were performed similarly ANOVA, i.e., for total dataset and for selected treatments to evaluate effect of PI and egg turning. For data obtained in CEDROB S. A. hatchery, the basic unit used for calculations was a single hatching tray (168 eggs × 32 trays on one trolley = one treatment group). The results were presented as P-values for F-test in two-way ANOVA. Relationships between egg weight and chicks weight were evaluated using simple linear regression. The regression lines were presented graphically as well regression equations, and coefficients of determinations (R2) were presented. All variables followed normal distribution; only parametric statistical procedures were applied as well means and standard deviations were presented. All results for one flock collected in two dates (for flocks A of 49 and 52-wk-old and B of 70 and 73-wk-old) are presented as mean values, because the difference between the trials was not significant. Therefore, for each flock (A and B), the chicken BW calculations were made on the basis of 6 replications (3 for each of the trials). The statistical analyses were performed by using Statistica 12.0 software (Statsoft, INC, 2014). RESULTS AND DISCUSSION Embryonic Development The embryos from older hens, as well as those developing in pre-incubated and turned eggs, were at a higher stage of development in 48 h of incubation compared to the embryos developing in non-treated eggs (Table 1 and Figure 1). The use of PI alone (P = 0.580) did not affect the embryos’ qualification in 48 h of incubation to a higher stage of development, but the application of turning of eggs during the storage had a significant impact on this feature (P = 0.025). The relationships observed in 48 h of incubation between the developmental stage of embryos, hens’ age, and the modifications of egg storage conditions were slightly weakened in 72 h of incubation (Table 1 and Figure 2). A significant effect on the development of embryos up to 72 h of incubation was noted in case of the turning of eggs during the storage (P = 0.034) and in combination with PI (P = 0.040). The age of hens did not affect the embryonic development stage in 72 h of incubation (P = 0.052). Table 1. Effect of pre-incubation and eggs turning of storage time on embryonic development at 48 and 72 h of incubation.1 Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* 1Results of laboratory study. 2Values of total 20 embryos in each group in both trial. 3According to Hamburger and Hamilton (1951). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. *Significant effect in generalized linear model with logit link function at 0.05 probability level. 4Effects of the factors and their interaction were evaluated on all treatments. 5Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 6Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 1. Effect of pre-incubation and eggs turning of storage time on embryonic development at 48 and 72 h of incubation.1 Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* 1Results of laboratory study. 2Values of total 20 embryos in each group in both trial. 3According to Hamburger and Hamilton (1951). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. *Significant effect in generalized linear model with logit link function at 0.05 probability level. 4Effects of the factors and their interaction were evaluated on all treatments. 5Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 6Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large A more advanced embryonic development stage in 48 h of incubation in eggs from older hens was probably related to the fact that older hens tend to produce eggs in which the embryo is more developed at the time of laying (Fasenko et al., 1992). It results from the fact that the production of eggs decreases with an age due to individual series shortening. As a consequence, older hens lay more eggs in the early stages of the series. In the study conducted by Reijrink et al. (2009), embryos in the eggs from the beginning of a particular series, which are numerous in the case of older hens, were already characterized the formed hypoblast, in contrast to embryos from the late stages of the series where hypoblast was at the stage of formation. The occurrence of this phenomenon is explained by the fact that the first egg in the series remains in the F1 follicle phase about 16 h longer than the next eggs, which causes a faster beginning of development (Scott and Warren, 1936), and that the embryos in the first eggs of the series later start the embryonic pause as a consequence of prolonged stay in the shell gland (Berg, 1945). It is interesting, why an effect of hen age on embryonic development was not noticeable already in 72 h of incubation (Table 1). It can be assumed that other environmental factors at some point begin to slow down the development of embryos in the eggs of older hens. According to Reijrink et al. (2008) this may be associated with a greater difficulty for embryos in regulating the pH of the environment in eggs from older hens when the quality of egg whites and egg shells deteriorates progressively. It is difficult to clearly indicate in which moment of incubation the prevalence of embryos in the eggs of older hens gives way to those developing in eggs from younger hens. The results obtained in the present study indicate that this is a period between 48 and 72 h of incubation. However, an analysis of the embryonic development stage did not allow to include any of the 160 embryos to the HH20 stage (Table 1), i.e., embryos 70–72 h according to Hamburger and Hamilton (1951). This was mainly due to the fact that no clearly observed wings- and leg-buds were observed (Figure 2), despite that the analyzes were performed exactly after 72 h of incubation. However, many environmental factors like the genotype or nutrition of reproductive flocks can modify early embryonic development (Novo et al., 1997; Tona et al., 2004a). Therefore, in future studies, an effect of faster development of embryos from older hens may no longer be observed even in 48 h of incubation. Since the aim of pre-incubation of the eggs is mainly to allow the embryos to fully form the hypoblast before the beginning of the proper incubation, Fasenko et al. (2001a) and Reijrink et al. (2009) formulated the hypothesis that it is important primarily for embryos from young hens. In this study, no statistical differences were observed in the embryonic development stage under the influence of PI in the eggs of young and old hens (Table 1). Thus, it should be considered whether this lack of statistical differences is an evidence that PI actually stimulates the development of embryos in the eggs of young hens, since as previously reported, they are at a lower stage at the time of eggs laying. On the example of turkey embryos, Fasenko et al. (2001a) showed that a single PI lasting 6 or 7 h allowed to finish area pellucida formation and start hypoblast formation, but only 12 or 14 h of PI enabled the embryo transition to a higher stage of development. Since the turkey embryos are less advanced in the development at the time of egg laying than the hen embryos, in can be supposed that the SPIDES used in this study, which included a total of 8 h PI, allowed embryos from younger hens to form a hypoblast, but there was too little time for embryos from older hens with fully formed hypoblasts to pass to the next stage according to the bridging method of Hamburger and Hamilton (1951). In the case of this experiment, the significant effect of the turning of eggs during the storage was found to be an interesting result, both for embryos in 48 and 72 h of incubation (Table 1). In previous reports, particular attention was paid to the need to turning change the position of eggs in later incubation periods, which is necessary for the proper development of embryonic membranes (Deeming, 1989) and the orientation of the embryo before the hatching (Lundy, 1969; Elibol and Brake, 2008). Little attention was paid to the analysis of an effect of turning of eggs during the storage (Lundy, 1969; Elibol et al., 2002; Mahmud and Pasha, 2008). The studies published to this day do not explain whether the turning of eggs during the storage may affect the initial stage of embryonic development. The present study indicates that egg turning twice a day, i.e., 24 times during the 12 d storage period, contributes to the faster development of embryos in the initial incubation period. Incubation Duration and Hatching Synchrony Analyzing the conditions in which mass egg incubation is currently taking place in hatching plants, it can be concluded with complete conviction that the length of incubation time is of little importance compared to achieving the best possible synchronization of hatching, thus reducing the time between hatching the first and the last chick. It has been proven that the length of incubation, except that it is species-specific, can vary depending on the age and genotype of the mother (Vieira et al., 2005), egg weight (Ruiz and Lunam, 2002), as well as time and conditions of eggs storage before incubation (Fasenko, 2007). All these factors are now taken into account in commercial hatcheries, where the aim is to simultaneously incubate eggs from the same reproductive flocks and treated equally at the time of storage (Van de Ven, 2012). Despite this, the observed HW is between 24 and 48 h (Van de Ven et al., 2011; Tong et al., 2013), thus, far from observed in nature in precocial birds - 3 to 24 h, which clutch consists of at least several eggs (Vince, 1964; Eichholz and Towery, 2010). This difference can be explained by the fact that several eggs in a natural clutch are not much compared to a few tens of thousands of eggs set to the hatching apparatus. However, it should be remembered that the birds lay eggs for a few days before they start the incubation, so a single clutch consists of eggs “stored” for several days until the last egg, which is a signal to start hatching. The results confirm that the most important factor determining the length of incubation time and synchronization of hatch was the length of time the eggs were stored. Although this factor was not directly taken into account in the statistical analysis, the chicks from the COI groups (5 d storage), both in the case of young and old hens, hatched at the earliest in 483 and 482 h, respectively (Table 2). The time between the hatching of the first and the last chick was also the shortest in the B COI group (12 h) and not much longer, only by 6 h, in the A COI group (Figure 3). In general, the age of hens itself did not affect the incubation time (P = 0.723), as was the use of PI alone (P = 0.116). Chicks from PI eggs hatched after a relatively long incubation period: 485 h in the A SPIDES group, and significantly later, i.e., after 489 h in the B SPIDES group (Table 2). However, it should be noted that the chicks in both groups started the hatching late, in the A SPIDES group the first chicks were registered in 477 h of incubation, and in the B SPIDES group in 479 h of incubation. However, the hatching in the B SPIDES HW group was very short, only 13 h, and in the A SPIDES group it was relatively long, as much as 21 h (Figure 3). Dymond et al. (2013) demonstrated previously an effect of SPIDES on shortening the incubation time, but with a 4 h PI × 4 d of 21 d of cool storage system, thus providing twice as much PI hours as in the present study. Longer egg storage time in the study conducted by Dymond et al. (2013) could also be the reason why the authors obtained a much longer incubation time: 499–508 h, even in the SPIDES group. Also shortening the PI time to 6 or 12 hours in 21 d cool storage in the study by Dymond et al. (2013) resulted in a relatively long incubation period of 508 and 507 h, respectively, compared to the present study. Previously, Reijrink et al. (2010) also reported shortening of the incubation time but using 24 h PI during 14 d cool storage. It may be expected that 8 h PI in 12 d during storage time that has been applied in the study was a too short period of time to have a significant impact on the shortening of the incubation time and hatching synchronization. An effect of the turning of egg during the storage (P < 0.001) on incubation time and the interaction of all analyzed factors (P < 0.001) was observed in this study (Table 2). This is mainly due to the fact that the incubation time in the NCOI groups was relatively long for eggs from both young (488 h) and older (498 h) hens. In the case of B NCOI HW group it was prolonged to 22 h, and the first chick was observed in 478 h, in the A NCOI HW group it was synchronized since it lasted only 15 h, but the first chick was hatched only in 480 h of incubation (at the latest compared to all other groups) (Figure 3). However, the turning of eggs during the storage was primarily important in the case of incubation of eggs from older hens, where the incubation time in the B NSP group lasted only 482 h, and in the A NSP group up to 488 h. In both HW groups it was comparatively long: 28 h in the B NSP group, and 23 h in the A NSP group (Table 2; Figure 3). The available literature lacks information on an effect of turning of eggs during the storage on the length of incubation period and synchronization of hatch, which makes it difficult to discuss this topic. Thus, the results obtained in the present study seem to be the more interesting, but it should be noted that the duration of incubation and HW are modified by many factors at almost all stages of embryonic development. A recent study suggests that the synchronization of hatching occurs primarily in the piping time through the physical contact of eggs (receiving movements of “neighboring” embryos) (Reyna and Burggren, 2017), vocalizations (Brua, 2002) and even chemical communication (Webster et al., 2015). Therefore, only a simultaneous analysis of all these factors could clearly prove the action or lack of action of factors occurring much earlier, for example in the period preceding the incubation. Figure 3. View largeDownload slide Distribution of hatch in relation to the egg treatment in storage time, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. HW = hatch window—time between the early-hatching and late-hatching chicks. Figure 3. View largeDownload slide Distribution of hatch in relation to the egg treatment in storage time, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. HW = hatch window—time between the early-hatching and late-hatching chicks. Table 2. Results (means and ±SD) of pre-incubation and eggs turning of storage time on incubation duration.1 Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* 1Results of laboratory study. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–cDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 2. Results (means and ±SD) of pre-incubation and eggs turning of storage time on incubation duration.1 Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* 1Results of laboratory study. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–cDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Hatchability Characteristics Table 3 shows the results of the general incubation analysis carried out in the conditions of the hatchery. The number of eggs remaining for further incubation after the first candling (6 d) was dependent on all the analyzed factors (P <0.05) except for PI alone (P = 0.386). This index was referred to as an apparent fertility, because its percentages refer to the sum of eggs that are actually unfertilized and embryos dead up to 6 d of incubation. The age of the hens could significantly determine both egg fertilization and early embryonic mortality. This study confirms the deterioration of these indices in older hens, which has been proven many times before (Elibol et al., 2002). However, the applied modifications of egg storage conditions before the incubation could only have an impact on the number of dead embryos. The number of eggs from young hens remaining for further incubation was high in all groups (≥ 93.0%). Only the lower index in the NCOI group was confirmed statistically (P < 0.05). In the case of eggs from older hens, better (P < 0.05) apparent fertility characterized the SPIDES and NCOI groups as compared to COI and NSP. Hatching from set and apparently fertilized eggs was significantly lower (P < 0.001) in the case of eggs from older hens compared to eggs from younger hens mainly due to the higher (P < 0.001) number of eggs not hatched in the last incubation phase. The use of PI during the storage increased hatchability from set (P = 0.008) and apparently fertilized eggs (P = 0.009) and reduced the number of eggs not hatched (P = 0.022), contrary to the applied turning of eggs during the storage, which produced no effect on these indices (P > 0.05). The interaction of age × treatment in group B resulted in a higher (P < 0.001) hatch from the set eggs, but was not significant for hatching from apparently fertilized eggs, later mortality of embryos and the number of eggs not hatched (P > 0.05). Table 3. Results (means and ±SD) from hatchery of pre-incubation and eggs turning of storage time on fertility, hatchability, late embryonic mortality2, and chicks quality.1 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 1Results of field study. 1Values of 10,692 eggs in each group in both trials. 2Embriony died between 6 and 18 d of incubation. 3Embriony died during hatching. 4% Chicks qualified to rearing by hatchery staff. 5% Chicks not suitable for rearing; disqualified by hatchery staff. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 6Effects of the factors and their interaction were evaluated on all treatments. 7Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 8Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 3. Results (means and ±SD) from hatchery of pre-incubation and eggs turning of storage time on fertility, hatchability, late embryonic mortality2, and chicks quality.1 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 1Results of field study. 1Values of 10,692 eggs in each group in both trials. 2Embriony died between 6 and 18 d of incubation. 3Embriony died during hatching. 4% Chicks qualified to rearing by hatchery staff. 5% Chicks not suitable for rearing; disqualified by hatchery staff. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 6Effects of the factors and their interaction were evaluated on all treatments. 7Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 8Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Lower rates of fertilization, hatchability, and higher embryonic mortality at various incubation periods of older hens’ eggs are caused by a number of biological factors such as decreased sperm retention in the uterovaginal sperm host glands (Fasenko et al., 1992) and deteriorating egg quality (Reijrink et al., 2008). In this study, 2 × 4 h PI during 12 d of eggs storage allowed for an improvement of hatchability, mainly from eggs of older hens. This result is consistent with previous reports by Yassin et al. (2008) and Reijrink et al. (2010), who also improved hatching from eggs from older hens stored for 11 d and treated with PI. In the present study, although the effect of PI on hatching results was more significant in the case of eggs from older hens, it was also beneficial in case of the eggs from younger hens. This is evidenced primarily by the fact that hatching of eggs set and apparently fertilized in the A SPIDES group was significantly higher (P < 0.05) compared to the A NCOI group, and also higher (P < 0.05) in the case of hatching from apparently fertilized eggs and comparable in case of hatching from the set eggs compared to the A NSP group. Lower indices compared to the COI group were most likely associated with a longer storage period. Therefore, the obtained results indicate the general beneficial effect of PI on the hatchability indexes of eggs stored for a long time. It can be assumed, however, that with the age of hens, the beneficial effect of PI is related to the impact on other mechanisms of embryonic development, such as enabling to achieve hypoblast stage of the embryos safe for longer storage in younger hens’ eggs, and counteracting the deteriorating possibility of pH regulating by embryo on the eggs of older hens. The turning of eggs during the storage, as opposed to previously discussed indices (early embryonic development, time and synchronization of hatching), was neither positive nor negative in the case of hatchability results. Only apparent fertilization was advantageously dependent on the turning of eggs during the storage, but this concerned only the eggs from young hens where the lowest (P < 0.05) value was observed in the A NCOI group. Elibol et al. (2002) demonstrated that the turning of eggs during the storage may positively affect the hatching results in case of both eggs from young and old hens, but the effect is greater when the eggs are generally characterized by a low fertility rate and high survival rate of embryos. Egg fertilization in the experiment of Elibol et al. (2002) was at the level of 50.3–83.7% for the flock at the age of 62 wks, and therefore much lower compared to the apparent fertilization of eggs from hens from group B (70—73 wks) in this study: 86.2—90.5%. It can therefore be assumed that the generally high hatching quality of the eggs was the reason for the lack of influence of the turning of eggs during the storage. Earlier, Lundy (1969) demonstrated that high quality hatching eggs are less sensitive to the change in their position during the storage. Chicks Quality and Juvenile Growth Table 3 shows the percentage of chickens in the first grade of quality, based on the classification made in commercial conditions by the hatchery staff, and Table 4 shows the quality of chicks evaluated according to the methodology of Tona et al. (2003a). Generally, the chicks from the COI groups, i.e., hatched from eggs stored 5 d, were the best quality. Significant (P < 0.05) effect on the quality of chicks in case of the eggs stored 12 d in both analyzes was noted for an age of hens and the use of PI during egg storage. Chicks from younger hens and those that hatched in the SPIDES groups were characterized by better quality. Chicks from older hens usually have higher BW which is associated with higher egg weight and a strong correlation between these traits (Pinchasov, 1991; Willemsen et al., 2008). These results were confirmed in this study (Table 5 and Figure 4). For this reason, BW was not taken into account when evaluating the quality on the Tona's scale when chicks from flocks of different ages are compared, especially that according to Dymond et al. (2013) it shows positive correlations only with BW in the first 2—3 wks of life. In turn, BW in this study does not correlate with BW at the age of slaughter (Figure 4). According to Robinson et al. (1991) and Fasenko et al. (1992) poorer hatchability of eggs from older hens is associated with aging of the preovulatory oocyte and changes in the composition of yolk, but it cannot be excluded that it may contribute to poorer quality of chicks for which these changes are not fatal in the embryonic period. Contrary to this study, Reijrink et al. (2010) did not show any effect on the quality of day-old chicks under the influence of PI during egg storage. However, the authors took into account other characteristics of the chicks, except for navel quality, such as body dimensions, rectal temperature and the weight of selected organs. This could be the reason for obtaining different results. In the present study, the better quality of the chicks in the A and B SPIDES groups resulted primarily from giving them more points for the appearance of down, activity and no changes in the area of the navel. Poor quality of the navel and down also contributed to the lower score of chicks in other groups, especially in the B NCOI group, where low scores were also awarded for activity and legs. It seems that the better quality of the chicks from the SPIDES groups could have been caused by the hatching process itself. Although the chicks in both groups hatched late (Table 2), they started the hatching late (Figure 3). As a consequence, the chicks in both groups stayed in the hatching apparatus about several hours after the hatching (up to 504 h of incubation). This time is enough to dry the down and straighten the legs so that the chicks can move freely, and at the same time do not cause dehydration due to too long staying at a high temperature without an access to water. Figure 4. View largeDownload slide Correlation results between egg weight and chicks hatching weight (higher up) and chicks hatching weight and chicks weight in 42 d (under) for treatment groups, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation, R2 = coefficients of determinations * significant and NS not significant effect in ANOVA at 0.05 probability level. Figure 4. View largeDownload slide Correlation results between egg weight and chicks hatching weight (higher up) and chicks hatching weight and chicks weight in 42 d (under) for treatment groups, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation, R2 = coefficients of determinations * significant and NS not significant effect in ANOVA at 0.05 probability level. Table 4. Results (means and ± SD) of pre-incubation and eggs turning of storage time on chicks quality.1,2 Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* 1Results of laboratory study. 2According to Tona et al. (2003a). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 3Effects of the factors and their interaction were evaluated on all treatments. 4Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 5Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 4. Results (means and ± SD) of pre-incubation and eggs turning of storage time on chicks quality.1,2 Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* 1Results of laboratory study. 2According to Tona et al. (2003a). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 3Effects of the factors and their interaction were evaluated on all treatments. 4Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 5Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 5. Means (±SD) for eggs weight and chicks body weight and comparisons based on analysis of variance and Duncan's multiple range test.1 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 1Results of laboratory study and experimental farm. Number of chicks: A COI = 126, A NSP = 120, A SPIDES = 123, A NCOI = 116, B COI = 114, B NSP = 108, B SPIDES = 114, B NCOI = 109. EW = egg weight. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–dDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 5. Means (±SD) for eggs weight and chicks body weight and comparisons based on analysis of variance and Duncan's multiple range test.1 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 1Results of laboratory study and experimental farm. Number of chicks: A COI = 126, A NSP = 120, A SPIDES = 123, A NCOI = 116, B COI = 114, B NSP = 108, B SPIDES = 114, B NCOI = 109. EW = egg weight. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–dDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large The turning of eggs during the storage did not affect the quality of the chicks evaluated by the hatchery staff (Table 3), but significantly affected the chicken improvement according to the Tona's method (Table 4). This result is a consequence of the lowest score given to the chicks from the A and B NCOI groups. Few reports of an effect of turning of eggs during incubation indicate the positive effects of such treatment, which is explained by a better orientation of the embryo before the hatching, restriction of embryo or embryonic membrane adhesion to the shell membranes and stimulation of timely closure of chorioallantis at the sharp end of the egg (Wilson, 1991; Tona et al., 2003b). It seems, however, that the positive effect of turning of eggs during the storage on chicks quality could not be caused by the same factors. Nevertheless, the chicks from the A and B NCOI groups showed the worst quality of down, navel, and the remains of membranes and shells integrated with the body as well as the reluctance to move were the most frequent among them compared to the chicks from other groups. The last three defects negatively affect the evaluation of the chicks in the Tona's score, but they can and probably are neglected at the mass hatching in the hatchery, which was a probable cause of the differences in the evaluation done experimentally and industrially. Mechanisms that have contributed to an improved quality of chicks under the influence of turning of eggs during the storage are unknown and require further research, especially that this is the first report on this topic according to the authors’ knowledge. Analysis of the course of chicks growth demonstrated that age of chicks affected the hatching BW, which was strongly correlated in all experimental groups with egg weight, and BW in 7 and 35 d, but had no effect on BW in 42 d (Table 5 and Figure 4). However, hatching BW of chicks from older hens was significantly higher, as much as 12 g on average (∼ 1/5 hatching BW) compared to hatching BW of the chicks from young hens, and average BW in 42 d was similar, higher for chicks from older hens, only 60 g on average (∼2% BW in 42 d). Particular attention should be paid to BW of the chicks in the A and B NCOI groups. Although the chicks in the A NCOI group had a significantly lower hatching BW compared to the B NCOI group chicks (by 14.5 g at similar variability; 2.2—2.3 g), the BW of the chicks from young hens in 42 d was higher by 158 g on average, and although this is not a significant difference, considering the hatching BW, it can be concluded that chicks from young hens show better growth potential. These results are consistent with previous reports by Tona et al. (2004b), in which there was no correlation of hatching BW with BW in 42 d in broilers, and chicks from younger hens obtained slightly higher BW in 42 d compared to BW of the chicks from older hens, which showed a significantly higher hatching BW. The use of PI and the turning of eggs during the storage did not have a significant positive or negative effect on BW of the chicks in 42 d of life (P > 0.05). However, a significant effect was observed for age × treatment interaction (P = 0.011). Chicks that hatched from older hens’ eggs subjected to PI and eggs turning during the storage (B NSP and B SPIDES) reached a higher BW (P < 0.05) compared to B NCOI chicks. However, this dependence did not occur in the case of chicks hatched from the eggs of younger hens (Table 5). This is probably the consequence of a significantly better quality of chicks at the time of hatching (Table 4). Dymond et al. (2013) showed that the use of 6 or 12 h PI during egg storage did not affect the hatching BW of the chicks, but the chicks from SPIDES groups were heavier than those hatched from eggs without PI applied to 4 wk of life, the authors did not conduct the observation further. In conclusion, the present study showed that the use of 2 × 4 h PI in 12 d egg storage period could have an effect on the initial acceleration of embryonic development in eggs of young hens, contributing to the levelling of embryonic development stage in eggs from young and old hens to 72 h of incubation. The eight-hour PI did not affect the length of the incubation period, HW and BW of the chicks at 42 d of life, but increased the hatchability of the eggs set and apparently fertilized and reduced the number of not hatched eggs, and also affected the quality of the chicks. Turning of eggs every 12 h during the storage had a positive effect on the development of embryos, shortening the incubation time and quality of chicks, but had no effect on hatchability and BW indices up to 42 d of life. The obtained results recommend the use of 2 × 4 h PI with simultaneous turning of eggs every 12 h during the storage to improve the results in industrial hatcheries. In addition, the applied modifications can be used without any harm in the case of an incubation of eggs from both young reproductive flocks and in later laying phase. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

# Short periods of incubation, egg turning during storage and broiler breeder hens age for early development of embryos, hatching results, chicks quality and juvenile growth

Poultry Science, Volume 97 (9) – Sep 1, 2018
13 pages

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
DOI
10.3382/ps/pey163
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29762735
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### Abstract

ABSTRACT An effect of modification of storage conditions of the eggs of broiler breeder flocks at the age of 49-, 52- and 70-, 73-wks of life on an early embryonic development, hatching time and synchronization, hatchability rates, chicks quality and broiler growth was investigated. The eggs were divided into 4 experimental groups: COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = were treated with 4 h pre-incubation at 30°C and 50–55% air humidity, delivered at 5 and 10 d over of 12 d of storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Eggs from older hens were characterized by poorer hatchability and poorer chicks quality. The use of 2 × 4 h pre-incubation in 12 d of eggs storage could have an effect on the initial acceleration of embryonic development in eggs of young hens, contributing to the alignment of embryos development in eggs from young and older hens to 72 h of incubation. Pre-incubation had no effect on the length of incubation period, hatching window, but it increased the hatchability of the set and apparently fertilized eggs and decreased the number of eggs not hatched, and also improved chicks quality. Eggs turning by 90° every 12 h during the storage positively affected the embryonic development, shortening the incubation time and the quality of chicks, but had no effect on hatchability rates and body weight in 42 d of life. Based on the obtained results, it can be concluded that the applied modifications can be effective in counteracting the negative effects of storage of hatching eggs from both young and older birds. INTRODUCTION In commercial hatcheries, obtaining a large number of simultaneously hatched chicks is often only possible after a period of eggs storage for several days. However, it has been proven that the length of eggs storage time is negatively correlated with hatchability rates (Fasenko et al., 2001a,b; Dymond et al., 2013) and the quality of the chicks (Becker, 1960; Tona et al., 2003a). Standard methods of counteracting these phenomena were eggs storing no longer than 7 d at a temperature below physiological zero, i.e., 20–21°C (Edwards, 1902). According to Fasenko et al. (2001a) under these conditions, blastodermal cells of the embryos exhibit mitotic activity but embryo development is inhibited. According to Reijrink et al. (2008), such a solution is not enough in case of longer eggs storage, since there is still an increase in the number of necrotic cellular indicators contributing to early mortality of the embryos. Therefore, it is important to examine and refine techniques aimed to counteract the reduction in hatching ability of eggs stored for more than 7 d. Several methods of eggs storage conditions modification in order to prolong the period in which they retain their high ability to hatch have been described in the literature. Pre-incubation, i.e., temporary heating of eggs, which is supposed to imitate the natural conditions that the birds provide to the eggs before they start to hatch them, turned out to be the most effective method (Meijer and Siemers, 1993). Fasenko et al. (2001a,b) demonstrated that a single pre-incubation (PI) allows the embryos to finish the formation of hypoblasts, which increases the ability of hatching even with 14 d of eggs storage. Dymond et al. (2013) modified this method by analyzing an effect of several PIs during the storage (SPIDES—short periods of incubation during egg storage). In turn, Elibol and Brake (2008) showed that the change in the position of eggs during the storage significantly reduces early mortality, which leads to improved hatching results. The aim of the study was to determine the applicability of SPIDES and turning of eggs during the storage and the age of broiler breeder hens on the improvement of hatching indices in industrial conditions and on the growth of the chicks. MATERIALS AND METHODS Experimental Design Two independent trials were conducted using eggs produced by younger (A) and older (B) broiler breeder flocks that were used in trials 1 (August 2017), and 2 (September 2017), respectively. Each of the two trials covered the production part carried out at the Poultry Hatching Plant belonging to CEDROB S.A. (Ujazdówek, PL), and the laboratory part carried out at the Poultry Breeding Division, University of Life Sciences (Warsaw, PL). Due to the practical conditions in a commercial hatchery, and analysis of the development of chicken embryos below 13 d of incubation, this experiment not require application to and consent of the National Ethical Commission at the Ministry of Science and Higher Education in Poland (Directive2015/266/EC; Public Information Biulletin, 2017). Breeder Flock The study was carried out using the eggs from two broiler breeder flocks Ross 308. Flock A in 49- and 52-wk-old and flock B in 70- and 73-wk-old. Birds were fed restricted diets according to standard Ross guidelines (Aviagen, 2016). At the time the beginning of the experiment, the number of birds in flock A was 51,239, where male: female ratio was 1:7. The number of birds in flock B was 42,900, and male: female ratio was 1:10, and 20% of cocks in the 63 wk of life, were replaced with younger ones (21 wk of life). The light schedule for both flocks included 13 h of light and 11 h of darkness. The conditions for both flocks maintaining were standard: litter with a fully automated system of feeding, watering and eggs collecting. Eggs Storage Profiles and Experimental Classes Eggs after collection were laid with the large end up (LEU) on the hatching trays, which were then put on trolleys. The eggs were stored in the farm storehouse for approximately 12 h (16°C and 75% RH). The eggs were then transported using adapted trolleys with shock absorbing wheel, under controlled microclimate conditions (15–18°C and 65–75% RH). Transport to the CEDROB S. A Hatchery lasted about 2 hours, after which the eggs were placed in the hatching egg storehouse under the same conditions as on the farm. Experimental eggs (1 trolleys × 4 treatment × 2 flock × 2 trial = 86,016 eggs) were stored together with other commercial eggs in a storehouse holding once 6 million eggs. There was 5376 eggs on one trolley (32 trays x 168 eggs), which constituted one experimental group. Eggs were stored 5 and 12 d using SPIDES consisting of only two PIs, not four as described by Dymond et al. (2013), but with the concurrent introduction of turning of eggs during storage: ⇒ COI = control incubation treatment were eggs storage 5 d, at turning every 12 h ⇒ NSP = non-SPIDES control eggs were eggs storage 12 d, at turning every 12 h ⇒ SPIDES = were treated with 4 h PI at 30°C and 50–55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h ⇒ NCOI = negative control incubation were eggs storage 12 d, no turning and no PI. The turning of eggs during the storage consisted in setting the angle of trays inclination by 90° every 12 h, so the eggs were always in the 45° inclination from the vertical plane, but with the change of the side plane (up-down). PI was carried out in setter (Jamesway, Inc. P120–129,024, Cambridge, Canada), to which entire egg trolleys were introduced and then returned to the storehouse. Incubation The eggs were placed in the setter (Jamesway, Inc. P120–129,024, Cambridge, Canada). Incubations were carried out at parameters 37.5–38.5°C and 50–60% RH. Egg turning (by 90°) during the incubation was carried out at a frequency of 12 h, the CO2 level was monitored from 5 d of incubation (3.500–4.500 ppm). One incubator contained at one time 4 trolleys with eggs arranged in 2 rows. The trolleys were moved every 24 hours clockwise to eliminate the effect of different distance from the fan. Eggs candling was performed in the 6 d of incubation, all eggs rejected were considered unfertilized. In the 18 d incubation, a second candling was carried out on the basis of which the late mortality of the embryos was determined. After an candling in 18 d, the eggs were placed in breeding baskets and placed in hatcher (Jamesway, Inc. P40–43,008, Cambridge, Canada). The hatching of the chicks was carried out in the horizontal position of the eggs at 37.6°C and 65% RH. The chicks were removed exactly 504 h after the start of incubation. The hatching was carried out using a separator (Chich/Shell Separators, KL, London, Ontario, Canada) which is used for shells separation as well as counting and vaccination of the chicks. Apparent fertile eggs (%), hatchability of set eggs (%) and hatchability of fertile eggs (%) were calculated on the basis of the results obtained, where: \begin{eqnarray*} &&{Apparent\,fertile\,eggs\,}\\ && = \frac{\begin{array}{@{}l@{}} number\,of\,set\,eggs\, - \,number\,of\,rejected\,eggs\,\\ during\,candling\,at\,6\,d\,of\,incubation \end{array}}{{number\,of\,set\,eggs}}\\ \quad && \times\, 100 \end{eqnarray*} s \begin{eqnarray*} &&{Hatchability\,of\,set\,eggs}\,\\ && = \,\frac{number\,of\,hatched\,chicks}{number\,of\,set\,eggs}\times\, 100 \end{eqnarray*} \begin{eqnarray*} && Hatchability\,of\,apparent\,fertile\,eggs\,\\ && = \,\frac{number\,of\,hatched\,chicks}{number\,of\,apparent\,fertile\,eggs} \times\, 100 \end{eqnarray*} Additional 100 eggs for each treatment group (total for both trials: 1600 eggs with possibly similar weight: 55.0–62.0 g for flock A and 67.0–74.0 g for flock B), treated in the same manner in the storehouse as the eggs incubated at the hatchery, were transported to the laboratory of Poultry Breeding Division, University of Life Sciences (Warsaw, PL). The transport took about 1.5 h (119 km). The eggs were transported on trlleys adapted to transport hatching eggs with a mean of transport (Mercedes-Benz, Daimler AG, Germany). The cars are equipped with full suspension and climate control determining the microclimae conditions inside the zone with hatching eggs (15–18°C and 65–75% RH). Then, all the eggs were numbered and placed in the setter with humidity pump (OvaEasy 380 Setter, Brinsea, Weston, UK). Incubations were carried out at 37.6–38.0°C and 50–65% RH, but without the possibility of CO2 level control. The eggs were turned automatically every by 90° 12 h. At 48 and 72 h of incubation, 15 eggs (240 eggs in the entire experiment) were removed from each group to control the development of embryos. The remaining 70 eggs from each group (1120 eggs in the entire experiment) were removed from the incubators and manually candled (OvaScope Brinsea, Weston, UK) after 18 d of incubation removing the eggs unfertilized and not hatched fetuses. The remaining eggs were placed in hatcher with clear, double-glazed observation door (OvaEasy 380 Hatcher, Brinsea, Weston, UK). Determination of Embryonic Characteristics In each experimental group, 10 embryos were analyzed in 48 and 72 h of incubation. The remaining 5 out of 15 eggs for this part of the study constituted a reserve. The embryos were isolated following the methodology described by Gupta and Bakst (1993). It consisted in applying a filter ring (paper disc) on the perivitelline layer so that the entire embryo was located in the central part of the ring. Then, after moistening the ring with protein residues for better integration, one of the periphery of the ring was held with tweezers and the vitelline membrane was cut with scissors on its outer edge. The filtration ring together with the PL on which the embryo was located was transferred to the microscope slide by placing the inside up. After gentle rinsing with deionized water to remove the remaining yolk, the specimen was viewed using a stereoscopic microscope (Olympus SZX10, Shinjuku, Japan). A microscope image was taken for each embryo and its developmental stage was classified according to the scale described by Hamburger and Hamilton (1951). In order to compare the degree of embryonic development in 48 h of incubation, the number of somites, the size and shape of the heart (if it was visible), brain development, primary optic vesicles and left head flexion advancement were noted in the SEM image (Figure 1). To analyze the degree of embryos development in 72 h of incubation, the following traits were used: number of somites pairs, body length measured from the tail tip to mesencephalon tip, wing- and leg buds presence, heart development, brain development, pigmentation of the eye and head flexion advancement (Figure 2). Figure 1. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 48 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50–55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 1. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 48 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50–55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 2. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 72 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Figure 2. View largeDownload slide Broiler chick embryos stereoscopic microscope image of 72 h of incubation, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. Hatching Time and Chicks quality The hatching of the chicks was carried out individually at 37.6°C and 65% RH. The equipment of the hatching apparatus with a transparent door made it possible to precisely determine the time of hatching of the first chicks in a given experimental group. From that moment, each hatching apparatus was checked every 1 h. Hatched chicks were recorded (hatching time and egg number), weighed and tagged on the right wing with an individual chick tag. Next, the chicks were transferred to the collective hatching apparatus (R COM Maru, Bandury, UK) in which they stayed until the end of hatching. Hatching time monitoring allowed to calculate the percentage of chicks hatched in particular time intervals and the total time of hatching referred to as hatch window (HW = time between first and last hatched chick). The quality of chicks hatched in the laboratory was determined in accordance with the methodology described by Tona et al. (2003a). This method involves the point evaluation of several characteristics of the hatchling according to their meaning, in which the maximum sum of points is 100. These characteristics include: activity, down and appearance, retracted yolk, eyes, legs, navel area, remaining membrane, and remaining yolk. The quality of chicks hatched in the hatchery was evaluated in a standard manner due to their large numbers. All chicks that were qualified in the hatchery for the rearing were determined as first grade chicks. The other chicks, i.e., those that were removed from the separator and considered unsuitable for rearing, were qualified as the second grade. Generally, with the practical distribution of the chicks to the first and second categories, the three characteristics are considered: down appearance: chicks with wet down, or the apparent deficiencies of down are classified in the second category; navel appearance: open, visible remains of the yolk sac and the distinct discoloration of the navel area disqualifies the chick; other visible defects such as Loco genetic defects, or non-genetic innate variations, e.g., lack or additional limbs, also disqualify the chicks (hatchery personnel, unpublished data). Chicks Growth Chicks hatched in the laboratory of Poultry Breeding Division, University of Life Sciences (Warsaw, PL) were transported to the experimental farm RZD Wilanów-Obory Goździe owned by the University of Life Sciences (Warsaw, PL). The chicks were reared in experimental groups in separate compartments without gender distribution (with an initial stock density of 11–14 chicks/m2) up to 42 d of life. From each treatment, the chickens were divided into 3 replications. In each replication depending on the numbers of hatched chicks, reared 18 to 23 chickens. The light schedule was as follows: 24L: 0D for the first 2 d, then reduced to 16L: 8D in 8 d and maintained in this schedule until the end of rearing. The temperature was consistent with the relevant guidelines for broiler chickens: 35°C for 1st wk then successively reduced for 18°C in 6th wk. Chicks were fed ad libitum a commercially produced wheat-maize-soya-based diets in a 3-stage system: 0–14 d, starter: 12.5 MJ of energy, 21.9% crude protein; 15–35 d, grower: 12.8 MJ of energy, 20.8% crude protein; 36–42 d, finisher: 13.2 MJ of energy, 19.3% crude protein. Body weight (BW) of the chicks was controlled individually every 7 d, up to 42 d of life. Only those chickens that survived until the last weighing were included in the analysis. Mortality in all groups was low and did not exceed 1.80% (data not shown). Statistical Analysis Comparison of means between various treatments of eggs and flock age was conducted based on two-way analysis of variance (ANOVA) and Duncan's multiple range test at 0.05 significance level. The first factor in ANOVA was flock age with two levels and the second factor was treatment with four levels. Effects of the factors and their interaction were evaluated on all treatments or only for selected treatments to evaluate effects of egg turning and pre-incubation. Effect of egg turning was evaluated using dataset for NSP and NCOI treatments, while effect of pre-incubation was determined using dataset for NSP and SPIDES treatments. Analysis of variance was performed according to linear model: \begin{equation*} {Y_{ijk}} = \mu + {\alpha _i} + {\boldsymbol{\beta }_j} + {\left( {\alpha \boldsymbol{\beta }} \right)_{ij}} + {\varepsilon _{ijk}} \end{equation*} where: Yijk—is observed value of the trait, —is effect of ith flock age, βj—is effect of jth treatment, (αβ)ij—is interaction of flock age and treatment, —is error term. Effects of flock age and treatment were treated as fixed and independent (not nested). Evaluation of effects of flock age and treatment and their interaction for binomial data advances in embryonic development in 48 and 72 h of incubation (according to Hamburger and Hamilton (1951)) was conducted using generalized linear model with logit link function. The ANOVA analyses were performed similarly ANOVA, i.e., for total dataset and for selected treatments to evaluate effect of PI and egg turning. For data obtained in CEDROB S. A. hatchery, the basic unit used for calculations was a single hatching tray (168 eggs × 32 trays on one trolley = one treatment group). The results were presented as P-values for F-test in two-way ANOVA. Relationships between egg weight and chicks weight were evaluated using simple linear regression. The regression lines were presented graphically as well regression equations, and coefficients of determinations (R2) were presented. All variables followed normal distribution; only parametric statistical procedures were applied as well means and standard deviations were presented. All results for one flock collected in two dates (for flocks A of 49 and 52-wk-old and B of 70 and 73-wk-old) are presented as mean values, because the difference between the trials was not significant. Therefore, for each flock (A and B), the chicken BW calculations were made on the basis of 6 replications (3 for each of the trials). The statistical analyses were performed by using Statistica 12.0 software (Statsoft, INC, 2014). RESULTS AND DISCUSSION Embryonic Development The embryos from older hens, as well as those developing in pre-incubated and turned eggs, were at a higher stage of development in 48 h of incubation compared to the embryos developing in non-treated eggs (Table 1 and Figure 1). The use of PI alone (P = 0.580) did not affect the embryos’ qualification in 48 h of incubation to a higher stage of development, but the application of turning of eggs during the storage had a significant impact on this feature (P = 0.025). The relationships observed in 48 h of incubation between the developmental stage of embryos, hens’ age, and the modifications of egg storage conditions were slightly weakened in 72 h of incubation (Table 1 and Figure 2). A significant effect on the development of embryos up to 72 h of incubation was noted in case of the turning of eggs during the storage (P = 0.034) and in combination with PI (P = 0.040). The age of hens did not affect the embryonic development stage in 72 h of incubation (P = 0.052). Table 1. Effect of pre-incubation and eggs turning of storage time on embryonic development at 48 and 72 h of incubation.1 Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* 1Results of laboratory study. 2Values of total 20 embryos in each group in both trial. 3According to Hamburger and Hamilton (1951). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. *Significant effect in generalized linear model with logit link function at 0.05 probability level. 4Effects of the factors and their interaction were evaluated on all treatments. 5Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 6Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 1. Effect of pre-incubation and eggs turning of storage time on embryonic development at 48 and 72 h of incubation.1 Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* Embryonic develpoment2,3 Flock age Treatment 48 h of incubation 72 h of incubation HH11 HH12 HH13 HH14 HH18 HH19 HH20 A COI n 0 6 10 4 4 16 0 NSP n 2 7 7 4 7 13 0 SPIDES n 2 8 8 2 6 14 0 NCOI n 5 10 3 2 11 9 0 B COI n 1 2 11 6 5 15 0 NSP n 3 7 5 5 5 15 0 SPIDES n 3 6 6 5 4 16 0 NCOI n 6 10 2 2 12 8 0 P-values based on complete dataset (all treatments) Effect of Age4 0.035* 0.052 Effect of Treatment4 0.022* 0.040* Age x Treatment4 0.685 0.770 P-values based on subsets of the data (selected treatments) Effect of PI5 0.580 0.601 Effects of eggs turning6 0.025* 0.034* 1Results of laboratory study. 2Values of total 20 embryos in each group in both trial. 3According to Hamburger and Hamilton (1951). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. *Significant effect in generalized linear model with logit link function at 0.05 probability level. 4Effects of the factors and their interaction were evaluated on all treatments. 5Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 6Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large A more advanced embryonic development stage in 48 h of incubation in eggs from older hens was probably related to the fact that older hens tend to produce eggs in which the embryo is more developed at the time of laying (Fasenko et al., 1992). It results from the fact that the production of eggs decreases with an age due to individual series shortening. As a consequence, older hens lay more eggs in the early stages of the series. In the study conducted by Reijrink et al. (2009), embryos in the eggs from the beginning of a particular series, which are numerous in the case of older hens, were already characterized the formed hypoblast, in contrast to embryos from the late stages of the series where hypoblast was at the stage of formation. The occurrence of this phenomenon is explained by the fact that the first egg in the series remains in the F1 follicle phase about 16 h longer than the next eggs, which causes a faster beginning of development (Scott and Warren, 1936), and that the embryos in the first eggs of the series later start the embryonic pause as a consequence of prolonged stay in the shell gland (Berg, 1945). It is interesting, why an effect of hen age on embryonic development was not noticeable already in 72 h of incubation (Table 1). It can be assumed that other environmental factors at some point begin to slow down the development of embryos in the eggs of older hens. According to Reijrink et al. (2008) this may be associated with a greater difficulty for embryos in regulating the pH of the environment in eggs from older hens when the quality of egg whites and egg shells deteriorates progressively. It is difficult to clearly indicate in which moment of incubation the prevalence of embryos in the eggs of older hens gives way to those developing in eggs from younger hens. The results obtained in the present study indicate that this is a period between 48 and 72 h of incubation. However, an analysis of the embryonic development stage did not allow to include any of the 160 embryos to the HH20 stage (Table 1), i.e., embryos 70–72 h according to Hamburger and Hamilton (1951). This was mainly due to the fact that no clearly observed wings- and leg-buds were observed (Figure 2), despite that the analyzes were performed exactly after 72 h of incubation. However, many environmental factors like the genotype or nutrition of reproductive flocks can modify early embryonic development (Novo et al., 1997; Tona et al., 2004a). Therefore, in future studies, an effect of faster development of embryos from older hens may no longer be observed even in 48 h of incubation. Since the aim of pre-incubation of the eggs is mainly to allow the embryos to fully form the hypoblast before the beginning of the proper incubation, Fasenko et al. (2001a) and Reijrink et al. (2009) formulated the hypothesis that it is important primarily for embryos from young hens. In this study, no statistical differences were observed in the embryonic development stage under the influence of PI in the eggs of young and old hens (Table 1). Thus, it should be considered whether this lack of statistical differences is an evidence that PI actually stimulates the development of embryos in the eggs of young hens, since as previously reported, they are at a lower stage at the time of eggs laying. On the example of turkey embryos, Fasenko et al. (2001a) showed that a single PI lasting 6 or 7 h allowed to finish area pellucida formation and start hypoblast formation, but only 12 or 14 h of PI enabled the embryo transition to a higher stage of development. Since the turkey embryos are less advanced in the development at the time of egg laying than the hen embryos, in can be supposed that the SPIDES used in this study, which included a total of 8 h PI, allowed embryos from younger hens to form a hypoblast, but there was too little time for embryos from older hens with fully formed hypoblasts to pass to the next stage according to the bridging method of Hamburger and Hamilton (1951). In the case of this experiment, the significant effect of the turning of eggs during the storage was found to be an interesting result, both for embryos in 48 and 72 h of incubation (Table 1). In previous reports, particular attention was paid to the need to turning change the position of eggs in later incubation periods, which is necessary for the proper development of embryonic membranes (Deeming, 1989) and the orientation of the embryo before the hatching (Lundy, 1969; Elibol and Brake, 2008). Little attention was paid to the analysis of an effect of turning of eggs during the storage (Lundy, 1969; Elibol et al., 2002; Mahmud and Pasha, 2008). The studies published to this day do not explain whether the turning of eggs during the storage may affect the initial stage of embryonic development. The present study indicates that egg turning twice a day, i.e., 24 times during the 12 d storage period, contributes to the faster development of embryos in the initial incubation period. Incubation Duration and Hatching Synchrony Analyzing the conditions in which mass egg incubation is currently taking place in hatching plants, it can be concluded with complete conviction that the length of incubation time is of little importance compared to achieving the best possible synchronization of hatching, thus reducing the time between hatching the first and the last chick. It has been proven that the length of incubation, except that it is species-specific, can vary depending on the age and genotype of the mother (Vieira et al., 2005), egg weight (Ruiz and Lunam, 2002), as well as time and conditions of eggs storage before incubation (Fasenko, 2007). All these factors are now taken into account in commercial hatcheries, where the aim is to simultaneously incubate eggs from the same reproductive flocks and treated equally at the time of storage (Van de Ven, 2012). Despite this, the observed HW is between 24 and 48 h (Van de Ven et al., 2011; Tong et al., 2013), thus, far from observed in nature in precocial birds - 3 to 24 h, which clutch consists of at least several eggs (Vince, 1964; Eichholz and Towery, 2010). This difference can be explained by the fact that several eggs in a natural clutch are not much compared to a few tens of thousands of eggs set to the hatching apparatus. However, it should be remembered that the birds lay eggs for a few days before they start the incubation, so a single clutch consists of eggs “stored” for several days until the last egg, which is a signal to start hatching. The results confirm that the most important factor determining the length of incubation time and synchronization of hatch was the length of time the eggs were stored. Although this factor was not directly taken into account in the statistical analysis, the chicks from the COI groups (5 d storage), both in the case of young and old hens, hatched at the earliest in 483 and 482 h, respectively (Table 2). The time between the hatching of the first and the last chick was also the shortest in the B COI group (12 h) and not much longer, only by 6 h, in the A COI group (Figure 3). In general, the age of hens itself did not affect the incubation time (P = 0.723), as was the use of PI alone (P = 0.116). Chicks from PI eggs hatched after a relatively long incubation period: 485 h in the A SPIDES group, and significantly later, i.e., after 489 h in the B SPIDES group (Table 2). However, it should be noted that the chicks in both groups started the hatching late, in the A SPIDES group the first chicks were registered in 477 h of incubation, and in the B SPIDES group in 479 h of incubation. However, the hatching in the B SPIDES HW group was very short, only 13 h, and in the A SPIDES group it was relatively long, as much as 21 h (Figure 3). Dymond et al. (2013) demonstrated previously an effect of SPIDES on shortening the incubation time, but with a 4 h PI × 4 d of 21 d of cool storage system, thus providing twice as much PI hours as in the present study. Longer egg storage time in the study conducted by Dymond et al. (2013) could also be the reason why the authors obtained a much longer incubation time: 499–508 h, even in the SPIDES group. Also shortening the PI time to 6 or 12 hours in 21 d cool storage in the study by Dymond et al. (2013) resulted in a relatively long incubation period of 508 and 507 h, respectively, compared to the present study. Previously, Reijrink et al. (2010) also reported shortening of the incubation time but using 24 h PI during 14 d cool storage. It may be expected that 8 h PI in 12 d during storage time that has been applied in the study was a too short period of time to have a significant impact on the shortening of the incubation time and hatching synchronization. An effect of the turning of egg during the storage (P < 0.001) on incubation time and the interaction of all analyzed factors (P < 0.001) was observed in this study (Table 2). This is mainly due to the fact that the incubation time in the NCOI groups was relatively long for eggs from both young (488 h) and older (498 h) hens. In the case of B NCOI HW group it was prolonged to 22 h, and the first chick was observed in 478 h, in the A NCOI HW group it was synchronized since it lasted only 15 h, but the first chick was hatched only in 480 h of incubation (at the latest compared to all other groups) (Figure 3). However, the turning of eggs during the storage was primarily important in the case of incubation of eggs from older hens, where the incubation time in the B NSP group lasted only 482 h, and in the A NSP group up to 488 h. In both HW groups it was comparatively long: 28 h in the B NSP group, and 23 h in the A NSP group (Table 2; Figure 3). The available literature lacks information on an effect of turning of eggs during the storage on the length of incubation period and synchronization of hatch, which makes it difficult to discuss this topic. Thus, the results obtained in the present study seem to be the more interesting, but it should be noted that the duration of incubation and HW are modified by many factors at almost all stages of embryonic development. A recent study suggests that the synchronization of hatching occurs primarily in the piping time through the physical contact of eggs (receiving movements of “neighboring” embryos) (Reyna and Burggren, 2017), vocalizations (Brua, 2002) and even chemical communication (Webster et al., 2015). Therefore, only a simultaneous analysis of all these factors could clearly prove the action or lack of action of factors occurring much earlier, for example in the period preceding the incubation. Figure 3. View largeDownload slide Distribution of hatch in relation to the egg treatment in storage time, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. HW = hatch window—time between the early-hatching and late-hatching chicks. Figure 3. View largeDownload slide Distribution of hatch in relation to the egg treatment in storage time, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. HW = hatch window—time between the early-hatching and late-hatching chicks. Table 2. Results (means and ±SD) of pre-incubation and eggs turning of storage time on incubation duration.1 Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* 1Results of laboratory study. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–cDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 2. Results (means and ±SD) of pre-incubation and eggs turning of storage time on incubation duration.1 Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* Flock age Treatment N Incubation duration (h) A COI 126 483 ± 3.2c NSP 121 488 ± 5.4a,b SPIDES 123 485 ± 5.7b NCOI 118 488 ± 3.9a,b B COI 114 482 ± 3.2c NSP 109 482 ± 5.5c SPIDES 115 489 ± 3.1a NCOI 111 489 ± 4.7a P-values based on complete dataset (all treatments)  Effect of Age2 0.723  Effect of Treatment2 <0.001*  Age x Treatment2 <0.001* P-values based on subsets of the data (selected treatments)  Effect of PI3 0.116  Effects of eggs turning4 <0.001* 1Results of laboratory study. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–cDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Hatchability Characteristics Table 3 shows the results of the general incubation analysis carried out in the conditions of the hatchery. The number of eggs remaining for further incubation after the first candling (6 d) was dependent on all the analyzed factors (P <0.05) except for PI alone (P = 0.386). This index was referred to as an apparent fertility, because its percentages refer to the sum of eggs that are actually unfertilized and embryos dead up to 6 d of incubation. The age of the hens could significantly determine both egg fertilization and early embryonic mortality. This study confirms the deterioration of these indices in older hens, which has been proven many times before (Elibol et al., 2002). However, the applied modifications of egg storage conditions before the incubation could only have an impact on the number of dead embryos. The number of eggs from young hens remaining for further incubation was high in all groups (≥ 93.0%). Only the lower index in the NCOI group was confirmed statistically (P < 0.05). In the case of eggs from older hens, better (P < 0.05) apparent fertility characterized the SPIDES and NCOI groups as compared to COI and NSP. Hatching from set and apparently fertilized eggs was significantly lower (P < 0.001) in the case of eggs from older hens compared to eggs from younger hens mainly due to the higher (P < 0.001) number of eggs not hatched in the last incubation phase. The use of PI during the storage increased hatchability from set (P = 0.008) and apparently fertilized eggs (P = 0.009) and reduced the number of eggs not hatched (P = 0.022), contrary to the applied turning of eggs during the storage, which produced no effect on these indices (P > 0.05). The interaction of age × treatment in group B resulted in a higher (P < 0.001) hatch from the set eggs, but was not significant for hatching from apparently fertilized eggs, later mortality of embryos and the number of eggs not hatched (P > 0.05). Table 3. Results (means and ±SD) from hatchery of pre-incubation and eggs turning of storage time on fertility, hatchability, late embryonic mortality2, and chicks quality.1 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 1Results of field study. 1Values of 10,692 eggs in each group in both trials. 2Embriony died between 6 and 18 d of incubation. 3Embriony died during hatching. 4% Chicks qualified to rearing by hatchery staff. 5% Chicks not suitable for rearing; disqualified by hatchery staff. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 6Effects of the factors and their interaction were evaluated on all treatments. 7Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 8Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 3. Results (means and ±SD) from hatchery of pre-incubation and eggs turning of storage time on fertility, hatchability, late embryonic mortality2, and chicks quality.1 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 Flock age Treatment Apparent fertility, %2 Hatchability of set eggs, % Hatchability of apparent fertile eggs, % Late3 embryonic mortality, % of apparent fertile eggs, % No hatched chicks4, % of apparent fertile eggs, % First grade chicks5, % A COI 95.0 ± 1.80a 91.8 ± 3.14e 96.6 ± 2.81b 0.117 ± 0.25a 3.25 ± 2.83a 99.3 ± 2.41a NSP 94.8 ± 1.69a 88.3 ± 3.66c,d 93.1 ± 3.45a 0.235 ± 0.44a,b 6.63 ± 3.34b 98.9 ± 2.08a SPIDES 94.3 ± 2.05a 89.9 ± 2.69d 95.3 ± 2.60b 0.218 ± 0.38a,b 4.44 ± 2.65a 99.6 ± 3.11a NCOI 93.0 ± 2.58b 86.5 ± 4.03c 93.0 ± 3.62a 0.441 ± 0.55b 6.55 ± 3.52b 99.1 ± 1.77a B COI 86.2 ± 2.60d 80.5 ± 3.41a 93.4 ± 3.78a 0.496 ± 0.61b 6.07 ± 3.74b 98.7 ± 1.02a NSP 86.3 ± 3.44d 79.9 ± 5.46a 92.5 ± 3.77a 0.448 ± 0.56b 7.07 ± 3.69b 98.7 ± 2.11a SPIDES 89.8 ± 1.79c 83.3 ± 3.38b 92.7 ± 3.01a 0.292 ± 0.41a,b 7.00 ± 2.99b 98.6 ± 2.36a NCOI 90.5 ± 2.43c 83.4 ± 3.91b 92.1 ± 3.23a 0.410 ± 0.64b 7.48 ± 3.33b 98.6 ± 4.00a P-values based on complete dataset (all treatments)  Effect of age6 <0.001* <0.001* <0.001* 0.011 <0.001* 0.101  Effect of treatment6 <0.001* 0.001 <0.001* 0.266 <0.001* 0.344  Age x Treatment6 <0.001* <0.001* 0.074 0.110 0.103 0.190 P-values based on subsets of the data (selected treatments)  Effect of PI7 0.386 0.008* 0.009* 0.060 0.022* 0.056  Effects of eggs turning8 0.013* 0.286 0.692 0.393 0.790 0.833 1Results of field study. 1Values of 10,692 eggs in each group in both trials. 2Embriony died between 6 and 18 d of incubation. 3Embriony died during hatching. 4% Chicks qualified to rearing by hatchery staff. 5% Chicks not suitable for rearing; disqualified by hatchery staff. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 6Effects of the factors and their interaction were evaluated on all treatments. 7Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 8Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Lower rates of fertilization, hatchability, and higher embryonic mortality at various incubation periods of older hens’ eggs are caused by a number of biological factors such as decreased sperm retention in the uterovaginal sperm host glands (Fasenko et al., 1992) and deteriorating egg quality (Reijrink et al., 2008). In this study, 2 × 4 h PI during 12 d of eggs storage allowed for an improvement of hatchability, mainly from eggs of older hens. This result is consistent with previous reports by Yassin et al. (2008) and Reijrink et al. (2010), who also improved hatching from eggs from older hens stored for 11 d and treated with PI. In the present study, although the effect of PI on hatching results was more significant in the case of eggs from older hens, it was also beneficial in case of the eggs from younger hens. This is evidenced primarily by the fact that hatching of eggs set and apparently fertilized in the A SPIDES group was significantly higher (P < 0.05) compared to the A NCOI group, and also higher (P < 0.05) in the case of hatching from apparently fertilized eggs and comparable in case of hatching from the set eggs compared to the A NSP group. Lower indices compared to the COI group were most likely associated with a longer storage period. Therefore, the obtained results indicate the general beneficial effect of PI on the hatchability indexes of eggs stored for a long time. It can be assumed, however, that with the age of hens, the beneficial effect of PI is related to the impact on other mechanisms of embryonic development, such as enabling to achieve hypoblast stage of the embryos safe for longer storage in younger hens’ eggs, and counteracting the deteriorating possibility of pH regulating by embryo on the eggs of older hens. The turning of eggs during the storage, as opposed to previously discussed indices (early embryonic development, time and synchronization of hatching), was neither positive nor negative in the case of hatchability results. Only apparent fertilization was advantageously dependent on the turning of eggs during the storage, but this concerned only the eggs from young hens where the lowest (P < 0.05) value was observed in the A NCOI group. Elibol et al. (2002) demonstrated that the turning of eggs during the storage may positively affect the hatching results in case of both eggs from young and old hens, but the effect is greater when the eggs are generally characterized by a low fertility rate and high survival rate of embryos. Egg fertilization in the experiment of Elibol et al. (2002) was at the level of 50.3–83.7% for the flock at the age of 62 wks, and therefore much lower compared to the apparent fertilization of eggs from hens from group B (70—73 wks) in this study: 86.2—90.5%. It can therefore be assumed that the generally high hatching quality of the eggs was the reason for the lack of influence of the turning of eggs during the storage. Earlier, Lundy (1969) demonstrated that high quality hatching eggs are less sensitive to the change in their position during the storage. Chicks Quality and Juvenile Growth Table 3 shows the percentage of chickens in the first grade of quality, based on the classification made in commercial conditions by the hatchery staff, and Table 4 shows the quality of chicks evaluated according to the methodology of Tona et al. (2003a). Generally, the chicks from the COI groups, i.e., hatched from eggs stored 5 d, were the best quality. Significant (P < 0.05) effect on the quality of chicks in case of the eggs stored 12 d in both analyzes was noted for an age of hens and the use of PI during egg storage. Chicks from younger hens and those that hatched in the SPIDES groups were characterized by better quality. Chicks from older hens usually have higher BW which is associated with higher egg weight and a strong correlation between these traits (Pinchasov, 1991; Willemsen et al., 2008). These results were confirmed in this study (Table 5 and Figure 4). For this reason, BW was not taken into account when evaluating the quality on the Tona's scale when chicks from flocks of different ages are compared, especially that according to Dymond et al. (2013) it shows positive correlations only with BW in the first 2—3 wks of life. In turn, BW in this study does not correlate with BW at the age of slaughter (Figure 4). According to Robinson et al. (1991) and Fasenko et al. (1992) poorer hatchability of eggs from older hens is associated with aging of the preovulatory oocyte and changes in the composition of yolk, but it cannot be excluded that it may contribute to poorer quality of chicks for which these changes are not fatal in the embryonic period. Contrary to this study, Reijrink et al. (2010) did not show any effect on the quality of day-old chicks under the influence of PI during egg storage. However, the authors took into account other characteristics of the chicks, except for navel quality, such as body dimensions, rectal temperature and the weight of selected organs. This could be the reason for obtaining different results. In the present study, the better quality of the chicks in the A and B SPIDES groups resulted primarily from giving them more points for the appearance of down, activity and no changes in the area of the navel. Poor quality of the navel and down also contributed to the lower score of chicks in other groups, especially in the B NCOI group, where low scores were also awarded for activity and legs. It seems that the better quality of the chicks from the SPIDES groups could have been caused by the hatching process itself. Although the chicks in both groups hatched late (Table 2), they started the hatching late (Figure 3). As a consequence, the chicks in both groups stayed in the hatching apparatus about several hours after the hatching (up to 504 h of incubation). This time is enough to dry the down and straighten the legs so that the chicks can move freely, and at the same time do not cause dehydration due to too long staying at a high temperature without an access to water. Figure 4. View largeDownload slide Correlation results between egg weight and chicks hatching weight (higher up) and chicks hatching weight and chicks weight in 42 d (under) for treatment groups, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation, R2 = coefficients of determinations * significant and NS not significant effect in ANOVA at 0.05 probability level. Figure 4. View largeDownload slide Correlation results between egg weight and chicks hatching weight (higher up) and chicks hatching weight and chicks weight in 42 d (under) for treatment groups, where: A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55%, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation, R2 = coefficients of determinations * significant and NS not significant effect in ANOVA at 0.05 probability level. Table 4. Results (means and ± SD) of pre-incubation and eggs turning of storage time on chicks quality.1,2 Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* 1Results of laboratory study. 2According to Tona et al. (2003a). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 3Effects of the factors and their interaction were evaluated on all treatments. 4Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 5Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 4. Results (means and ± SD) of pre-incubation and eggs turning of storage time on chicks quality.1,2 Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* Flock age Treatment N Chicks with score 100, % Average score of all chicks Average score of chicks with score < 100 A COI 126 81.7b 99.1 ± 0.66a 95.4 ± 1.64a NSP 121 79.3b 97.5 ± 2.75a,b 87.5 ± 3.48b,c SPIDES 123 88.5a 99.0 ± 1.34a 91.8 ± 4.92b NCOI 118 79.6b 96.3 ± 3.12b 82.0 ± 5.22d B COI 114 65.8c 96.7 ± 2.51b 90.4 ± 4.67b NSP 109 53.2d,e 92.4 ± 3.64c 83.8 ± 5.63d SPIDES 115 57.4d 95.5 ± 1.10b 89.4 ± 3.45b NCOI 111 49.5e 85.6 ± 5.97d 71.9 ± 5.52e P-values based on complete dataset (all treatments) Effect of Age3 0.021* 0.001* Effect of Treatment3 0.009* <0.001* Age x Treatment3 0.064 0.054 P-values based on subsets of the data (selected treatments) Effect of PI4 0.001* <0.001* Effects of eggs turning5 0.033* <0.001* 1Results of laboratory study. 2According to Tona et al. (2003a). A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–eDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means. *Significant effect in ANOVA at 0.05 probability level. 3Effects of the factors and their interaction were evaluated on all treatments. 4Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 5Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 5. Means (±SD) for eggs weight and chicks body weight and comparisons based on analysis of variance and Duncan's multiple range test.1 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 1Results of laboratory study and experimental farm. Number of chicks: A COI = 126, A NSP = 120, A SPIDES = 123, A NCOI = 116, B COI = 114, B NSP = 108, B SPIDES = 114, B NCOI = 109. EW = egg weight. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–dDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large Table 5. Means (±SD) for eggs weight and chicks body weight and comparisons based on analysis of variance and Duncan's multiple range test.1 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 Flock age Treatment EW (g) Chicks BW (g) Hatching 7 d 14 d 21 d 28 d 35 d 42 d A COI 59.6 ± 3.2c 40.6 ± 2.4c 135 ± 18b 346 ± 63a 649 ± 124a,b 1124 ± 206a-c 1722 ± 287a 2579 ± 396a NSP 54.6 ± 2.1d 38.8 ± 2.0d 118 ± 11c 292 ± 36b 580 ± 79c 1060 ± 149c 1526 ± 207b 2150 ± 329c SPIDES 54.7 ± 1.8d 39.3 ± 3.0c,d 126 ± 19c 319 ± 65a,b 664 ± 95a 1201 ± 178a,b 1691 ± 231a 2464 ± 293a,b NCOI 55.1 ± 2.3d 39.2 ± 2.6c,d 126 ± 12c 321 ± 49a,b 656 ± 98a 1196 ± 182a,b 1713 ± 274a 2491 ± 342a,b B COI 71.3 ± 3.2a 49.8 ± 2.3a 146 ± 20a 345 ± 53a 627 ± 105a-c 1097 ± 161b,c 1776 ± 248a 2623 ± 350a NSP 66.8 ± 2.5b 47.8 ± 2.5b 138 ± 16a,b 345 ± 51a 682 ± 105a 1205 ± 168a 1752 ± 241a 2472 ± 334b SPIDES 68.0 ± 2.4b 48.0 ± 3.1b 136 ± 13b 313 ± 29a,b 595 ± 68c,b 1124 ± 112a-c 1655 ± 207a,b 2497 ± 321b NCOI 67.6 ± 2.2b 48.5 ± 2.6b 144 ± 15a,b 333 ± 52a 635 ± 100a-c 1138 ± 173a-c 1780 ± 311a 2333 ± 485c P-values based on complete dataset (all treatments) Effect of age2 <0.001* <0.001* <0.001* 0.051 0.862 0.869 0.031* 0.240 Effect of treatment2 — 0.002* 0.001* 0.024* 0.827 0.306 0.077 0.001* Age x Treatment2 — 0.930 0.342 0.021* <0.001* 0.004* 0.081 0.011* P-values based on subsets of the data (selected treatments) Effect of PI3 — 0.746 0.186 0.311 0.378 0.900 0.163 0.352 Effects of eggs turning4 — 0.269 0.011* 0.355 0.436 0.307 0.040* 0.182 1Results of laboratory study and experimental farm. Number of chicks: A COI = 126, A NSP = 120, A SPIDES = 123, A NCOI = 116, B COI = 114, B NSP = 108, B SPIDES = 114, B NCOI = 109. EW = egg weight. A = 49-, 52- wk of breeder hens life; B = 70-, 73- wk of breeder hens life; COI = eggs storage 5 d, at turning every 12 h; NSP = eggs storage 12 d, at turning every 12 h; SPIDES = 4 h pre-incubation at 30°C and 50—55% RH, delivered at 5 and 10 d over of 12 d of cool storage, and turning every 12 h; NCOI = eggs storage 12 d, no turning and no pre-incubation. a–dDifferent letters in columns indicate significant differences based on Duncan's post-hoc comparisons of means *Significant effect in ANOVA at 0.05 probability level. 2Effects of the factors and their interaction were evaluated on all treatments. 3Effect of eggs turning was evaluated using dataset only for NSP and NCOI treatments. 4Effect of pre-incubation was evaluated using dataset only for NSP and SPIDES treatments. View Large The turning of eggs during the storage did not affect the quality of the chicks evaluated by the hatchery staff (Table 3), but significantly affected the chicken improvement according to the Tona's method (Table 4). This result is a consequence of the lowest score given to the chicks from the A and B NCOI groups. Few reports of an effect of turning of eggs during incubation indicate the positive effects of such treatment, which is explained by a better orientation of the embryo before the hatching, restriction of embryo or embryonic membrane adhesion to the shell membranes and stimulation of timely closure of chorioallantis at the sharp end of the egg (Wilson, 1991; Tona et al., 2003b). It seems, however, that the positive effect of turning of eggs during the storage on chicks quality could not be caused by the same factors. Nevertheless, the chicks from the A and B NCOI groups showed the worst quality of down, navel, and the remains of membranes and shells integrated with the body as well as the reluctance to move were the most frequent among them compared to the chicks from other groups. The last three defects negatively affect the evaluation of the chicks in the Tona's score, but they can and probably are neglected at the mass hatching in the hatchery, which was a probable cause of the differences in the evaluation done experimentally and industrially. Mechanisms that have contributed to an improved quality of chicks under the influence of turning of eggs during the storage are unknown and require further research, especially that this is the first report on this topic according to the authors’ knowledge. Analysis of the course of chicks growth demonstrated that age of chicks affected the hatching BW, which was strongly correlated in all experimental groups with egg weight, and BW in 7 and 35 d, but had no effect on BW in 42 d (Table 5 and Figure 4). However, hatching BW of chicks from older hens was significantly higher, as much as 12 g on average (∼ 1/5 hatching BW) compared to hatching BW of the chicks from young hens, and average BW in 42 d was similar, higher for chicks from older hens, only 60 g on average (∼2% BW in 42 d). Particular attention should be paid to BW of the chicks in the A and B NCOI groups. Although the chicks in the A NCOI group had a significantly lower hatching BW compared to the B NCOI group chicks (by 14.5 g at similar variability; 2.2—2.3 g), the BW of the chicks from young hens in 42 d was higher by 158 g on average, and although this is not a significant difference, considering the hatching BW, it can be concluded that chicks from young hens show better growth potential. These results are consistent with previous reports by Tona et al. (2004b), in which there was no correlation of hatching BW with BW in 42 d in broilers, and chicks from younger hens obtained slightly higher BW in 42 d compared to BW of the chicks from older hens, which showed a significantly higher hatching BW. The use of PI and the turning of eggs during the storage did not have a significant positive or negative effect on BW of the chicks in 42 d of life (P > 0.05). However, a significant effect was observed for age × treatment interaction (P = 0.011). Chicks that hatched from older hens’ eggs subjected to PI and eggs turning during the storage (B NSP and B SPIDES) reached a higher BW (P < 0.05) compared to B NCOI chicks. However, this dependence did not occur in the case of chicks hatched from the eggs of younger hens (Table 5). This is probably the consequence of a significantly better quality of chicks at the time of hatching (Table 4). Dymond et al. (2013) showed that the use of 6 or 12 h PI during egg storage did not affect the hatching BW of the chicks, but the chicks from SPIDES groups were heavier than those hatched from eggs without PI applied to 4 wk of life, the authors did not conduct the observation further. In conclusion, the present study showed that the use of 2 × 4 h PI in 12 d egg storage period could have an effect on the initial acceleration of embryonic development in eggs of young hens, contributing to the levelling of embryonic development stage in eggs from young and old hens to 72 h of incubation. The eight-hour PI did not affect the length of the incubation period, HW and BW of the chicks at 42 d of life, but increased the hatchability of the eggs set and apparently fertilized and reduced the number of not hatched eggs, and also affected the quality of the chicks. Turning of eggs every 12 h during the storage had a positive effect on the development of embryos, shortening the incubation time and quality of chicks, but had no effect on hatchability and BW indices up to 42 d of life. The obtained results recommend the use of 2 × 4 h PI with simultaneous turning of eggs every 12 h during the storage to improve the results in industrial hatcheries. In addition, the applied modifications can be used without any harm in the case of an incubation of eggs from both young reproductive flocks and in later laying phase. 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Poultry ScienceOxford University Press

Published: Sep 1, 2018

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